Annurev - Iy.12.040194.004313) J. Banchereau F. Bazan D. Blanchard F. Briè J. P. Galizzi - The CD40 Antigen and Its Ligand

Annu. Rev. Immunol.
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THE CD40 ANTIGEN AND ITS

LIGAND
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J. Banchereau, F. Bazan', D. Blanchard, F. Briere,

J.P. Galizzi, C. van Kooten, Y.l. Liu, F. Rousset, S. Saeland
Schering-Plough, Laboratory for Immunological Research, Dardilly,

France; ** DNAX Research Institute, Palo Alto, California
KEY WORDS:
CD40, CD40-ligand, lymphocyte activation, B cell(T cell inter
actions, hyper IgM syndrome
Abstract
CD40 is an integral membrane protein found on the surface of B lym

phocytes, dendritic cells, follicular dendritic cells, hematopoietic pro
genitor cells, epithelial cells, and carcinomas. It is a 45-50 kDa glycoprotein
of 277 aa, which is a member of the tumor necrosis factor receptor super
family. The CD40 gene maps to h uman chromosome 20q 1 1 -2-q1 3-2. CD40
binds to a ligand (CD40-L) which is an 35 kDa glycoprotein of 261 aa,
a member of the tumor necrosis factor s uperfamily. The CD40-L gene
maps to h uman chromosome Xq24. This CD40-L is expressed on activated
T cells, mostly CD4 + but also some CD8 + as well as basophils/mast cells.
The CD40-L is defective in the X-linked hyper- IgM syndrome.
Cross-linking of CD40 with immobilized anti-CD40 or cells expressing
CD40-L induces B cells to proliferate strongly, and addition of I L-4 or
IL-1 3 allows the generation of factor-dependent long-term normal h uman
B cell lines and the secretion of IgE following isotype switching. Addition
of IL- l O results in very high immunoglobulin production with limited cell
proliferation. IL- I O induces naive B cells to produce IgG3, IgGI, and
IgA l , and further addition ofTGFfJ permits the secretion ofIgA2. Several
evidences suggest that CD40-dependent activation of B cells is important
for the generation of memory B cells within the germinal centers: (i) CD40
activated germinal center B cells cultured in the presence of IL-4 acquire
a memory B cell phenotype, (ii) CD40 activated B cells can undergo isotype
switching, (iii) the deficit of CD40-L results in the hyper-IgM syndrome
characterized by lack of germinal centers in secondary lymphoid organ
881
0732-0582/94/041 0-088 I $05.00
882
BANCHEREAU ET AL

follicles and lack of IgG, IgA, and IgE, and (iv) CD 40-L positive T
cells are present in secondary follicles. Thymic epithelial cells, activated
monocytes, and dendritic cells express CD40 antigen which may be
involved in an enhanced cytokine production by these cells, allowing an
amplification of T cell proliferation. Finally, as other members of the
tumor necrosis factor receptor family have been shown to bind several
ligands, it is possible that CD40 may bind other ligands that may trigger
CD40 on d ifferent cell types such as hematopoietic cells or epithelial cells.
INTRODUCTION
The CD40 antigen was independently identified in 198 5 and 1 98 6 by
monoclonal antibodies reacting with carcinomas and B cells (antibody
S2C6, antigen p50) ( 1 ) and showing costimulatory effects for B lymphocyte
(antibody G28-5, antigen Bp50) (2). This antigen was designated as CDw40
at the Third International Workshop on leukocyte antigens in Oxfo rd in
1 986, and as CD40 at the Fourth Workshop in Vienna in 1 989. A cDNA
encoding CD40 was isolated in 1 989 (3), and this sequence demonstrated
a relationship with the human low affin ity nerve growth factor receptor
(LNGFR). These molecules are now considered as part of the tumor
necrosis factor receptor superfamily. Cross-linking of CD40, in con
junction with IL-4, was then found to induce B cells to undergo long-term
B cell growth, as well as isotype switching (4-6). In 1 992, expression
cloning using CD40 Fc fusion protein allowed the isolation of a CD40ligand (CD40-L) expressed on activated T cells (7), an observation which
led to the demonstration of the key role o f CD40-L/CD40 interactions in
T cell-dependent B cell activation by many groups (8). The CD40-L is one
of the members of the recently identified tumor necrosis factor superfamily.
In 1 99 3, a genetic alteration of the CD 40-L was shown to be responsible
for the X-linked hyper-IgM syndrome, which is characterized by the lack
of circulating IgG and IgA and the absence of germinal centers (9). While
the function of CD40 has principally been studied on mature B lympho
cytes, more recent studies show the presence of b iologically functional
CD40 on other cell types, such as epithelial cells ( 1 0), monocytesj
macrophages (1 1 ), and hematopoietic progenitors ( 1 2, l 3).
CD40 ANTIGEN
Modular Structural Design and Evolutionary Relationship of

CD40
The CD40 antigen is a phosphorylated glycoprotein which migrates in

SDS polyacrylamide gel electrophoresis as a 48 kDa polypeptide under

CD40 AND ITS LIGAND
883
both reducing and nonreducing conditions. It is a hydrophobic mo lecule

with an acidic Pi of 3 .2. A significant proportion of CD40 from the Burkitt
lymphoma Raji cells and normal B cells is in a dimeric form, whereas such
dimers are virtually absent from carcinoma lines or EBV-transformed cells
(14, 1 5).
A cDNA encoding CD40 was isolated by expression cloning from a
library of the Burkitt lymphoma Raji (3). The mature molecule is com
posed of 277 aa with a 1 93 aa extracellular domain, a 22 aa transmembrane
domain, and a 62 aa intracellular tail. While the extracellular segment of
CD40 displayed significant similarity to the analogous protein domain of
the p75 low-affinity, nerve growth factor receptor (LNGFR) ( 1 6), the
intracellular chain did not betray a relationship to any other characterized
molecule. Human and murine CD40 molecules share 62% amino acid
identity in the extracellular domains and 78% identity in the intracellu lar
extensions ( 1 7).
The p75 LNGFR is the founding father of a superfamily of receptor
lik e molecules that share a common binding domain composed oftandemly
repeated cysteine rich modules ( 1 8) (Figure I, Figure 2, Table 1). It is
remarkable that this nascent gro up of receptors is more appropriately
named after two later additions: the p55 and p75 receptors for t umor
necrosis factor (TNFR I and TNFR2, respectively) ( 1 9-2 1 ) that bind the
related cytokines tumor necrosis factor-ex (TNF-ex; also known as cachec
tin) and lymphotoxin (LT; also known as TNF-f3) (22, 23). LNGFR acts
principally to recruit a n umber of neurotrophin ligands, including NGF,
to the cell surface, aiding in the formation of the signaling complex formed
by dimeric trk receptors that possess intracellular tyrosine kinase domains
(24, 25). LNGFR does not apparently play a role in signal transduction
(26). In contrast, the two TNFRs are both active signalling receptors and
bind ligands with a "TNF fold" that is unrelated to the neurotrophin
structure (27). S ubsequent additions to the TNFR superfamily display
conserved interactions to TNF-like cytokine ligands (28), cementing the
TNFR moniker for the cognate receptors; perhaps the LNGFR molecule
represents an "escaped " TNFR analogous to the tissue factor cell s urface
tether for coagulation Factor VII that is s urprisingly related to the hemato
poietic cytokine receptor sup erfamily (29).
In addition to the LNGFR, CD40, and the two TNF receptors, the
TNFR superfamily currently includes CD27, a molecule expressed on T
cells and activated B cells (30, 3 1) ; CD30, an activation molecule of T cells
and B cells initially detected on Reed Sternberg cells (32); OX40, a rat
activated T cell antigen (33); 4-1BB, another T cell molecule (34);
Fas/Apol, a lymphocyte antigen whose triggering induces apoptosis (3537); and a receptor-like protein from an expressed open reading frame
884
BANCHEREAU ET AL
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Comparative alignment of the Cys-repeat domains of TNFR superfamily
molecules. Conserved Cys residues are in reverse lettering; other conserved amino acids are
in bold on in grey blocks. A summary line of residues important for folding is located at the
bottom of the alignment. Also in this area, the disulfide links indicated by the X-ray structure
of the TNFRI soluble fragment (49) are drawn for the domain alignment. Note that the C l
C2 and C4-C6 links create two similar sized loops that are designated as separate folding
modules. In contrast, the C3-C5 link is dispensable for folding and is missing from several
superfamily receptors. The sequences are grouped by repeat type (numbered from the N
termini of chains as rJ-r4. Sequences are marked with prefixes to designate the species of
origin: human (h), mouse (m), rat (r), or chicken (c). The poxviral receptors are from myxoma
(myx), shope fibroma (sf), cowpox (cpx), variola (var), and vaccinia (vac) viruses. The lone
fungal sequence is the ECPI protein from Cladosporium fulvum. It is best to view this
alignment with Figure 2 in mind-several receptors lack entire repeats or repeat modules.
isolated from a human chromosome 12-specific library of heterogeneous

