Rapp DB 00-01

TABLE DES MATIERES
Page PREAMBULE__________________________________________________________________________ 2 EFFECTIFS ET BUDGET DE LINSTITUT DE BIOCHIMIE ____________________________________ 4 ENSEIGNEMENTS DISPENSES PAR LINSTITUT DE BIOCHIMIE _____________________________ 8 THESES DE DOCTORAT ________________________________________________________________ 9 RECHERCHES A LINSTITUT DE BIOCHIMIE Leishmania parasites: biology, interaction with host macrophages, and vaccine studies Laboratoire J. Maul et D. Rivier________________________________________________________ 10 Immunobiology of infection with an intracellular parasite: Leishmania major Laboratoire J. Louis (Centre de recherche et de formation en immunologie de lOMS)_______________ 16 Structural and functional studies of virulence factors in the human protozoan parasite Leishmania Laboratoire N. Fasel _________________________________________________________________ 21 Antibodies and peptabodies for tumor targeting Laboratoire J.P. Mach ________________________________________________________________ 26 Immune responses to viruses and protein-hapten antigens Laboratoire H. Acha-Orbea ____________________________________________________________ 33 Mucosal vaccines against oncogenic microbial pathogens Laboratoire J.P. Kraehenbuhl___________________________________________________________ 37 Apoptosis mediated by TNF receptor family members Laboratoire J. Tschopp________________________________________________________________ 43 Malaria: in the search of antigen for the development of protective vaccines Laboratoire G. Corradin_______________________________________________________________ 50 Biochemistry of lymphocyte membrane proteins Laboratoire C. Bron __________________________________________________________________ 54 Molecular mechanisms of T lymphocyte antigen recognition Laboratoire S. Valitutti _______________________________________________________________ 59 Protein and peptide chemistry facility Laboratoire E. Servi__________________________________________________________________ 63 Protein analysis facility Laboratoire M. Quadroni ______________________________________________________________ 67
PUBLICATIONS ______________________________________________________________________ 70
Institut de biochimie, Facult de mdecine, Universit de Lausanne, Ch. des Boveresses 155, CH-1066 Epalinges, Suisse/Switzerland Tl. +41-21-692 57 00, fax +41-21-692 57 05, e-mail: prenom.nom@ib.unil.ch Site web: http://www.unil.ch/ib
PREAMBULE
Ltude du systme immunitaire et des mcanismes biochimiques de sa rgulation demeure un axe de recherche important lInstitut de Biochimie (IB), ainsi quen tmoignent les 134 publications originales et revues produites par les divers laboratoires durant ces deux annes coules. Soulignons en particulier les travaux du laboratoire du Centre OMS qui, en collaboration avec plusieurs quipes internationales, ont mis en vidence les effets opposs que la cytokine IL-4 peut exercer sur la diffrentiation des lymphocytes T en cellules effectrices Th1 et Th2 et les mcanismes cellulaires et molculaires responsables de ces effets (rf. 11). Dautre part, les recherches de base orientes vers le dveloppement de vaccins contre diverses maladies virales (HIV, HPV) ou parasitaires (Malaria, Leishmaniose) ont progress et sont pour certaines entres dans des phases dtudes cliniques destines tester des substances vaccinales obtenues par synthse ou recombinaison gntique. Ainsi, par exemple, un peptide de synthse du Plasmodium falciparum, qui induit une forte rponse immunitaire humorale et cellulaire chez ltre humain (rf. 63), fait actuellement lobjet dune tude clinique soutenue par la European Malaria Vaccine Initiative et sinscrit dans un effort de coopration internationale entre onze pays. Progressivement cependant, la pertinence biologique des questions abordes dans les recherches de lIB a dpass le cadre de limmunologie. Ainsi, ltude des voies biochimiques de signalisation cellulaire, en particulier celles qui sont contrles par les facteurs de la famille TNF (Tumor Necrosis Factor), est devenue un sujet central lIB comme le reflte limportance des ressources et des publications consacres ce domaine. Parmi les contributions faites au cours de cette dernire priode, citons la mise en vidence du facteur BAFF (rf. 94, 95), un nouveau membre de la famille des TNF, essentiel pour la survie et la diffrenciation des lymphocytes B. Ces travaux ouvrent dimportantes perspectives pour la comprhension et le traitement de maladies autoimmunes et de lymphomes. Les projets qui concernent ltude des voies biochimiques qui sous-tendent les mcanismes de signalisation, de prolifration, de diffrenciation ou dapoptose cellulaire relvent la fois de 2
limmunologie, de linflammation et de loncologie exprimentale. Ils renforcent donc les thmatiques du Centre de Recherche Biomdicale et accroissent le potentiel de coopration entre les instituts partenaires du Centre, lInstitut Suisse de Recherche Exprimentale sur le Cancer (ISREC), lInstitut Ludwig de Recherche sur le Cancer (ILRC) et lInstitut Suisse de Bioinformatique (ISB). Plusieurs groupes de lIB ont ds lors t associs au Programme de Recherche National (PRN) Oncologie molculaire, de la recherche fondamentale aux approches thrapeutiques dont la conduite a t confie ds janvier 2001 lISREC, centre dexcellence dans ce domaine. Ces exemples de lvolution des projets de recherche de lIB refltent galement le renforcement de lorientation vers des questions dintrt clinique et thrapeutique intensifiant ainsi la collaboration avec des services du CHUV, du HUG et dautres centres hospitaliers universitaires. La convergence des intrts scientifiques du consortium dinstituts dEpalinges facilite la concertation lors du recrutement de nouveaux collaborateurs, de la mise en place de platesformes technologiques ou de lacquisition dune instrumentation coteuse. La complmentarit scientifique recherche dans les thmatiques des divers partenaires a pour but daborder des programmes de recherche plus ambitieux, datteindre un niveau de qualit des contributions scientifiques qui puisse assurer la participation un rseau de coopration internationale et crer des conditions susceptibles dattirer des jeunes chercheurs talentueux. La diversification des projets de recherche, des modles exprimentaux et des techniques dinvestigation opre par lIB dans le cadre des thmatiques du Centre de Recherche Biomdicale permet de surcrot de maintenir lexpertise requise pour assurer avec comptence les nombreux enseignements de biochimie dont notre institut a la responsabilit, en particulier en Facult de mdecine. Les progrs rapides de la biologie, de la gntique et de la technologie molculaire ont un impact direct sur la comprhension et le traitement des maladies; ils exigent plus que jamais une rflexion permanente sur ladquation de nos enseignements en mdecine. La pluridisciplinarit du Centre offre prcisment les conditions qui nous permettent de
PREAMBULE
garder un ventail de comptences dans plusieurs domaines de la biochimie et nous confre ainsi la capacit dassurer cette responsabilit primordiale. A cet gard, il convient une fois encore de souligner la coopration souvent bnvole de lensemble de la communaut scientifique du Centre dans la ralisation de cette mission. Lattrait et le bnfice pour les tudiants sont manifestes puisque le Centre accueille en permanence prs de 70 doctorants de lUNIL, de Suisse ou de ltranger. Cest dans ce contexte que sinscrit le premier dune srie de remplacements au sein du corps enseignant de lIB: le Dr. Gian-Paolo Dotto, actuellement professeur la Harvard Medical School, a t appel succder au Prof. JeanPierre Mach. Par ses intrts pour les mcanismes molculaires de la croissance et de la diffrenciation des cellules pithliales, lhomostasie de la peau et la biologie des cellules souches, le Dr. Gian-Paolo Dotto renforcera le potentiel de coopration scientifique de lIB avec lISREC et divers services cliniques, en particulier du CHUV. Il entrera en fonction en septembre 2002. Au bnfice dimportants subsides de recherche et libr de ses tches universitaires, le Prof. Jean-Pierre Mach poursuit ses travaux axs sur le dveloppement de mthodes immunologiques destines lidentification, le dpistage prcoce et le traitement de tumeurs cancreuses; travaux qui lui ont valu une rputation internationale. Dautre part, les Drs Walter Hunziker et Salvatore Valitutti, tous deux professeurs assistants lIB, au bnfice de bourses START, Leenaards et SCORE A, ont quitt lIB suite leur nomination des postes professoraux sdentariss, respectivement lInstitute of Molecular and Cell Biology de lUniversit de Singapour et lINSERM, Institut Claude de Preval de lUniversit Paul Sabatier de Toulouse. On peut regretter le dpart de ces deux chercheurs faisant partie de la relve acadmique et esprer que la
cration lUniversit de Lausanne de nouveaux postes de professeurs assistants avec prtitularisation conditionnelle permettra notre Institut dtre mieux soutenu dans une politique de relve acadmique applique depuis de nombreuses annes et dviter le dpart de chercheurs talentueux ltranger. Laccs des chercheurs toutes les techniques les plus modernes dinvestigation de la gntique molculaire, de la structure des protines et de la morphologie est un paramtre indispensable la progression efficace des projets de recherche. Cest donc galement dans la mise jour de ces technologies de pointe et dans lacquisition des savoir-faire spcifiques et dune instrumentation coteuse que la politique concerte entre les instituts trouve sa pleine justification. Dans ce contexte, lIB a entrepris, au cours de ces deux dernires annes, avec le soutien de la Facult de mdecine et de lUniversit dans le cadre du projet Science, Vie et Socit, la mise en place dune plate-forme de protomique destine terme tre accessible la communaut scientifique de Lausanne (p. 67-69). Enfin, dans notre dernier rapport, nous faisions tat de la progression des travaux dinstallation dune premire unit de transfert de technologie manant du Centre de Recherche Biomdicale. Depuis plus dune anne, le Biople est oprationnel et abrite six entreprises de technologie biomdicale. Grce au soutien de lEtat de Vaud et lintrt manifest entre autres par les Dpartements de lEconomie et des Infrastructures, cette premire unit, qui contribue la symbiose entre la recherche biomdicale fondamentale et la technologie qui en dcoule, devrait tre le catalyseur de dveloppements plus importants sur le site de Vennes dont lUniversit et les Hospices Cantonaux Vaudois seront deux partenaires dterminants. C. Bron
EFFECTIFS ET BUDGET DE LINSTITUT DE BIOCHIMIE
Personnel rtribu par lUNIL: 47.4 EPT (quivalent plein temps) * Personnel rtribu par des fonds privs: 33.7 EPT 1. EFFECTIFS DE LINSTITUT DE BIOCHIMIE (dcembre 2001) Corps enseignant Professeur associ (50% Institut Ludwig de Recherche sur le Cancer) Professeur ordinaire - Directeur Professeur associ Professeur associ Professeur ordinaire (50% ISREC) Professeur extraordinaire (OMS) Professeur honoraire Professeur ordinaire Matre denseignement et de recherche Professeur ordinaire
H. Acha-Orbea C. Bron G. Corradin N. Fasel J.P. Kraehenbuhl J. Louis * J.P. Mach J. Maul D. Rivier J. Tschopp Corps intermdiaire
S. Corradin-Betz * M. Quadroni P. Schneider E. Servi * M. Thome Miazza Privat-Docents
Charge de recherche Matre-assistant Professeur assistant remplaant Cheffe de projet Matre-assistante
F. Buchegger S. Demotz S. Valitutti Assistant(e)s
Postdoctorant(e)s R. Audran * C. Beghdadi * V. Blancheteau * C. Bochud * K. Burns Doctorant(e)s N. Acestor L. Agostini B. Brissoni V. Brossard * V. Cesson * H. Chavez * Stagiaires B. Valzasina * 4 A. Didierlaurent B. Favier * O. Gaide * G. Ibrahim B. Jaccard F. Martinon V. Meraldi S. Monnerat J. Romero C. Schaff F. Sierro Q.G. Steiner A. Tinel J. Wagner R. Zaru * N. Debard * M. Delgado M.-A. Doucey D. Legler S. Masina O. Micheau M. Mpandi L. Otten * K. Peter * B. Robert * S. Rothenberger M. Thurau *
Personnel Technique
Laborantin(e)s G. Badic D. Bonnet * C. Desponds * K. Fournier * Aide laborantine L. Morgado * Apprenti(e)s S. Brunetti (IBCM) Services Gnraux Service de synthse et danalyse de peptides N. Lvy * (laborantine) E. Servi * (chimiste) F. Penea (laborantine) Atelier D. Roy Laverie L. Skupienski R. Cornaz S. Zutter S. Levrand * N. Lvy * N. Olivos * F. Prevel * L. Rodrigues * A. Tardivel * M. Corthsy * S. Hertig M. Rousseaux A. Ransijn M. Reinhardt C. Mattmann
Secrtariat S. Aslan (75%) F. Flejszman (80%) * M. Jayet Herzstein Bibliothque F. Sacco (25%) Magasin central et Travaux pratiques E. Margot U. Margot (50%) Hte sabbatique
A. Kummer
Free University Hospital, Dpt de pathologie, Amsterdam, Pays-Bas
2. EFFECTIFS DU CENTRE OMS DE RECHERCHE ET DE FORMATION EN IMMUNOLOGIE J. Louis * F. Tacchini-Cottier * H. Voigt A. Gumy R. Chakour * A. Grand * Y. Hauyon * F. Flejszman * Professeur extraordinaire - Directeur Chercheur Postdoctorante Doctorant Doctorant Laborantin Laborantine Secrtaire
3. EFFECTIFS DE LA FONDATION INSTITUT SUISSE DES VITAMINES M. Bui* M. Desbes * D. Schtz-Geser * Cheffe de laboratoire Laborantine Laborantine 5
4. MEMBRES DU PERSONNEL AYANT QUITTE LINSTITUT DE BIOCHIMIE, LE CENTRE OMS ET LA FONDATION ISV EN 2000-2001 Corps enseignant (avril 2000) S. Valitutti (novembre 2000)
W. Hunziker
Corps intermdiaire
M. Kovacsovics (septembre 2001) Assistant(e)s
Postdoctorant(e)s A. Aseffa F. Bender J.L. Bodmer D. Deperthes V. Estienne L. Hathaway R. Jones Doctorant(e)s A. Bonelo S. Cloutier G. Fotopoulos H. Himmelrich N. Holler M. Hunn Diplmant(e)s V. Crivelli K. Giddey Stagiaires M. Bazzaro G. Bonomi A. Dohrman V. Dutoit F. Estvez C. Hetz J. Khaldoune S. Janssens O. Kalyuzhnity (septembre 2000) (novembre 2000) (aot 2001) (juin 2001) (mai 2000) (mars 2001) (juillet 2000) (mai 2001) (mai 2001) M. Kaufmann S. Lauriault C. Perrier N. Restrepo B. Rodriguez R. Rodriguez E. Rojas A. Schmid M. Sissoko (juin 2001) (aot 2001) (octobre 2001) (septembre 2001) (novembre 2001) (avril 2001) (mars 2000) (octobre 2001) (octobre 2001) (mai 2000) (mai 2000) L. Marguerat K. Soudani (avril 2000) (octobre 2000) (mars 2000) (mars 2000) (septembre 2000) (mars 2000) (octobre 2000) (janvier 2000) D. Penna A. Praetor B. Saucy I. Segura H. Zangger (juin 2001) (aot 2000) (octobre 2000) (juin 2001) (aot 2001) (dcembre 2000) (aot 2001) (septembre 2001) (mars 2000) (juillet 2000) (septembre 2000) (septembre 2000) S. Lens F. Niedergang J. Peli M. Reichert F. Rharbaoui V. Rochat H. Takatsuka (dcembre 2000) (janvier 2001) (septembre 2000) (septembre 2000) (janvier 2001) (juin 2001) (mars 2001)
Personnel technique
Laborantin(e)s C. Aletti R. Castillo J. Clerc L. Martin Apprenti(e)s I. Bassi Stagiaires K. Ingold M. Gonzalez (mai 2001) (novembre 2000) A. Thiocone (juillet 2000) (juin 2000) (aot 2001) (fvrier 2001) (septembre 2000) (janvier 2001) C. Mossu S. Reynard S. Valitutti (juin 2000) (septembre 2001) (avril 2001)
Services Gnraux
Comptabilit D. Reichen (octobre 2001)
Photo- Infographie P. Gschwend (aot 2001)
Dpart la retraite
Magasin central et Travaux pratiques E. Margot (dcembre 2001)
5. BUDGET 2001
Industrie 7% Fonds institutionnels 11% UNIL 51% FNRS et autres fonds 31%
UNIL (Etat de Vaud): CHF 5'134629 Ressources extrieures: CHF 4'782503
ENSEIGNEMENT
INSTITUT DE BIOCHIMIE - ENSEIGNEMENTS
Nombre dheures/an C* S* TP* 1er cycle Cours gnral I Cours gnral II Cours intgrs Cours gnral III Cours gnral IV 2me cycle - modules Biologie molculaire et cellulaire - Structure des protines Biochimie cellulaire Immunologie molculaire 12 138 72 19 32 254 56 40 36 76 44 54 105 160
Etudiants T*
Nombre tudiants
mdecine (1e anne) biologie (1e anne) mdecine (2e anne) mdecine (2 anne) biologie (2 anne) pharmacie (2e anne) pharmacie (2e anne)
e e
172 106 110 110 65 23 23
biologie (5e semestre) 168 224 biologie (3 /4 anne) biologie (3 /4 anne)