n uclear cDNAs (38). This latter protein appears most closely sim ilar to
CD40 antigen in its extracellular binding domain (X % identity) and is an
intriguing target for a CD40 l igand-related TNF-fold cytokine. Two novel
GLY'IQDK
885
CD40 AND ITS LIGAND
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TNFR-like proteins appear to encode species homologs of superfamily

receptors: A human receptor induced by lymphocyte activation (lLA) is
likely the human counterpart to mouse 4- I BB (39); the human CD30
antigen appears to have suffered a duplication of binding domains in
comparison to the recently described mouse CD30 (40),
Viral homo logs of the TNFRs were first described by Smith et al (21 ),
who noticed a s ignificant resemblance between the extracellular domains
of several TNFR superfamily members and the protein product of the
transcriptionally active open reading frame T2 in the Shope Fibroma virus
genome (SFV-T2). This poxvirus protein is a secreted receptor for both
TNF-IX and LT (41 ) and reveals a novel aspect of viral subversion of the
host immune system (42, for review). A close homolog of SFV-T2 has
been characterized from Myxoma virus (MYX-T2) (43). The Vaccinia
virus genome contains an open reading frame, Sa1F1 9R, that appears to
encode a fragment of a poxvirus T2-like protein, and can be extended by
jumping reading frames (44). More recent homologs of this poxvirus

uperfalllily ytoki"e
uperfamily Rceptors
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Figure 2_ Schematic organization of receptor and cytokine superfamilies: The re

ceptor supergroup comprises twelve molecules, ten from mammalian cells: LNGRF,
TNFR1 and TNFR2, the TNFR homolog from human chromosome 12 (TNFRh), C040,
OX-40, 4-1SS, Fas, C02? and C030 antigens. A single, representative poxvirus TNFR
homolog chain is noted (from myxoma virus: ref). The small, single-repeat fungal
TNFR homolog is ECP1 protein from Cladosporium fulvum. Repeats are drawn as
diamonds (red, purple, yellow and cyan denote repeats 1-4, respectively) that are
divided into two halves (see box of the TNFR Cys-repeat fold) that likely form disulfide
linked loops. Ser-Thr-Pro rich chain segments that typically link the repeat domains
to the transmembrane segment are drawn as wavy lines-note that the poxvirus and
fungal receptor homologs are soluble proteins. Two types of cytoplasmic domain
homologies are noted by blue or orange colored boxes. The TNFR2 chain does not
resemble either motif. The known cytokine ligands of the TNFR1, TNFR2, C040,
C02? and C030 are drawn as folded sheets with opposing red and grey faces. A
separate box shows the -strand construction of the fold-the grey (inner) sheet is
formed by strands 6"-8-I-O-G, while the red (outer) sheet comprises strands 8'-C'
C-H-E-F. Note that all ligands, save LT, are membrane anchored by N-terminal hy
drophobic segments.
The bottom box shows a decomposition of structural features in the receptor and
cytokine chains. A composite receptor is shown divided into four repeats each of
which show a variation {)f the Cys-repeat pattern: disulfide links are shown above.
Repeats are also colored (red, purple, yellow and cyan) as in the receptor superfamily
figure above. Intron positions are mapped to the receptor chain and show some
correlation to repeat and subrepeat structure. The composite ligand shows the position
of the eleven -strands with the few identical amino acids in the alignment of Fig. 5
noted above the chain-all of these contribute to the hydrophobic core. Orange and
green stars note the location of residues in contact with two distinct receptor chains
in the trimer complex of LT and TNFR1.

00
00
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Table 1
Members of the TNF receptor and cytokine superfamily analyzed in the present work
M olecule
Chain length (aa)
Gene structure
Chromosome location
References
t:C
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Z
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Receptors
LNGFR
TNFR I
TNFR II
TNFR h
CD40
OX-40
4-1 BB
Fas
CD27
CD30
Poxvirus Rh
ECP 1
Cytokines
TNFo:
LT
LT-f3
CD40-L
CD27-L
CD30-L
4-1 BB-L
:r:
399; 396; 397 (h; r; c)

434; 433 (h; m)
493; 452 (h; m)
408 (h)
258; 286 (h; m)
262 (r)
229; 229 (h; m)
319; 306 (h; m)
240; 230 (h; m)
577; 480 (h; m)
310; 309; 337; 330
(myx; sf; cpx; var)
65
6 exons (h)
10 exons
233; 235 (h;

205; 202 (h;
244 (h)
260; 261 (h;
193 (h)
234; 239 (h;
309 (m)
4 exons (h; m)
4 exons (h; m)
4 exons (h)
4 exons (h)
m)
m)
m)
m)
17q21-22 (h)
12pI 3; 6 (h; m)
1; 4 (h; m)
12p (h)
20qlI-q13; 2 (h; m)
single viral orfs
(16)
(19, 20, 61, 62)
(21)
(38)
(3, 17, 63, 66, 67)
(33)
(34, 39, 139a)
(35-37)
(30, 31, 64)
(30, 40)
(41, 43-46)
3 exons (cf)
(47)
9 exons
partial (m)
1; 4 (h; m]
IOq24.1; 19 (h; m)
12q I 3 (h)
I p36 (h)
6
6
6
Xq26-q27 (h)
19p I 3.3 (h)
9q33; 4 (h; m)
17 (m)
(131)
(132)
(137)
(7, 117, 119, 127-129)
(138)
(139)
(I 39a)
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CD40 AND ITS LIGAND
887
branch of the TNFR superfamily include the Variola virus G4R (45) and
Cowpox virus crmB (46) gene products.
The most intriguing member of the TNFR superfamily is the fungal
pathogenic protein ECP I isolated from Cladosporium julvum, a tomato
pathogen (47). ECPI is a secreted protein of 65 residues that appears to
contain a single, characteristic TNFR-like cysteine repeat of 46 amino acid
length; ECP l is suggested to play a role in suppressing the tomato p lant
defense response by b inding plant cytokine-like molecules secreted in the
face of fungal attack (48). This economical molecule may represent the
minimal binding structure of a TNFR homolog and is a functional analog
of the poxvirus T2 factors.
It is instructive to examine the modular protein architecture of the
TNFR superfamily from the vantage point of the recently described x
ray crystal structure of the complex formed by soluble b inding domains
of p55 TNFR I and a LT trimer (49). Early comparisons ofTNFR super
family members highlighted the striking division of the extracellular bind
ing domains into three or four imperfect repeats of ,.... 40 residues, anchored
by a superimposable pattern of six cysteines ( 1 6, 1 8-2 1 , 30, 32, 33). While
the disulfide bridging pattern was unknown, each repeat was assumed to
form an independently folding entity with three main intrachain disulfide
bridges-covariant loss of Cys 3 and 5 in several receptor modules led to
the suggestion that these were likely linked ( 1 8) . The protein fold of the
TNFR repeat module is shown to be a tandem arrangement of "tethered
loops, " where the N-terminal loop is fixed by a Cys l-Cys2 link, and the
C-terminal loop features an analogous Cys4-Cys6 bridge (49) (see Figure
2). The non-essential Cys3-Cys5 link ties the stalk connecting the two loop
structures to the second loop (49) . A structural subdivision nevertheless
does seem to indicate that the TNFR modular fold arose from the union
of two smaller folding units (48). This finding explains the occasional,
puzzling truncations of exact half-repeats in several superfamily receptors:
TNFRI repeat 4, OX-40 repeat 3, human CD30 repeat 4, and CD27 repeat
3 -a half-repeat corresponds to either the N- or C-terminal loop structure
that is presumably capable of correct folding. The comparative alignment
of available TNFR modules ( 1 8 ; see also Figure 1) also h ighlights the
conservation of additional residues, notably an aromatic amino acid
located 5 residues after Cys I that in the crystal structure is shown to be
important for the interaction of N- and C-terminal loops (49). The stacked
repeat modules form an elongated, slightly bent rod that intercalates in
the groove between two LT subunits in the packed trimer. In addition, the
key receptor-ligand interactions involve residues in repeats 2 and 3 (49).
Together, the three receptor subunits encage the LT trimer and scru
pulously avoid. contact between each other.