e e e e
40 23 23
* C = cours; S = sminaires; TP = travaux pratiques; T = travail personnel. 3me cycle Enseignements postgradus des Instituts du Centre de Recherche d'Epalinges (BIL): 2000 - Immunology - Cell division and cell death H. Acha-Orbea M. Peter (ISREC) et J. Tschopp 27 participants 35 participants
Enseignement postgradu sous l'gide de l'OMS, organis en collaboration et avec le soutien de la Direction suisse du Dveloppement et de la Coopration internationale (DDC) Advanced WHO course on immunology, vaccinology and biotechnology applied to infectious diseases. Epalinges, September 13 to October 28, 2000. (J. Louis, F. Tacchini-Cottier and C. Aletti), 19 participants. Cours OMS de spcialisation en immunologie, vaccinologie et biotechnologie appliques aux maladies infectieuses Epalinges, 10 mai au 23 juin 2001. (J. Louis, F. Tacchini-Cottier et C. Aletti), 16 participants. Advanced WHO course on immunology, vaccinology and biotechnology applied to infectious diseases. Epalinges, September 13 to October 27, 2001. (J. Louis, F. Tacchini-Cottier and Y. Hauyon), 16 participants.
THESES DE DOCTORAT
2000 Antoine Attinger Etude du comportement des lymphocytes T activs par un superantigne bactrien. Les nouveaux rcepteurs de la mort cellulaire programme. Signalisation par le rcepteur FAS. Transport Intracellulaire et Structure Oligomrique du Rcepteur aux IgG, homologue aux Molcules de Classe I du Complexe Majeur d'Histocompatibilit, FcRn. Rgulation de la Protine Kinase C: Rle des lipides, des protines et de la localisation intracellulaire. FIST-3, une protine qui interagit avec Fas, un rcepteur induisant la mort cellulaire, et qui bloque des facteurs de transcription.
Jean-Luc Bodmer Nils Holler Asja Praetor
Brangre Saucy
Vronique Steiner
2001 Grigorios Fotopoulos L'infection et le franchissement par transcytose de monocouches de cellules pithliales par le virus VIH-1 dpend de leur tat de diffrenciation et de l'expression des rcepteurs aux chimiokines. Rle des anticorps dirigs contre la protine majeure d'enveloppe (gp52) dans la prvention systmique et mucosale contre l'infection par le rtrovirus de la tumeur mammaire de la souris. La dgradation de ZAP-70 fait partie des mcanismes impliqus dans le contrle de la rponse immunitaire par les lymphocytes T.
Michel Mpandi
Doris Penna
LEISHMANIA PARASITES: BIOLOGY, INTERACTION WITH HOST MACROPHAGES, AND VACCINE STUDIES
Jacques Maul et Denis Rivier
Denis Rivier graduated in medicine and biological sciences from the University of Lausanne in 1964 and 1972. He did an MD at the Institute of Pharmacology of the University of Bern. He studied mucosal immune reponses in the Institute of Biochemistry (University of Lausanne) and in the gastrointestinal department (University of California, San Diego). He was appointed Research associate in 1986. His recent interests are in Leishmania vaccine and immunomodulators.
Jacques Maul graduated in Biological Sciences from the University of Lausanne in 1967. He then did a PhD at the Swiss Institute for Experimental Cancer Research, on the cytotoxic mechanisms of T lymphocytes. He then moved to the Wistar Institute in Philadelphia, where he studied macrophage differentiation, before joining the WHO Laboratories at the Institute of Biochemistry, Lausanne, to work on macrophage interaction with Leishmania parasites. He was appointed full professor in 1980. His recent interests have focused on nitric oxide production by human macrophages and on macrophage downregulation by Leishmania.
Group members 2000-2001 Florence Prevel Adriana Ransijn Josiane Smith Clerc Sally Corradin-Betz Technician Technician Technician (09.2000) Research associate Maria Delgado Michel Mpandi Iris Segura Sbastien Brunetti Postdoctoral fellow Postdoctoral fellow Ph.D. student (06.2001) Apprentice
LEISHMANIA PARASITES: BIOLOGY, INTERACTION WITH HOST MACROPHAGES, AND VACCINE STUDIES Leishmania is a protozoan parasite which can infect man, causing leishmaniasis. Leishmaniasis is a serious disease which affects millions of people worldwide, particularly in tropical and subtropical countries (Fig.1). Existing drugs are toxic and vaccination is not available. Leishmania is an obligate intracellular microorganism which resides within phagolysosomes of macrophages (m) (Fig. 2), cells that help our body fight infection and are responsible for destruction of many microbes. However, for reasons that are under investigation, m do not easily kill Leishmania. 10
MacMARCKS (McM) is an abundant m protein believed to be involved in processes such as cell division, migration and phagocytosis. Recently, the observation was made that infection of m by Leishmania parasites results in downregulation of McM (Fig. 3). Our laboratory is investigating this interesting phenomenon. In addition, an experimental model of vaccination in the mouse is used to test the effect of various novel adjuvants on the immune response. Finally, our lab is investigating the capacity of nicotine conjugates on protein carriers to be used as potential vaccines against smoking addiction. Fig. 1 Fig. 2 Fig. 3
A case of cutaneous leishmaniasis (Credit : Dr. R. Behin)
Leishmania in macrophages
(a) McM (green) in macrophages (b) Disappearance of McM in Leishmania-infected macrophages
1. Leishmania, MacMARCKS, "trojan" peptides, and macrophage apoptosis McM degradation by Leishmania gp63.
Infection by Leishmania modulates m signaling pathways leading to inhibition of m functions. In particular, PKC-dependent signaling, a process perhaps critical to parasite establishment in the host, appears to be strongly affected. If the mechanisms whereby Leishmania downregulates PKC activity could be determined, new tools to combat the disease might be designed. MARCKS (Myristoylated Alanine-Rich C Kinase Substrate) and MacMARCKS (McM; macrophage MARCKS) are major protein kinase C (PKC) substrates postulated to regulate the structure of the actin cytoskeleton and thereby participate in characteristic m responses such as
phagocytosis, secretion and membrane recycling. We therefore examined the effect of infection by Leishmania on McM. Leishmania was found to dramatically reduce McM levels in activated murine m, apparently due to proteolytic degradation. This effect could be ascribed to the L e i s h m a n i a surface metalloproteinase leishmanolysin (PSP, gp63). Cleavage was found to occur within a specific 20-amino acid sequence of McM called the effector domain (ED), also known as the phosphorylation site domain, since it contains serine residues that may be subject to PKC-dependent phosphorylation. Strikingly, rMcM degradation by gp63 in a cell-free system was inhibited by prior PKC-dependent phosphorylation or by deletion of its phosphorylation site domain, clearly identifying this region as being the target of gp63 proteolytic activity.
11
Inhibition of gp63 by McM ED peptides.
Determining the role of gp63 in Leishmania physiology has been hampered by the lack of specific, non-toxic inhibitors of the protease. We postulated that such inhibitors could be designed, based on the ED sequence of McM (McMED) that is recognized by the enzyme (see above). Indeed, McM degradation by gp63 in a cell-free assay was efficiently blocked by addition of free ED peptides. As phosphorylation of ED had been found to render the molecule insensitive to gp63 mediated degradation, we wondered if a phosphorylated McMED peptide might indeed be an even better inhibitor of gp63 than the nonphosphorylated peptide. Phosphorylated peptides are unstable however ; we therefore constructed a "pseudo-phosphorylated" peptide in which 3 phosphorylatable serine residues were substituted by aspartate (hence the designation of this peptide by the acronym "3D"), which because of its charge and size mimics to some extent phosphoserine. Interestingly, this 3DMcMED peptide was ten fold more efficient than the unmodified peptide as an inhibitor of McM degradation by gp63. These observations might serve as basis for developing more potent and specific inhibitors of gp63 and possibly of the parasite itself (see Section Effects of cell permeable...). Tat-McM peptides and m apoptosis.
peptides do enter both m and other cells, and their effect on the parasite are being investigated. Surprisingly however, exposure of murine or human m to TatTD-McMED resulted in rapid apoptosis of the cells. The same effect was observed on other cell types, such as HeLa cells (fibroblasts). Apoptosis correlated with cleavage of poly-ADP ribose polymerase (PARP) and activation of caspases 3, 7 and 9 (but not of caspase 8), characteristic of the stress-induced pathway of apoptosis. However, no release of cytochrome c was observed, except at late time points following exposure to TatTD-McMED. DNA laddering was also demonstrated, and cell death could be prevented by the caspase inhibitor ZVAD-fmk, confirming that death is a result of apoptosis. In addition, rapid and profond disruption of actin stress fibers in fibroblasts as revealed by phalloidin staining could be observed (see Fig 4). Interestingly, in all of these experiments, the 3D analog of McMED coupled to Tat (TatTD-3DMcMED) was much less toxic. In attempts to understand the mechanisms by which TatTD-MCMED induces apoptosis, we tested analogs including a scrambled peptide identical to the TatTD-McMED in amino acid composition but containing an altered ED sequence. Surprisingly, the scrambled peptide was as effective as TatTDMcMED suggesting that charge and not sequence is responsible for its effect on cells. These results suggest that the recently described capacity of McMED to bind and sequester PIP2 v i a electrostatic interactions may be relevant to the apoptotic response of the cells. McM in human macrophages.
For any agent aimed at interfering with the activity of an intracellular microorganism, one major difficulty is to reach the microbe in its intracellular compartment. Fortunately, a newly recognized set of molecules may help us do just that. Indeed, the HIV protein Tat contains a highly basic eleven amino acid region known as the transduction domain (TD) which allows the protein to cross cell membranes by an as yet uncharacterized mechanism. Several similar sequences have been described in unrelated proteins and the corresponding peptides have been termed "trojan peptides" based on their ability to allow other peptides or proteins synthesized in tandem with the trojan moiety to cross the plasma membrane. We have now synthesized a series of Tat TD peptides (TATTD) including TatTD-McMED and an analog containing the 3DMcMED sequence. These
McM is also expressed in human macrophages. When cells of the monocytoid line U937 were differentiated with vitamin D3 plus retinoic acid, strong downregulation of McM expression was observed. As for murine macrophages, McM is reinduced by stimulation with LPS or a phagocytic stimulus. The intracellular localization of McM was investigated by immunofluorescence and confocal microscopy. McM appeared as a population of small vesicles throughout the cytoplasm but concentrated to some degree at the cell periphery. Although McM was previously proposed to cycle between the plasma membrane and lysosomes in a PKC-dependent manner (as demonstrated for MARCKS), antibodies against
12
Fig. 4 : HeLa cells stained with phalloidin to reveal actin stress fibers
Control cells : actin fibers are clearly visible
Same cells, but treated with Same cells, treated with TatTD-McMED(10 M) for TatTD-McMED(10 M) for Note disappearance of stress fibers. entry occurs by a nonreceptor mediated mechanism as previously shown for Tat-mediated transduction in other systems. These and other results suggest that Leishmania were undergoing apoptosis in analogy to mammalian cells treated with TatTD-McMED although it remains unclear as to whether or not apoptosis as such exists in protozoan parasites. Attempts to demonstrate DNA fragmentation in peptide-treated promastigotes have been unsuccessful to date. Higher concentrations of TatTD-McMED were required to observe effects than those required to induce apoptosis in mammalian cells. However, a strain of L. mexicana which lacks surface gp63 was significantly more sensitive to TatTD-McMED than its reconstituted counterpart or wildtype L. mexicana. This suggests that surface gp63 cleaves TatTD-McMED before it enters the parasites, thus decreasing its toxic effects. Moreover, the enhanced sensitivity of gp63-deficient parasites indicates that inhibition of gp63 activity is not involved in the toxic effects of TatTD-McMED. Considering the multiple effects which TatTDMcMED might exert intracellularly (PIP2 binding or sequestration, PKC activation or inhibition, actin or calmodulin binding, etc), it is likely that the effects of TatTD-McMED on parasites are due to pathways unrelated to gp63. Recently, a novel peptide capable of forming a noncovalent
McM and the lysosomal marker LAMP1 gave distinct patterns of staining with or without pretreatment by the PKC activator PMA. An association was also reported between McM and dynamitin, a subunit of the dynactin complex which is a positive regulator of the microtubuledependent motor protein dynein. When we costained cells with antibodies against McM and dynamitin, a similar distribution of the two proteins was indeed observed. However, McM and dynamitin did not appear to colocalize to a significant degree. Effects of cell permeable analogs of McMED on parasite viability and function.
Inasmuch as TatTD-McMED induced apoptosis in various cell types, we examined the effect of TatTD-MCMED on Leishmania promastigotes. Parasites treated with TatTD-McMED showed a striking decrease in motility as well as decreased incorporation of [3H]thymidine and increased staining with a membrane impermeant dye which becomes fluorescent upon binding to DNA. Confocal microscopy with FITC analogs confirmed that TatTD-MCMED (but not McMED alone) entered the parasites. Moreover, the effects of TatTD-McMED on [3H]thymidine incorporation were equally pronounced when peptide was added to parasites at 0 or 26 C suggesting that
13
complex with diverse peptides and proteins was reported to induce transduction of these molecules. Experiments using this peptide are in progress to determine if it will allow the transduction of McMED in parasites and macrophages. This technology may be of value in investigating the mechanisms by which TatTDMcMED affects cell function(s). For example, transduction of purified calmodulin might be able to reverse the TatTD-McMED -induced effects if calmodulin-dependent processes are involved. Moreover, it will be of interest to determine if transduction of full length recombinant McM occurs and is able to mimic certain effects of the McMED alone. 2. In search of an anti-Leishmania vaccine In the search for a Leishmania vaccine, the design of immunoadjuvants potentiating preferentially the TH1 response is important, since immune protection against this parasite is mediated largely by CD4+ T cells of TH1 lineage. Microorganisms are known to be a source of compounds (CpG motifs, lipid A from the wall of Gram-negative bacteria, etc.) that stimulate immune responses. To study in details the immunoadjuvant potential of some of these molecules, different analogs and derivatives of lipid A have been tested and shown to strongly stimulate specific T-cell proliferation and antibody production in mice immunized against purified proteins of Leishmania. Studies of the effects of these molecules on the course of L e i s h m a n i a infection are in progress. Furthermore, to test for activity and study the mechanisms of action of these different analogs, in vitro models of immunization against Leishmania are being developed, using murine spleen cells and human peripheral blood mononuclear cells.
3. A vaccine against smoking addiction ? Cigarette smoking constitutes a major public health problem in both developed and developing countries, where treatment of the pathologies associated with tobacco consumption represents an ever increasing financial burden for health authorities. Nicotine is a strongly addictive drug and smoking creates a state of dependence in many individuals. Although different ways of combating nicotine addiction are available (nicotine patches, chewing gums, psychological help, etc.), these often fail and other avenues must be explored. One such approach consists in developing a vaccine capable of eliciting humoral antibodies that might inhibit nicotine from accessing its receptors in the brain, thus rendering the drug physiologically inert. This concept is also being tested in several other laboratories as an aid in the fight against other drugs of abuse such as cocaine and heroin. Mice immunized with nicotine conjugated to different protein carriers do produce antibodies capable of recognizing the hapten coupled to bovine serum albumin, as well as free nicotine. Even intra-nasal instillation of nicotine-carrier conjugates is capable of eliciting anti-nicotine serum IgG as well as secretory IgA antibodies, which might have the advantage of blocking nicotine already at its port of entry, i.e. the mucosal surfaces. The presence of antibodies appears to markedly alter the distribution of the drug in the immunized animals, particularly by restricting its access to the brain. These experiments are a prerequisite for clinical studies which should be initiated in the near future.
Recent publications Marzio, R., Jirillo, E., Ransijn, A. Maul, J., Corradin S.B. (1997). Expression and function of the early activation antigen CD69 in murine macrophages. J. Leukoc. Biol. 62 (3), 349-355. Maul, J., Ransijn, A. (1997). Leishmania spp.: Mechanisms of toxicity of Nitrogen oxidation products. Exp. Parasitol. 87, 98-111.
Le Roy, D., Heumann, D., Glauser, M.P., Maul, J., Smith, J., Betz Corradin, S. (1998). Nitric oxide production in experimental gram-negative infection: studies with cytokine receptor-deficient mice. Shock 10, 37-42. Bertholet, S., Tzeng, E., Felley-Bosco, E., Maul, J. (1999). Expression of the inducible NO synthase in human monocytic U937 cells allows high output nitric oxide production. J. Leukoc. Biol. 65, 50-58.
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Corradin, S., Maul, J., Ransijn, A., Sturzinger, C., Vergeres, G. (1999). Down-regulation of MARCKS-related protein (MRP) in macrophages infected with Leishmania. J. Biol. Chem. 274, 16782-16787. Corradin, S., Ransijn, A., Corradin, G., Roggero, M.A., Schmitz, A.A., Schneider, P., Maul, J., Vergeres, G. (1999). MARCKS-related protein (MRP) is a substrate for the Leishmania major surface protease leishmanolysin (gp63). J. Biol. Chem. 274, 25411-25418. Rivier, D., Bovay, P., Shah, R., Didisheim, S., Maul, J. (1999). Vaccination against Leishmania
major in a CBA mouse model of infection: role of adjuvants and mechanism of protection. Paras. Immunol. 21, 461-473. Solioz, N., Blum-Tirouvanziam, U., Jacquet, R., Rafati, S., Corradin, G., Maul, J., Fasel, N. (1999). The protective capacities of histone H1 against experimental murine cutaneous leishmaniasis. Vaccine 18, 850-859. Bertholet, S. Mauel, J. (2000). Human monocytic U937 cells transfected with human hepatic inducible nitric oxide synthase exhibit leishmanicidal activity. J. Leukoc. Biol. 67, 34.
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IMMUNOBIOLOGY OF INFECTION WITH AN INTRACELLULAR PARASITE: LEISHMANIA MAJOR
Jacques Louis
Jacques Louis received his MD in Lige, Belgium in 1967. After specializing in Internal Medicine, he carried out a postdoctoral fellowship with Drs W.O. Weigle and J.M. Chiller in the Department of Experimental Pathology, Sripps Clinic and Research Foundation, La Jolla, California (19711975). He joined the WHO Immunology Research and Training Center at the Institute of Biochemistry in 1976. He was appointed Director of the WHO Center in 1985.
Group members 2000-2001 Alexandre Grand Yazmin Hauyon Christine Mossu Consolee Aletti Fabienne Tacchini-Cottier Aseffa Armidie Technician Technician Technician (06.2000) Technician (08.2001) Senior scientist Postdoctoral fellow (12.2000) Heike Voigt Reza Chakour Alain Gumy Hayo Himmelrich Franoise Flejszman Rolf Cornaz Postdoctoral fellow MD/Ph.D student Ph.D student Ph.D student (03.2000) Secretary Apprentice
IMMUNOBIOLOGY OF INFECTION WITH AN INTRACELLULAR PARASITE: LEISHMANIA MAJOR Previous work by our group demonstrated an early burst of IL-4 transcripts in the draining lymph nodes of mice susceptible to infection with L. major. The cognate recognition of a single epitope of the LACK antigen of L. major was shown to drive this rapid IL-4 response by a restricted CD4+ T cell population expressing the V 4 V 8 TCR chains. The importance of these cells and the IL-4 they produce rapidly for Th2 development and susceptibility to infection was established. We have now documented the functional plasticity, in terms of cytokines production, of these LACK-reactive cells, strongly suggesting that they are not differentiated Th2 memory cells. The early IL-4 production by V4V8 T cells was found to be under the regulatory influence of IL-2 and CD25 regulatory T cells. The rapid IL-4 response to L. major, which was never found in resistant mice, could be induced in these mice by neutralization of IL-12 or IFN- at the initiation of infection. Remarkably, early IL-4 mRNA expression in anti-IL-12 or IFN- treated resistant mice also occurred in CD4+ cells expressing the V4V8 TCR chains and reactive to LACK. Together these results indicate that the genetically determined susceptibility to L. major in mice stems from a deficiency in mechanism(s) able of down-regulating the early IL-4 response by LACK-reactive V4V 8 T cells rather than from a greater frequency of these cells. In contrat to its well documented role in Th2 maturation, IL-4 appears to also instruct Th1 development in some systems. Using the murine model of infection with L. major we showed that when present during the initial activation of dendritic cells (DC) by L. major, IL-4 instructed DCs to produce IL-12 and promoted Th1 development. This Th1 response established resistance to L. major even in susceptible mice. When present later during the period of T cell priming, IL-4 induced Th2 differentiation and progressive leishmaniasis even in resistant mice. These results show that the opposite effects that IL-4 can have on Th cell development depend upon the cells targeted for IL-4 signaling. 16
The mechanism underlying T helper (Th) subsets differentiation in vivo. The elimination of several pathogenic microorganisms by their hosts depends upon the differentiation of nave CD4+ T cells into effector cells secreting cytokines decisive for protection. Control of intracellular pathogens depends upon the activity of CD4+ T cells that produce activating cytokines such as Interferon- (Th1 cells) and containment of extracellular microorganisms usually requires CD4+ T cells producing other cytokines such as Interleukin 4 (Th2 cells). Over the past two years, our group has continued to focus on the study of the cellular and molecular mechanisms underlying genetically determined differences in the differentiation of CD4+ Th subsest in vivo. We have used the murine model of infection with an intracellular parasite: Leishmania major. In this model, genetically determined resistance and susceptibility to infection are clearly related to the development of polarized Th1 and Th2 responses, respectively. Several factors have been reported to play a role in Th subsets differentiation. Among those factors, cytokines themselves have been described as the most important stimulus. Interleukin 12 (IL-12) is the main cytokine instructing Th1 cell maturation whereas Interleukin 4 (IL-4) plays a crucial role in Th2 cell development. Th2 cell development in susceptible mice following infection with L. major: IL-4 is required. We have previously demonstrated a burst of IL-4 mRNA expression in the draining lymph node of BALB/c mice within 16 hours after infection with L. major. Importantly, this IL-4 production occurred during the period in which neutralizing IL-4 antibodies was capable of redirecting protective Th1 development in BALB/c mice. After this initial IL-4 mRNA burst, IL-4 mRNA expression returned to base line values before the occurrence of a second and permanent wave of IL-4 transcripts which reflects the establishment of a Th2 response. The cognate recognition of a single epitope of the Leishmania homolog of mammalian RACK1, designated LACK was demonstrated to drive this early IL-4 response by
a restricted population of MHC class II restricted CD4+ T cells that express the V4V8 TCR chains. The importance of V4V8 CD4+ T cells for subsequent Th2 cell development following infection with L. major was studied in BALB/c mice rendered deficient in V4 cells as a result of neonatal exposure to MMTV (SIM). V4 CD4+ T cell-deficient BALB/c mice did not exhibit early IL-4 mRNA expression after inoculation with L. major and Th2 cell differentiation was abrogated in these mice. Strikingly, the presence of these cells was required for establishment of progressive disease. The requirement of LACKreactive V4V8 CD4+ for Th2 cell development and susceptibility to L. major in BALB/c mice stems from their capacity to produce rapidly IL-4 in response to infection and it appears that they are not essential at the effector phase of the Th2 response. Functional plasticity of LACK-reactive V4V8 CD4+ T cells in terms of cytokine production. The kinetics of IL-4 production by LACKreactive T cells suggests that these cells were at an activated stage prior to antigen exposure. In contrast to nave CD4+ T cells, differentiated cells have been shown to exhibit cell cycleindependent gene expression that is stable and inheritable. The previously demonstrated possibility to modulate the rapid IL-4 response to L. major by exogenous IL-12 or IFN- suggested that the LACK-reactive V4V8 cells were not yet mature Th2 cells. Experiments were then designed to assess whether the lineage-specific gene expression of these cells was irreversibly fixed or could be redirected. The functional plasticity of LACK-reactive V4V8 cells was studied using BALB/c mice inoculated with L. major shortly after infection with MMTV(SIM), i.e. before deletion of V4+ cells. These mice failed to produce the early IL-4 response to L. major and instead exhibited an IFN- response that also occurred within the same population of LACK-specific V4V8 cells. Strikingly, neutralization of IFN- in these mice restored the production of IL-4 by these cells. These data strongly suggest that the functional properties of LACK-reactive cells are not irreversibly fixed in BALB/c mice, indicating that these cells are not differentiated memory cells.
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Regulation of rapid IL-4 production by LACK-reactive V4V8 CD4+ T cells in BALB/c mice. Interleukine-2 Early IL-2 responses following infection of BALB/c mice with L. major were also recently observed in our laboratory and kinetics analysis of this response revealed that it precedes the early peak of IL-4 transcription. Strikingly, the early IL-4 response by LACK-reactive V4V8 CD4+
T cells was found IL-2 dependent. Interestingly, neutralization of IL-2 in BALB/c mice abrogated the early IL-4 response to L. major/LACK and redirected protective Th1 response. Although IL2 was found to be produced by CD4+, expressing various TCR chains, and B cells following infection with L. major, recent and still preliminary data strongly indicates that IL-4 production by LACK-reactive V4V8 cells requires IL-2 production (in an autocrine fashion) by the same subset of cells in response to the same antigen.
Figure 1: Activation of a highly restricted population of V 4V 8 CD4+ T cells after recognition of a single antigen of L. major, LACK, results in a rapid production of IL-4. The IL-4 produced instructs the differentiation of CD4+ T cell precursors, with specificities for different antigens from L. major, towards the Th2 phenotype. IL-2 production (in an autocrine fashion) by the LACK-specific V4V8 CD4+ T cells is required for induction of the early burst of IL-4 transcription.
Antigen presenting cells Analysis of the accessory cells requirement for the expression of IL-4 transcripts in V4V8 CD4+ T cells following infection with L. major or injection of LACK in BALB/c mice has further and unexpectedly demonstrated that B cells were essential. A B cell-dependent enhancement of IL4 production by T cells has also been described in other systems and B cell-deficient mice have recently been shown to mount enhanced Th1 responses. Experiments aiming at determining whether the early IL-4 response by V4V8 CD4+ T cells requires cognate recognition of their specific epitope on B cells are currently in progress. The hypothesis that simultaneous recognition by T cells of their specific epitope on B and professional APC (i.e. dendritic cells) could signal B cells to produce cytokine(s) able to 18
interfere with the Th1 inducing signals from DC in the close vicinity is attractive and being experimentally tested. CD25 regulatory T cells: In the past few years, the concept that subpopulations of T cells are specialized in the suppression of immune responses has been revisited and considerable attention has been given to a minor subpopulation of CD4+ T cells constitutively expressing CD25, the chain of the IL-2 receptor. Both in mice and human, these CD25+ T cells, named regulatory T cells, have been shown capable of suppressing the proliferation of other T cell populations. Results of experiments recently completed have shown that CD4+CD25+ regulatory T cells somehow control the magnitude of the early IL-4 response
to L. major by LACK-reactive V4V8 CD4+ T cells in BALB/c mice. Furthermore, depletion of CD4+CD25+ regulatory T cells prior to infection with L. major was observed to exacerbate the development of lesions in BALB/c mice. Similar results were obtained using an adoptive cell transfer system in SCID mice reconstituted with BALB/c T cells. Using such system it was shown that CD4+CD25+ regulatory cells inhibit the proliferation of CD4+ cells following infection with L. major. Polymorphonuclear leukocytes It appears that polymorphonuclear leukocytes exert some role in CD4+ T cell differentiation in BALB/c mice infected with L. major. Indeed, the transient depletion of PMN, which normally accumulated in the site of the lesion very rapidly, totally prevented the expression of the early IL-4 burst in response to infection in BALB/c mice. Such treatment also resulted in inhibition of Th2 cell maturation at later times during infection, which was paralleled to partial resolution of the lesions. The cellular and molecular bases for this effect are being investigated. Together these results strongly indicate that the early IL-4 response to L. major responsible for Th2 cell development and susceptibility to L. major in BALB/c mice can be the target of regulatory processes. Why are resistant C57BL/6 mice unable to mount early IL-4 responses following infection with L. major? In contrast to susceptible BALB/c mice, C57BL/6 and other resistant mice do not mount an early IL4 response following infection with L. major or injection of LACK. Therefore, we had hypothesized that the LACK-specific V4V8 CD4+ T cells represent a unique subpopulation in BALB/c mice that produce great amounts of IL-4. Alternatively, a higher frequency of these cells in BALB/c mice could account for the ability of the initial IL-4 response to LACK to exceed the threshold required for Th2 cell development. Both of these hypotheses are unlikely because of our recent findings demonstrating that neutralization of either IL-12 or IFN- at the initiation of infection also allows the expression of this rapid IL-4 response to L. major in resistant
C57BL/6 mice. Strikingly, this early IL-4 response in resistant mice also occurs in CD4+ T cells that express the V4V8 TCR chains and that are specific for the LACK antigen. Further, the ability of treatment with anti-IFN- to allow the expression of a rapid IL-4 response to L. majoring resistant C57BL/6 mice was shown experimentally to account for its effect on the subsequent development of IL-4 producing T cell resulting in progressive disease in these otherwise resistant mice. Together with previous data showing that treatment of BALB/c mice with recombinant IL-12 suppresses the early IL-4 response to L. major, these results indicate that an impairment in mechanism(s) that down regulate the early IL-4 response by V4V8 T cells might underlie the susceptibility of BALB/c mice. The use of the murine model of infection with L. major to study the contrasting effects that IL-4 can exert on Th subset differentiation. Several recent reports indicate that, paradoxically, IL-4 may also promote Th1 cell development. For instance, IL-4-deficient mice have been shown by other to be defective in developing Th1-responses when infected with certain strains of L. major or Candida albicans (C. albicans). Similarly, a critical role for IL-4 and STAT6 was demonstrated in the development of efficient Th1-responses against haptens, and auto-, alloand even tumor-antigens. While the effects of IL-4 on Th2 differentiation are well described (also see above), the mechanisms underlying the opposite effects of IL-4 on the instruction of Th1 responses remain enigmatic. In collaboration with the group of Martin Rcken, we have directly shown that when present during the initial activation of APC by L. major in vivo, IL-4 instructs the differentiation of CD4+ T cells toward a Th1 phenotype and establishes resistance to L. major in susceptible BALB/c mice. In sharp contrast, when also present during the period of T cell priming, IL-4 induces Th2 differentiation and susceptibility to L. major, even BALB/c mice rendered resistant to infection as a result of deletion of LACK-reactive V4V8 CD4+ T cells. Thus, the effects of IL-4 on APC on the one hand, and on recently activated T cells on the other hand, lead to differentiation of effector Th 19
cells with opposite functions. These results support the concept that the opposite effects that IL-4 can exert on Th subset differentiation depends upon the nature of the cells targeted for IL-4 signaling. Since receptors for IL-4 and most other cytokines are expressed by a variety of
cells, it is likely that, beyond cytokine concentration, the kinetics of cytokine action, including consecutive or preferential interactions with distinct cell subtypes, significantly contribute to their pleiotropic effects in vivo.
Recent publications Himmelrich, H., Launois, P., Maillard, I., Biedermann, T., Tacchini-Cottier, F., Locksley, R.M., Rcken, M. and Louis, J.A. (2000). In BALB/c mice, IL-4 production during the initial phase of infection with Leishmania major is necessary and sufficient to instruct Th2 cell development resulting in progressive disease. J. Immunol. 164, 4819-4825. Tacchini-Cottier, F., Zweifel, C., Belkaid, Y., Vasei, M., Launois, P., Milon, G. and Louis, J.A. (2000). An Immunoregulatory function for neutrophils during the induction of a CD4+ Th2 response in BALB/c mice infected with Leishmania major. J. Immunol. 165, 2628-2636.
Maillard, I., Launois, P., Himmelrich, H., AchaOrbea, H., Diggelmann, H., Locksley, R. and Louis, J.A. (2001). Functional plasticity of the LACK-reactive V4-V8 CD4+ T cells normally producing the early IL-4 instructing Th2 cell development and susceptibility to Leishmania major in BALB/c mice. Eur. J. Immunol. 31, 1288-1296. Biedermann,+ T., Zimmermann+, S., Himmelrich+, H., Gumy+, A., Egeter, O., Sakrauski, A., Seegmuller, I., Voigt, H., Launois, P., Levine, A.D., Wagner, H., Heeg, K., Louis, J.A., and Rcken, M. (2001). Interleukin 4 instructs Th1 responses and resistance to Leishmania major in susceptible BALB/c mice. Nature Immunol. 2, 1054-1060. (+ contributed equally; share senior authorship)
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STRUCTURAL AND FUNCTIONAL STUDIES OF VIRULENCE FACTORS IN THE HUMAN PROTOZOAN PARASITE LEISHMANIA
Nicolas Fasel
Nicolas Fasel is an associate professor at the Medicine Faculty of Lausanne. After studying biology at the University of Fribourg (Switzerland) and obtaining a doctoral degree at the Swiss Institute for Experimental Cancer Research working on mouse mammary tumor virus, he took up a post-doctoral position at the University of California Los Angeles working on immunoglobulin gene regulation. On his return to Switzerland, he studied posttranslational modification of cell surface antigens. As an independant researcher of the Dr.Max Clotta Research Foundation for five years he had the opportunity to establish his own group investigating the molecular biology of protozoan parasites. Nicolas Fasel is also one of the founder member of Dictagene, a start-up biotechnology company.
Group members 2000-2001 Myriam Corthsy Chantal Desponds Carole Beghdadi-Rais Slavica Masina Nathalie Acestor Technician Technician Postdoctoral fellow Postdoctoral fellow Ph.D student Hedna Chavez Sverine Monnerat Cdric Schaff Haroun Zangger Ph.D student Ph.D student Ph.D student Ph.D student (08.2001)
STRUCTURAL AND FUNCTIONAL STUDIES OF VIRULENCE FACTORS IN THE HUMAN PROTOZOAN PARASITE LEISHMANIA Currently, the world-wide prevalence of leishmaniasis is 12 million cases, with 350 million people living in risk areas. Since the mid-1980s, with the spread of the HIV epidemy to areas that are traditionally endemic for leishmaniasis, together with the emergence of drug resistant parasites, a dramatic increase in the number of Leishmania infections has been reported. Leishmania protozoan parasites differentiate from non infective to infective promastigote forms in the alimentary tract of the sandfly vector, and then transform into nonmotile intracellular amastigotes in host macrophages. These transformations are necessary for the successful transmission of the parasite into the host. Understanding the molecular events underlying these developmental changes in the parasite will lead to the identification of new target molecules which could be used in chemotherapy and vaccine development, thus contributing significantly to the control of leishmaniasis in the Old and New World. The regulation of gene expression in Leishmania is poorly understood. At the level of regulation of gene expression, trypanosomatids are unusual organisms. In contrast to higher eukaryotes, in which gene expression is controlled mainly at the level of transcription initiation, gene expression in trypanosomatids is primarily regulated at the post-transcriptional level. Protein encoding genes are arranged into polycistronic transcription units which are processed to monocistronic mRNAs by trans-splicing. This peculiar RNA processing generates multiple mRNAs that are differentially regulated during parasite development. During the last few years, our research has focused upon analysing the structure and function of genes expressed in the infectious stages of the parasite and testing their protective capacities in a murine model of cutaneous leishmaniasis. 21
1. Organisation of the genome and gene expression in Leishmania Histone H1 and its locus Among various genes exhibiting significantly higher levels of expression in the infectious stages of the parasite, we have focused upon analysing the regulation of sw3, a gene which encodes histone H1 in Leishmania. The deduced amino acid sequence of Leishmania major sw3 cDNA revealed the presence of characteristic histone H1 amino acid motifs. However, the open reading frame was of an unusually small size for histone H1 (105 amino acids) because it lacked the coding potential for the central hydrophobic globular domain of linker histones present in other eukaryotes. We provided biochemical and immunocytochemical evidence that the sw3 protein was indeed a L. major nuclear histone H1, and that it was differentially expressed during the life cycle of the parasite. Together, these observations suggested that the different levels of this histone H1 protein could influence the varying degrees of chromatin condensation during the life cycle of the parasite. We characterized the genomic organisation of the locus and identified two copies of histone H1 in the genome of Leishmania. Using pulsed-field gel electrophoresis, the sw3 gene has been localized to a band corresponding to chromosome 27 providing evidence that the H1 gene cluster is present on only one chromosome. This work was included in the Leishmania genome network to establish a complete chromosomal organisation of Leishmania major, thus providing an essential tool for mapping and sequencing the whole genome. The histone H1 locus (30kb) was completely sequenced and additional open reading frames were identified inbetween the two histone H1 copies. We are currently analyzing the coding potential of this 30kb locus. The analysis is performed at the transcription and posttransciptional level. Our results show that addional genes are expressed inbetween the two H1 copies at the RNA and protein level. In contradiction with the transcription modrl proposed in trypanosomatidae, both DNA strands are transcribed and used to generate mRNAs. Our preliminary data had shown that histone H1 in Leishmania like in lower eukaryotes, does not express an histone H1 with hydrophobic globular domain. Based on the sequence of the locus we 22