888
BANCHEREAU E T AL
A notable superstructural motif in the TNFRI rods is a spiralling

"staircase " formed by the successive stacking of disulfide links per
pendicular to the rod axis (49). The gross spatial architecture of the
CD40/CD40 ligand interaction is schematically shown in Figure 9 and
should help in the following structural discussion on CD40 and its ligand.
This energetically favorable packing arrangement places the disulfide
bridges in a preferred hydrophobic core location (50); it has been observed
in other small, disulfide rich proteins like toxins and defensins (5 1-53), and
the cystine-knot family of NGF/TGFP/pDGF folds (28). These disulfide
staircases may appear again in the binding domains of other cytokine
receptors in spite of distinct fo lding topologies; notable candidates include
cysteine-rich modules present in the EGF and insulin receptor extracellular
segments (54) and the TGF-p family signalling receptors (55).
The cysteine repeat pattern of TNFRs resembles an eight-cysteine motif
in laminin-like modules (33, 56). These latter laminin repeats are believed
to fold in a manner similar to EGF, with an "extra " disulfide-bridged loop
at the C-terminus (57). An X-ray crystallographic structural analysis of a
representative fragment of laminin containing a three-repeat segment of
chain that binds the protein nidogen appears to be in progress (58) and
may soon reso lve the matter.
The modular organization of the TNFR binding structures invites the
question of an underlying genetic organization-do the repeats, or half
repeats, correspond to exon-encoded folding units as observed for other
protein modules (59) ? Several gene structures have been elucidated for
superfamily members: the mouse LNGFR gene (60), the human and mouse
TNFRI genes (61 , 62), the mouse CD40 (63), human CD27 (64), the
fungal ECP l (47), and a fragment of the mouse Fas gene (65). The CD40
murine gene is fairly typical of this collection with nine exons that span
1 6.3 kb of genomic D NA (63). A diagrammatic i llustration of exon boun
daries is presented in Figure 2. While there is no consistent subdivision of
repeats into exons for any given gene, the composite picture that emerges
suggests that type 1 introns (which intercut codons after the first nucleotide
base) typically map to the dividing boundaries of repeats, or half-repeats.
Type 1 modules are fairly common in the diverse collection of small protein
motifs found in mosaic cell surface molecules (57).
The mouse CD40 gene is located on the distal region of chromosome 2
which is syntenic to human chromosome 20q I l -q13. Accordingly the
human CD40 gene was mapped to chromosome 20 by using human-rodent
somatic cell hybrids (66) and to 20 q ll-20q1 3-2 by in situ hybridization
(67). It is obvious that superfamily receptor genes have been dispersed
throughout the genome. Table 1 shows the chromosomal localization of
other receptor genes.
CD40 AND ITS LIGAND
889
Cells Expressing CD40
C D40 was early considered as a pan B cell antigen (68).

C D40 is detected on all B cells isolated from adult and cord blood, tonsils
and spleens (Figure 3A) . It is expressed on resting virgin sIgD+ sIgM+ B
cells in the primary follicles and the mantle zone of secondary follicles
within the peripheral lymphoid organs (Figure 4). The density of C D40 is
identical on naive B cells centroblasts and centrocytes, composi ng the
germinal centers of secondary follicles, and on the C D38 - sIgD - memory
B cells (69).
Plasmablasts isolated from tonsils express C D40 (P Merville et aI, manu
script in preparation), whereas roughly half of the antibody secreting cells
circulating in the blood eight days after vaccination have lost C D40 (70).
The fully differentiated p lasma cells of mucosal lamina propria and bone
marrow do not express C D40. Polyclonal activators such as anti-IgM,
anti-CD20, or anti-Bgp95 antibodies or phorbol esters slightly upregulate
C D40 expression on B cells (3, 71). IFNy (3) and IL-4 (72) are also able
to increase C D40 levels on B cells. The human C D40 gene is expressed as
a single 1 .4 kb species in B cells. The murine C D40 gene is expressed in B
lymphocytes as two mRNA species, a predominant one of l.7 kb and a
minor one of 1 .4 kb, which are generated by alternative usage of poly
adenylation signals in the 3' untranslated region. The activation of murine
B lymphocytes preferentially increases the level of the smaller transcripts
(17). Anti-human C D40 antibodies react with the C D40 antigen from
macaques and baboons (73, 74).
Virtually all chronic lymphocytic leukemia B cells and non Hodgkin's
lymphoma cells express C D40. Burkitt lymphoma cell lines and EBV
transformed B cell lines all display C D40 (1). The p lasmacytoma cell line
RPMI 8226 displays low levels of C D40 while U266 cells do not. Most
IL-6-dependent myeloma cell lines are C D40 positive (B Klein, personal
communication).
Numerous EBV-transformed B cell lines release soluble C D40 (sCD40)
spontaneously, as detected with a specific ELISA (75). Supernatants of
Staphylococcus aureus strain Cowan I-activated normal B cells cultured
in the presence of IL-4 or IL-2 also contain sC D40. This is in line with the
described soluble forms of the other members of the TNF-R family, which
include sTNFR l and sTNFR2 (76, 77), sLNGFR (78), and sC D27 (79).
Although the mechanisms of sC D40 release have not yet been studied, it
is possible that these molecules originate from proteolytic cleavage as
shown for sCD27 (80), and as indicated by the lack of alternatively spliced
C D40 mRNA. sCD40 of EBV cell line supernatants is able to bind to the
C D40-L expressed on activated T cells.

B LYMPHOCYTE
890
BANCHEREAU ET AL
CD40
CD19
sIgM
sIgD

CD40
CDlO
CDl9
sIgM
CD40
CD34
CDIO
CDl9
1\
Figure 3 Expression of CD40 on B lineage cells and hematopoietic progenitor cells. Total
B cells isolated from tonsils (panel A), B cell precursors (CD I 9 + CDl O + sIg - ) obtained
from mid-term fetal bone marrow (232) (panel B), and CD34+ progenitors separated from
full-term umbilical cord blood (233) (panel C), were stained with the anti-CD40 mAb 89
(72). In parallel, and as indicated, cells were monitored for expression of CD19, CDlO,
CD34, sIgM, and sIgD. Histograms represent log of FITC-fiuorescence analyzed on a fiow
cytometer. Dotted lines correspond to negative control staining with an unrelated murine
mAb.
In human fetal and adult bone marrow,

CD40 is detectable on the majority of B cell precursors (BCP), which
express CDl9 and CDl O but lack surface Ig (Figure 3B; 1 3 ,8 1-83). CD40
HEMATOPOIETIC PROGENITORS

CD40 AND ITS LIGAND
89 1
is acquired early in B cell ontogeny, as it is present on BCP expressing the

CD34 progenitor cell antigen ( 1 3) and has been reported on fetal liver
CDI 9 + cells prior to rearrangements at the IgH locus (pro-B cells) (8 1 ).
CD40 is also expressed on malignant BCP in B lineage acute lym
phoblastic leukemias at various maturation stages, ranging from pro-B
cells to pre-B cells expressing cytoplasmic J1 chain (8 1 , 84). About 28 to
44% of BCP-ALL cases have been shown to express CD40 (81, 82). Among
the positive BCP-ALL, CD40 is present only on a proportion of the
leukemic cells, a feature that has been associated with clonogenic capacity
(81).
Finally, CD40 is not restricted to early B lineage cells in normal hemato
poiesis; it is found on most cord blood CD34 + progenitors, which lack a
CDI9 + CDIO+ subset (Figure 3C), and on the majority of bone marrow
CD34 + cells ( 1 3). In this context, CD40 expression is more heterogeneous
on CD34 + progenitors than on mature B cells (Figure 3A and 3C). CD40
expression is lost during myeloid development in cultures of CD34+ cells
( 1 3). It is important to further define the CD34+ CD40+ population, and
to investigate whether the most primitive nonlineage committed CD34+
cells, characterized by lack of CD38 antigen (85), express CD40.
Immunohistological analysis (68) on
tonsil and spleen sections has shown high levels of CD40 on interdigitating
dendritic cells in the T cell-rich areas of secondary lymphoid organs
(Figure 4). These cells derive from skin/mucosal Langerhans cells which
only weakly express CD40 (86). However, following culture, Langerhans
cells express CD40 at high levels. Dendritic cells can be generated in vitro
by culturing CD34 + hematopoietic progenitor cells in the presence of GM
CSF /IL-3 and TNFIX (87). These cells, which resemble Langerhans cells
as they express CD l a and display Birbeck granules, express CD40 at high
levels and may thus represent cells at a stage of differentiation between
Langerhans cells and interdigitating dendritic cells. In fact, dendritic cells
isolated from peripheral blood also express CD40 and may represent
Langerhans cells homing towards secondary lymphoid organs (88).
Primary human monocytes freshly isolated or cultured for 48 hr show
low but detectable CD40 surface protein ( 1 1). GM-CSF, IL-3, and IFNy
strongly upregulate their CD40 expression.
DENDRITIC CELLS AND MONOCYTES
CD40 is expressed in the CD45-negative stromal

cell population of human thymus ( 1 0). Immunohistology shows CD40
expression on cortical and medullary thymic epithelial cells, as well as
thymic interdigitating cells and B cells (Figure 4). Expression of CD40 is
specifically maintained on cultured thymic epithelial cells but not on thymic
OTHER CELL TYPES
896
BANCHEREAU ET AL
CD40-L in NK cells and purifie d m on ocytes (1 27). Northern blot analysis

of the B cell line Daudi and the histiocytic lymphoma U937 have shown
specific hybridizati on signals at 3.7 and 1 .7 kb, suggesting expression of
CD40-L related m olecules in B cells and monocytes (1 29).
CD40-Ligand Is a Member of an Emerging Cytokine Family
The initial characterizati on of the m ouse CD40-L did n ot easily yield i ts

chain similarity to the TNF superfamily (7). Subsequent analyses showed
that an 200 aa domain which formed the major extracellular domain of
CD40-L could be aligned with available TNF-O( and LT sequences in a
structurally sound manner (27, 1 1 9, 1 29) (Figure 2, Figure 5). The region
in questi on comprises the bioactive, receptor-binding, gl obular porti on of
TNF-like molecules that folds, by example of TNF-O( ( 1 30, 1 3 1) and LT
(49, 1 3 2), into a barrel-like structure reminiscent of viral capsid proteins.
This distinctive TNF fold consists of two packed sheets of eight major
antiparallel f3-strands linked in a "f3-jellyroll" topology with an N-terminal
loop insertion that contains two additional, short f3-strands (1 3 1 , 1 32, 1 33).
In this manner, and following a s tandard n omenclature for viral capsid
proteins (1 30, 1 33), the "inner " sheet which is primarily involved in trimer
contacts is formed by s trands B '-B-I-D-G in correct spatial order, and the
"outer" sheet analogously consists of s trands C'-C-H-E-F (see Figure 2).
While there is no detectable sequence similarity with viral capsid proteins,
detailed structural comparisons u tilizing the TNF three-dimensi onal coor
dinates show excellent backbone superposition, similar types of sidechain
contacts, conserved amino acids, and vari ous geometric matches with
available fj-jellyroll folds ( 1 34).
The concept of structural conservation in spi te of amino acid divergence
has prompted the constructi on of detailed CD40-L m odels ( 1 35, 1 36)
utilizing homology modeling techni ques and the available protein frame
works of TNF-O( and L T. These m odels show that the sparse amino
acid matches between CD40-L and TNF-O( or LT are important for the
construction of the fJ- jellyroll core fold, and also predict that CD40-L is
capable of trimerization. Gaps in the protein alignment partiti on to vari
able loops in the model and two cysteines (residues X and Y) are suggested
to form a disulfide link analogous to the Cys l 45-Cys 1 77 link in TNF-a
(1 35).
Recent additions to the TNF cytokine superfamily have been a LT-fj
molecule capable of forming heterotrimers with L T (1 37), and the cytokine
ligands for CD27 (1 38), CD30 (1 39), and 4- 1 BB (139a). It is interesting
to n ote that all TNF superfamily cytokines, with the exception of LT, are
produced as type II membrane-tethered m olecules ( 1 39). Secreted LT has
an alternative membrane-bound form when complexed with a p33 factor


Spleen
Tonsil
Thymus
Figure 4. Expression of CD40 antigen on frozen sections of human spleen, tonsil

and thymus: Expression of CD40 was analysed with anti-CD40 antibody G28.5 (a
kind gift of professor E. A. Clark) using the APAAP method. Spleen: CD40 staining
on B cells in follicle (F) and marginal zone (MZ). Very strong CD40 staining is observed
on interdigitating cells in the periarteriolar lymphocytic sheath (PALS) around the
central arteriole (CA). Tonsil: CD40 staining on follicular mantle (FM) B cells, germinal
center (GC) B cells and follicular dendritic cells. Again, very strong CD40 staining is
seen on interdigitating cells in the extrafollicular area. Thymus: CD40 staining on
thymic epithelial cells both in the cortex (CT) and the medulla (M). Very strong CD40
staining is observed on dendritic cells in the medulla.
892
BANCHEREAU ET AL
fibroblasts. IL-IO(, TNFO(, and IFNy signi ficantly upregulate CD40 levels
and 1 .4 kb CD40 transcripts on cultured epithelial cells.
CD40 is also expressed on follicular den dritic cells (FDC) of secondary
lymphoid organs, as shown by immunohistology analysi s on tissue sections
(Figure 4) and by flow cytometry on freshly i solated FDC (89), and on
FDC cultured in the presence of GM-CSF (90).
Immunohistological analysi s performed on many different tissues (9 1 )
has also indicated staining o f various cell types with the anti-CD40 mAb
G28. 5 : endothelial cells (mixed pattern of reactivity +/ ), smooth
muscle cells (+/ ), cardiac myocytes (+), epidermi s (weakly +), gastro
intestinal mucosa (+), gallbladder mucosa (+), bronchus mucosa (+),
salivary gland ductal and acinar cells ( +), pancreas ductal cells, mammary
ductal and acinar cells (+/ - ), sweat gland (weakly +), prostate gland
cells (weakly +), thyroid gland ( + ), parathyroid gland ( + )
Basophils isolated from the blood of chronic myeloid leukemia patients
have been reported to express CD40, shown by the binding of mAb B-El O
(92).
The CD40 antigen was initially i dentified with the mAb S2C6, which
was generated from mice immunized with a urinary bladder carcinoma
(93). Subsequently, CD40 antigen has been identifie d on carcinomas of
other origins such as colon, prostate, breast, and lung, as well as on
melanomas (94). CD40 has also been detected on T cell lines transformed
with HTLV I and II (95). Accordingly baboon T cell lymphomas, which
display HTLV I, are CD40 positive (74). Thus, CD40 is expressed on cells
with high proliferation potential such as hematopoietic progenitors, B
lymphocytes, and epithelial cells, and cells able to present antigen such as
dendritic cells, activated monocytes, B lymphocytes, and follicular den
dritic cells.