identified an open reading frame with the coding potential for histone H1 with a globular domain. The mRNA coding for this polypeptide would be synthesized by alternative trans-splicing. Preliminary results show that histone H1 with a globular domain could exist. If confirmed, these results would indicate that we identified an ancestral histone H1, but it would also suggest that the coding potential of the Leishmania genome could be larger than estimated thus far. Transcriptional organization of Leishmania genome In the course of our analysis concerning the coding potential of the sw3 regions, we detected the presence of overlapping open reading frames on both the sense and antisense DNA strands of regions comprising the sw3.0 and sw3.1 genes. When a search was carried out for antisense expression in L. major parasites, it revealed the presence of two poly(A)+ RNA transcripts, a predominant one at 0.8 kb and a less abundant one at 2.5 kb. Both transcripts were shown to be developmentally regulated but in a pattern different from the histone H1 expression pattern. Two products of 19kDa (462.0 polypeptide) and 36kDa (462.1 polypeptide) were detected by immunoblotting using an antibody raised against a common amino acid peptide sequence. Although encoded in different regions of the locus, the two antisense polypeptides had the same C-terminal sequence and both coding regions included a sw3 gene segment in their 3' end sequence. Thus, the 462.1 polypeptide contains the complete sequence of the 462.0. A similar transcriptional organisation and translational pattern seem to be present in other Leishmania species since two transcripts and two polypeptides have been detected using strand specific probes and specific antibodies respectively. Transfection of a cosmid carrying a single L. major sw3.0 gene into L. guyanensis was sufficient to obtain production of two polypeptides and confirmed that complementary DNA strands are used to synthesize histone H1 and an antisense polypeptide. This would add complexity to the gene regulation and expression in Leishmania. Our preliminary results indicate that a specific AT rich region could served as an entry site for RNA polymerase II and be involved in this anti-sense process.
Programmed death in the intracellular stage of Leishmania In the course of our studies on the expression of histone H1 and differential chromatin condensation during the life cycle of the parasite, we observed a DNA fragmentation pattern at specific stages of the parasite life cycle. This was reminiscent of cells undergoing apoptosis. In addition to a condensation status which increased during differentiation towards the infectious stages of the life cycle,we observed DNA laddering corresponding to a typical internucleosomal cleavage only in the infectious stages of the life cycle (metacyclic and amastigote) but not in the non infectious stage. The degradation was even more pronounced in the amastigote nuclei. These data prompted us to test for the presence of a specific DNase. We showed that this nuclease was present in very high levels in the amastigote stage, at a low level in metacyclic and barely detectable in procyclic parasites. Death of the amastigote in the macrophage was confirmed by specific cell death assays. However, we were not able to demonstrate any caspase synthesized by the amastigote. Our current data study demonstrate that under a variety of stress conditions such as serum deprivation, heat shock and nitric oxide, cell death can be induced leading to genomic DNA fragmentation into oligonucleosomes. DNA fragmentation was observed, without induction, in the infectious stages of the parasite, and correlated with the presence of internucleosomal nuclease activity, visualisation of 45 to 59 kDa nucleases and detection of TUNEL-positive nuclei. DNA fragmentation was not dependent on active effector downstream caspases nor on the lysosomal cathepsin L-like enyzmes CPA and CPB. These data are consistent with the presence of a caspase-independent cell death mechanism in Leishmania, induced by stress and differentiation that differs significantly from metazoa. Identification of virulent Leishmania Viannia species markers in
World Leishmania species. Our approach is based on the comparison of the proteomes of various clones exhibiting different metastatic phenotypes and the isolation of specific genes which can then be used as probes, or to produce recombinant proteins and corresponding antibodies. In preliminary studies to identify molecular factors associated with metastasis, clones derived from the Leishmania Viannia guyanensis metastatic strain WHI/BR/78/M5313 have been isolated. They exhibited different phenotypes in the animal and were classified as highly metastatic clones, and infrequently or non-metastatic clones. The proteomes of these different clones has been obtained and is currently compared to the proteome of representative metastatic and nonmetastatic strains. The gene(s) that are associated with metastatic behavior in the hamster model or metastatic lesions in the human host will be identified and their effect after transfection into non metastatic strains will be tested in animal models. Polypeptides identified by the proteomic approach may play an important role in pathogenicity and have potential as diagnostic markers as well as antigens for the production of vaccines. 2. The immunogenicity and the protective capacities of antigens highly expressed in the infectious stages of Leishmania against cutaneous leishmaniasis Histone H1 as a protective antigen against cutaneous leishmaniasis Polypeptides expressed at high level in the infectious stages of the parasites could have potential immunoprotective capacities. Histone H1 and cysteine proteinases are such polypeptides under current investigation. We investigated the protective potential of the nuclear antigen histone H1 encoded by the sw3 gene. This protein is developmentally regulated during the life cycle of Leishmania major being overexpressed in the metacyclic infective promastigote, as well as in the amastigote. We investigated the protective capacity of histone H1 isolated from parasites by perchloric acid extraction, a recombinant H1 protein produced in E. coli and also as long synthetic H1 peptides. BALB/c mice, which are susceptible to infection with L. major, were immunized with these molecules and the immune response was investigated. Histone H1, in all 23
Little is known about the pathogenesis of mucocutaneous leishmaniasis, especially regarding the dissemination of the infection from the site of inoculation to secondary sites (metastasis). Our project is to elucidate the genetic basis of this metastatic process in New
forms, elicited levels of protection ranging from 60 to 100 %, even in the absence of adjuvant. These results indicated that nuclear-derived immunogens of an intracellular parasite could serve as vaccines in murine cutaneous leishmaniasis. To determine which part of the Sw3 protein was involved in protection, we used a series of 20-mer overlapping synthetic peptides, spanning the entire deduced sequence of the Sw3 protein. Furthermore, either recombinant or synthetic peptides corresponding to histone H1 were able to specifically induce the proliferation of PBLs from naturally infected donors, as well as the secretion of g-IFN. These observations argue in favour of a thorough examination of the possibility of including the nuclear antigen described here in a cocktail vaccine against human leishmaniasis. Identification of Leishmania major cysteine proteinases as targets of the immune response in humans In this study performed with Dr. Rafati (Pasteur Instiute in Tehran), we studied the identification of two parasite polypeptides recognized by human sera of patients infected with Leishmania major. Isolation and sequencing of the two genes encoding these polypeptides revealed that one of the genes is similar to the L. major cathepsin Llike gene family CPB, whereas the other gene codes for the L. major homologue of the cysteine proteinase a (CPA) of L. mexicana. By restriction enzyme digestion of genomic DNA, we show that the CPB gene is present in multiple copies in contrast to the cysteine proteinase CPA gene which could be unique. Specific antibodies directed against the mature regions of both types expressed in Escherichia coli were used to analyze the expression of these polypeptides in different stages of the parasite's life cycle.
Polypeptides of 27 and 40 kDa in size, corresponding to CPA and CPB respectively, were detected at higher level in amastigotes than in stationary phase promastigotes. Purified recombinant CPs were also used to examine the presence of specific antibodies in sera from either recovered or active cases of cutaneous leishmaniasis patients. Unlike sera from healthy uninfected controls, all the sera reacted with recombinant CPA and CPB. This finding indicates that individuals having recovered from cutaneous leishmaniasis or with clinically apparent disease have humoral responses to cysteine proteinases demonstrating the importance of these proteinases as targets of the immune response and also their potential use for serodiagnosis. A protective cocktail vaccine against murine cutaneous leishmaniasis with DNA encoding cysteine proteinases of Leishmania major The protection elicited by the intramuscular injection of two plasmid DNAs encoding Leishmania major cysteine proteinase type I (cpb) and type II (cpa) was evaluated in a murine model of experimental cutaneous leishmaniasis. Mice were immunized either separately or with a cocktail of the two plasmids. Only when the cpa and cpb genes were co-injected, protection against challenge with parasites was achieved. Analysis of the immune response showed that protected animals developed a specific Th1 immune response to Leishmania lysate and this response was associated with an increase of IFNg production which persisted at least for 14 weeks after challenge. Similarly, this is the first report demonstrating that co-injection of two genes expressing different antigens induces a protective response whereas the separate injection of cysteine proteases genes is not protective.
Recent publications Noll, T., Desponds, C., Jacquet, R., Belli, S., Fasel, N. J. (1997). Histone H1 expression varies during Leishmania major development. Mol. Biochem. Parasitol. 84, 215-227.
Rais-Beghdadi, C., Roggero, M.A., Fasel, N., Reymond, C.D. (1998). Purification of recombinant proteins by chemical removal of the affinity tag. Appl. Biochem. Biotechnol. 74, 95103.
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Belli, S., Fasel, N. (as members of the Leishmania genome network) (1998). The complete chromosomal organisation of the reference strain of the Leishmania genome project, L. major "Friedlin". Parasitol. Today 14, 301-303. Belli, S., Formenton, A., Noll, Jacquet, R., Desponds, C., Fasel, genomic organisation of the sw3 histone H1 in Leishmania. Exp. 151-160. T., Ivens, A., N. (1999). The locus encoding Parasitol. 9 1 ,
van Bemmelen, M., Rais-Beghdadi, C., Desponds, C., Reymond, C. D., Fasel, N. (2000). Expression and one step purification of Plasmodium proteins in Dictyostelium. Mol. Biochem. Parasitol. 111, 377-390. Rafati, S., Salmanian, A. H., Hashemi, K., Schaff, C., Belli, S., Fasel, N. (2001). Identification of Leishmania major cysteine proteinases as targets of the immune response in humans. M o l . Biochem. Parasitol. 113, 35-43. Rafati, S., Salmanian, A. H., Taheri, T., Vafa, M., Fasel, N. (2001). A protective cocktail vaccine against murine cutaneous leishmaniasis with DNA encoding cysteine proteinases of Leishmania major. Vaccine 19, 3369-3375.
Solioz, N., Blum-Tirouvanziam, U., Jacquet, R., Rafati, S., Corradin, G., Maul, J., Fasel, N.J. (1999). The protective capacities of a nuclear antigen against leishmaniasis. Vaccine 18, 850859.
25
NEW STRATEGY OF TUMOR TARGETING WITH ANTIBODIES
Jean-Pierre Mach
Jean-Pierre Mach received his medical degree at the University of Geneva and trained from 1963 to 1967 as a postdoctoral fellow at the Massachussetts General Hospital in Boston. He specialized in the field of tumor markers at the Department of Biochemistry of the University of Lausanne where he became professor in 1976. From 1978 to 1986, he was associate director of the Lausanne branch of the Ludwig Institute. He received a joint appointment at ISREC and the Department of Biochemistry in 1988 to pursue his program on antibody-mediated tumor targeting.
Group members 2000-2001 Karine Fournier David Deperthes Faza Rharbaoui Bruno Robert Technician Postdoctoral fellow (03.2000) Postdoctoral fellow (01.2001) Postdoctoral fellow (12.2001) Valrie Cesson Sylvain Cloutier Valrie Crivelli Laurent Marguerat Ph.D student Ph.D student (03.2000) Diploma student (05.2000) Diploma student (04.2000)
NEW STRATEGY OF TUMOR TARGETING WITH ANTIBODIES The goal of our group has been for many years to produce anti-tumor marker antibodies and peptabodies capable of specifically targeting diagnostic or therapeutic radionuclides on cancer cells. During the last two years, in collaboration with the Lausanne branch of the Ludwig Institute, we have developed a novel tumor targeting approach. We demonstrated that Fab fragments from different anti-tumor marker antibodies, when chemically conjugated to major histocompatibility complexes (MHC) containing viral peptides, can target these very active T lymphocyte antigens on human cancer cells. Then, the presence of the new viral antigens oligomerized on the surface of cancer cells can induce the efficient lysis of the target cells by viral peptidespecific human cytotoxic T lymphocytes. 1. Antibody-MHC viral peptide conjugates for cancer cells targeting Bruno Robert and Jean-Pierre Mach, in collaboration with Philippe Guillaume, Immanuel Luescher, Marie-Agns Doucey and Pedro Romero from the Ludwig Institute. Antibodies are large soluble proteins, which have the property of binding with high specificity to almost any kind of native antigen or marker present on bacteria, viruses, or tumor cells. Monoclonal antibodies (mAbs) binding with high affinity to tumor markers can be selected by the classical hybridoma method or by the phage display technology. We and others have shown, 26 both in experimental models and in clinical studies, that intravenously injected radiolabeled mAbs can target tumors with a good degree of specificity. However, despite some recent encouraging clinical results obtained with antiErbB-2 mAbs (Herceptin) and anti-CD20 mAbs (Rituximab) in the therapy of breast carcinoma and B cell lymphoma patients, respectively, antibodies are not very efficient in killing tumor cells. In contrast, CD8 cytotoxic T lymphocytes (CTL) have the capacity, as a major natural effector function, to efficiently kill cells that are infected by a virus. For that purpose, they possess specific
receptors recognizing processed antigens in the form of short peptides expressed in the context of the MHC. In recent years, several groups have shown that some human cancer cells may express specific peptides and can be lyzed by CTL. Thus, major efforts are being made to boost or to induce CTL anti-tumor responses by various strategies of vaccination. However, despite some welldocumented encouraging clinical results, the CTL response against tumor antigens has remained relatively weak. Furthermore, one of the obstacles to active T lymphocyte immunotherapy is that tumor cells can escape CTL recognition by deletion of their MHC. A new bridge between antibody and T lymphocyte attack on cancer cells Our idea was to combine the advantage of the above-mentioned high binding and targeting properties of antibodies with the powerful cytotoxic capacity of CD8 T lymphocytes. For
that purpose, we prepared chemical conjugates between Fab fragments from anti-tumor marker antibodies and soluble MHC peptide complexes containing antigenic viral peptides such as the common Influenza Flu matrix peptide. HLA-A2/Flu-matrix peptide complexes (HLAA2/Flu-ma-peptide) were coupled to Fab fragments from mAbs directed against cell surface markers, such as carcinoembryonic antigen (CEA), ErbB-2 or CD20. These conjugates were first shown to be able to target the antigenically active HLA-A2/Flu-ma-peptide on cancer cells expressing the relevant tumor marker, by flow cytometric analysis, using a fluorescent labeled anti-HLA-A2 mAb. We then asked the question: will these tumor cells, originally HLA-A2 negative, but now coated with HLA-A2/Flu-ma-peptide, become susceptible to lysis by HLA-A2 restricted and Flu-ma-peptide specific CTL, as expected from the schematic diagram below (Fig. 1).
Figure 1. Schematic diagram illustrating the mechanism of induction of CTL effector function by anti-tumorassociated antigen Fab-HLA-A2/Flu conjugates oligomerized on the surface of tumor target cells. Note that the free conjugates should not bind to T cell receptor (TCR) due to the low affinity of the monomeric MHC/peptide complex-TCR interaction. The unique advantage of the conjugate is that their antibody site (in red) can associated with highly expressed non-MHC-restricted, tumor-associated antigens, while their MHC/peptide site (in blue and yellow), when coated at high density on the target cell surface, can induce the binding and effector function of specific cytotoxic T lymphocytes. Thus, the Fab-MHC/peptide conjugates do make a functional bridge between antibody and T cell recognition of tumor cells (ref. 10).
The results, obtained in a classical 4 hour 51Cr release assay, showed that a very efficient and sensitive lysis of the CEA expressing colon carcinoma cells LoVo was obtained, when the cells were preincubated with anti-CEA-HLAA2/Flu peptide conjugate (Fig. 2, upper panel), while control CEA-negative breast carcinoma cells SK-BR-3 coated with the same conjugate
were not lyzed. In contrast, when ErbB-2 expressing SK-BR-3 cells were preincubated with anti-ErbB-2-HLA-A2/Flu peptide conjugate, they were lyzed, while control ErbB-2 negative Daudi cells were not (middle panel). Finally, when Daudi cells were preincubated with anti-CD20HLA-A2/Flu-ma peptide, they were lyzed, while LoVo colon carcinoma cells were not (lower 27
panel). The results in figure 2 demonstrate not only the specificity, but also the sensitivity of CTL-mediated cancer cells lysis induction by antibody-MHC/viral peptide conjugates. Indeed, 50% of cancer cell lysis was induced with conjugate concentrations as low as 0.5 to 10ng per ml, overall the conjugates were active at 5 to 10 picomolar range (ref. 10).
Perspectives and future clinical applications The theoretical advantages of this immunotherapy strategy are: First, that the monomeric MHCpeptide complexes conjugated to antibody fragments will not bind to circulating T lymphocytes due to their low affinity for T cell receptor (TCR); thus, only when oligomerized on target tumor cells (as schematically described on figure 1), can they induce the activation and cytotoxic activity of peptide-specific T lymphocytes due to multiple binding sites. This represents a major progress compared to conjugates between an antibody and a tetramer of MHC/peptide that we reported last year (ref. 6), or the peptide directly coupled to antibodies that we tested in 1998 (ref. 1). Second, the size of the conjugate of about 95 kDa is optimal for in vivo tumor localization, as we have demonstrated for F(ab)2 fragments from anti-CEA mAbs. Third, the antibody-MHC will target multiple copies of the same selected antigenic peptide to the surface of the tumor cells, without the limitation of autologous antigenic peptides, which have to compete with a host of normal cellular peptides for expression on MHC. F o u r t h , the oligomerization of antibody-MHC peptide conjugates on tumor cells will provide binding sites, not only for the TCRs from specific CTL, but also for CD8 molecules, which have been shown to play an essential role in the interaction with MHC peptide complexes. Overall, tumor cells targeted with antibody-MHC conjugates containing selected immunodominant viral peptides should be killed by viral peptide specific CTL, as efficiently as cells infected by the virus. For potential clinical application, we could consider to give to cancer patients a strong vaccination or boost with a common attenuated virus, such as Influenza, Cytomegalo, or EpsteinBarr virus, followed a week later by the intravenous injection of a conjugate consisting of anti-tumor antibody fragment coupled to autologous MHC containing the relevant immunodominant viral peptide. Thus, we can expect that the patient anti-viral cytotoxic lymphocytes will attack the cancer cells targeted with the conjugate (a patent application for the new immunotherapy strategy has been deposited in the U.S.A).
Figure 2. Demonstration of the specificity and sensitivity of the cytotoxic T lymphocyte lysis of cancer cells by antibody-MHC/viral peptide conjugates. Titration of increasing concentrations, ranging from 0 to 1 g/ml (abscissa), of the three antitumor marker Fab HLA-A2/Flu conjugates, which were incubated for 1 h at 37C with different tumor target cell lines, followed by the addition of specific CTL for a 4 h 51Cr release assay. (A) Anti-CEA Fab-HLA-A2/Flu was incubated with LoVo cells (filled squares) or SKBR-3 cells (open circles). (B) Anti-ErbB-2 Fab-HLAA2/Flu was incubated with SK-BR-3 cells (filled circles), LoVo cells (open squares) or Daudi cells (open diamonds). C. Anti-CD20 Fab-HLA-A2/Flu was incubated with Daudi cells (filled diamonds) B lymphoma cells and with SK-BR-3 (open circles) carcinoma cells. Results are expressed as % specific target cell lysis. The conjugate concentration, which induces 50% specific lysis, is indicated in each panel (ref. 10).
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2. Activation of the complement alternative pathway on the surface of human cells by transfection of recombinant properdin. Bernard Vuagnat, Jean-Marc Le Doussal and Jean-Pierre Mach Human blood normally contains grams of circulating toxin precursors: the proteins of the complement system. The important role of complement in the innate and acquired immunity against microorganisms, as well as in various immunopathologies, has been well documented. However, the few attempts to direct the large potential cytotoxicity of the complement system towards invading tumor cells, namely by coupling the cobra venom factor to antibodies, have reached until now a limited success. We propose here a new strategy based on the activation of the alternative pathway of complement. Complement activation is a cascade of enzymatic cleavage and conformational changes of proteins, driven either by antibody aggregation classical pathway, or by the abnormal biochemical composition of bacterial or parasite cell surfaces alternative pathway In its resting steady-state, complement is activated at a low rate, but activated components are rapidly inactivated, spontaneously, or by either fluid-phase, or cellsurface regulatory proteins. Properdin is a serum glycoprotein that stabilizes the labile component C3 convertase (C3b-Bb) of the alternative pathway. Thanks to its oligomeric nature, properdin specifically upregulates the alternative pathway on the surface of bacteria in the fluid phase. Here we have demonstrated that human cells displaying properdin on their membrane can activate autologous alternative pathway. The cDNAs encoding human properdin and the transmembrane domain of human platelet derived growth factor receptor were fused together and expressed in human embryo kidney cells (HEK293). Transfected cells displayed properdin at their surface as shown by flow cytometry. In contact with human serum at 37C, the membrane bound properdin molecules triggered the alternative pathway-mediated C3 deposition. SDS-PAGE analysis showed C3 covalently bound to various membrane proteins, but not to properdin itself. Furthermore, properdin expressed on transfected cells can bind, at 4C,
the hydrolyzed form of C3 (C3i) present in serum or added in the purified form. C3 binding was restricted to the cells expressing properdin, it was inhibited by an anti-properdin mAb and did not require serum properdin. Bound C3 allowed further C5, C7 and C9 deposition, as well as cell lysis after blocking CD59 function. In contrast, wild type cells, or cells displaying a truncated form of properdin (deleted from its 6th thrombospondin-like repeat) did not activate the alternative pathway. We hypothesized that properdin expressed on cell surface activates the alternative pathway by stabilizing bystander C3b and/or by capturing serum C3iBb convertase. Our results suggest that properdin could be used for retargeting autologous complement to tumor cells (ref. 4). 3. Selection of peptides and synthesis of pentameric peptabody molecules reacting specifically with ErbB-2 receptor on the surface of cancer cells. Mehdi Houimel, Alexey Terskikh, Pascal Schneider and Jean-PierreMach The Her-2/ErbB-2 is a member of the epidermal growth factor (EGF) receptor family, which is overexpressed on the surface of tumor cells and has been shown to have a clear implication in the malignant phenotype. Thus, ErbB-2 represents one of the rare oncoproteins directly accessible on the surface of tumor cells. Major efforts have been made to identify the natural ligand of ErbB2, or to raise antibodies against this particular receptor. The production of monoclonal antibodies (mAb) against ErbB-2 has been successfully achieved by several groups and a humanized mAb, called Herceptin has been approved for therapy of patients with ErbB-2overexpressing carcinomas. However, the search for an ErbB-2 ligand has led to controversial results, and the current consensus opinion is that a specific natural ligand for ErbB-2 (analogous to EGF or the neuregulins for ErbB-1, ErbB-3 or ErbB-4, respectively) does not exist. Despite this absence of known ligand, ErbB-2 was shown to play a central role in both normal development and malignant cell growth. ErbB-2 possesses a cytoplasmic autophosphorylating tyrosine kinase domain, which is activated following heterodimerization with ligand-occupied ErbB-1, ErbB-3 and ErbB-4. 29
In view of this puzzling apparent absence of natural ligand for ErbB-2, it appears interesting to select peptide sequences, which could substitute for the natural ligand and act as agonists or antagonists for this receptor. For this purpose, we used the extracellular domain of recombinant ErbB-2, that we have fused to the Fc, hinge, CH2 and CH3 domains of human IgG1, to select receptor binding peptides from two phage display libraries recently developed in our laboratory. By screening the constrained and unconstrained hexapeptide libraries, we isolated 37 phage clones, which bound specifically to ErbB-2 extracellular domain. Among them, we found 6 constrained and 10 linear different hexapeptide sequences. Interestingly, in the linear sequence, 5 consensus motifs, all with a common methionine and a positively charged residue at positions 1 and 3, respectively, were identified (ref. 8). Furthermore, our group has recently shown that the functional affinity of selected peptides for their target receptor was highly increased by fusion with the coil-coiled domain of cartilage oligomeric matrix protein (COMP) via a semirigid hinge region, resulting in a high avidity pentameric recombinant molecule called peptabody. Thus, 3 of the selected hexapeptides were used to synthesize new peptabody molecules. The 3 peptabodies bound specifically to the ErbB-2 extra cellular domain, as determined by enzyme-linked immunosorbent assay and BIA-core analysis and to tumor cells overexpressing ErbB-2, as shown by flow cytometry. Interestingly, one of the free selected linear peptides and all 3 peptabodies inhibited the proliferation of tumor cells overexpressing ErbB2. In conclusion, a novel type of ErbB-2-specific ligand was described, that might complement presently available monoclonal antibodies (ref. 8) (The newly identified peptide sequences were submitted for a patent protection). 4. Synthesis of high avidity streptabody molecules, by tetramerization of in vivo biotinylated single chain Fv fragments on streptavidin. Sylvain M. Cloutier, David Deperthes, Laurent Marguerat and Jean-Pierre Mach, in collaboration with the Urology Research Unit at the Department of Urology from the CHUV. 30
The phage display represents a powerful method for the isolation of antibody fragments from highly diverse nave human antibody repertoires. However, the affinity of the selected antibodies is usually low (refs. 2 and 7) and current methods of affinity maturation are complex and timeconsuming. We have described an easy way to increase the functional affinity (avidity) of single chain variable fragments (scFvs) by tetramerization on streptavidin, following their intracellular sitespecific biotinlylation by the enzyme BirA. Expression vectors have been constructed that enable addition of the 15 amino-acid biotin acceptor domain on selected scFvs. Different domains were cloned at the C-terminus of scFv in the following order: a semi-rigid hinge region (of 16 residues), the biotin acceptor domain and a histidine tail. Two such recombinant scFvs directed against the carcinoembryonic antigen (CEA) were previously selected from a human non-immune phage display library and a murine immune library. The scFvs were synthesized in Escherichia coli carrying the plasmid encoding the BirA enzyme, and purified from the cytoplasmic extracts by Ni-NTA affinity chromatography. Purified biotinylated scFvs were then tetramerized on the streptavidin molecule to create a streptabody (StAb). The avidity of various forms of anti-CEA StAbs, tested on purified CEA by competitive assays and surface plasmon resonance showed an increase of more than one log, as compared with the monomeric scFv counterparts. Furthermore, the percentage of direct binding of 125I-labeled StAb on CEA-Sepharose beads, as well on CEAexpressing cells, showed a dramatic increase for the tetramerized scFv (>80%), as compared with the monomeric scFv (<20%). Interestingly, the percentage binding of 125I-labeled anti-CEA StAbs to CEA-expressing colon carcinoma cells was definitely higher (>80%) than that obtained with a reference high affinity murine anti-CEA mAb (30%). Another advantage of using scFvs in a StAb format was demonstrated by Western blot analysis, where tetramerized anti-CEA scFv could detect a small quantity of CEA at a concentration 100-fold lower than the monomeric scFv. Our results support the idea that the biotin acceptor domain separated by a hinge, as
described here, should be systematically added, either within the phagemid used for construction of scFv libraries, or in the vectors used for expression of soluble form of scFv, to produce scFvs, which can be directly biotinylated in vivo and used for assembly in the StAb format (ref. 5). 5. Improved tumor localization of radiolabeled Iododeoxyuridine in human glioblastoma xenografts by preinjection of Fluorodeoxyuridine. Yves M. Dupertuis, Franz Buchegger and JeanPierre Mach, in collaboration with Nicolas de Tribolet, Neurosurgery Department from the HUG and CHUV, Daniel O. Slosman and Angelika Bischof Delaloye, Nuclear Medicine Service from the HUG and the CHUV. In previous years, in collaboration with Franz Buchegger and Angelika Bischof Delaloye, we performed pioneer work in the study of in vivo tumor localization of radiolabeled antibodies and fragments, which contributed to the development of a new treatment modality called radioimmunotherapy (ref. 3). Thanks to this personal experience, we were able to initiate the first radioimmunotherapy trial of B cell lymphoma in Switzerland. This trial is now progressing at the clinical level, in collaboration with Industry. Furthermore, the first radiolabeled antibodies for B cell lymphoma treatment has been recently approved by the FDA. Here, we report on the improvement of a completely different approach of tumor targeting with a radiolabeled molecule The concept of using radiolabeled Iododeoxyuridine as a marker of tumor cells, is not new. Indeed, it is known that the Van der Waals radius of the iodine atom is similar and can replace the 5-methyl group of thymidine on the pyrimidine ring, thus its incorporation is increased in dividing cells. What is more original is our demonstration of increased Recent publications 1. Eberl, E., Jiang, S., Yu, Z., Schneider, P., Corradin, G. and Mach, J.-P. (1998). An antiCD19 antibody coupled to a tetanus toxin peptide induces efficient Fas ligand (FasL)-mediated cytotoxicity of a transformed human B cell line by specific CD4+ T cells. Clin. Exp. Immunol. 114, 173-178.
tumor cell uptake of radiolabeled Iododeoxyuridine after preincubation in vitro or injection in vivo of Fluorodeoxyuridine, which is known to inhibit the biosynthesis of thymidine. The results showed that coincubation of Fluorodeoxyuridine substantially increased the incorporation of 125Iododeoxyuridine in the 3 glioma cells lines tested. The biodistribution and tumor localization 24 hours after i.v. injection of 125 Iododeoxyuridine was measured in nude mice xenografted with glioma cells, and pretreated or not with Fluorodeoxyuridine. The results showed that an i.v. injection of 10 mg/kg of Fluorodeoxyuridine 1 hour before 125 Iododeoxyuridine increased the uptake radiolabeled pyrimidine 4.8 to 6.8 fold in the 3 human glioma xenografts tested. The tumor uptake was found to increase up to 13.6 fold with a stepwise decrease of Fluorodeoxyuridine to 1.1 mg/kg. There was also an increased uptake in rapidly dividing normal tissues, such as bone marrow, spleen and intestine but to a lower extent (1.7 to 5.8 fold). There was almost no increase of uptake in quiescent tissues. Scintigraphy of glioma xenografted mice was performed at different times after i.v. injection of Fluorodeoxyuridine followed by 125 Iododeoxyuridine and the tumor imaging was greatly improved. Background activity could be greatly reduced by p.o. administration of KC104, in addition to potassium iodide (ref. 9). We conclude that Fluorodeoxyuridine preadministration may improve positron or single photon emission tomography with cell division tracers, such as radio-Iododeoxyuridine and possibly other thymidine analogues. Furthermore, the increased tumor uptakes of 125 Iododeoxyuridine observed are encouraging for the improvement of a radiotherapy strategy of gliomas by local injection of different forms of radiolabeled pyrimidine molecules.
2. Waibel, R., Alberto, R., Willuda, J., Finnern, R., Schibli, R., Stichelberger, A., Egli, A., Abram, U., Mach, J.-P., Plckthun, A., and Schubiger, P. A. (1999). Stable one-step technetium-99m labeling of His-tagged recombinant proteins with a novel Tc(I)-carbonyl complex. Nat. Biotechnol. 17, 897-901. 31
3. Buchegger, F., Roth, A., Allal, A., Dupertuis, Y.M., Slosman, D.O., Bischof Delaloye, A. and Mach, J.-P. (2000). Radioimmunotherapy of colorectal cancer liver metastases: Combination with radiotherapy, Annals New York Acad. Sci. 910, 263-269. 4. Vuagnat, B,, Mach, J.-P. and Le Doussal, J.-M. (2000). Activation of the alternative pathway of human complement by autologous cells expressing transmembrane recombinant properdin. Mol. Immunol. 37, 467-478. 5. Cloutier, S.M., Couty, S., Terskikh, A., Marguerat, L., Crivelli, V., Pugnires, M,, Mani, J.C., Leisinger, H.J., Mach, J.-P. and Deperthes D. (2000). Streptabody, a high avidity molecule made by tetramerization of in vivo biotinylated, phage display-selected scFv fragments on streptavidin. Mol Immunol. 37, 1067-77. 6. Robert B, Guillaume P, Luescher I, Romero P and Mach JP (2000). Antibody-conjugated MHC class I tetramers can target tumor cells for specific lysis by T lymphocytes. Eur J Immunol. 30, 3165-3170.
7. Houimel, M., Corthsy-Theulaz, I., Fisch, I., Wong, C., Corthsy, B., Mach, J.-P. and Finnern, R. (2001). Selection of human single chain Fv antibody fragments (scFv) binding and inhibiting Helicobacter pylori urease. Tumor Biol. 22, 36-44. 8. Houimel ,M,, Schneider, P., Terskikh, A. and Mach J.-P. (2001). Selection of peptides and synthesis of pentameric peptabody molecules reacting specifically with ErbB-2 receptor. Int J Cancer 92, 748-55. 9. Dupertuis, Y.M., Vazquez, M., Mach, J.-P., De Tribolet, N., Pichard, C., Slosman, D.O. and Buchegger F. (2001). Fluorodeoxyuridine improves imaging of human glioblastoma xenografts with radiolabeled iododeoxyuridine. Cancer Res. 61, 7971-7977. 10. Robert, B., Guillaume, P., Luescher, I., Doucey, M.-A., Cerottini, J.-C., Romero, P. and Mach, J.-P. (2001). Redirecting anti-viral CTL against cancer cells by surface targeting of monomeric MHC class-I viral peptide conjugated to antibody fragments. Cancer Immunity 1, 2 (available on the web: http://www.cancerimmunity.org).
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IMMUNE RESPONSES TO VIRUSES AND PROTEIN-HAPTEN ANTIGENS
Hans Acha-Orbea
Hans Acha-Orbea studied biochemistry at the ETH in Zurich and received a Ph.D. in biochemistry in 1984 with H. Hengartner, H. Zuber and R.Zinkernagel. After a postdoctoral fellowship with H. O. MacDevitt at Stanford University in California, USA he came to the Ludwig Institute of Cancer Research, Lausanne Branch as an Assistant Member and START fellow in 1989. Since 1994 he has a joint appointment as an associate member at the Ludwig Institute for Cancer Research and Associate Professor at the Institute of Biochemistry. The main focus of his laboratory concerns virus-host interactions, immune response and immunologic tolerance. Group members 2000-2001 Maria Bochatay Christine Lavanchy Leo Scarpellino Eva Grdby Technician Technician Technician (11.2001) Postdoctoral fellow Luc Otten Jean-Marc Waldburger Quynh-Giao Steiner Julian Wagner Postdoctoral fellow Postdoctoral fellow Ph.D student Ph.D student
IMMUNE RESPONSES TO VIRUSES The main interests of the lab are 1) interactions between viruses and the host immune system, and 2) the molecular and cellular mechanisms of immune responses to T dependent antigens in vivo. For this purpose we utilize either viruses or classical hapten-carrier conjugates as immunogens. Recombinant viruses expressing foreign genes are utilized to analyze their effect on the immune response. In addition, we utilize transgenic and knockout mice to analyze the impact of the transgenes or knockouts on different phases of the immune response. 1. Virus host interaction Viruses have developed a large variety of tools and strategies to evade or exploit the immune response. The understanding of these mechanisms gives insights into the in vivo immune response. In the last years we have mostly been working with the retrovirus mouse mammary tumor virus (MMTV) and started working now with other viruses such as adenovirus and vaccinia virus. MMTV has found a strategy to exploit the immune response by infecting antigen presenting cells and expression of a superantigen (SAg) at the cell surface. There is a high frequency of SAg-reactive T cells in nave animals since the less polymorphic lateral part of the T cell receptor beta chain is involved in interaction with the SAg. The reactive cells can be detected with T cell receptor V-specific antibodies. This induces an extremely strong classical immune response by dendritic cell priming of nave T cells followed by SAg-mediated T cell help to infected B cells and long-lasting immune responses. Instead of eliminating the virus, this immune response is a key step for the establishment of a chronic infection. With each division the number of infected B cells doubles and the SAg-induced normal immune response leads to long-lived infected memory and effector B cells. The life cycle of MMTV with the questions addressed in our laboratory are shown in figure 1. A deeper understanding of antigen priming, the ensuing long-term lymph node reaction, the emigration of
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1. Virus uptake via gut