Signal Transduction Through CD40
The TNFR superfamily is primarily defined by common structural motifs

in the extracellular binding domains-there is no equivalent organizing
principle for the diverse cytoplasmic extensions ( 1 8). This is an important
distinction: perhaps the TNFR superfamily is a grouping of composite
receptors with related binding folds grafted onto a diverse collection of
intracellular signalling domains. After all, most growth factor/cytokine
receptors operate by a common signalling paradigm irrespective of their
cytoplasmic mass. Receptors rely on the ligand-induced association of
extracellular domains to drive the concommitant association of intra
cellular structures, independently of whether these cytoplasmic domains
are enzymes (i.e. tyrosine or serine kinases) or not (96, 97). The TNFR
intracellular domains clearly fall into the latter category--by analogy to

CD40 AND ITS LIGAND
893
the T cell antigen receptor complex (9S) or the hematopoietic superfamily

receptors (99, 100), there may be a reduced number of protein motifs that
act as binding epitopes for intracellular signalling molecules like kinases.
Perhaps a cytoplasmic grouping is possible: it has been noticed that the
cytoplasmic domains of Fas, TNFR1, and LNGFR show a distant but
significant similarity (35, 37). The more chain-economical domain ofCD40
has also been proposed to belong to this group (35, 37), but further
comparison suggests a fortuitous alignment. The structural integrity of t he
intracellular domain of Fas is required for apoptosis as assayed by both
site-directed mutagenesis and deletion analysis (101), or by naturally occur
ring variants involved in murine lymphoproliferative disorders (1 02). The
Fas gene in mice is also the structural gene for lymphoproliferation (ipr)
mutation (102, 103 , 104). While the key ipr mutation causes a rearrange
ment in gene intron 2 (and no viable mRNA is detectable), the allelic iprcg
defect involves a single amino acid change in the Fas cytoplasmic domain,
converting Ile225 Asn (102-104). The Fas-similar domain in TNFRI
mediates TNF-directed cell cytotoxic activity ( l05). Most recently, t he
homologous domain in the LNGFR was shown to be also involved in the
induction of apoptosis (103). Together, these observations suggest that a
subgroup of TNFR superfamily receptors share a common signalling
domain. That CD40 is not included in this group makes some biological
sense: while TNFR I and Fas require triggering by antibodies or ligands
to induce cell death, CD40 acts instead as a survival factor (4). Some
functional analogy between CD40 and LNGFR was observed in t hat
LNGFR constitutively induces cell death unless triggered by NGF or a
monoclonal antibody (106).
Of the remaining TNFR superfamily members, a cytoplasmic domain
homology has been noticed only between CD27 and 4-1BB (31). Inter
estingly, this region of 4-1BB displays a short sequence bracketed by two
cysteines that resembles the kinase binding motifs of CD4 and CDS ( 1 07,
l OS). Indeed, Kim et al ( 109) have recently s hown that the mouse T cell
antigen 4-1BB directly associates with the tyrosine kinase p56\Ckl through
these motifs. CD40,OX-40,CD30,and the TNFR homolog from human
chromosome 12 in turn show a short stretch of sequence similarity centered
on a Glu-(AspjGlu)-Gly-Lys motif at the C-terminal end of their respective
cytoplasmic domains. TNFR2 remains unclassified in this organizational
scheme. Further dissection of this multitude of domains may uncover their
role in TNFR superfamily signal transduction (110).
The importance of the CD40 intracellular domain in signal transduction
has been directly inferred by the deleterious nature of a Thr234 Ala
mutation (1 11). Cross-linking of CD40 on nonresting human B lym
phocytes i nduces tyrosine p hosphorylation of four distinct substrates (S4).

894
BANCHEREAU ET AL
Activation of protein tyrosine kinases (PTK) appears important for the

transduction of CD40 signals because PTK inhibitors block B cell aggre
gation (112). As the CD40 cytoplasmic segment does not contain any
enzymatic domain, it is likely that the CD40 mediated protein tyrosine
kinase activity (113) occurs through activation of separate kinases. Accord
ingly, engagement of CD40 on the Daudi cell line induces activation of
the src type kinase, iyn, following its increased phosphorylation (113). In
contrast, anti-CD40 alters neither the phosphorylation nor the activity
of jyn, another kinase of B lymphocytes. CD40 engagement results in
phosphorylation of the 85 kDa subunit of phosphatidyl inositol 3 kinase
(PI3K), while it does not affect its 110 kDa subunit. CD40 cross-linking
induces an increased activity ofPI3K which catalyzes the phosphorylation
of phosphoinositols on the 3' moiety and which plays a key role in mito
genesis. Finally, CD40 ligation induces within one minute increased phos
phorylation ofPLCy2 (phospholipase C). PLCyl , which is present in lower
amounts in B cells (114) does not appear to be phosphorylated in response
to CD40 ligation. Phosphorylation of PLCy2 is consistent with anti-CD40
induced IP3 production (84).
CD40-LIGAND
Expression of CD40-Ligand on Activated T Cells

Fusion proteins made from the extracellular domain of human CD40 and
the Fc region of human IgGI have been used to identify CD40-L. CD40L is expressed on activated EL-4 thymoma cells, activated mature T cells
but not on resting T cells (7, 115-123, 123a). CD40-L can be detected on
T cells very early (1-2 hr) after activation. The expression of CD40-L is
primarily on CD4+ T cells, although a small population of CD8+ cells
also acquires CD40-L. The CD40-L can be induced on THO, THI and
TH2 cells. The functional expression of CD40-L, as detected with the
CD40-chimera is inhibited after co-culture with B cells (120). This effect
can be explained by a downregulation of CD40-L mRNA and by the
release of sCD40 which binds to CD40-L. As a consequence, T cells stain
positive with a polyclonal anti-CD40 antiserum (75). The capacity of B
cells to release CD40 in cultures was confirmed by a specific ELISA.
Studies performed with monoclonal antibodies specific for the CD40-L
(122, 124), isolated for their ability to block T cell-dependent B cell
activation, confirm the expression of CD40-L observed with the CD40
fusion protein. Immunohistochemistry demonstrates that human CD40L is expressed on CD3 + CD4 + T lymphocytes of the mantIe zone and
germinal center light zone of secondary follicles in all peripheral lymphoid
tissues. CD40-L positive cells can be identified within the interfolIicular T

CD40 AND ITS LIGAND
895
cell rich areas of secon dary lymphoi d tissues and the medulla and cortex
of normal thymus.
Immunohi stochemistry on murine spleen i solated three to four days,
following immunization with the thymus-dependent antigen KLH ,demon
strates an increase of CD4 + CD40-L + T cells in and around the terminal
arterioles, and on the periphery of the outer periarteriolar lymphoi d
sheath. Double immunohistochemical analysi s reveals that the B cells
producing the specific antibodies are juxtaposed to CD40-L+ T cells (1 25).
The latter study failed to show the presence of CD40-L + T cells in germinal
centers. This difference with human data may be linked either to species
or to the reagents as the anti-murine CD40-L may not detect CD40-L
saturated with sCD40.
Lung mast cells and blood basophils also express CD40-L which i s
functional a s shown b y the ability o f basophils t o induce B cells t o secrete
IgE ( l 25a).
Characterization of a CD40-Ligand cDNA
Murine EL-4 thymoma cells can induce the proliferation of resting human
B lymphocytes (1 26). A cDNA that encodes CD40-L (7) was i solated from
the EL-4 line following enrichment of cells binding to a CD40-Fc fusion
protein. The murine cDNA predicts a polypeptide which has 260 aa con
sisting of a 22 aa cytoplasmic domain, a 24 aa transmembrane domain,
and a 2 14 aa extracellular domain with four cysteines. Murine CD40-L i s
a type I I membrane protein which has a n extracellular carboxy-terminus.
A human CD40-L cDNA has been i solated by screening stimulated
human blood T cell libraries with the murine CD40-L probe ( 1 1 7 , 1 1 9 ,1 27,
128). Another group had independently isolated a TNF-related activation
protein (TRAP) from activated human T cells, which turned out to be the
human homolog to murine CD40-L ( 1 29). The cDNA for human CD40L encodes a polypeptide of 261 aa. The human CD40-L has a 22 aa
cytoplasmic domain, a 24 aa transmembrane domain, and a 2 1 5 aa extra
cellular domain with five cysteines. The murine and human CD40-L dis
play a conserved N-linked glycosylation site in the extracellular domain
and the human CD40-L displays an additional, but probably not utilized,
glyco sylation site in the cytoplasmic domain. The two sequences exhibit
78 % aa i dentity. There is 75% i dentity in the extracellular domain, 96%
between the transmembrane region, and 81 % between the cytoplasmic
domains.
Northern blot analysis of activated T cells demonstrates the presence of
two mRNA species of 2 . 1 and 1.4 kb (117, 127) that differ in the length of
the 3' untranslated ends. CD40-L mRNA has been detected in activated
CD4+ and CD8+T cells. RT-PCR analysi s also indicates the presence of

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Sequence and structural alignment ofTNF superfamily cytokines. The known X- ray structures ofTNF-a, LT and the model construction
of CD40-L (135, 136) were used to accurately align the available sequences of human (h) and mouse (m) TNF-like cytokines. J3-strands derived
from the TNF-(l( and LT structures are boxed and labeled according to standard viral capsid nomenclature: B- C- D-E-F-G-H-I, in such a manner
that strands B-I- D-G and C-H-E-F form two opposing J3-sheets of a flattened barrel structure. Note a small insertion of sequence (large boxed
region) between strands B and C that forms threc short J3-strands labeled 8', B, and C. The line beneath the sequences denotes the residue
environment from TNF-a and LT X-ray structures: d marks highly exposed (i.e. solvent accessible) residues, are moderately exposed amino
acids, *residues are buried in the hydrophobic core of the fold while t denotes residues buried in the trimer interface. Above the sequences are
noted residues potentially in contact with receptors by homology to the determined L T- TNFR I receptor complex: & and $ symbols separate these
interactions to the two receptor subunits that contact each ligand subunit in the trimer.
Figure 5

898
BANCHEREAU ET AL
( 140) that was revealed to be LT-f3 ( 1 37). These membrane-tethered homo

or heteromeric cytokines bind to specific TNFR superfamily receptors that
are displayed on the surface of neighboring cells, signaling the direct,
contact-mediated immunoregulation of one cell type by another. However,
as aptly demonstrated by the X-ray complex of LT and TNFRI proteins,
soluble forms of these cytokines are perfectly capable of inducing receptor
association, and subsequent intracellular signalling.
A comprehensive alignment of representative TNF-C( and LT molecules
with the LT-13 chain and the ligands for CD27, CD30, and CD40 illustrates
the great divergence of amino acid sequences within the TNF superfamily
and is a graphic measure of the plasticity of the TNF fold (Figure 2). How
can we decide which sequence variations are covariantly absorbed by the
core fold, and which contribute to changes in receptor specificity? Several
groups have tried to answer this question by exhaustive mutagenic studies
of the structure and function of TNF-C( and LT ( 1 4 1-144) prior to the
determination of the complex structure. Van Ostade et al ( 1 45) have
remarkably arrived at the identification of a single residue change in human
TNF-C( (Arg32 ---t Trp) that drastically lowers the binding affinity for
TNFR2 but retains wild-type binding to TNFR I . As each receptor has
distinct signalling pathways (23), this finding has important therapeutic
potential ( 1 46). In addition, these studies may reveal the structural basis
of receptor promiscuity for TNF-like ligands: clearly the distantly related
LT and LT-f3 molecules can bind to the same receptor subunits ( 1 37, 1 40).
Similar situations may exist with other TNF-like cytokines, reminiscent of
the multitude of promiscuous couplings that are found between hemato
poietic cytokines and their receptors ( 1 47, 1 48).
FUNCTIONAL CONSEQUENCES OF CD40

ENGAGEMENT
Mature B Lymphocytes
For simplifying the description of the effects of CD40 ligation on human B

cells, we use the classical model where B cells, following antigen encounter,
undergo activation, proliferation, and differentiation in a stepwise fashion
(149).
PROLIFERAnON OF B LYMPHOCYTES
Resting B cells
cultured in the presence of anti-CD40 antibody increase their size (72) and
form homotypic aggregates ( 1 1 2, 1 50-1 53). Studies with antibodies have
shown that this aggregation involves not only LFAI-ICAM l , but also the
recently identified pair CD23/CD21 ( 1 54, 1 55). This latter interaction is
inhibited by the anti-CD23 antibody MHM6 and the anti-CD2 1 antibody
ACnyAnON AND

CD40 AND ITS LIGAND
899
BU32, while antibodies directed to other epitopes of CD23 and CD2 1 are
inefficient. Interestingly, fucose-2-P04 also blocks CD40 induced homo
typic aggregation in accordance with the lectin-like nature of CD23. Acti
vation of B lymphocytes through CD40 also stimulates their adhesiveness
to endothelial cells in a VLA-4 dependent fashion ( 1 56). Soluble anti
CD40 ( 1 57) and CD40-L transfected cells ( 1 58) are able to prevent apop
totic death of germinal center B cells. Triggering B cell CD40, either by
cross-linking with monoclonal anti-CD40 antibody presented by a murine
fibroblastic Ltk-cell iine transfected with FcyRIIjCDw32 (CD40 system)
(4, 5) or with CD40-L transfected cells, results in an increased expression of
CD23, class II antigens and B7jB B l ( 1 2 1 , 1 24, 1 59-1 6 1 ) . CD38+ CD44germinal center B cells cultured in the CD40 system (CDw32 + L cells +
anti-CD40 antibody) in the presence of IL-4, acquire the phenotype of
memory CD3S- CD44+ B cells (YJ Liu et aI, manuscript in preparation).
Finally, CD40 cross-linking induces B cells to produce IL-6 and IL- l O
( 1 62, 1 63).
Anti-CD40 antibodies have been isolated for their ability to co stimulate
with either anti-IgM antibodies or phorbol esters (2, 7 1 , 72). In a soluble
form, some anti-CD40 antibodies can induce DNA replication in resting
B cells although this does not result in sustained proliferation ( 1 50, 1 64,
1 65). B cells cultured with soluble CD40-1igand in a monomeric form enter
into limited DNA synthesis, but a trimeric form of soluble CD40 ligand
obtained through various constructions results in quite significant DNA
synthesis particularly in combination with anti-Ig ( 1 1 5, 1 1 9, 1 66). Culture
of resting B cells in the CD40 system results in strong and long lasting B
cell DNA replication (4). At least 70-80% of B cells enter into the G 1
phase of cycle and 50--60% into the S phase, permitting B cell numbers to
increase by three- to four-fold over two weeks. These culture conditions
allow the proliferation of various B cell subpopulations including mantle
zone sIgD + sIgM + B cells, sIgD- sIgM + /- B cells, CD5 + and CD5 - B
cells ( 1 67). Furthermore, quite significant DNA synthesis is observed in
leukemic B cells, such as non-Hodgkin B cell lymphomas and chronic
lymphocytic leukemia B cells ( 1 68). Transient expression of transfected
murine and human CD40-L into various cell lines allows short-term pro
liferation of murine and human B lymphocytes ( 1 1 7, 1 19, 1 2 1 , 1 27, 1 28,
1 59, 1 61). L cells stably expressing human CD40-L can induce a pro
liferation of B lymphocytes at least as important as that obtained using
CDw32 L cells and anti-CD40 (Figure 6).
DNA synthesis of B cells in response to soluble anti-CD40 antibodies
and either anti-IgM or phorbol esters is further boosted by IL-4 (72, 1 50,
1 69). Addition of IL-4 to B cells cultured in the CD40 system results in
their sustained proliferation (4), and the total B cell population can expand
900
BANCHEREAU ET AL
cpm
CDw32 L-celJs
80000
60000
40000