Epithelium Peyer's Patch
2. Infection of AgPresenting Cells

TI B cell activation g) Mammary tumor development after integration in vicinity of protooncogene
5. Infection of the mammary gland
B cell
f) Migration
DC
a) Antigen-presentation
CD8 + T cell
3. SAg-Priming of CD4 + T Cells

TD B cell activation b) T cell help SAg V CD4+ T cell proliferation d) Deletion of Sag-reactive T cells in nondraining lymphoid organs
Viral spread
CD4 + T cell
B cell
B cell
A P C
V MHC class II
4. Chronic immune response

Sag-reactive TH cells remain in GC of the draining lymphoid organ. e) Memory
c) B cell differentiation
day 4-7: extrafollicular B cell differentiation mostly IgG2a ,weakly neutralizing day 12->120: follicular B cell differentiation: germinal centers mostly IgG 1, neutralizing
Hans Acha-Orbea Adapted from Immunol. Rev. 168, 287, 1999
the cells from the lymph node to other tissues and the tolerance mechanisms preventing cells from making a fulminant immune response are important features for efficient therapy. The role of the draining lymph node in chronic immune responses Lymph nodes draining the site of tumors or infection often show a strong antigen-specific immune response. In MMTV infection we found germinal centers and increased levels of SAgreactive CD4+ T cells in the draining lymph node up to 1 year after infection. This finding was surprising since previous studies indicated induction of anergy in SAg-reactive T cells. In other lymphoid compartments SAg reactive T cell 34
became slowly deleted. Neutralizing antibodies were detectable throughout life. We studied the role of the draining lymph node in this chronic immune response by removing the draining lymph node at different time points after footpad MMTV injection. At all time points the virus achieved a chronic systemic infection and a complete deletion of SAg-reactive T cells. Therefore systemic spread and peripheral deletion was not dependent on the draining lymph node. Neutralization, on the other side, was strongly reduced shortly after removal of the draining lymph node. Therefore the initial site of antigen priming seems to control the maintenance of a strong neutralizing antibody response. These results suggest that the chronic immune response of CD4+ T cells and B cells is dependent on the
presence of the draining lymph node. We are currently investigating the role of the neutralizing antibodies on viral spread and mammary cancer development as well as on long-term neutralization. 2. Immune response: from antigen priming to the germinal center reaction The immune response in a lymph node develops as outlined in figure 2. Nave T cells encounter antigen in the paracortex of the lymph node presented by dendritic cells. After differentiation into effector cells cytotoxic T cells leave the
production of rather low affinity or, alternatively, to formation of germinal centers. Such germinal centers are seeded by 1-3 B cells that start dividing and mutating their immunoglobulin V regions to reach more than 10000 cells after a few days. Only the highest affinity B cells are selected in this highly competitive environment to pick up some antigen from the follicular dendritic cells and to present it to germinal center T cells. These cells receive cognate T cell help to develop either into plasma or memory B cells. The events have been analyzed in many knockout, transgenic and normal mice. To obtain a clearer picture on the role of the numerous cytokines, chemokines and cell surface molecules at precise time points of the immune response we expressed soluble molecules that interfere or stimulate specific receptors on the B and T cell surface. We utilize B-7 family members and their receptors, TNF and TNFR family members, chemokines and their inhibitors etc. that can be expressed continuously at precise time points of the immune response. The effect on the immune response is then analyzed by immunohistology, FACS analysis, affinity maturation etc. A special interest is being put in the characterization of stroma cells in lymph node and spleen. Their role in the immune response is poorly understood. We are currently cloning stroma-specific genes with special interest in genes specifically expressed by follicular dendritic cells. We generated a knockout mouse that has normal MHC class II expression on all the bone marrowderived cells but lacks inducible expression on all the other tissues. These mice are currently investigated for immune tolerance and immune response in skin, mucosa and peripheral lymphoid tissues.
Follicular and extrafollicular B cell differentiation

T Cell B Cell Dendritic Cell Antigen
EF
F: Follicle EF: Extrafollicular differentiation
lymph node whereas a proportion of antigenprimed CD4+ T cells initiate T cell-B cell collaboration in the interfollicular regions of the lymph node. B cells then differentiate along either an extrafollicular pathway of differentiation, that leads to quick isotype switched antibody
Recent publications Wehrli, N., Legler, D.F., Finke, D., Toellner, K.M., Loetscher, P., Baggiolini, M., MacLennan, I.C.M., Acha-Orbea, H. (2001). Changing responsiveness to chemokines allows medullary plasmablasts to leave lymph nodes. Eur. J. Immunol. 31, 609-616. Attinger, A., MacDonald, H.R., Acha-Orbea, H. (2001). Lymphoid environment limits superantigen and antigen-induced T cell proliferation at high precursor frequency. Eur. J. Immunol. 31, 884-93.
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Finke, D., Baribaud, F., Diggelmann, H., AchaOrbea, H. (2001). Extrafollicular plasmablast B cells play a key role in carrying retroviral infection to peripheral organs. J. Immunol. 166, 6266-6275. Waldburger, J.M., Suter, T., Fontana, A., Reith, W.*, Acha-Orbea, H*. (2001). Selective abrogation of MHC class II expression on extrahematopoietic cells in mice lacking promotor IV of the CIITA gene. J. Exp. Med. 194: 393-406. * equal contribution Finke, D., Acha-Orbea, H. (2001). Differential migration of in vivo primed B and T lymphocytes to lymphoid and non-lymphoid organs. Eur. J. Immunol. 31: 2603-2611. Attinger, A., Acha-Orbea, H., MacDonald, H.R. (2000). Cell autonomous rather than environmental factors control bacterial superantigen-induced T cell anergy in vivo. J. Immunol. 165, 1171-1174. Ardavin, C., Martin, P., Ferrero, I., Azcoitia, I., Anjure, F., Diggelmann, H., Luthi, F., Luther, S., Acha-Orbea, H. (1999). B cell response after MMTV infection: extrafollicular plasmablasts represent the main infected population and can transmit viral infection. J. Immunol. 162, 25382545. Baribaud, F., Maillard, I., Vacheron, S., Brocker, T., Diggelmann, H., Acha-Orbea, H. (1999). Role
of dendritic cells in the immune response induced by the mouse mammary tumor virus superantigen. J. Virol. 73, 8403-8410. Toellner, K.M., Luther, S., Sze, D.M.-Y., Choy, R.K.-W., Taylor, D.R., MacLennan, I.C.M., Acha-Orbea, H. (1998). T helper 1 (Th1) and Th2 characteristics start to develop during T cell priming and are associated with an immediate ability to induce immunoglobulin class switching. J. Exp. Med. 187, 1193-1204. Luther, S., Gulbranson-Judge, A., Acha-Orbea, H., MacLennan, I.C.M. (1997). Viral superantigen drives functional extrafollicular and follicular B cell differentiation leading to virusspecific antibody production. J. Exp. Med. 185, 551-562. Reviews: Acha-Orbea, H., Finke, D., Attinger, A., Schmid, S., Wehrli, N., Vacheron, S., Xenarios, I., Toellner, K.M., MacLennan, I.C.M., Luther, S. (1999). Interplays between mouse mammary tumor virus and the cellular and humoral immune response. Immunol. Rev. 168, 287-303. Finke D., Acha-Orbea, H. (2001). Immune response to murine and feline retroviruses. In: Retroviral Immunology: Immune response and restoration, Eds. G. Pantaleo & B.D. Walker, Humana Press Inc. Totowa, NJ. pp125-157.
36
MUCOSAL VACCINES AGAINST ONCOGENIC MICROBIAL PATHOGENS
Jean-Pierre Kraehenbuhl
Jean-Pierre Kraehenbuhl received his MD at the University of Lausanne in 1967. He carried out a postdoctoral fellowship with Dr. G. Palade at Rockefeller University and became Assistant Professor at Yale University School of Medicine. In 1975, he returned to the Institute of Biochemistry, University of Lausanne and joined ISREC in 1982 as a senior staff member to work on immunoglobulin transepithelial transport and mucosal immunity.
Group members 2000-2001 Monique Reinhardt Hiltrud Stubbe Corinne Tallichet-Blanc Daniela Finke Jean-Claude Sirard Nathalie Debard Technician Technician Technician Junior faculty member Senior postdoctoral fellow Postdoctoral fellow Lucy Hathaway Florence Niedergang Martin Rumbo Arnaud Didierlaurent Grigorios Fotopoulos Frdric Sierro Postdoctoral fellow (09.2000) Postdoctoral fellow (01.2001) Postdoctoral fellow Ph.D student Ph.D student (09.2000) Ph.D student
MUCOSAL VACCINES AGAINST ONCOGENIC MICROBIAL PATHOGENS Most microorganisms infect humans through mucosal tissues. Some viruses and bacteria can eventually cause tumors. Helicobacter pylori causes persistent infection and inflammation in the stomach, which is a risk factor for gastric adenocarcinoma and malignant lymphomas. The human papilloma virus (HPV) chronically infects the female genital mucosa where some strains in association with other cofactors induce cervical cancers. Skin tumors (Kaposi sarcomas) are associated with AIDS. The prevention or the cure of such infections can protect against cancers. Vaccines against H. pylori, HPV and human immunodeficiency virus (HIV) are developed in Lausanne as a team effort between our group and groups at the University Hospital. Our objectives are: 1. understand how antigens, microorganisms and candidate vaccines are sampled and processed by mucosal tissues, 2. analyze the development mucosa-associated lymphoid tissue, 3. elucidate how immune effector molecules (antibodies) and cells (cytotoxic T lymphocytes) protect mucosal surfaces, 4. design efficient mucosal vaccines that elicit long lasting mucosal and systemic humoral and cellular immune responses. Antigen sampling in mucosal tissues The airways, the gut and the genital tract are covered by epithelia that protects the host from environmental pathogens by innate and adaptive immune mechanisms. Depending on the structure of the epithelial barriers, different antigen sampling strategies have evolved. Dendritic cells of hematopoeitic origin migrate into all epithelia where they sample antigens for subsequent presentation in local or distant organized lymphoid tissue. The simple epithelia over 37
lymphoid follicles in the gut and the airways, including the tonsils, the appendix, the Peyers patches, contain specialized resident M cells that transport antigens across the epithelial barrier and deliver samples to underlying lymphoid tissues. Thus, dendritic cells can take up antigens either directly in the environment via extensions able to open the junctions between the epithelial cells or once the antigens have crossed the epithelial barrier. Dendritic cells and M cells constitute the major portals of entry for pathogenic organisms, including viruses, bacteria, and parasites. Dendritic cells have the capacity to open the tight junctions and to sample antigens across epithelia. They express tight junction proteins such as occludin, claudin 1 and zonula occludens 1, which enable them to establish junctions with neighboring epithelial cells thus preserving the integrity of the epithelial barrier. Moreover, the fine regulation of the expression of occludin enables dendritic cell to migrate out the epithelium after antigen uptake.
The gastrointestinal tract, including the anorectal region, is considered as a major entry site for HIV-1, where dendritic and M cells are particularly abundant. Using a human colon carcinoma cell line, we have shown that HIV-1 can infect monolayers of poorly, but not fully, differentiated enterocytes. The CXCR4 or the CCR5 chemokine receptors together with galactosylceramide mediate infection of epithelial cells as well as transcytosis by HIV-1 across enterocytes and M cells. Transcytosis results in productive infection of underlying lymphocytes and monocytes. The stage of differentiation and the chemokine receptor expression profile of intestinal epithelial cells are rate limiting in HIV1 transport through an intact epithelium and the subsequent infection. HIV-1 infection may potentially generate favorable environmental conditions in the intestinal tract for virus replication. On the basis of these considerations, the role of epithelial infection in establishing a reservoir in the intestinal tract needs careful reevaluation.
M cells
Lumen
Epithelium
Target cells
CD4+Tcells monocyte monocyte
Figure 1 Model for HIV-1 penetration across the epithelial barrier HIV-1 viruses ( ) bind to receptors expressed on the apical surface of epithelial cells, enter and cross the epithelial monolayer. For binding, uptake, infection of epithelial cells and transcytosis through the epithelium, both a glycolipid ( )and chemokine receptors ( ) are required. Lack of a chemokine receptor restricts HIV entry into epithelial cells. Uptake of particles ( ) as found in M is not sufficient to mediate HIV-1 infection. HIV-1 integrates and replicates inside the host epithelial cells and is released, probably by budding, into the serosal compartment of the mucosa.
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Differentiation of the follicle-associated epithelium Like the intestinal epithelial cells of the villi, the epithelial cells of the follicle-associated epithelium (FAE) arise from precursor stem cells located in the crypts that proliferate, and differentiate as they migrate to the apex. Epithelial differentiation along the crypt to villus axis requires interaction between epithelial and
mesenchymal cells for the elaboration of the basal lamina. The cells present in the lymphoid tissue also contribute to the formation of the follicle associated epithelium and M cells (figure 2). The membrane lymphotoxin on activated B lymphocytes mediates in part M cell formation. We have developed custom made gut DNA microarrays to identify which genes are specifically activated in the follicle associated epithelium compared to the crypt and villus associated epithelium. B cell-deficient mice
WT
Figure 2: The follicle associated epithelium over a Peyers patch of a wild type (WT) and a B lymphocyte deficient mice. B lymphocytes control the size of the epithelium. M cells (white cells) are reduced in number in the absence of B lymphocytes Microarrays are hybridized with cDNA from undifferentiated and differentiated human intestinal cells to study the changes in gene expression participating in the cell differentiation. RNA extracted from these cells serve as a template to make fluorescently-labelled cDNA probes that are used to probe a microarray consisting of DNA representing different genes spotted onto a glass slide. Among the genes induced differentiation, one finds the homeotic transcription factor cdx-2, which in turn controls several differentiation genes.
Figure 3: Microarray hybridized with cDNA from human intestinal cells Genes preferentially expressed in undifferentiated cells appear in green and those present in differentiated cells in red.
With these techniques we are identifying the genes that are selectively activated along the follicle-associated epithelium and in M cells. This
information is crucial for the design of vaccine delivery systems that efficiently target M cells to trigger strong mucosal immune responses. 39
Antigen presentation in mucosal tissues We have previously reported efficacy of Salmonella vaccine strains in the development of immune responses able to prevent or cure infection such as H. pylori and HPV. As a tool to study the fate of antigens in Peyers patches, we use Salmonella
typhimurium. To trace the bacteria in vivo, we designed bacteria that express the green fluorescent protein (GFP). Dendritic cells were identified as the first Peyers patch cells that internalized bacteria, after transport through the M cells of the follicle-associated epithelium.
Figure 4: Uptake of Salmonella by dendritic. Left: Bone marrow derived dendritic cells in the absence of bacteria. Middle: dendritic cells infected with wild type Salmonella (note the membrane ruffles). Right: dendritic cells infected with Salmonella deleted for invasion virulence genes We further characterized the interactions between Salmonella and dendritic cells in vitro, (Figure 4) and found that dendritic cells efficiently internalize, and present antigens derived from attenuated Salmonella that are defective for invasion of mammalian cells. By contrast, these attenuated Salmonella are unable to enter and survive in macrophages and recombinant antigens are not presented by this type of cells. Thus, dendritic cells may play a major role in the induction of an immune response against a recombinant Salmonella vaccine. whereas macrophages would be more implicated in the clearance of such bacteria. Role of antibodies in protection of mucosal surfaces. To study the effect of neutralizing antibody responses on mucosal infection we use the mouse mammary tumor virus (MMTV) as a model. The target cells of early MMTV infection are B lymphocytes which become activated, infected, and can present a retroviral superantigen to specific T cells. We have immunized rats with 40 MMTV to generate neutralizing antiviral monoclonal antibodies. The antibodies were able to prevent superantigen-specific T cell responses following oral, nasal or systemic challenges. However, they failed to prevent infection of B lymphocytes, the classical target cells for MMTV infection. This suggest that neutralizing antibodies can inhibit the amplification of retroviral infection in mucosal tissues but cannot prevent uptake of viral particles by B cells. We are currently studying whether B cell infection in the presence of neutralizing antibodies contributes to virus dissemination and chronic infection. Mucosal vaccine design Our objectives are to develop live vectors for delivery and presentation of antigens in MHC class I pathway and for generation of CTL in mucosal sites, especially for vaccination against oncogenic pathogens. CD8-mediated immunity is induced by intravacuolar microbes including S. typhimurium, Leishmania s p e c i e s or Mycobacteria. We confirmed that CTL are
stimulated against ovalbumin produced by S . typhimurium. However, this process is not only poorly efficient but is dependent on the nature and the level of antigen. Efforts are currently conducted to improve stimulation of CTL by Salmonella producing ovalbumin as a model antigen by either screening for mutants highly efficient in delivery of antigen in MHC class I pathway or by using inducible bacterial promoters. The epithelial and dendritic cell in vitro systems that we have developed are now used to screen Salmonella strain that efficiently cross M cells triggering the recruitment of dendritic cells but not inflammatory cells, that are targeted and taken up by dendritic cells and that induce both strong CTLs and neutralizing antibodies. In parallel we test the immunogenicity of Salmonella vaccines specific for the clade B and C of HIV-1 in humanized HLA-A2 transgenic mice using novel neutralizing antibody assays and tetrameric complexes specific for various HIV-1 specific CTLs epitopes expressed in Salmonella. The immunogenicity of different attenuated Salmonella strains is compared to that of two Recent publications Hopkins, S., Niedergang, F., Corthsy-Theulaz, I. E., and Kraehenbuhl, J. P. (2000). A recombinant Salmonella typhimurium vaccine strain is taken up and survives within murine Peyer's patch dendritic cells. Cell. Microbiol. 2, 56-68. Stubbe, H., Berdoz, J., Kraehenbuhl, J. P., and Corthsy, B. (2000). Dimeric IgA is superior to monomeric IgA and IgG carrying the same variable domain in preventing Clostridium difficile toxin A damaging of T84 monolayers. J. Immunol. 164, 1952-1960. Schulte, R., Kernis, S., Klinke, S., Bartels, H., Preger, S., Kraehenbuhl, J. P., Pringault, E., and Autenrieth, I. B. (2000). Translocation of Yersinia enterocolitica across reconstituted intestinal epithelial monolayers is triggered by Yersinia invasin binding to beta 1 integrins apically expressed on M-like cells. Cell. Microbiol. 2, 173-185. Coste, A., Sirard, J. C., Johansen, K., Cohen, J., and Kraehenbuhl, J. P. (2000). Nasal immunization of mice with virus-like-particles
vaccinia strains (modified Ankara vaccinia virus and NYVAC) and a recombinant parvovirus (Adenovirus-associated virus (AAV) carrying the same CTLs epitopes. Several phase I trials are planned to test different routes of vaccine administration. This work is carried out in the frame of the EuroVac cluster, the European effort for a HIV/AIDS vaccine. Part of this work was done in collaboration with Hans Acha Orbea (Institute of Biochemistry and Ludwig Institute), Giuseppe Pantaleo and Franois Spertini, (Allergy and Immunology, CHUV), Denise Nardelli-Haefliger and Pierre de Grandi (Gynecology, CHUV), Irne CorthsyTheulaz (Nestl, Vers chez les Blancs), Pierre Michetti (Beth Israel Hospital, Harvard Medical School, Boston), Frank Heppner and Adriano Aguzzi, (Institute of Neuropathology, University of Zurich), Sophie Kernis and Eric Pringault (Institut Pasteur, Paris), Armelle Phalipon and Philippe Sansonetti (Institut Pasteur, Paris), Marian Neutra (Children Hospital, Harvard Medical School, Boston), Anna Rescigno and Paola Ricciardi-Castagnoli (Cellular and Molecular Pharmacology Centre, Milano). protects offspring against rotaviral diarrhea. J. Virol. 74, 8966-8971 . Michetti, M., Kelly, C. P., Kraehenbuhl, J. P., Bouzourene, H., and Michetti, P. (2000). Gastric mucosal alpha(4)beta(7)-integrin-positive CD4 T lymphocytes and immune protection against Helicobacter infection in mice, Gastroenterology 119, 109-118. Niedergang, F., Sirard, J. C., Tallichet Blanc, C., and Kraehenbuhl, J. P. (2000). Entry and survival of Salmonella typhimurium in dendritic cells and presentation of recombinant antigens do not require macrophage-specific virulence factors, Proc. Natl. Acad. Sci. (USA) 97, 14650-14655. Sierro, F., Pringault, E., Simon Assman, P., Kraehenbuhl, J. P., and Debard, N. (2000). Transient expression of M cell phenotype by enterocyte-like cells of the follicle-associated epithelium of mouse Peyer's patches. Gastroenterology 119, 734-743. Debard, N., Sierro, F., Browning, J., and Kraehenbuhl, J. P. (2001). The role of mature lymphocytes and lymphotoxin on the 41
development of the follicle-associated epithelium and M cells in mouse Peyer's patches. Gastroenterology 120, 1173-1182. Nardelli-Haefliger, D., Benyacoub, J., Lemoine, R., Hopkins-Donaldson, S., Potts, A., Hartman, F., Kraehenbuhl, J. P., and De Grandi, P. (2001). Nasal vaccination with attenuated Salmonella typhimurium strains expressing the Hepatitis B nucleocapsid: Dose response analysis. Vaccine 19, 2854-2861. Heppner, F.L., Christ, A.D., Klein, M.A., Prinz, M., Fried, M., Kraehenbuhl, J.P. and Aguzzi, A. (2001). Transepithelial prion transport by M cells. Nat Med 7, 976-977. Rescigno, M., Urbano, M., Valzasinam, B., Francolini, M., Bonasio, R., Kraehenbuhl, J. P., Granucci, F., and Ricciardi-Castagnoli, P. (2001). Dendritic cells express occludin and claudin, open the tight junctions and sample bacteria preserving epithelial barrier integrity. N a t . Immunol. 2, 361-367.
Hathaway, L. J., and Kraehenbuhl, J. P. (2000). The role of M cells in mucosal immunity. Cell. Mol. Life Sci. 57, 323-332 Niedergang, F., and Kraehenbuhl, J. P. (2000). Much ado about M cells. Trends Cell Biol. 10, 137-141. Kraehenbuhl, J. P., and Neutra, M. R. (2000). Epithelial M cells: structure and function. Annu. Rev. Cell Develop. Biol. 16, 301-332. Neutra, M. R., and Kraehenbuhl, J. P. (2001). The regional immune response to microbial pathogens. In Immunology of Infectious Diseases, S. E. H. Kaufmann, A. Sher, and R. Ahmed, eds. (ASM Press). Neutra, M. R., Sansonetti, P., and Kraehenbuhl, J. P. (2001). Interactions of Microbial pathogens with intestinal M cells. In Infections of the Gastrointestinal Tract, B. M.J, P. D. Smith, J. I. Ravdin, H. B. Greenberg, and L. Guerrant, eds. (New York, Raven Press).
42
SIGNALING PATHWAYS LEADING TO APOPTOSIS AND INFLAMMATION
Jrg Tschopp
Jrg Tschopp graduated in biophysics in 1979 at the University of Basel studying T4 phage assembly in the laboratory of Jurgen Engel at the Biocentre. He then joined the group of MllerEberhard at the Scripps Clinic in La Jolla to work on the Membrane Attack Complex of complement. In 1982, he joined the Institute of Biochemistry as assistant professor, where he was promoted to full professor in 1989. His work has focused on mechanisms of target cell death induced by cytotoxic T cells, in particular on signaling pathways triggered by death receptors that lead to apoptosis.
Group members 2000-2001 David Bonnet Sylvie Hertig Laurence Martin Chantal Mattmann Natalia Olivos Sverine Reynard Aubry Tardivel Alain Kummer Magdalena Kovacsovics Pascal Schneider Margot Thome Miazza Vincent Blancheteau Jean-Luc Bodmer Technician Technician Technician (01.2001) Technician Technician Technician (09.2001) Technician Sabbatical host Research Associate (09.2001) Research Associate Research Associate Postdoctoral fellow Postdoctoral fellow (09.2001) Kimberly Burns Susanne Lens Olivier Micheau Vronique Rochat Hisakazu Takatsuka Mathias Thurau Laetitia Agostini Brian Brissoni Olivier Gaide Nils Holler Fabio Martinon Antoine Tinel Barbara Valzasina Postdoctoral fellow Postdoctoral fellow (12.2000) Postdoctoral fellow Postdoctoral fellow (06.2001) Postdoctoral fellow (03.2001) Postdoctoral fellow Ph.D student Ph.D student MD/Ph.D student Ph.D student (10.2000) Ph.D student Ph.D student Ph.D student
SIGNALING PATHWAYS LEADING TO APOPTOSIS AND INFLAMMATION Apoptosis is a naturally occurring process of cell suicide that plays a crucial role in the development and maintenance of multicellular organisms by eliminating superfluous or unwanted cells. This occurs during development, normal cell turn over, hormone-induced tissue atrophy, and pathological processes such as in Alzheimer's disease. Cells undergoing apoptosis show characteristic morphological changes, including shrinkage of the cell and its nucleus, plasma membrane blebbing, chromatin condensation and DNA fragmentation (Figure 1). All mammalian cells appear to constitutively express the basic machinery that mediates apoptotic cell death (a family of cysteine proteases, the caspases), but the initiation of apoptosis is carefully regulated. Signals diverse as viral infection, extracellular survival factors, cell interactions, and hormones may act to either suppress or promote the activation of the death program. One of the most fascinating problems in immunology is understanding how the host organism detects the presence of infectious agents and disposes of the invader without destroying self tissues. IL-1 is a key inflammatory cytokine that mediates profound effects in virtually every organ system of the body. It activates the rapidly acting innate immune response leading to inflammation and places the organism in a state of readiness to deal with injury or infection. 43
Fig. 1: Cytotoxic T cell (left), attacking a tumor cell and inducing apoptosis.
Our research objectives are: The elucidation of signaling pathways that lead to apoptosis and cell survival, in particular those triggered by members of the TNF receptor family (Fas, TRAIL-R, TNF-R, BCMA, TACI, BAFF-R). Their implication in diseases such as cancer, graft-versus-host diseases and autoimmunity will be evaluated. The identification of proteins and signaling pathways that lead to inflammation (NF-kB, Toll-receptors, IL1R).
1. Death receptors, a subfamily of TNF receptors inducing apoptosis The 'death receptors', as their name implies, link extracellular signals to apoptosis. They are members of the TNF-receptor family and are characterized by the presence of a protein-protein interaction motif of about 80 amino acids called the 'death domain' (DD) in the cytplasmic tail. The best studied signaling pathway is the one triggered by Fas (Figure 1). Clustering of Fas upon FasL binding recruits the adaptor protein FADD which in turn binds procaspase-8, resulting in the formation of the 'death inducing signaling complex' (DISC). Procaspase-8 is cleaved autocatalytically and cleaves other caspases further downstream. Some of the caspases cleave other substrates, the death substrates -for example structural proteins (actin, fodrin), signaling proteins (Akt-1) and the inhibitor of a nuclease (ICAD)-eventually leading to the biochemical and morphological changes of the apoptosis.
Fig. 2: Following the binding of the homotrimeric Fas ligand, Fas oligomerizes and recruits the adaptor protein FADD to form the death-inducing signalling complex (DISC), causing the activation of caspase-8. Caspase-8 in turn activates other effector caspases such as caspase-3. The death signal can be amplified through cleavage of Bid and activation of the APAF/caspase-9 complex via cytochrome c.
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Fas signals induce apoptosis and necrosis Cell death is achieved by two fundamentally different mechanisms, apoptosis and necrosis. Apoptosis is dependent on caspase activation, while the caspase-independent necrotic signaling pathway remains largely uncharacterized. Triggering of Fas and TRAIL-R2 by its respective ligands (FasL/TRAIL) is known to rapidly induce cell death leading to apoptosis. However, we found that Fas kills activated primary T cells efficiently in the absence of active caspases, resulting in necrotic morphological changes, late mitochondrial damage but no cytochrome c release. This Fas ligand-induced caspase-independent death is absent in T cells that are deficient in either FADD or RIP. RIP is also required for necrotic death induced by TNF and TRAIL. Importantly, in contrast to its role in NF-B activation, RIP requires its kinase activity for death signaling. Thus, Fas, TRAIL and TNF receptors can initiate cell death by two alternative pathways, one relying on caspase-8, the other dependent on the kinase RIP. FIST, a novel kinase, is recruited to Fas Several proteins have been identified that bind to the cytoplasmic death domain of Fas such as FADD, RIP and Daxx, which couples Fas to the Jun N-terminal kinase pathway. We have identified a 130 kDa kinase designated FIST/HIPK3 as a novel Fas-interacting protein. Binding to Fas is mediated by a conserved sequence in the C-terminus of the protein. FIST/HIPK3 is widely expressed in mammalian tissues and is localized both in the nucleus and in the cytoplasm. In transfected cell lines, FIST/HIPK3 causes FADD phosphorylation, thereby promoting FIST/HIPK3-FADD-Fas interaction. Whereas Fas ligand-induced activation of Jun N-terminal kinase is impaired by overexpressed active FIST/HIPK3, cell death is not affected. Thus, Fas-associated FIST/HIPK3 modulates one of the two major signaling pathways of Fas. Identification of novel receptors and signaling proteins involved in apoptosis Based on the observation that most proteins implicated in the death signaling pathways contain DD, the related DED or CARD domains,
we have cloned and characterized a number of novel pro- and anti-apoptotic proteins. These include various receptors for TRAIL and several signaling proteins such as RIP-2 and Bcl-10 which are crucial signaling proteins leading to NF-kB activation and thus resistance to apoptosis. We also identified a novel CARD-containing protein and interaction partner of Bcl-10, named Carma1. Carma1 is predominantly expressed in lymphocytes and represents a new member of the membrane-associated guanylate kinase family, Carma1 induces phosphorylation of Bcl10 and activation of the transcription factor NF-kappaB. 2. Inhibitions of death receptor pathways Since Fas expression is widespread and the cellular machinery to undergo apoptosis is present in all nucleated cells, cells need to keep undesired suicide or fratricide under control. Despite the expression of abundant surface Fas, cells are frequently resistant to FasL, indicating that the Fas signaling pathway is blocked intracellularly. Identification of FLIPs as potent inhibitor of death receptors Since Fas-induced apoptosis is a critical element in the elimination of virally infected cells it is not surprising that viruses have evolved particularly elegant strategies to evade this facet of the immune response. We have identified a new family of viral and mammalian inhibitors (FLIPs) which interfere with Caspase-8 recruitment to the DED of FADD.
Fig. 3: Structure of FLIPs. The longest form, FLIPL or FLIPa contains two DEDs and a caspase-like domain with significant homology to caspase-8 (or-10). In contrast to caspase-8, however, FLIPL lacks key features that are required for substrate catalysis. The proteolytic activity of caspases
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is dependent on a catalytic diad composed of a Cys and a His residue. Both amino acids are absent in FLIP and replaced by Tyr and Arg, respectively rendering FLIP catalytically inactive. The shortest variant identified, FLIPs corresponds in length to the v-FLIP and comprises 2 DED3 only.
inhibitor of death-receptor-induced apoptosis but it also mediates the activation of NF-kappaB and Erk by virtue of its capacity to recruit adaptor proteins involved in these signaling pathway. Expression of FLIP Activation of the transcription factor NF-kappaB is a major effector of the inducible resistance to death receptor-mediated apoptosis. Previous evidence indicates that the combined transcriptional activation of TRAF-1, TRAF-2, IAP-1, and IAP-2 is required to suppress cell death by tumor necrosis factor. We found that NF-kappaB activation upregulates FLIP, resulting in increased resistance to Fas ligand or TNF. Resistance to either FasL- or TNF-induced apoptosis is overcome when cells are incubated in the presence of a protein synthesis inhibitor which leads to the rapid downregulation of. Our findings suggest that FLIP is an important mediator of NF-kappaB-controlled antiapoptotic signals. Moreover we found that p53 enhances the degradation of FLIP via a ubiquitinproteasome pathway. Thus, p53-mediated downregulation of FLIP may explain the potent sensitization of human cancer cells to the apoptotic suicide program induced by wild-type p53 gene transfer. Bcl-Rambo The Bcl-2 family of proteins plays a central regulatory role in apoptosis. We have identified a novel, widely expressed Bcl-2 member which we have named Bcl-rambo. Bcl-rambo shows overall structural homology to the anti-apoptotic Bcl-2 members containing conserved Bcl-2 homology (BH) motifs 1, 2, 3, and 4. Unlike Bcl-2, however, the C-terminal membrane anchor region is preceded by a unique 250 amino acid insertion containing two tandem repeats. In mammalian cells, Bcl-rambo was localized to mitochondria, and its overexpression induces apoptosis that is specifically blocked by the caspase inhibitors, IAPs, whereas inhibitors controlling upstream events of either the 'death receptor' (FLIP, FADD-DN) or the 'mitochondrial' pro-apoptotic pathway (Bcl-x(L)) had no effect. Surprisingly, the Bcl-rambo cell death activity was induced by its membrane-anchored C-terminal domain and not by the Bcl-2 homology region. Thus, Bclrambo constitutes a novel type of pro-apoptotic
FLIPs are able to protect cells from apoptosis induced by Fas, TNF-R1, TRAMP, and TRAILreceptors. Two distinct splice variants of cellular FLIP are found: The 55 kDa FLIP protein (FLIPL) is expressed in many tissues. In lymphatic tissues an additional 25 kDa FLIP species (FLIPS) is detectectable. The expression of FLIP in T lymphocytes is dependent on the degree of activation.and correlates with the lymphocytes' resistance to FasL. Activation-induced cell death (AICD) of lymphocytes is an important mechanism of selftolerance. AICD is mediated by Fas and is enhanced by IL-2. We found that IL-2 upregulates FasL and decreases transcription and expression of FLIP, rendering T cells sensitive to apoptosis. The finding that IL-2 promotes T cell death seems paradoxical, considering that its most clearly established function is as growth and survival factor. The unregulated accumulation of lymphocytes seen in mice lacking IL-2, however, suggests that the major obligatory function is lymphocyte homeostasis. Fas/FLIP signals induce proliferation Fas signals do not always result in apoptosis but can also trigger a pathway that leads to proliferation. We investigated the level at which the two conflicting Fas signals diverge and the protein(s) that are implicated in switching the response. Under conditions in which proliferation of CD3-activated human T lymphocytes was increased by recombinant FasL, there was activation of the transcription factors NF-kappaB and AP-1 and recruitment of the caspase-8 inhibitor and FADD-interacting protein FLIP. Fas-recruited FLIP interacted with TNF-receptor associated factors 1 and 2, as well as with the kinases RIP and Raf-1, resulting in the activation of the NF-kappaB and extracellular signal regulated kinase (Erk) signaling pathways. In T cells these two signal pathways are critical for interleukin-2 production. Increased expression of FLIP in T cells resulted in increased production of interleukin-2. Thus, FLIP is not simply an 46
Bcl-2 member that triggers independently of its BH motifs.
cell
death
3. Members of the TNF family that stimulate cell survival Members of the tumor necrosis factor (TNF) family induce pleiotropic biological responses, including not only cell death but also cell growth and differentiation. We characterized two novel structurally related members of the TNF family designated APRIL and BAFF. APRIL, a ligand that supports tumor cell growth APRIL is expressed at low abundance in normal tissues, but high levels of mRNA are detected in transformed cell lines, and in human cancers of colon, thyroid, and lymphoid tissues in vivo. The addition of recombinant APRIL to various tumor cells stimulates their proliferation. Moreover, APRIL-transfected NIH-3T3 cells show an increased rate of tumor growth in nude mice compared with the parental cell line. We identified B cell maturation antigen (BCMA) and transmembrane activator and calcium modulator and cyclophilin ligand (CAML) interactor (TACI), two predicted members of the TNF receptor family, as receptors for APRIL. A soluble form of BCMA, which inhibits the proliferative activity of APRIL in vitro, decreases tumor cell proliferation in nude mice. Growth of HT29 colon carcinoma cells is blocked when mice are treated once per week with the soluble receptor. These results suggest an important role for APRIL in tumorigenesis and point towards a novel anticancer strategy. BAFF controls B cell growth The second member that we have identified, designated BAFF is expressed by T cells and dendritic cells. Membrane-bound BAFF is processed and secreted through the action of the protease furin. BAFF induces proliferation antiimmunoglobulin M-stimulated peripheral blood B lymphocytes. Moreover, increased amounts of immunoglobulins are found in supernatants of germinal center-like B cells costimulated with BAFF. Mice transgenic for BAFF have vastly increased numbers of mature B and effector T
cells, and develop autoimmune-like manifestations. This phenotype is reminiscent of certain human autoimmune disorders and suggests that dysregulation of BAFF expression may be a critical element in the chain of events leading to autoimmunity.
Fig. 4: Immunohistochemistry of frozen spleen of control and TACI:Fc (BAFF-deficient) transgenic mice. Serial sections were double stained with B cells (anti B220, brown), T cells (anti-CD3, purple). Note the highly reduced numbers of B cells.
There is an absolute requirement for BAFF in normal B cell development. Examination of secondary lymphoid organs from BAFF-deficient mice reveals an almost complete loss of follicular and marginal zone B lymphocytes (Fig. 4). BAFF therefore plays a crucial role in B cell development. Although BAFF is already known to bind two receptors, BCMA and TACI, we have identified a third receptor for BAFF that we have termed BAFF-R. BAFF-R binding appears to be highly specific for BAFF, suggesting a unique role for this ligand-receptor interaction. Consistent with this, the BAFF-R locus is disrupted in A/WySnJ mice, which display a B cell phenotype qualitatively similar to that of the BAFF-deficient mice. Thus, BAFF-R appears to be the principal receptor for BAFF-mediated mature B cell survival. 4. Inflammation: The IL-1 receptors and activation of NF-kB The cytokine interlenkin-1 (IL-1) is a key regulator of immune and inflammatory responses and a determining cytokine in the pathogenesis of inflammatory diseases such as rheumatoid arthirits. IL- 1 induces a variety of inflammatory proteins such as acute phase proteins, cytokines and adhesion molecules whose genes are mainly regulated by activation of NF-kB and AP- 1. The receptor system and signal transduction pathway
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that leads to the changes in gene expression has only relatively recently begun to be elucidated. An intriguing theme has emerged in that there are a number of IL-1 receptor like molecues in mammals, insects and plants which are all involved in the responses to infection. These systems utilize remarkably conserved molecular components, many of which contain homologous protein-protein interaction domains such as the Toll/interleukin-1 receptor (TIR) domain or the death domain (DD). MyD88 is an adapter protein in IL-1 signal transduction IL- 1 signaling is initiated by formation of a complex composed of type I interleukin- 1 receptor I (IL-1RI) and IL-1R associated protein (IL-lRAcP) (IL-lRs) which contain cytoplasmic TIR domains. Binding of IL-1 to the IL-1RI induces recruitment of the serine/threonine IL-lRassociated kinase (IRAK) which contains a Nterminal DD. Subsequently, IRAK becomes highly phosphorylated leading to its dissociation from the IL-lRs and association with TRAF6, the
link to the NF-KB/JNK activating machinery. We found that with its dual domain organization MyD88 (a N-terminal DD and a C-terminal TIR) has ideal properties to function as an adapter linking Toll and death modules. Overexpression of MyD88 induces activation of the c-Jun Nterminal kinase. Characterization of Tollip, a crucial component of the IL-1R signalling pathway We have identified a new component of the IL-1 signaling complex which we have named Tollip. We found that, before IL-1beta treatment, Tollip is present in a complex with IRAK, and that recruitment of Tollip-IRAK complexes to the activated receptor complex occurs through association of Tollip with IL-1RAcP. Corecruited MyD88 then triggers IRAK autophosphorylation, which in turn leads to rapid dissociation of IRAK from Tollip (and IL-1Rs). As overexpression of Tollip results in impaired NF-kappaB activation, we conclude that Tollip is an important constituent of the IL-1R signalling pathway.
Recent publications Kataoka, T., Holler, N., Micheau, O., Martinon, F., Tinel, A., Hofmann, K. and Tschopp, J. (2001). Bcl-rambo, a novel Bcl-2 homologue that induces apoptosis via its unique C-terminal extension. J. Biol. Chem. 276, 19548-54. Thompson, J. S., Bixler, S. A., Qian, F., Vora, K., Scott, M. L., Cachero, T. G., Hession, C., Schneider, P., Sizing, I. D., Mullen, C., Strauch K., Zafari M., Benjamin C.D., Tschopp J., Browning J.L. and Ambrose C. (2001). BAFF-R, a newly identified TNF receptor that specifically interacts with BAFF. Science 293, 2108-11. Holler, N., Zaru, R., Micheau, O., Thome, M., Attinger, A., Valitutti, S., Bodmer, J. L., Schneider, P., Seed, B. and Tschopp, J. (2000). Fas triggers an alternative, caspase8independent cell death pathway using the kinase RIP as effector molecule. Nat. Immunol. 1, 489-502.