20000
none
IL2
IL4
IUO none
IL2
IL4
IL 1 0
mAb89
CD40-ligand L-celJs
cpm
100000
80000
60000
40000
20000
none
IL2
IL4
IL 10 none
IL2
IL4
IL 1 0
mAb89
Figure 6 B cell proliferation induced either by CDw32-L cells and anti-CD40 mAb 89 (CD40
system) or by CD40-Ligand-L cells. Purified total tonsil B cells (20 x 1 031200 III well) were
cultured with irradiated L cells (8000 Rad; 4 x 103lwell) which were stably-transfected with
either FcyRIIICDw32 (Panel A) or with CD40-L (Panel B). Cultures were performed without
or with IL-2 (40 Vlml), IL-4 ( 1 00 Vlml) or IL- I O ( 1 00 nglml) in the absence or presence of
anti-CD40 antibody mAb89, as indicated. B cell proliferation was determined at day 5 of
culture by the addition of 3H-thymidine, present during the last 1 6 hr of the culture period.
Indicated is the mean cpm observed in triplicate cultures. Note that the Mab89, in a soluble
form, is able to inhibit CD40-L induced B cell proliferation while it is a powerful stimulator
in an immobilized form.
up to lOOO-fold. This results in the generation of factor-dependent long

term normal B cell lines which are negative for Epstein-Barr viral infection.
B cell clones can be generated that contain several hundred cells. IL-4 also
strongly enhances the proliferation of B cells cultured in the presence of
cells expressing CD40-L (Figure 6) ( 1 1 7, 1 59). In recent studies, we have
been able to show that L cells stably expressing CD40-L are able to induce

CD40 AND ITS LIGAND
90 1
the multiplication of B cells over two weeks provided IL-4 is added to

cultures. IL- 1 3, a cytokine which shares homology with IL-4 ( 1 70, 1 7 1 )
can also induce a strong and long lasting proliferation o f B cells stimulated
in the CD40 system or with CD40-L transfected cells (1 27).
IL-l and IFNy enhance the DNA synthesis observed in the CD40 system
or with CD40-L transfected cells, with or without IL-4 ( 1 59, 1 72). In our
hands, IL-2 poorly enhances the proliferation of B cells cultured in the
CD40 system or with CD40-L transfected cells, while another study indi
cates that IL-2 is able to stimulate the proliferation of B cells activated
with CD40-L transfected cells ( 1 59). Cells cultured in the CD40 system
with IL-4 express CD 1 9 , CD20, CD40, sIg, high levels of CD23, and
HLA class II antigens. Surprisingly, a quite significant proportion of cells
cultured for three weeks still express sIgD ( l 72a). Addition of anti-IgM
antibody or SAC particles, which enhance DNA synthesis ( 1 73), does not
down-regulate sIgD expression (our unpublished results).
Both viral and human IL- l O enhance the proliferation ofB cells cultured
in the CD40 system or with CD40-L transfected cells, as determined both
by tritiated thymidine incorporation and increased viable cell numbers
( 1 59, 1 74) (Figure 6). IL- l 0 appears to be almost as efficient as IL-4 during
the first week of culture, but proliferation slows down thereafter and
eventually ceases after 1 4 days. The combination of IL-4 and IL- l O is
additive and results in a 60-100 fold expansion of viable B cells over
two weeks. IL- I O upregulates the expression of CD25jTac on anti-CD40
activated B cells, and accordingly addition of IL-2 strongly enhances
B cell proliferation ( 1 75). In fact, the production of IL-I 0 by CD40-L
transfected CVl cells could explain the response of B cells to IL-2 (1 59)
which is only marginal in our studies. B cells cultured in IL- I O differ
microscopically from those cultured in IL-4 in that loose aggregates are
observed early on, which then yield cultures mostly composed of single
large cells.
Human and murine B cells cultured
with anti-CD40 antibody, with or without CDw32 L cells or with CD40L transfected cells, produce marginal amounts of immunoglobulins ( 1 2 1 ,
1 59, 1 6 1 , 1 72). However, coculture o f human B cells with SAC particles,
anti-CD40 antibody, and CDw32 L cells results in the production of very
large amounts of IgM, IgG, and IgA without IgE (1 76). Mantle zone
sIgD + sIgM + B cells secrete only IgM, whereas sIgD - sIgM + j - B cells
secrete IgG and IgA and lower amounts of IgM. This indicates that the
concommitant triggering of sIg and CD40 results in differentiation of
human B lymphocytes independently of exogenous cytokines.
Addition of IL-4 to cells cultured in the CD40 system only results in a
DIFFERENTIATION OF B LYMPHOCYTES

902
BANCHEREAU ET AL
slight increase in the production of IgM and IgG and in the secretion of
large amounts of IgE (1 72). In fact, IgE production results from isotype
switching as highly purified naive slgD+ B cells produce as much IgE
as isotype committed slgD - B cells. In contrast to long-term B cell
proliferation, the production of IgE does not require the presence of
CDw32 L cells (6, 1 77-1 79). Cells transfected with CD40-L can also induce
murine and human B lymphocytes to secrete IgE in response to IL-4 (1 17,
1 2 1 , 1 59, 1 6 1). Addition of lFNy or IFNa: to CD40 activated B cells fails
to inhibit IL-4-induced IgE production. Indeed the inhibitory effects of
interferons on IL-4-induced IgE production by mononuclear cells ( 1 80)
may to be due to downregulation of CD40-L on IL-4 activated T cells
( 1 23, 1 28). TGFp and TNF!X are able respectively to block and to stimulate
the production of IgE by CD40-activated B cells ( 1 8 1 ) . B cells stimulated
through their CD40 antigen secrete IgE and IgG4 in response to IL- 1 3, as
a result of isotype switching ( 1 27, 1 82).
Addition of lL- l O to CD40-activated B lymphocytes results in the pro
duction of considerable amounts of IgM, IgG, and IgA without any IgE
( 1 59, 1 74). In fact, cells cultured in the CD40 system in the presence of lL10 differentiate into plasma cells expressing large amounts of intra
cytoplasmic immunoglobulins. IL- l O induces anti-CD40 activated tonsil
B cells to secrete IgG I , IgG2, and IgG3. Purified human B lymphocytes
cultured in the CD40 system in the presence of IL- I 0 produce IgG and
IgM antibodies able to bind to antigens such as tetanus toxoid (Table 2).
The combination of soluble anti-CD40 or soluble CD40-L and IL- l O is,
however, insufficient to allow production of bacteriophage-specific anti
body by memory slgD- B cells from immunized individuals (I 82a). Specific
antibody can however be detected provided bacteriophages are added to
cultures, a finding consistent with a preferential expansion of antigen
specific B cells and reminiscent of the comitogenic effect of anti-CD40 and
anti-Ig antibody (2, 72, 1 73). Interestingly, CD40 activated slgD+ JslgM +
B cells were found essentially to secrete IgM but also IgG 1 and IgG3 in
response to IL- l O, indicating that this cytokine may act as a switch factor
for certain IgG subclasses ( 1 83a). CD40 activated naive slgD+ , slgM+ B
cells cultured with IL- l O also produce low levels of lgA l , and addition of
TGFp induces large amounts of both IgAI and IgA2 subtypes, while
inhibiting IgM and IgG production ( 1 76) (F. Briere, manuscript in prep
aration). In contrast, TGFfl inhibits the production of lgM, IgG, and IgA
by isotype committed sIgD - B cells stimulated by IL- l O in the CD40
system. This strongly suggests that TGFfl may represent an IgA-switch
factor both in human and in mouse ( 1 83, 1 84). In line with these findings,
recent studies have indicated that TGFfl is able to induce !X I and !X2
germline transcripts in activated human B cells ( 1 85).
CD40 AND ITS LIGAND
903
Table 2 Production of tetanus toxoid specific antibody by B lym

phocytes activated in the CD40 system with cytokines'