Rochat-Steiner, V., Becker, K., Micheau, O., Schneider, P., Burns, K. and Tschopp, J. (2000). FIST/HIPK3. A Fas/Fadd-interacting serine/ threonine kinase that induces Fadd phosphorylation and inhibits Fas-mediated Jun NH(2)-terminal kinase activation. J. Exp. Med. 192, 1165-74 Rennert, P., Schneider, P., Cachero, T. G., Thompson, J., Trabach, L., Hertig, S., Holler, N., Qian, F., Mullen, C., Strauch, K Browning J.L, Ambrose C. and Tschopp J. (2000). A Soluble Form of B Cell Maturation Antigen, a Receptor for the Tumor Necrosis Factor Family Member APRIL, Inhibits Tumor Cell Growth. J. Exp. Med. 192, 1677-1684. Burns, K., Clatworthy, J., Martin, L., Martinon, F., Plumpton, C., Maschera, B., Lewis, A., Ray, K., Volpe, F.. and Tschopp, J. (2000). Tollip, a new component of the IL-1RI pathway, links IRAK to the IL-1 receptor. Nat. Cell Biol. 2, 34651.
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Bodmer, J. L., Holler, N., Reynard, S., Vinciguerra, P., Schneider, P., Juo, P., Blenis, J. and Tschopp, J. (2000). TRAIL receptor-2 signals apoptosis through FADD and caspase-8. Nat. Cell Biol. 2, 241-3. Schneider, P., MacKay, F., Steiner, V., Hofmann, K., Bodmer, J. L., Holler, N., Ambrose, C., Lawton, P., Bixler, S., Acha-Orbea, H., Valmori, D., Romero, P., Werner-Favre, C., Zubler, R. H., Browning, J. L. and Tschopp, J. (1999). BAFF, a novel ligand of the tumor necrosis factor family, stimulates B cell growth. J. Exp. Med. 189, 174756. Viard, I., Wehrli, P., Bullani, R., Schneider, P., Holler, N., Salomon, D., Hunziker, T., Saurat, J. H., Tschopp, J. and French, L. E. (1998). Inhibition of toxic epidermal necrolysis by blockade of CD95 with human intravenous immunoglobulin. Science 282, 490-493. Hahne, M., Kataoka, T., Schroter, M., Hofmann, K., Irmler, M., Bodmer, J. L., Schneider, P., Bornand, T., Holler, N., French, L. E., Sordat, B., Rimoldi, D. and Tschopp, J. (1998). APRIL, a new ligand of the tumor necrosis factor family, stimulates tumor cell growth. J. Exp. Med. 188, 1185-1190. Thome, M., Hofmann, K., Burns, K., Martinon, F., Bodmer, J. L., Mattmann, C. and Tschopp, J. (1998). Identification of CARDIAK, a RIP-like kinase that associates with caspase-1. Curr. Biol. 8, 885-888.
Burns, K., Martinon, F., Esslinger, C., Pahl, H., Schneider, P., Bodmer, J. L., Di Marco, F., French, L. and Tschopp, J. (1998). MyD88, an adapter protein involved in interleukin-1 signaling. J. Biol. Chem. 273, 12203-12209. Schneider, P., Thome, M., Bodmer, J. L., Hofmann, K., Kataoka, K., Holler, N. and Tschopp, J. (1997). TRAIL-receptors 1 (DR4) and 2 (DR5) signal FADD-dependent apoptosis and activate NF-kB. Immunity 7, 831-836. Irmler, M., Thome, M., Hahne, M., Schneider, P., Hofmann, K., Steiner, V., Bodmer, J. L., Schroter, M., Burns, K., Mattmann, C., Rimoldi, D., French, L. E. and Tschopp, J. (1997). Inhibition of death receptor signals by cellular FLIP. Nature 388, 190-195. Thome, M., Schneider, P., Hofmann, K., Fickenscher, H., Meinl, E., Neipel, F., Mattmann, C., Burns, K., Bodmer, J.-L., Schrter, M., Scaffidi, C., Krammer, P. H., Peter, M. E. and Tschopp, J. (1997). Viral FLICE-inhibitory proteins (FLIPs) prevent apoptosis induced by death receptors. Nature 386, 517-521. Hahne, M., Rimoldi, D., Schrter, M., Romero, P., Schreier, M., French, L. E., Schneider, P., Bornand, T., Fontana, A., Lienard, D., Cerottini, J.-C. and Tschopp, J. (1996). Melanoma cells express Fas(Apo-1/CD95) ligand: Implications for tumor immune escape. Science 274, 13631366.
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MALARIA: IN THE SEARCH OF ANTIGEN FOR THE DEVELOPMENT OF PROTECTIVE VACCINES
Giampietro Corradin
Giampietro Corradin graduated in chemistry at the University of Padua and received his PhD degree in chemistry from the University of California, Santa Barbara, after completion of a thesis on the structure and function of cytochrome c. After a post-doctoral position in biochemistry at Dartmouth Medical School, Hanover, New Hampshire, he continued his training in molecular immunology at the National Jewish Hospital, Denver, Colorado. He joined the Institute of Biochemistry in 1979 where he is Associate Professor.
Group members 2000-2001 Luis Rodrigues Leonor Morgado Rgine Audran Katrin Peter Vincent Brossard Martina Bazzaro Anilza Bonelo Technician Assistant Technician Postdoctoral fellow Postdoctoral fellow Ph.D student (12.2001) Ph.D student (09.2000) Ph.D student (03.2000) Francisco Estvez Ghada Ibrahim Valentin Meraldi Jackeline Romero Karin Giddey Aly Thiocone Alexandre Schmid Ph.D student (05.2000) Ph.D student Ph.D student Ph.D student Diploma student (05.2000) Research assistant (07.2000) Trainee (10.2001)
MALARIA: IN THE SEARCH OF ANTIGENS FOR THE DEVELOPMENT OF PROTECTIVE VACCINES Malaria is a world-wide parasitic disease which affects millions of people especially young children and pregnant women. Among the measures directed toward the prevention of this disease, vaccines represent a cost-effective approach. Identification of molecules important in the elicitation of a protective immune response and their use in experimental animals to be followed by testing in human volunteers has been a constant goal of our laboratory. Malaria is a parasitic disease transmitted during the blood meal of infected mosquitoes which inoculate sporozoites into the mammalian host. Within minutes, sporozoites invade hepatocytes and develop into merozoites intracellularly by asexual schizogony. The merozoites then invade red blood cells, producing the various symptoms associated with the disease. The life-cycle is completed when gametocytes are ingested during the blood meal of the mosquito vectors. Protective immunity against malaria can be obtained by immunizing mice and humans with irradiation-attenuated sporozoites. This immunity is the result of the effect of neutralizing antibodies recognizing free sporozoites in the blood stream and of CD4+ and CD8+ T cells which prevent the development of the parasite hepatic forms. Experiments performed in B cell deficient mice have demonstrated that, despite the absence of anti-sporozoite antibodies, protection is induced by irradiated sporozoite immunization. This suggests that T cells specific for proteins present in the intracellular hepatic stage play a predominant role in protection. Therefore, one of the aim in malaria vaccine research is to mimic the protective immune response induced by injection of irradiated sporozoites.
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The impossibility of culturing sporozoites outside the mosquitos renders this vaccination approach impracticable on a large scale. Thus, considerable effort has be devoted to the identification of highly antigenic sporozoite derived antigens which can substitute for the whole sporozoite in the design of anti-malaria vaccines. Our approach to mimic the immune response observed upon immunization with irradiated sporozoites involves the use of long polypeptides containing various B, T-helper, and T-cytotoxic epitopes. The use of longer fragments of the protein will also provide epitopes capable of binding to the various MHC class I and II molecules, thus overcoming the MHC restriction observed in response to smaller epitopes. Implicated in the protective effect observed with irradiated sporozoites is an abundant surface molecule, the circumsporozoite (CS) protein. The recombinant CS protein has been shown to inhibit sporozoite liver invasion. The region of the CS protein responsible for binding to the hepatocytes has been located in the C-terminus. In particular, region 2 segment (RII, aa 342-363 of the NF-54 sequence) from different parasite strains or species is highly conserved and is homologous to other mammalian proteins such as thrombospondin. Both the N- and C-terminal fragments of P. falciparum parasites are recognized by human sera from donors living in endemic areas and are very immunogenic in Aotus monkeys. In addition we have shown that mice immunized with a synthetic peptide corresponding to the C-terminal domain of the CS protein from P. berghei were efficiently protected from sporozoite challenge. The immunogenic and/or protective properties of long polypeptides covering the flanking regions of the CS protein of Plasmodium berghei (Pb) and P. yoelii (Py) were studied in vivo in BALB/c mice. Peptides PbCS 21-91, PbCS 242-310, PyCS 20-138 or PyCS 277-345 were injected in combination with Incomplete Freund's Adjuvant (IFA) or aluminum hydroxide (Alum). Two doses were sufficient to generate a specific proliferative response in the presence of the immunizing antigen, a high level of antibodies directed to the synthetic peptides and the native protein on the infecting sporozoite, and also a high level of
cytotoxic activity. Interestingly, immunization with the long peptides induced an efficient cytotoxic response not only in the draining lymph nodes and spleen, but also in the liver. This result is very encouraging, since the liver appears to be the first organ in which the immune system may encounter the parasites. Furthermore, an infective challenge with P. berghei sporozoite-bearing mosquitoes performed 10-15 days after the peptide boost resulted in 50-100% protection. Peptides corresponding to the N- and C- terminal ends of the CS protein of P. falciparum have also been tested as immunogens in mice. Both antibody production and lymphocyte proliferation were observed; moreover, a strong cytotoxic activity was also detected, which led us to the description of a new CTL epitope in the CS protein. This has led to the exploration of the best combination of antigen and adjuvant for an optimal elicitation of an immune response in experimental animals first and then in humans. Among the adjuvant systems tried, biodegradable microspheres made of poly-lactate and glycolate are of particular interest since they hold the promise of obtaining a strong immune response with a single injection. Malaria specific cytotoxic response in humans The role of antigen specific CD8+ T-lymphocytes in mediating protection against sporozoiteinduced malaria has been well established in murine models. In humans, indirect evidence has accumulated suggesting a similar protective role for antigen-specific CD8+ T-lymphocytes. Nevertheless, the low frequency of circulating specific cells together with the lack of sensitive and reliable methods for the quantitation of class I restricted CD8+ T-lymphocytes have hampered the direct assessment of their function in malaria exposed individuals. We determined the optimal conditions for the evaluation of CD8+ Tlymphocytes recognizing single epitopes in humans, using the model peptide of the influenza virus matrix protein 58-66 presented by the HLAA*0201 molecule. A combination of short term cell culture and IFN-ELISPOT, allowed the identification of new CD8+ T-lymphocyte epitopes derived from various proteins of preerythrocytic stages of Plasmodium falciparum. Some of these epitopes were also strongly 51
recognized in classical chromium release assay after repeated in vitro stimulation of peripheral blood lymphocytes. The frequency of peptide specific IFN- spot forming cells was found to be higher in donors coming from a region of high malaria endemicity in Burkina Faso compared to donors coming from a low endemic region or donors which had not been exposed to malaria. We conclude that the IFN- ELISPOT assay is a sensitive technique to monitor antigen-specific CD8+ T-lymphocyte responses in human malaria which may help in the improvement and assessment of the efficacy of malaria subunit vaccines. Phase I clinical trial Based on the results described above we have undertaken a phase I clinical trial of vaccination in normal volunteers in order to evaluate the safety and immunogenicity of peptide 282-383 corresponding to the C-terminal region of the CS protein from P. falciparum NF-54 strain. No adverse reactions were observed upon repeated intramuscular administration of the peptide in alum or Montanide 720. Immunization resulted in resulted in the elicitation of high titer specific antibodies as well as specific helper and cytotoxic T cell responses. A phase II trial is planned for 2002.
Malaria cycle
Recent publications Renggli, J., Hahne, M., Matile, H., Betschart, B., Tschopp, J., Corradin, G. (1997). Elimination of P. berghei liver stages is independent of Fas (CD95/Apo-I) or perforin-mediated cytotoxicity. Parasite Immunol. 19, 145-148. Roggero, M.A., Servis, C., Corradin, G. (1997). A simple and rapid procedure for the purification of synthetic polypeptides by a combination of affinity chromatography and methionine chemistry. Febs Letters 408, 255. Men, Y., Tamber, H., Audran, R., Gander, B., Corradin, G. (1997). Induction of a cytotoxic T lymphocyte response by immunization with a malaria specific CTL peptide entrapped in biodegradable polymer microspheres. Vaccine 15, 1405. Audran, R., Men, Y., Johansen, P., Gander, B., Corradin, G. (1998). Enhanced immunogenicity of microencapsulated tetanus toxoid with stabilizing agents. Pharm. Res. 15, 1111-1116.
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Roggero, M.A., Weilenmann, C., Bonelo, A., Audran, R., Renggli, J., Spertini, F., Corradin, G., Lopez, J.A. (1999). Plasmodium Falciparum CS C-terminal fragment: preclinical evaluation and phase-I clinical studies. Parassitologia 41, 421424. Roggero, M.A., Meraldi, V., Lopez, J.A., Eberl, G., Romero, J.C., Matile, H., Betschart, B., Corradin, G., Renggli, J. (2000). The synthetic, oxidized C-terminal fragment of the Plasmodium berghei ciucumsporozoite protein elicits a high protective response. Eur. J. Immunol. 30, 26792685. Bonelo, A., Valmori, D., Triponez, F., Tiercy, J.M., Mentha, G., Oberholzer, J., Champagne, P., Romero, J.F., Esposito, F., Nebi, I., Barbey, C., Romero, P., Herrera, S., Corradin, G., Lopez, J.A. (2000). Generation and characterization of malaria-specific human CD8+ lymphocyte clones: effect of natural polymorphism on T cell recognition and endogenous cognate antigen presentation by liver cells. Eur. J. Immunol. 30, 3079-3088. Gonzalez, J.M., Peter, K., Esposito, F., Nebi, I., Tiercy, J.M., Bonelo, A., Arvalo-Herrera, M., Valmori, D., Romero, P., Herrera, S., Corradin, G., Lopez, J.A. (2000). HLA-A*0201 restricted CD8+ T-lymphocyte responses to malaria : identification of new Plasmodium falciparum
epitopes by IFN-g ELISPOT. Parasite Immunol. 22, 501-514. Romero, J.F., Eberl, G., MacDonald, H.R., Corradin, G. (2001). CD1d-restricted NK T cells are dispensable for specific antibody responses and protective immunity against liver stage malaria infection in mice. Parasite Immunol. 23, 267-269. Lopez, J.A., Weilenman, C., Audran, R., Roggero, M.A., Bonelo, A., Tiercy, J.M., Spertini, F., Corradin, G. (2001). A synthetic malaria vaccine elicits a potent CD8+ and CD4+ T lymphocyte immune response in humans. Implications for vaccination strategies. Eur. J. Immunol. 31, 1989-1998. Perlaza, B.L., Sauzet, J.P., Toure Balde, A.T., Brahimi, K., Tall, A., Corradin, G., Druilhe, P. (2001). Long synthetic peptides encompassing the Plasmodium falciparum LSA3 are the target of human B and T cells and are potent inducers of B helper, T helper and cytolytic T cell responses in mice. Eur. J. Immunol. 31, 2200-2209. Peter, K., Men, Y., Pantaleo, G., Gander, B., Corradin, G. (2001). Induction of a cytotoxic Tcell response to HIV-1 proteins with short synthetic peptides and human compatible adjuvants. Vaccine 19, 4121-4129.
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BIOCHEMISTRY OF LYMPHOCYTE MEMBRANE PROTEINS
Claude Bron
Claude Bron studied biochemistry at the University of Lausanne and received his Ph.D in 1967. He then joined the laboratory of Dr M.D. Poulik at the Child Research Center (Wayne State University, Detroit, Michigan) to work on the structure and function of red cell membrane. In 1970, he returned to the Institute of Biochemistry as Assistant Professor and set up a laboratory centered on the biochemistry of lymphocyte membrane proteins. He was promoted full professor of Biochemistry in 1974 and became chairman of the Institute in 1988 upon retirement of Prof. Henri Isliker. His present interests have focused on the role of membrane lipid rafts in glycolipid anchored and transmembrane receptormediated lymphocyte signaling. Group members 2000-2001 Rosa Castillo-Arias Sandra Levrand Marga Rousseaux Florent Bender Technician (02.2001) Technician Technician Postdoctoral fellow (08.2001) Marie-Agns Doucey Daniel Leger Sylvia Rothenberger Stphanie Zutter Postdoctoral fellow Postdoctoral fellow Postdoctoral fellow Apprentice
BIOCHEMISTRY OF LYMPHOCYTE MEMBRANE PROTEINS A number of functionally diverse cell surface proteins are anchored in the plasma membrane by a complex phosphatidylinositol-containing glycolipid (GPI) with a structure well conserved in evolution. Such membrane proteins differ from conventional cell surface proteins in that they lack transmembrane and intracellular regions. This peculiar mode of association with the membrane provides GPI-anchored proteins with distinct properties such as increased membrane mobility, novel mechanisms of shedding and intercellular protein transfer. In addition, cell surface cross-linking of GPI-anchored molecules triggers transmembrane signaling events. This is of particular importance for leukocytes which express a large number of GPI-anchored proteins whose perturbation initiates cellular activation or modulates activation signals delivered by transmembrane receptors. Although the exact physiological significance of this mode of membrane anchoring is not yet understood, several recently described properties of GPI-anchored proteins point toward a unifying concept according to which specialised plasma membrane domains called lipid rafts which are the preferred location of GPIanchored and dualy acylated signaling molecules, could function as " message centers " for the cell and may play a role in the reception, conversion and transmission of external stimuli. Lipid rafts are tightly packed, liquid-ordered domains enriched in sphingolipids and cholesterol that resist solubilisation in non-ionic detergent (Fig. 1). Over the past years, our laboratory has studied the structural and biochemical features required for the post-translational maturation and surface expression of GPI-anchored molecules. Experiments undertaken more recently were aimed at clarifying some questions which bear on the functional significance of lipid rafts particularly in the initiation of the signaling ability displayed by GPI-anchored proteins.
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Fig. 1 Identification and purification of membrane lipid rafts of T lymphocytes.
1. Thy-1 binds to integrin 3 on astrocytes and triggers formation of focal contact sites Thy-1 is a glycosyl phosphatidylinositol (GPI)anchored glycoprotein of the immunoglobulin superfamily (IgSF) expressed in various cell types, particularly those of the T cell lineage and the neuronal system. In neurons, Thy-1 expression is developmentally regulated, whereby both initial appearance and ultimate distribution are controlled to ensure that Thy-1 is excluded from regions of axonal growth. Thy-1 expression is preferentially initiated toward the end of axon extension, consistent with the idea that it might participate in stabilizing existing neuronal connections and inhibiting future neurite outgrowth. However Thy-1 function remains largely obscure in the absence of a defined ligand. Astrocytes, ubiquitous cells of the brain, express a putative Thy-1 ligand that prevents neurite outgrowth. We identified ligand molecule for Thy-1 and investigated the consequences of Thy1 binding for astrocyte function. Thy-1 has been implicated in cell adhesion and, indeed, all known Thy-1 sequences were found to contain an integrin binding, RGD-like sequence. Thy-1 interaction with 3 integrin on astrocytes was demonstrated in an adhesion assay using a thymoma line (EL-4) expressing high levels of Thy-1. EL-4 cells bound to astrocytes five times more readily than EL-4-f, control cells lacking Thy-1. Binding was blocked by either anti-Thy-1 or anti-3 antibodies, by RGD-related peptides, or by soluble Thy-1-Fc chimeras. However,
neither RGE/RLE peptides nor Thy-1(RLE)-Fc fusion protein inhibited the interaction. Immobilized Thy-1-Fc, but not Thy-1(RLE)-Fc fusion protein supported the attachment and spreading of astrocytes in a Mn2+-dependent manner. Binding to Thy-1-Fc was inhibited by RGD peptides. Moreover, vitronectin, fibrinogen, denatured collagen (dcollagen), and a kistrinderived peptide, but not fibronectin, also mediated Mn2+-dependent adhesion, suggesting the involvement of 3 integrin. The addition of Thy-1 to matrix-bound astrocytes induced recruitment of paxillin, vinculin, and focal adhesion kinase (FAK) to focal contacts and increased tyrosine phosphorylation of proteins such as p130Cas and FAK. Furthermore, astrocyte binding to immobilized Thy-1-Fc alone was sufficient to promote focal adhesion formation and phosphorylation on tyrosine. In conclusion, we demonstrated that Thy-1 binds to 3 integrin and triggers tyrosine phosphorylation of focal adhesion proteins in astrocytes, thereby promoting focal adhesion formation, cell attachment, and spreading. 2. Cell surface insertion of GPI-anchored proteins into lipid rafts (in collaboration with Beat Imhof, Dept. Patholology, Faculty of Medicine) Isolated GPI-anchored proteins have the capacity to integrate with cell surface membranes and to exert their function. Thus a recombinant GPIGreen Fluorescence Protein (GFP) was constructed and shown to selectively insert into lipid rafts upon exogenous addition to cultured cells. This alternative approach to gene transfer might be particularly suitable for labeling rafts of specific T cell clones and for studying the dynamics of lymphocyte lipid raft mobility and clustering in the plane of the membrane upon antigen stimulation (Fig. 2). The usefulness of such cell surface engineering of GPI-anchored proteins is presently explored in a murine model of cancer immune therapy based on targeting cytotoxic lymphocytes to tumors. In fact, invasion of tumors by T lymphocytes is not efficient due to the absence of adhesion mechanisms leading to tumor specific homing. 3 is an integrin which is upregulated in tumor-associated blood vessels of small caliber in 55
invasive breast carcinoma. A chimeric ligand for 3 on angiogenic endothelial cells of vascularized tumors was created to facilitate tumor homing. This ligand, termed KISS31, consists of the soluble kistrin from the viper venom which binds specifically to integrin 3 and IIb3, and the N-terminal region of PECAM-1 (CD31) the platelet endothelial cell adhesion molecule which among other adhesion properties interacts heterotypically with integrin 3. Lymphocytes stably expressing KISS31 as a transgene bound recombinant soluble 3 integrin in vitro and mediated homing to vascularized tumor in vivo. We generated a GPIlinked KISS31 fusion protein. Cells expressing this recombinant GPI-KISS 31 as a transgene at the plasma membrane, bound soluble 3 in vitro. Primary human and mouse lymphocytes were then incubated with purified KISS31-GPI. This exogenous insertion into the plasma membrane (cell surface painting) was stable and fully functional when tested by solid phase adhesion assay. In addition, cell surface painted primary lymphocytes with KISS 31-GPI mediated specific homing to vascularized tumors in vivo.
receptor (TNFR) family. In this study we analyze the structural elements of the EB virus LMP1 required for insertion into membrane rafts of infected cells and investigate the biological significance of such a localization in view of the fact that LMP1 can bind TNF receptor associated factors, a property which might provide the virus with a means of interfering with cell apoptosis. This 386 amino acid protein consisting of a short cytoplasmic N-terminal domain, six transmembrane regions and a long cytoplasmic Cterminal segment is targeted to lipid rafts in transfected HEK 293 cells. The endogenous TNFR-associated factor 3 binds LMP1 and is recruited to lipid rafts upon LMP1 expression. An LMP1 mutant lacking the C-terminal 55 amino acids (C55) behaves like the wild-type (wt) LMP1 with respect to membrane localization. In contrast, a mutant with a deletion of the 25 Nterminal residues (N25) does not concentrate in lipid rafts but still binds TRAF3, demonstrating that the N-terminal segment is crucial for targeting the protein to lipid rafts. Moreover, N25 inhibits wt LMP1-mediated induction of the transcription factors NF- B and AP-1. Morphological data indicate that N25 hampers wt LMP1 plasma membrane localization and blocks LMP1 function (Fig. 3).
Fig. 2 3. Functional significance of the localisation of latent membrane protein 1 (LMP1) of Ebstein-Barr virus (EBV) in membrane rafts of lymphocytes The latent membrane protein 1 (LMP1) encoded by the Epstein-Barr virus acts like a constitutively activated receptor of the tumor necrosis factor Fig. 3 4. Role of lymphocyte membrane rafts in TCR-mediated signaling (in collaboration with I.F. Luescher, Ludwig Institute for Cancer Research, S.Valitutti, INSERM, Toulouse, France)
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Specific T-cell activation is initiated by interaction of the T-cell receptor (TCR) with antigenic peptides presented by major histocompatibility complex (MHC) molecules on the surface of antigen-presenting cells (APC). Engagement of the TCR by cognate MHC peptide results in the rearrangement of signaling molecules at the site of T-cell contact with the APC. This process is crucial for signaling, because it induces apposition of protein kinases with their substrates at the contact site. Specialized subdomains of the T-cell membrane participate in the initiation of this process by being recruited to the site of TCR engagement. These membrane microdomains, also called lipid rafts or detergent-insoluble glycolipid-enriched membranes (DIG), contain various acylated proteins involved in the early steps of T-cell activation, such as the Src kinases p56lck (Lck) and Fyn, the adaptor protein LAT (linker for activation of T cells), and the coreceptors CD4 and CD8. CD8 plays a crucial role in cytolytic T cell activation and development. By binding to TCRassociated MHC molecules, CD8 greatly strengthens TCR-ligand interaction on cells. Because CD8 can associate with Lck, as well as with LAT, this coordinate binding recruts these two important signaling molecules to engaged TCR. We examined the role of CD8 and rafts in the activation of cloned CD8+ CTL by soluble MHC-peptide complexes and observed that monomeric complexes co-engaging TCR and CD8 promote association of TCR/CD3 with CD8/Lck and that cross-linking of these adducts induces the initial and essential activation of Lck in rafts. Palmitoylation and myristoylation of Lck, Fyn, LAT, CD4 and CD8 are essential for their partitioning in lipid rafts and for contribution to TCR signaling. Mutation or deletion of the acylation sites of these molecules compromises their functional integrity. In addition, 2bromopalmitate and 1-hydroxymyristate, analogues of palmitate and myristate, respectively, have been shown to prevent localization of these molecules to lipid rafts and
hence T-cell activation. Likewise, prolonged incubation of T cells in the presence of polyunsaturated fatty acids has been shown to inhibit protein acylation and TCR signaling. On the basis of these observations, we investigated whether insertion of palmitoylcontaining lipids into T-cell membrane interferes with the molecular sorting and TCR signaling in lipid rafts, without disrupting these microdomains. In fact exogenous dipalmitoylphosphatidylethanolamine (DPPE) partitions in lipid rafts and inhibits CTL activation induced by sensitized APC or soluble MHC/peptide antigen complexes tetramers. In contrast, the closely related dioleoyl-phosphatidylethanolamine (DOPE), which contains unsaturated oleic acid in place of saturated palmitic acid, failed to partition in lipid rafts and had no effect on CTL activation. Moreover, we provide evidence that DPPE prevents activation-induced translocation of acylated molecules to lipid rafts and hence induction of TCR signaling (Fig. 4).
Fig. 4 More recently we showed that CTL adhesion in extracellular matrix proteins is required for efficient activation of the CTL effector function by soluble antigen complexes. Hence the convergence of the 1 integrin and the antigenmediated specific signals leads to the formation of large clusters of lipid rafts associated with the cell cytoskeleton in the focal adhesion areas thus contributing to amplify the activation process.
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Recent publications Romagnoli, P., Bron, C. (1997). Phosphatidylinositol-based glycolipid-anchored proteins enhance proximal TcR signaling events. J. Immunol. 158, 5757-5764. Romagnoli, P., Bron, C. (1999). Defective TCR signaling events in glycosylphosphatidylinositoldeficient T cells derived from paroxysmal nocturnal hemoglobinuria patients. Int. Immunol. 11, 1411-1422. Leyton, L., Quest, A.F.G., Bron, C. (1999). Thy1/CD3 coengagement promotes TCR signaling and enhances particularly tyrosine phosphorylation of the raft molecule LAT. Mol. Immunol. 36, 755-768. Felley-Bosco, E., Bender, F.C., CourjaultGautier, F., Bron, C., Quest, A.F.G. (2000). Caveolin-1 down-regulates inducible nitric oxide synthase via the proteasome pathway in human colon carcinoma cells. Proc. Natl. Acad. Sc. USA 97, 14334-14339. Bender, F.C., Reymond, M.A., Bron, C., Quest, A.F.G. (2000). Caveolin-1 levels are down-
regulated in human colon tumors and ectopic expression of caveolin-1 in colon carcinoma cell lines reduces cell tumorigenicity. Cancer Res. 60, 5870-5878. Doucey, M.A., Legler, D.F., Boucheron, N., Cerottini, J.C., Bron, C., Luescher, I. (2001). CTL activation is induced by cross-linking of TcR/MHC-peptide CD8/p56lck adducts in rafts. Eur. J. Immunol. 31, 1561-1570. Legler, D.F., Doucey, M.A., Cerottini, J.C., Bron, C., Luescher, I.F. (2001). Selective inhibition of CTL activation by a dipalmitoyl-phospholipid that prevents the recruitment of signaling molecules to lipid raft. FASEB J. 15, 1601-1603. Leyton, L., Schneider, P., Labra, C.V., Regg, C., Hetz, C.A., Quest, A.F.G., Bron, C. (2001). Thy1 binds to integrin b3 on astrocytes and triggers formation of focal contact sites. Curr. Biol. 11, 1028-1038. Legler, D.F., Wiedle, G., Ross, F.P. and Imhof, B.A. (2001). Superactivation of integrin v3 by low antagonist concentrations. J.Cell Sc. 114, 1545-1553.
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MOLECULAR MECHANISMS OF T LYMPHOCYTE ANTIGEN RECOGNITION
Salvatore Valitutti
Salvatore Valitutti studied Medicine at the Catholic University in Rome, where he received his MD in 1986. After a postdoctoral fellowship with Daniela Corda at the Mario Negri Institute for Pharmacological Research, Italy, he joined Antonio Lanzavecchia laboratory at the Basel Institute for Immunology in 1991. In 1993 he become member of the Basel Institute for Immunology until May 1997, when he came to the Institute of Biochemistry, University of Lausanne as FNSR SCORE A fellow. The main focus of his laboratory concerns the physiological mechanisms of T lymphocyte antigen recognition. Salvatore Valitutti is now professor of Immunology at the University of Toulouse, France and responsible of a research group at the Unit INSERM 395 in Toulouse, France. Group members 2000-2001 Sabina Mller Valitutti Benoit Favier Technician (04.2001) Ph.D student Doris Penna Rossana Zaru Ph.D student (06.2001) Ph.D student
MOLECULAR MECHANISMS OF T LYMPHOCYTE ANTIGEN RECOGNITION T lymphocytes play a crucial role in the immune-surveillance against pathogens and neoplastic cells. The recognition of foreign antigens by helper or cytotoxic T lymphocytes initiates a complex cascade of events, eventually leading to the production of specific antibodies by B cells, or to the destruction of infected or neoplastic cells. T lymphocytes are activated by the engagement of their antigen receptors with peptide-MHC complexes displayed on the surface of the antigen presenting cells (APCs). The T cell antigen receptor (TCR) is a multimeric protein complex composed of six different subunits, , , , , and . The heterodimer is responsible for antigen recognition, while the associated CD3 chains (, , ) and the homodimer are necessary for the surface expression of the receptor complex and for signal transduction. Antigen recognition by the T cell is exquisitely sensitive. Indeed helper T cells can proliferate and produce cytokines in response to APCs displaying as few as 50-100 specific peptide-MHC complexes. Even more striking, cytotoxic T cells can reportedly kill target cells presenting a single antigenic complex. Such high sensitivity of T cell responses requires a strict control to avoid exaggerated T cell activation. A key element in this control is the rapid and long-lasting down-regulation of triggered TCRs. TCR down-regulation leads to the extinction of signaling in T-APC conjugates and affects T cell responsiveness to further antigenic stimulation. Our early studies on the relationship between the quality, the duration and the intensity of TCRmediated signal transduction and corresponding T cell responses led to the following observations: i) a small number of specific peptide-MHC complexes present on the APC surface can engage and trigger a large number of TCRs, eventually leading to their internalization and degradation in the lysosomes; ii) the level and the duration of TCR signaling control the type and
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magnitude of the T cell biological response. These observations have challenged current models of TCR triggering based on receptor cross-linking, introducing the variable "time" in the paradigm of T cell activation (Figure 1).
cell-surface molecule CD2 is rapidly recruited into this signaling domain, whereas TCRs progressively percolate from the entire T cell surface into the phosphorylation area. Remarkably, the highly expressed phosphatase CD45 is excluded from the signaling domain. These results indicate that physiological TCR triggering at the T cell-APC contact site is the result of a localized alteration in the balance between cellular kinases and phosphatases. We therefore provide experimental evidence to support current models of T cell activation based on CD45 exclusion from the TCR signaling area (Figure 2, O. Leupin et al. Curr. Biol., 2000, 10:277). We are presently interested in pursuing and extending our studies on the mechanism that control T lymphocyte activation by specific antigen, by focusing on the molecular dynamics occuring at the T cell-APC contact site. Role of CD2 in fine immunological synapse tuning of the
Fig 1. The serial engagement model of T cell activation. (From S.Valitutti and A. Lanzavecchia, Immunology Today, 1997, 18: 299, ref. 1)
Our current studies concern the assembly of TCR-coupled signaling cascade and the signal transmission from engaged TCRs to T cell biological response. 1. Definition of the three-dimensional structure of the T cell-APC immunological synapse We apply three innovative and complementary morphological approaches to the study of T cell/APC interaction: (i) high resolution tridimensional confocal microscopy of the T cellAPC contact area, and (ii) time lapse video recording of [Ca2+]i and of the dynamics of various GFP/fusion proteins of TCR/CD3- complex and signaling components in living T cells, (iii) measurement of lateral mobility of TCR and recruited signaling components using techniques based on photo-bleaching recovery. In a first study we visualized T cellAPC contact sites using confocal microscopy and threedimensional reconstruction of z-sections. We show the rapid formation of a specialized signaling domain at the T cellAPC contact site that is characterized by a broad and sustained area of tyrosine phosphorylation. The T lymphocyte 60
Even though the formation of a specialized signaling domain has been documented at the T cell-APC contact site, its role in T cell activation is still elusive. Our results show that blocking CD2/CD58 interaction in T cell-APC conjugates with anti-CD58 antibodies results in: i) block of CD2 recruitment to the contact site (Fig 1); ii) inhibition of CD45 exclusion; iii) inhibition of PKC recruitment to the T cell-APC contact site; iv) reduction of the sensitivity of T cell biological response to antigenic stimulation. Conversely, this treatment does not affect TCR internalization and polarizaton of T cell tubulin cytoskeleton versus the APC. Our observations also indicate that when T cells are stimulated with peptide/MHC complexes coated on polystyrene beads, tyrosine phosphorylation and calcium fluxes are clearly detectable in the absence of immunological synapse formation. Co-immobilization of antiCD2 antibodies with peptide/MHC complexes results in CD2 recruitment and in a marked CD45 exclusion. These observations indicate that a structured immunological synapse is not required for productive TCR engagement and signaling. Conversely, the formation of such specialized
PTyr CD2 APC CD2 APC PTyr APC
c
untreated
+ anti-CD58 mAbs
Fig. 2. CD2 recruitment in the area of contact requires its engagement by CD58. T cells were conjugated by centrifugation with peptide-pulsed EBV-transformed B cells, previously loaded for 10 minutes at 37o C with 0.5 M Orange-CMTMR (Molecular probes, Leiden, The Netherlands) (RED). EBV-B cells where either untreated (upper panels) or treated with anti-CD58 antibodies (lower panels). Immediately after conjugation the cells were gently re-suspended and laid on poly-L-lysine-coated slides for 5 minutes. Cells were fixed, permeabilized and stained with an anti-phosphotyrosine mAb (BLUE) and an anti-CD2 mAb (GREEN). The samples were mounted in 90% glycerol-PBS containing 2.5% 1-4-diazabicyclo (2.2.2) octane (DABCO; Fluka AG, Buchs, Switzerland) and were examined using a Carl Zeiss, LSM 510, confocal microscope (Carl Zeiss, Germany). z-sections were taken at a 0.2 M distance. Images' processing was performed using Imaris software (Bitplane, Zrich, Switzerland). In B, C , E and F either the green or the blue staining have been removed to better show CD2 and PTyr staining (R. Zaru et al. submitted for publication).
domains reduces T cell activation thresholds by amplifying TCR mediated signal transduction. This peculiar mechanism of signal amplification may have been developed, in parallel with serial TCR engagement, to ensure sensible detection of a low number of antigenic determinants at the cell surface (R. Zaru et al.: "TCR engagement and triggering in the absence of a large scale molecular segregation at the T cell-APC contact site", submitted for publication). Molecular dynamics living T lymphocyte T lymphocyte activation by specific antigen requires prolonged TCR occupancy and sustained signaling. This is accomplished by the formation of a specialized signaling domain, the immunological synapse, at the T cell-antigen presenting cell contact site. Surface receptors and signaling components are progressively recruited into this domain where they are organized in
defined three-dimensional structures. To better understand how TCRs are supplied to the signaling domain during the activation process, we measured (using confocal microscopy and photo-bleaching recovery techniques) lateral mobility of GFP-tagged TCRs on living Jurkat cell surface. We show that: i) surface expressed TCRs exhibit an intrinsic, actin cytoskeleton independent, lateral mobility which allows them to passively diffuse over the entire T cell surface within ~60 minutes; and ii) non-stimulated TCRs rapidly enter the signaling domain. Our results indicate that TCR lateral mobility per se is sufficient to ensure TCR supply to the immunological synapse in the course of sustained T cell activation. (B. Favier et al.:"T cell antigen receptor dynamics on the surface of living T cells". Int. Immunol., 2001, 13:1525, Fig. 3).
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Green fluorescence intensity
Remaining cell surface
Bleached area
B
Bleached area Remaining cell surface
250 Non-stimulated 200 150 100 50 0 0 40 80 120 250 Anti-V3 200 150 100 50 0 0 40 80 120
Seconds
Fig. 3. Rapid TCR/GFP photo-bleaching recovery. Cells were irradiated with a strong laser beam in the indicated region. Two T cells either non-stimulated (A) or conjugated with anti-TCRV3 coated beads (B) are shown either before or immediately after photo bleaching. Regions were drawn at the T cell surface in the bleached area and on the remaining cell surface and the GFP fluorescence values were plotted against time for non-stimulated (C) and forantibody stimulated (D) T cells (From B. Favier et al. Int. Immunol., 2001, 13:1525).
2. Understanding the mechanisms that control sustained signaling in antigenstimulated T lymphocytes We have shown that T cell activation to interleukin production requires a sustained [Ca2+]i increase, which results from serial TCR engagement and prolonged PTK activation (S. Valitutti et al., Nature, 1995, 375:148). We therefore investigated how inositol lipid metabolism can be activated for a prolonged time to ensure a sustained link between receptor triggering and downstream signaling effectors. Four lines of evidence indicate that an extensive phosphoinositide turnover induced by TCR and CD28 engagement allows this task to be accomplished: i) continuous phosphoinositide breakdown is required for a sustained [Ca2+]i increase in antigen-stimulated T cells; ii) TCR
triggering results in a continuous release of inositol phosphates from the cell membrane paralleled by a massive and sustained phosphoinositide re-synthesis due to free inositol re-incorporation; iii) TCR-induced phosphoinositide turnover is strongly increased by CD28 ligation; and iv) CD28 engagement augments and sustains the TCR induced [Ca2+]i increase. Our results show that the T cell pool of phosphoinositides is continuously re-formed during T cell-APC cognate interaction, thereby explaining how sustained receptor triggering can transduce an equally sustained [Ca2+]i increase. Importantly, our data identify a novel step in the signaling cascade where co-stimulation converges with TCR-generated signals to sustain and amplify the activation process (R. Zaru et al. Eur. J. Immunol., 2001, 32:2438).
Recent publications Leupin, O., Zaru, R., Laroche, T., Mller, S. and Valitutti, S. (2000). Exclusion of CD45 from the T-cell receptor signaling area in antigenstimulated T lymphocytes. Curr. Biol. 10, 277280.
Favier, B., Burroughs, N.J., Wedderburn, L. and Valitutti, S. (2001). TCR dynamics on the surface of living T cells. Int. Immunol. 13, 1525-1532. Zaru, R., Berrie, C.P., Iurisci, C., Corda, D. and Valitutti, S. (2001). CD28 co-stimulates TCR/CD3-induced phosphoinositide turnover in human T lymphocytes. Eur. J. Immunol. 31, 2438-2447.
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PROTEIN AND PEPTIDE CHEMISTRY LABORATORY
Ekaterini Servi
Catherine Servis received her Ph.D. at the University of Heriot-Watt in Edinburgh in 1986. The research of this thesis was conducted at the Max-Planck Institute for Immunogenetics in Tbingen and at the Biochemistry Department of Northwestern University in Chicago. Postdoctoral experience was carried out at the Basel Institute for Immunology (Hoffman-La-Roche), the Friedrich Miescher Institute in Basel (CibaGeigy) and the Institute of Organic Chemistry at the University of Lausanne. From 1992 till 2001 she was Matre Assistante at the Institute of Biochemistry at the University of Lausanne. She is responsible for the Peptide and Protein Chemistry Laboratory at the BIL Biomedical centre at Epalinges. Group members 2000-2001 Nicole Lvy SERVICES The objective of this facility is to provide information on selected aspects of peptide synthesis and protein structure and to create the resources to investigators to extend their capabilities on peptide synthesis, peptide/protein purification and mass spectroscopic analysis through the aid of protein chemists operating this facility. The range of services provided by the Peptide/Protein Chemistry Laboratory are listed below: Peptide synthesis (linear or branched e.g. MAPS) Peptide purification Peptide coupling to proteins Protein purification Peptide modification Amino acid analysis Mass spectroscopic analysis 1. Identification of naturally processed peptide antigens: Natural peptides extracted from antigen presenting cells by acid extraction are separated by HPLC and then identified by MS. 2. C-terminal sequencing: Peptides up to 3,000 da are digested by carboxypeptidases and the digestion products are identified by MS. EQUIPMENT Equipment currently in place in the Facility includes: Peptide synthesis is performed using an automated Applied Biosystems 431A peptide synthesizer (up to 0.2 mmol) and an ACT348 OMEGA Advanced ChemTech multiple synthesizer (0.025 and 0.1 mmol), both employing Fmoc chemistry. Standard synthesis provides 20-100 mg of peptide with a delay of two-three weeks. Quality control includes analytical HPLC and mass spectroscopic analysis. High Performance Liquid Chromatography (HPLC) is performed by three Waters HPLC systems for analytical or preparative HPLC and a Hewlett Packard HPLC system for analytical applications. Mass spectroscopic analysis is performed using a Voyager-DE RP Biospectrometry Workstation by Perseptive Biosystems. 63 Technician Florela Penea Technician
CHARGES Service Peptide synthesis per aa incorporation of special amino acids and groups The charges include HPLC and mass spectroscopic analysis. Peptide/protein analysis HPLC Amino Acid analysis MALDI-TOF MS Peptide/protein purification Purification BIL (SFr.) 30.Inquire Others (SFr.) 60.Inquire
200.200.200.Inquire
Inquire Inquire Inquire Inquire
RESEARCH PROJECTS 1. In vitro degradation assay as a means to analyze the production of peptide tumor antigens In collaboration with Frdric Lvy (Ludwig Institute for Cancer Research, Lausanne Branch) The proteasome plays a crucial role in the proteolytic processing of antigens presented to T cells in the context of MHC class I molecules. However, the rules governing the specificity of cleavage sites are still largely unknown. By using mass spectrometry we developed a new technique for the identification of degradation products. Using this technique and purified proteasome, it has been possible to rapidly and unambiguously identify degradation products corresponding to antigenic peptides. This technique has also allowed to analyze the production of a limited number of candidate peptide tumor antigens, which had been identified on the basis of their restricted expression pattern and binding motif for particular MHC class I molecules. We will further analyze several precursors of peptide tumor antigens and plan to include this test in the characterization of each newly identified potential peptide tumor antigen. Introduction of this new technique may prove important as several potential peptide tumor antigens have been shown to be destroyed by the proteasome. 2. Identification of new proteases responsible for the final trimming of MHC class Irestricted antigenic peptides In collaboration with Frdric Lvy and JeanCharles Cerottini (Ludwig Institute for Cancer Research, Lausanne Branch) Using a precursor peptide derived from several proteins we demonstrated that proteasomes generate the proper C-terminus of the antigenic peptide. However, in many instances the fully processed antigenic peptide is not produced; rather, the antigenic peptide carries an N-terminal extension of several amino acids. It is, thus, generally concluded that the C-terminus of antigenic peptides is directly produced by the proteasome, while the proper N-terminus results from the trimming of a longer precursor by another protease. The exact cellular localization of this protease is unknown. However, preliminary results have demonstrated that this protease can be enriched from specific cytosolic fractions of cells that normally process and present the antigenic peptide and is able, in vitro, to generate the appropriate N-terminus of the antigenic peptide. Our plans are twofold: i) to isolate and characterize this aminopeptidase and ii) to investigate whether the same protease is responsible for the N-terminal trimming of all model antigenic peptide precursors.
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To identify the aminopeptidase responsible for the final processing of a specific CTL-defined epitope, human cell cytosol is fractionated at a preparative scale. Each fraction is assayed for activity on the defined epitope precursor and analyzed by mass spectrometry. Fractions containing an activity trimming the N-terminus of the precursor to the correct size of the epitope, but leaving intact the C-terminal end of the precursor, are separated by 2-D gels. Individual protein spots are excised, digested with trypsin, the peptides are extracted, purified on microcolumns and subjected to analysis by mass spectrometry. The search programme Prowl is used for peptide mass fingerprinting. Our findings are summarized as follows: 1. We have identified two cytosolic peptidases that are mediating the N-terminal trimming of an antigenic peptide precursor. These peptidases, Tripeptidyl peptidase II (TPP II) and puromycin sensitive aminopeptidase (PSA), act sequentially on peptide fragments liberated by the proteasome and lead to the accumulation of a species displaying the final N-terminus of the antigenic peptide sequence. 2. We did not detect any proteolytic activity against the peptide precursor in membranes. We showed that proteasome and TPP II inhibitors blocked the presentation of the antigenic peptide RU134-42 by HLA-B51+ tumor cells.
3. The two identified peptidases, TPP II and PSA, have already been implicated, in different experimental conditions, in the MHC class I antigen processing pathway. 4. The fact that the same peptidase (PSA) has been identified using two different peptide precursors may not be coincidental and leads us to postulate that a limited number of peptidases will be responsible for the final editing of MHC class I ligands. At present, we do not know whether this sequence of action is identical in all cell types nor in situations where the expression of other peptidases is induced. 5. We directly searched for membrane-embedded or luminal peptidases that would mediate the final trimming of our precursor. Although we could detect proteolytic activity in our membrane preparation, we did not detect any N-terminal trimming of our precursor peptide. We conclude that the trimming of our precursor to the size of the antigenic peptide RU134-42 is directly made in the cytosol. 6. Our work has identified cellular components acting between the proteasome and the peptidespecific endoplasmic reticulum transporters (TAP1/2) and may thus constitute a potentially relevant target for the modulation of the MHC class I-restricted antigen processing pathway.
Recent publications Roggero, M.A., Servis, C. and Corradin, G. (1997). A simple and rapid procedure for the purification of synthetic polypeptides by a combination of affinity chromatography and methionine chemistry. FEBS Letters 408, 285288. Maryanski, J.L., Casanova, J-L., Falk, K., Gournier, H., Jaulin, C., Kourilsky, P., Lemonnier, F.A., Lthy, R., Rammensee, H-G., Rtzschke, O., Servis, C. and Lopez J-A. (1997). The diversity of antigen-specific TCR repertoires reflects the relative complexity of epitopes recognized. Human Immunol. 54, 117-128.
Roggero, M.A., Servis, C. and Corradin, G. (1998). Purification of Synthetic Polypeptides by Immobilised Metal Affinity Chromatography and Methionine Chemistry. In: Innovation and Perspectives in Solid Phase Synthesis and Combinatorial Libraries, Roger Epton, Ed., Mayflower Scientific, London, England. Valmori, D., Servis, C., Cerottini, J-C., Romero, P. and Levy, F. (1998). Influence of proteasome inhibitors and specific peptide sequence on the generation of a CTL-defined peptide derived from the tumor antigen MAGE-3. J. Exp. Medicine 189, 895-905.
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Crottet, P., Peitch M.C., Servis C., Kraehenbuhl J-P. and Corthsy B. (1999). Covalent Homodimers of mouse secretory component induced by epitope substitutions unravel the capacity of the polymeric Ig receptor to dimerise noncovalently in the absence of IgA ligand. J. Biol. Chem. 274, 31445-31455. Miconnet, I., Servis, C., Cerottini, J-C., Romero, P. and Levy, F. (2000). Amino acid identity and/or position determines the Proteasomal cleavage of the HLA-A*0201-restricted peptide tumor antigen MAGE 271-279. J. Biol. Chem. 275, 26892-26897.
Romero, P., Dutoit, V., Rubio-Godoy, V., Linard, D., Guillaume, P., Servis, C., Rimoldi, D., Cerottini, J-C. and Valmori, D. (2000). CD8+ T cell respone to NY-ESO-1: Relative antigenicity and in vitro immunogenicity of natural and analogue seqeunces. Clinical Cancer Res. 7, 766-772 Rimoldi, D., Rubio-Godoy, V., Dutoit, V., Linard, D., Salvi, S., Guillaume, P., Speiser, D., Stockert, E., Spagnoli, G., Servis, C., Cerottini, JC., Lejeune, F. Romero, P. and Valmori, D. (2000). Efficient simultaneous presentation of NY-ESO-1/LAGE-1 primary and non-primary open reading frame derived CTL epitopes in melanoma. J. Immunol. 165, 7253-7261.
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PROTEIN ANALYSIS FACILITY
Manfredo Quadroni
Manfredo Quadroni graduated in Biochemistry at the ETH Zrich and then received his PhD from the same institution for work centered on calcium-mediated signalling and protein phosphorylation. There he also got his training in 2D-electrophoresis and mass spectrometry in the laboratory of Dr. Peter James. During a postdoctoral stay at the University of British Columbia, Canada, he also became familiar with signal transduction in immunology. A second post-doc brought him back to ETH Zrich, where he worked on the development of methods for proteome analysis. He joined the Institute of Biochemistry in February 2000.
Group members 2000-2001 Bastienne Jaccard Ph.D student
RESEARCH PROJETS 1. Characterization of the components of apoptotic signalling complexes. (in collaboration with the group of J. Tschopp ) Death-Inducing Signalling Complexes (DISC's) are multimolecular assemblies forming around "death receptors" such as Fas and the receptor for TNF after binding of ligands of the TNF family that are presented on the surface of cytotoxic T lymphocytes (CTL's). Formation of the DISC's is essential for instructive apoptosis, the mechanisms by which CTL's eliminate tumour cells, virus-infected cells and superfluous immune cells. Only some of the pathways activated by DISC's are known, in part because of the limitations of the methods used for studying intermolecular interactions. We plan to study by proteomics techniques the DISC's of FasL (CD95L), TRAIL and other non-apoptotic molecules of the TNF family involved in immune system regulation and tissue development. We will isolate the complexes after stimulation of the cells and lysis, and separate the proteins by 2DPAGE. Single polypeptides will be digested and the proteolytic peptides analyzed by highsensitivity mass spectrometry to obtain mass profiles and partial sequence data. This should allow us to characterize new molecules that mediate the multiple functions that are attributed to the DISC complexes. We will aim at establishing experimental tools that can be used to analyze the signalling complexes of all the members of the TNF family of ligands. Such tools could then be modified to apply them to other signalling complexes recruited by cytokines, growth factors and regulatory molecules. One line of investigation will be through the use of the so-called "TAP-tag" (Tandem Affinity Purification), an expression system that allows the highly specific recovery of a protein of interest together with interacting partners. The TAP system has been developed for use in the yeast system but we are adapting it for use in mammalian cells.
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2. Whole-cell proteome analysis of Leishmania parasites in correlation with pathogenicity (in collaboration with the groups of N. Fasel, D.Rivier ) Proteomics is a good tool for analyzing differential global protein expression in unicellular organisms. Parasitic protozoans undergo dramatic changes in their physiology and cellular structures according to their life stage. These changes are essential for survival in different hosts and compartments (body fluids, cell cytoplasm) and for evading immune responses. We propose to apply proteomics techniques to study these changes in protozoan pathogens of the genus Leishmania. A model system is available, in which parasites can be cultured in vitro in conditions that mimic the extra- or intracellular environment. We will analyze and compare the total protein pool ("proteome") of the parasite in the pro(extracellular) and amastigote (intracellular) form to identify in the genomic sequence those proteins whose expression is reduced respectively increased while going from one form to the other. In addition we will compare different isolates with varying virulence levels to look for differences at the protein level. Polypeptides identified by one or the other approach may play an important role in pathogenicity and have a potential as diagnostic markers as well as antigens for the production of vaccines. So far, we have established the methodology for the analysis of Leishmania whole cell lysates by 2D-PAGE and are in the process of setting up master gels (reference images). Preliminary analysis of metasthatic vs nonmethastatic clones of L. viannia guayanensis were analyzed and found to differ in the expression of several proteins. Indeed, more than 10 spots were found to be unique to the methastatic strain and will be object of analysis soon. For this comparisons, the technique of "zoom gels" (gels covering narrow pH ranges) has been used and found to be very useful.
3. Establishment of the UNIL Protein Analysis Facility (PAF) Link : www.unil.ch/paf Contact : e-mail : wwwpaf@unil.ch Proteomics is a new discipline based on the fast, large scale identification and quantification of proteins. By mining the information contained in genomic databases, proteomics identify changes in protein expression, protein-protein interactions or protein modifications that correlate with various biological processes. The discipline is booming due to increases in sensitivity and throughput that make it now possible to analyse very complex systems. A project for the creation at the University of Lausanne of a proteomics core facility has been promoted during 2001 by the Faculty of Medicine and has now been taken in charge by the University. The unit is being set up and should officially start operation during the second half of 2002. The overall goal is to make complex and expensive techniques such as mass spectrometry (MS) available to academic researchers in and around Lausanne and at the same time to diffuse the know-how on proteomics technologies and how to exploit them. The service: the core of the service activities of the PAF will be to provide fast, high sensitivity identification of proteins by mass spectrometry (MS). In addition, the facility will offer advice and support on protein separation techniques, proteome-related bioinformatics and data management. Research: in parallel to the service activity, the PAF will also perform independent and collaborative academic research in the field of proteomics. In addition to addressing relevant biological problems, the research activity will also serve to implement new techniques for proteome analysis (such as ICAT, 2D-LC-MS, ) as these become available.
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Teaching: in collaboration with proteomics researchers at the University of Geneva, the PAF will offer postgraduate courses on selected proteomics technologies (i.e. separation techniques or biological mass spectrometry). These teaching activities will be primarily for academic researchers of the Lausanne area. Access: researchers of the UNIL and associated institutions (CHUV, ISREC, Ludwig Inst.) as well as of the EPFL will have privileged access to the services of the facility. Samples from the industry will also be accepted, if the capacity will permit it.
Funding: start of the activity has been possible thanks to public financing accorded to the Institute of Biochemistry (EMPD from the Canton Vaud) and a generous contribution of the Leenaards Foundation. Equipment: presently comprises i) a SCIEX QSTAR quadrupole-time of flight mass spectrometer coupled to an LC Packings Ultimate nanobore-HPLC ii) liquid handling robots ProGEST and ProMS from Genomic Solutions for automated in-gel digestion and MALDI sample preparation iii) a Perseptive Biosystems Voyager DE-RP MALDI-TOF mass spectrometer.
Recent publications Hoving, S., Mnchbach, M., Schmid, H., Signor, L., Lehmann, A., Staudenmann, W., Quadroni, M. and James, P. (2000). A method for the chemical generation of N-terminal peptide sequence tags for rapid protein identification. Anal Chem. 72(5), 1006-14. Schreiber, J.V., Quadroni, M. and Seebach, D. (1999). Sequencing of beta-peptides by mass spectrometry. Chimia 53(12), 621-626. Quadroni, M., James, P., Dainese-Hatt, P. and Kertesz, M.A. (1999). Proteome mapping, mass spectrometric sequencing and reverse transcription-PCR for characterization of the sulfate starvation- induced response in Pseudomonas aeruginosa PAO1. Eur J Biochem. 266, 986-96. Corti, C., L'Hostis, E., Quadroni, M., Schmid, H., Dainese, P, James, P. and Carafoli, E. (1999). Phosphorylation of Calmodulin by the tyrosine kinase fgr alters its biological activity. Eur. J. Biochem. 262 (3), 790-802. Brunati, A.M., Donella-Deana, A., James, P., Quadroni, M., Contri, A., Marin, O. and Pinna, L.A. (1999). Molecular features underlying the sequential phosphorylation of HS1 protein and its association with c-Fgr protein-tyrosine kinase. J. Biol. Chem. 274, 7557-7564.
Hummerjohann, J., Kuttel, E., Quadroni, M., Ragaller, J., Leisinger, T. and Kertesz, M.A. (1998). Regulation of the sulfate starvation response in Pseudomonas aeruginosa: role of cysteine biosynthetic intermediates. Microbiology, 144, 1375-1386. Quadroni, M., L'Hostis, E.L., Corti, C., Myagkikh, I., Durussel, I., Cox, J., James, P. and Carafoli, E. (1998). Phosphorylation of calmodulin alters its potency as an activator of target enzymes. Biochemistry 37(18), 6523-6532. Dainese Hatt, P., Quadroni, M., Staudenmann, W. and James, P. (1997). Concentration of, and SDS removal from proteins isolated from multiple two-dimensional electrophoresis gels. Eur. J. Biochem. 246, 336-43. Dainese, P., Staudenmann, W., Quadroni, M., Korostensky, C., Gonnet, G., Kertesz, M. and James, P. (1997). Probing protein function using a combination of gene knockout and proteome analysis by mass spectrometry. Electrophoresis 18, 432-42. Review Quadroni, M. and James, P. (1999). Proteomics and Automation. Electrophoresis 20(4-5), 66477.
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PUBLICATIONS 2000-2001
Articles 1. Astori, M., von Garnier, C., Kettner, A., Dufour, N., Corradin, G. and Spertini, F. (2000). Inducing tolerance by intranasal administration of long peptides in naive and primed CBA/J mice. J Immunol 165, 34973505. Attinger, A., Acha-Orbea, H. and MacDonald, H.R. (2000). Cutting edge: cell autonomous rather than environmental factors control bacterial superantigen-induced T cell anergy in vivo. J Immunol 165, 1171-1174. Attinger, A., MacDonald, H.R. and AchaOrbea, H. (2001). Lymphoid environment limits superantigen and antigen-induced T cell proliferation at high precursor frequency. Eur J Immunol 31, 884-893. Bachmann, M.F., Gallimore, A., Jones, E., Ecabert, B., Acha-Orbea, H. and Kopf, M. (2001). Normal pathogen-specific immune responses mounted by CTLA-4-deficient T cells: a paradigm reconsidered. Eur J Immunol 31, 450-458. Baribaud, F., Wirth, S., Maillard, I., Valsesia, S., Acha-Orbea, H. and Diggelmann, H. (2001). Identification of key amino acids of the mouse mammary tumor virus superantigen involved in the specific interaction with T-cell receptor V domains. J Virol 75, 7453-7461. Batten, M., Groom, J., Cachero, T.G., Qian, F., Schneider, P., Tschopp, J., Browning, J.L. and Mackay, F. (2000). BAFF mediates survival of peripheral immature B lymphocytes. J Exp Med 192, 1453-1466. Bender, F.C., Reymond, M.A., Bron, C. and Quest, A.F. (2000). Caveolin-1 levels are down-regulated in human colon tumors, and ectopic expression of caveolin-1 in colon carcinoma cell lines reduces cell tumorigenicity. Cancer Res 60, 5870-5878. Benedict, C.A., Norris, P.S., Prigozy, T.I., Bodmer, J.L., Mahr, J.A., Garnett, C.T., Martinon, F., Tschopp, J., Gooding, L.R. and Ware, C. F. (2001). Three adenovirus E3 proteins cooperate to evade apoptosis by tumor necrosis factor-related apoptosis-inducing ligand receptor-1 and -2. J Biol Chem 276, 3270-3278. Benedict, C.A., Norris, P.S., Prigozy, T.I., Bodmer, J.L., Mahr, J.A., Garnett, C.T.,
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Martinon, F., Tschopp, J., Gooding, L.R. and Ware, C.F. (2000). Three adenovirus E3 proteins cooperate to evade apoptosis by TRAIL receptor-1 and 2. J Biol Chem. Bertholet, S. and Maul, J. (2000). Human monocytic U937 cells transfected with human hepatic inducible nitric oxide synthase exhibit leishmanicidal activity. J Leukoc Biol 67, 3439. Biedermann, T., Zimmermann, S., Himmelrich, H., Gumy, A., Egeter, O., Sakrauski, A.K., Seegmller, I., Voigt, H., Launois, P., Levine, A.D., Wagner, H., Heeg, K., Louis, J.A. and Rcken, M. (2001). IL-4 instructs Th1 responses and resistance to Leishmania major in susceptible BALB/c mice. Nat Immunol 2, 1054-1060. Bodmer, J.L., Holler, N., Reynard, S., Vinciguerra, P., Schneider, P., Juo, P., Blenis, J. and Tschopp, J. (2000). TRAIL receptor-2 signals apoptosis through FADD and caspase8. Nat Cell Biol 2, 241-243. Bodmer, J.L., Meier, P., Tschopp, J. and Schneider, P. (2000). Cysteine 230 is essential for the structure and activity of the cytotoxic ligand TRAIL. J Biol Chem 275, 20632-20637. Bonelo, A., Valmori, D., Triponez, F., Tiercy, J.M., Mentha, G., Oberholzer, J., Champagne, P., Romero, J.F., Esposito, F., Nebie, I., Barbey, C., Romero, P., Herrera, S., Corradin, G. and Lopez, J.A. (2000). Generation and characterization of malaria-specific human CD8(+) lymphocyte clones: effect of natural polymorphism on T cell recognition and endogenous cognate antigen presentation by liver cells. Eur J Immunol 30, 3079-3088. Buchegger, F., Allal, A.S., Roth, A., Papazyan, J.P., Dupertuis, Y., Mirimanoff, R.O., Gillet, M., Plegrin, A., Mach, J.P. and Slosman, D.O. (2000). Combined radioimmunotherapy and radiotherapy of liver metastases from colorectal cancer: a feasibility study. Anticancer Res 20, 1889-1896. Buchegger, F., Roth, A., Allal, A., Dupertuis, Y.M., Slosman, D.O., Delaloye, A.B. and Mach, J.P. (2000). Radioimmunotherapy of colorectal cancer liver metastases: combination with radiotherapy. Ann N Y Acad Sci 910, 263269; discussion 269-270. Bullani, R.R., Huard, B., Viard-Leveugle, I., Byers, H.R., Irmler, M., Saurat, J.H., Tschopp,
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J. and French, L.E. (2001). Selective expression of FLIP in malignant melanocytic skin lesions. J Invest Dermatol 117, 360-364. Burns, K., Clatworthy, J., Martin, L., Martinon, F., Plumpton, C., Maschera, B., Lewis, A., Ray, K., Tschopp, J. and Volpe, F. (2000). Tollip, a new component of the IL-1RI pathway, links IRAK to the IL-1 receptor. Nat Cell Biol 2, 346-351. Chapuis, A.G., Paolo Rizzardi, G., D'Agostino, C., Attinger, A., Knabenhans, C., Fleury, S., Acha-Orbea, H. and Pantaleo, G. (2000). Effects of mycophenolic acid on human immunodeficiency virus infection in vitro and in vivo [see comments]. Nat Med 6, 762-768. Cloutier, S.M., Couty, S., Terskikh, A., Marguerat, L., Crivelli, V., Pugnieres, M., Mani, J.C., Leisinger, H.J., Mach, J.P. and Deperthes, D. (2000). Streptabody, a high avidity molecule made by tetramerization of in vivo biotinylated, phage display-selected scFv fragments on streptavidin. Mol Immunol 37, 1067-1077. Coste, A., Cohen, J., Reinhardt, M., Kraehenbuhl, J.P. and Sirard, J.C. (2001). Nasal immunisation with Salmonella typhimurium producing rotavirus VP2 and VP6 antigens stimulates specific antibody response in serum and milk but fails to protect offspring. Vaccine 19, 4167-4174. Coste, A., Sirard, J.C., Johansen, K., Cohen, J. and Kraehenbuhl, J.P. (2000). Nasal immunization of mice with virus-like particles protects offspring against rotavirus diarrhea. J Virol 74, 8966-8971. Cremesti, A., Paris, F., Grassme, H., Holler, N., Tschopp, J., Fuks, Z., Gulbins, E. and Kolesnick, R. (2001). Ceramide enables Fas to cap and kill. J Biol Chem 276, 23954-23961. Debard, N., Sierro, F., Browning, J. and Kraehenbuhl, J.P. (2001). Effect of mature lymphocytes and lymphotoxin on the development of the follicle-associated epithelium and M cells in mouse Peyer's patches. Gastroenterology 120, 1173-1182. Demotz, S., Moulon, C., Roggero, M.A., Fasel, N. and Masina, S. (2001). Native-like, long synthetic peptides as components of sub-unit vaccines: practical and theoretical considerations for their use in humans. Mol Immunol 38, 415-422.
26. Doucey, M.A., Legler, D.F., Boucheron, N., Cerottini, J.C., Bron, C. and Luescher, I.F. (2001). CTL activation is induced by crosslinking of TCR/MHC-peptide-CD8/p56lck adducts in rafts. Eur J Immunol 31, 1561-1570. 27. Dupertuis, Y.M., Vazquez, M., Mach, J.P., De Tribolet, N., Pichard, C., Slosman, D.O. and Buchegger, F. (2001). Fluorodeoxyuridine improves imaging of human glioblastoma xenografts with radiolabeled iododeoxyuridine. Cancer Res 61, 7971-7977. 28. Elzey, B.D., Griffith, T.S., Herndon, J.M., Barreiro, R., Tschopp, J. and Ferguson, T.A. (2001). Regulation of Fas ligand-induced apoptosis by TNF. J Immunol 167, 3049-3056. 29. Favier, B., Burroughs, N.J., Wedderburn, L. and Valitutti, S. (2001). TCR dynamics on the surface of living T cells. Int Immunol 13, 15251532. 30. Felley-Bosco, E., Bender, F.C., CourjaultGautier, F., Bron, C. and Quest, A.F. (2000). Caveolin-1 down-regulates inducible nitric oxide synthase via the proteasome pathway in human colon carcinoma cells. Proc Natl Acad Sci U S A 97, 14334-14339. 31. Finke, D. and Acha-Orbea, H. (2001). Differential migration of in vivo primed B and T lymphocytes to lymphoid and non-lymphoid organs. Eur J Immunol 31, 2603-2611. 32. Finke, D., Baribaud, F., Diggelmann, H. and Acha-Orbea, H. (2001). Extrafollicular plasmablast B cells play a key role in carrying retroviral infection to peripheral organs. J Immunol 166, 6266-6275. 33. Foyouzi-Youssefi, R., Arnaudeau, S., Borner, C., Kelley, W.L., Tschopp, J., Lew, D.P., Demaurex, N. and Krause, K.H. (2000). Bcl-2 decreases the free Ca2+ concentration within the endoplasmic reticulum. Proc Natl Acad Sci U S A 97, 5723-5728. 34. Franke, E.D., Sette, A., Sacci, J., Jr., Southwood, S., Corradin, G. and Hoffman, S. L. (2000). A subdominant CD8(+) cytotoxic T lymphocyte (CTL) epitope from the Plasmodium yoelii circumsporozoite protein induces CTLs that eliminate infected hepatocytes from culture. Infect Immun 6 8 , 3403-3411. 35. Fukazawa, T., Fujiwara, T., Uno, F., Teraishi, F., Kadowaki, Y., Itoshima, T., Takata, Y., Kagawa, S., Roth, J.A., Tschopp, J. and 71
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Tanaka, N. (2001). Accelerated degradation of cellular FLIP protein through the ubiquitinproteasome pathway in p53-mediated apoptosis of human cancer cells. Oncogene 20, 52255231. Gaide, O., Martinon, F., Micheau, O., Bonnet, D., Thome, M. and Tschopp, J. (2001). Carma1, a CARD-containing binding partner of Bcl10, induces Bcl10 phosphorylation and NF-kappaB activation. FEBS Lett 496, 121127. Gervaz, P., Blanchard, A., Pampallona, S., Mach, J.P., Fontolliet, C. and Gillet, M. (2000). Prognostic value of postoperative carcinoembryonic antigen concentration and extent of invasion of resection margins after hepatic resection for colorectal metastases. Eur J Surg 166, 557-561. Gonzalez, J.M., Peter, K., Esposito, F., Nebie, I., Tiercy, J.M., Bonelo, A., Arevalo-Herrera, M., Valmori, D., Romero, P., Herrera, S., Corradin, G. and Lopez, J. A. (2000). HLAA*0201 restricted CD8+ T-lymphocyte responses to malaria: identification of new plasmodium falciparum epitopes by IFNgamma ELISPOT. Parasite Immunol 22, 501514. Gross, N., Balmas, K., Beretta Brognara, C. and Tschopp, J. (2001). Expression of Fas (APO-1/CD95) and Fas ligand (FasL) in human neuroblastoma. Med Pediatr Oncol 36, 111-114. Hathaway, L.J. and Kraehenbuhl, J.P. (2000). The role of M cells in mucosal immunity. Cell Mol Life Sci 57, 323-332. Heppner, F.L., Christ, A.D., Klein, M.A., Prinz, M., Fried, M., Kraehenbuhl, J.P. and Aguzzi, A. (2001). Transepithelial prion transport by M cells. Nat Med 7, 976-977. Himmelrich, H., Launois, P., Maillard, I., Biedermann, T., Tacchini-Cottier, F., Locksley, R.M., Rcken, M. and Louis, J.A. (2000). In BALB/c mice, IL-4 production during the initial phase of infection with Leishmania major is necessary and sufficient to instruct Th2 cell development resulting in progressive disease. J Immunol 164, 48194825. Holler, N., Kataoka, T., Bodmer, J.L., Romero, P., Romero, J., Deperthes, D., Engel, J. Tschopp, J., and Schneider, P. (2000).
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Development of improved soluble inhibitors of FasL and CD40L based on oligomerized receptors. J Immunol Methods 237, 159-173. Holler, N., Zaru, R., Micheau, O., Thome, M., Attinger, A., Valitutti, S., Bodmer, J.L., Schneider, P., Seed, B. and Tschopp, J. (2000). Fas triggers an alternative, caspase-8independent cell death pathway using the kinase RIP as effector molecule. Nat Immunol 1, 489-495. Hopkins, S.A., Niedergang, F., CorthsyTheulaz, I.E. and Kraehenbuhl, J. P. (2000). A recombinant Salmonella typhimurium vaccine strain is taken up and survives within murine Peyer's patch dendritic cells. Cell Microbiol 2, 59-68. Hopkins-Donaldson, S., Bodmer, J.L., Bourloud, K.B., Brognara, C.B., Tschopp, J. and Gross, N. (2000). Loss of caspase-8 expression in highly malignant human neuroblastoma cells correlates with resistance to tumor necrosis factor-related apoptosisinducing ligand-induced apoptosis. Cancer Res 60, 4315-4319. Houimel, M., Corthsy-Theulaz, I., Fisch, I., Wong, C., Corthsy, B., Mach, J. and Finnern, R. (2001). Selection of human single chain Fv antibody fragments binding and inhibiting Helicobacter pylori urease. Tumour Biol 2 2 , 36-44. Houimel, M., Schneider, P., Terskikh, A. and Mach, J.P. (2001). Selection of peptides and synthesis of pentameric peptabody molecules reacting specifically with ErbB-2 receptor. Int J Cancer 92, 748-755. Huard, B., Schneider, P., Mauri, D., Tschopp, J. and French, L.E. (2001). T cell costimulation by the TNF ligand BAFF. J Immunol 167, 6225-6231. Irmler, M., Steiner, V., Ruegg, C., Wajant, H. and Tschopp, J. (2000). Caspase-induced inactivation of the anti-apoptotic TRAF1 during Fas ligand-mediated apoptosis. FEBS Lett 468, 129-133. Johansen, P., Estevez, F., Zurbriggen, R., Merkle, H.P., Glck, R., Corradin, G. and Gander, B. (2000). Towards clinical testing of a single-administration tetanus vaccine based on PLA/PLGA microspheres. Vaccine 19, 1047-1054. Kaptein, A., Jansen, M., Dilaver, G., Kitson, J.,
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Dash, L., Wang, E., Owen, M.J., Bodmer, J., Tschopp, J. and Farrow, S.N. (2000). Studies on the interaction between TWEAK and the death receptor WSL-1/TRAMP (DR3) [In Process Citation]. FEBS Lett 485, 135-141. Kataoka, T., Budd, R.C., Holler, N., Thome, M., Martinon, F., Irmler, M., Burns, K., Hahne, M., Kennedy, N., Kovacsovics, M. and Tschopp, J. (2000). The caspase-8 inhibitor FLIP promotes activation of NF-kappaB and Erk signaling pathways. Curr Biol 10, 640648. Kataoka, T., Holler, N., Micheau, O., Martinon, F., Tinel, A., Hofmann, K. and Tschopp, J. (2001). Bcl-rambo, a novel Bcl-2 homologue that induces apoptosis via its unique C-terminal extension. J Biol Chem 276, 19548-19554. Kausalya, P.J., Reichert, M. and Hunziker, W. (2001). Connexin45 directly binds to ZO-1 and localizes to the tight junction region in epithelial MDCK cells. FEBS Lett 505, 92-96. Kerneis, S., Caliot, E., Stubbe, H., Bogdanova, A., Kraehenbuhl, J.P. and Pringault, E. (2000). Molecular studies of the intestinal mucosal barrier physiopathology using co-cultures of epithelial and immune cells: a technical update. Microbes Infect 2, 1119-1124. Kettner, A., Hughes, G.J., Frutiger, S., Astori, M., Roggero, M., Spertini, F. and Corradin, G. (2001). Api m 6: a new bee venom allergen. J Allergy Clin Immunol 107, 914-920. Klein, M.A., Kaeser, P.S., Schwarz, P., Weyd, H., Xenarios, I., Zinkernagel, R.M., Carroll, M. C., Verbeek, J.S., Botto, M., Walport, M.J., Molina, H., Kalinke, U., Acha-Orbea, H. and Aguzzi, A. (2001). Complement facilitates early prion pathogenesis. Nat Med 7, 488-492. Legler, D.F., Doucey, M.A., Cerottini, J.C., Bron, C. and Luescher, I.F. (2001). Selective inhibition of CTL activation by a dipalmitoylphospholipid that prevents the recruitment of signaling molecules to lipid rafts. FASEB J 15, 1601-1603. Legler, D.F., Wiedle, G., Ross, F.P. and Imhof, B.A. (2001). Superactivation of integrin v 3 by low antagonist concentrations. J Cell Sci 114, 1545-1553. Leupin, O., Zaru, R., Laroche, T., Mller, S. and Valitutti, S. (2000). Exclusion of CD45 from the T-cell receptor signaling area in
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antigen-stimulated T lymphocytes. Curr Biol 10, 277-280. Leyton, L., Schneider, P., Labra, C.V., Ruegg, C., Hetz, C.A., Quest, A.F. and Bron, C. (2001). Thy-1 binds to integrin beta(3) on astrocytes and triggers formation of focal contact sites. Curr Biol 11, 1028-1038. Lopez, J.A., Weilenman, C., Audran, R., Roggero, M.A., Bonelo, A., Tiercy, J.M., Spertini, F. and Corradin, G. (2001). A synthetic malaria vaccine elicits a potent CD8(+) and CD4(+) T lymphocyte immune response in humans. Implications for vaccination strategies. Eur J Immunol 31, 1989-1998. Maillard, I., Launois, P., Himmelrich, H., Acha-Orbea, H., Diggelmann, H., Locksley, R. M. and Louis, J.A. (2001). Functional plasticity of the LACK-reactive V 4-V 8 CD4(+) T cells normally producing the early IL-4 instructing Th2 cell development and susceptibility to Leishmania major in BALB / c mice. Eur J Immunol 31, 1288-1296. Martinon, F., Hofmann, K. and Tschopp, J. (2001). The pyrin domain: a possible member of the death domain-fold family implicated in apoptosis and inflammation. Curr Biol 11, R118-120. Martinon, F., Holler, N., Richard, C. and Tschopp, J. (2000). Activation of a proapoptotic amplification loop through inhibition of NF- B-dependent survival signals by caspase-mediated inactivation of RIP. FEBS Lett 468, 134-136. Medana, I., Li, Z., Flugel, A., Tschopp, J., Wekerle, H. and Neumann, H. (2001). Fas ligand (CD95L) protects neurons against perforin-mediated T lymphocyte cytotoxicity. J Immunol 167, 674-681. Micheau, O., Lens, S., Gaide, O., Alevizopoulos, K. and Tschopp, J. (2001). NF B signals induce the expression of c-FLIP. Mol Cell Biol 21, 5299-5305. Michetti, M., Kelly, C.P., Kraehenbuhl, J.P., Bouzourene, H. and Michetti, P. (2000). Gastric mucosal 47-integrin-positive CD4 T lymphocytes and immune protection against helicobacter infection in mice. Gastroenterology 119, 109-118. Miconnet, I., Servis, C., Cerottini, J.C., Romero, P. and Lvy, F. (2000). Amino acid 73
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TABLE DES MATIERES

EFFECTIFS ET BUDGET DE LINSTITUT DE BIOCHIMIE

S. Corradin-Betz * M. Quadroni P. Schneider E. Servi * M. Thome Miazza Privat-Docents

Charge de recherche Matre-assistant Professeur assistant remplaant Cheffe de projet Matre-assistante

F. Buchegger S. Demotz S. Valitutti Assistant(e)s

EFFECTIFS ET BUDGET DE LINSTITUT DE BIOCHIMIE

Free University Hospital, Dpt de pathologie, Amsterdam, Pays-Bas

EFFECTIFS ET BUDGET DE LINSTITUT DE BIOCHIMIE

M. Kovacsovics (septembre 2001) Assistant(e)s

EFFECTIFS ET BUDGET DE LINSTITUT DE BIOCHIMIE

Comptabilit D. Reichen (octobre 2001)

Photo- Infographie P. Gschwend (aot 2001)

Magasin central et Travaux pratiques E. Margot (dcembre 2001)

UNIL (Etat de Vaud): CHF 5'134629 Ressources extrieures: CHF 4'782503

INSTITUT DE BIOCHIMIE - ENSEIGNEMENTS

172 106 110 110 65 23 23

biologie (5e semestre) 168 224 biologie (3 /4 anne) biologie (3 /4 anne)

Jean-Luc Bodmer Nils Holler Asja Praetor

Jacques Maul et Denis Rivier

A case of cutaneous leishmaniasis (Credit : Dr. R. Behin)

(a) McM (green) in macrophages (b) Disappearance of McM in Leishmania-infected macrophages

Inhibition of gp63 by McM ED peptides.

Control cells : actin fibers are clearly visible

IMMUNOBIOLOGY OF INFECTION WITH AN INTRACELLULAR PARASITE: LEISHMANIA MAJOR

IMMUNOBIOLOGY OF INFECTION WITH AN INTRACELLULAR PARASITE: LEISHMANIA MAJOR

IMMUNOBIOLOGY OF INFECTION WITH AN INTRACELLULAR PARASITE: LEISHMANIA MAJOR

IMMUNOBIOLOGY OF INFECTION WITH AN INTRACELLULAR PARASITE: LEISHMANIA MAJOR

IMMUNOBIOLOGY OF INFECTION WITH AN INTRACELLULAR PARASITE: LEISHMANIA MAJOR

NEW STRATEGY OF TUMOR TARGETING WITH ANTIBODIES

NEW STRATEGY OF TUMOR TARGETING WITH ANTIBODIES

NEW STRATEGY OF TUMOR TARGETING WITH ANTIBODIES

NEW STRATEGY OF TUMOR TARGETING WITH ANTIBODIES

NEW STRATEGY OF TUMOR TARGETING WITH ANTIBODIES

NEW STRATEGY OF TUMOR TARGETING WITH ANTIBODIES

NEW STRATEGY OF TUMOR TARGETING WITH ANTIBODIES

IMMUNE RESPONSES TO VIRUSES AND PROTEIN-HAPTEN ANTIGENS

IMMUNE RESPONSES TO VIRUSES AND PROTEIN-HAPTEN ANTIGENS

1. Virus uptake via gut

2. Infection of AgPresenting Cells

5. Infection of the mammary gland

3. SAg-Priming of CD4 + T Cells

4. Chronic immune response

Hans Acha-Orbea Adapted from Immunol. Rev. 168, 287, 1999

IMMUNE RESPONSES TO VIRUSES AND PROTEIN-HAPTEN ANTIGENS

Follicular and extrafollicular B cell differentiation

F: Follicle EF: Extrafollicular differentiation

IMMUNE RESPONSES TO VIRUSES AND PROTEIN-HAPTEN ANTIGENS

MUCOSAL VACCINES AGAINST ONCOGENIC MICROBIAL PATHOGENS

MUCOSAL VACCINES AGAINST ONCOGENIC MICROBIAL PATHOGENS

MUCOSAL VACCINES AGAINST ONCOGENIC MICROBIAL PATHOGENS

MUCOSAL VACCINES AGAINST ONCOGENIC MICROBIAL PATHOGENS

MUCOSAL VACCINES AGAINST ONCOGENIC MICROBIAL PATHOGENS

MUCOSAL VACCINES AGAINST ONCOGENIC MICROBIAL PATHOGENS

SIGNALING PATHWAYS LEADING TO APOPTOSIS AND INFLAMMATION

SIGNALING PATHWAYS LEADING TO APOPTOSIS AND INFLAMMATION

SIGNALING PATHWAYS LEADING TO APOPTOSIS AND INFLAMMATION

SIGNALING PATHWAYS LEADING TO APOPTOSIS AND INFLAMMATION

SIGNALING PATHWAYS LEADING TO APOPTOSIS AND INFLAMMATION

Bcl-2 member that triggers independently of its BH motifs.

SIGNALING PATHWAYS LEADING TO APOPTOSIS AND INFLAMMATION

SIGNALING PATHWAYS LEADING TO APOPTOSIS AND INFLAMMATION

MALARIA: IN THE SEARCH OF ANTIGEN FOR THE DEVELOPMENT OF PROTECTIVE VACCINES

MALARIA: IN THE SEARCH OF ANTIGEN FOR THE DEVELOPMENT OF PROTECTIVE VACCINES