% Wells with IgG anti-tetanus toxoid
No cytokine
IL2
IL4
ILIO
ILIO + IL4
ILIO+ IL2
5000 B cells per well
500 B cells per well
0
0
3
9
19
29
0
0
0
0
8
11
' Purified tonsillar B lymphocytes were cultured over irradiated CDw32-L

cells (8000 Rad; 4 x I O'/well) with 0.5 Jig/ml anti-CD40 Mab 89. Cultures
were performed without or with IL2 (40 V/ml) or ILIO (100 ng/ml) or their
combination. Wells were harvested after 10 days and the presence of anti
tentanus toxoid antibody was determined by standard ELISA. Positive wells
yielded an optical density equal to at least three times the background
observed without addition of culture supernatants.
IL-5 acts synergistically with IL-4 to induce CD40 activated murine B

cells to secrete IgG I and IgE, and to promote IgM and IgG3 secretion.
Surprisingly, the induction of antigen specific antibody responses of CD40L activated murine B cells requires the presence of antigen and IL-2, while
IL-4 and IL-5 are only poorly efficient (1 2 1 ). This suggests that triggering
of the antigen receptor may skew the cytokine response of CD40 activated
B cells. Thus the engagement of CD40 on B cells turns on their isotype
switching machinery, the specificity of which is subsequently provided by
cytokines.
B CELL ACTIVATION Many different
culture systems have been set up to elucidate the cell surface and soluble
molecules involved in the growth and differentiation of B lymphocytes.
Activated T cells can induce resting B cells to proliferate and differentiate
into Ig secreting cells ( 1 86, for a review). In particular, the mouse thymoma
EL-4 can activate resting human B cells to proliferate ( 1 26), an observation
which allowed the cloning of the ligand for CD40 (7). T cells activated
with immobilized anti-CD3 antibodies, which mimic T cell receptor
engagement, are able to induce normal B cells to proliferate and secrete
Igs in the complete absence of accessory cells or lectins that might favor
cellular interactions ( 1 87, 1 88). Cytokines produced by activated T cells
are involved in the growth and differentiation of B cells, but the cell contact
cannot be replaced by T cell supernatants. In contrast, fixed activated T
cells or their membrane enriched fraction can induce B cell proliferation,
and addition of cytokines can further enhance growth and can induce Ig
ROLE OF CD40 IN T CELL-DEPENDENT

904
BANCHEREAU ET AL
secretion ( 1 88-195). The CD40-Fc fusion protein inhibits B cell stimulation

induced by activated T cells and their membranes ( 1 1 6, 1 2 1), as well as
IL-4-induced IgE production by mononuclear cells (1 96). Monoclonal
antibodies blocking T cell---dependent B cell activation against murine ( 1 1 6)
and human (1 24) activated T cells were in fact specific for CD40-L. In line
with these findings, anti-CD40 antibody strongly block both proliferation
and Ig secretion of tonsillar B cells induced by T cells stimulated with
immobilized anti-CD3 ( 1 97, 1 98). Interestingly, activation of naive sIgD+
B cells is significantly less inhibited by anti-CD40 than that of sIgD - B
cells, suggesting that the differentiation of naive B cells may also occur
independently of CD40-CD40-L interactions. Accordingly, patients with
defective CD40-L display increased circulating IgM levels (see below).
B Lymphocyte Precursors
Soluble anti-CD40 antibodies neither stimulate the proliferation of normal

B cell precursors (BCP) nor alter the effect of known growth signals for
such cells ( 1 2, 82). However, they enhance tyrosine phosphorylation and
inositol 1 ,4,5,-trisphosphate production in fetal liver pro-B cells (84).
CD I 9 + CDl O + BCP can grow in the CD40 system provided IL-3 is added
( 1 2). This proliferation is further potentiated by IL-7 and IL- IO. However,
at variance with mature B cells, IL-4 does not induce proliferation of BCP
cultured in the CD40 system. BCP can also be induced to proliferate by
triggering their CD40 with CD40-L present on activated CD4+ T cell
clones ( l 98a). The signal provided by CD40-L is essential, as is demon
strated by the blocking of T cell---dependent BCP proliferation with the
anti-CD40 mAb 89 and the lack of stimulatory effect of T cell clones
lacking functional CD40-L obtained from a hyper-IgM patient. Activation
of BCP through CD40, either in the CD40 system or in the presence of T
cells, does not promote differentiation into mature sIg+ B cells. A small
proportion of immature sIgM+ cells emerge in CD40-dependent cultures,
but cells bearing other isotypes are not observed ( 1 2). Of interest, triggering
of CD40 can induce high-level surface membrane expression of CD23 on
BCP ( 1 2). As CD23 is involved in myelopoiesis ( 1 99) and T cell ontogeny
(200), it appears of interest to evaluate the role of this molecule in B
lymphopoiesis.
Other Cell Types
Recent studies indicate that CD40 is functional on cells other than B

lineage cells. Thymic epithelial cells are induced to secrete GM-CSF when
triggered with soluble anti-CD40 in conjunction with IL- l and most
notably IL- l and IFNI', the latter upregulating CD40 expression ( 1 0). This
effect occurs in the absence of cell proliferation.

CD40 AND ITS LIGAND
905
Monocytes stimulated by CD40-L transfected cells secrete low amounts

of IL-6 and IL-8 (1 1 ) . Addition of GM-CSF, IL-3, or IFNy, which up
regulate CD40 expression on monocytes, further boosts CD40-L-induced
secretion of IL-6 and IL-8 and allows secretion of TNFcx. Cross-linking of
monocyte CD40 using CD40-L transfected cells also results in the acti
vation of tumoricidal activity against a melanoma cell line.
T lymphocytes also appear to respond to CD40-L (20 1). CD40-L, on
transfected cells or in a soluble trimeric form, induces (i) resting T cells to
express CD25/Tac and CD40-L; (ii) activated T cells to secrete IFNy,
TNFa, and IL-2, and (iii) activated T cells to proliferate. Both CD4 + and
CD8 + T cells appear to proliferate in an IL-2-independent fashion. It is
proposed that CD40-L binds to CD40 antigen which is expressed at very
low density on T cells (201 ).
ROLE OF CD40 IN ANTIGEN-DRIVEN IMMUNE

RESPONSES
Mutated CD40-Ligand in the X-Linked Hyper-IgM Syndrome
Several immunodeficiencies mapped to loci distributed throughout the

X chromosome include: X-linked agammaglobulinemia, X-linked severe
combined immunodeficiency, Wiskott-Aldrich syndrome, X-linked lym
phoproliferative syndrome, and X-linked hyper IgM syndrome (202, 203,
for a review). Males affected with hyper-IgM syndrome are susceptible to
various infections (204). These patients do not make antibodies to exogen
ous antigens but make a variety of autoantibodies. Their serum has slightly
or vastly elevated concentrations of polyclonal IgM and IgD, but no
detectable IgA or 19B and very low levels of IgG. The secondary lymphoid
organs of these patients display no germinal centers although they have
normal levels of circulating B cells and plasma cells producing IgM and
IgD in lymphoid tissues and the gastrointestinal tract. Immunization of
these patients with bacteriophage 1 74, a thymus-dependent antigen,
results in a poor humoral immune response that is restricted to the IgM
isotype (205). These features indicate a defect in the generation of memory
B cells. It is important to note that these patients often also suffer from
neutropenia.
The gene mutated in the hyper-IgM syndrome was mapped to chro
mosomal region Xq24-27 close to HPRT (206). The localization of the
CD40-L gene in region Xq26-3-Xq27- 1 ( 1 29, 1 36, 207) led to the demon
stration that the defect in the hyper-IgM syndrome is due to point
mutations or deletions in the gene encoding the CD40-L (9, 1 36, 207-2 1 0).
Activated T cells from these patients do not bind CD40 fusion proteins,
although cells from some of these patients stain with polyclonal antibody

906
BANCHEREAU ET AL
specific for the CD40-L. This suggests either expression of the truncated
CD40-L on the cell surface or expression of a conformationally altered
protein unable to bind CD40. mRNA transcripts for the CD40-L have
been sequenced in 1 3 patients, and 1 2 displayed either point mutations (9
cases) or deletions (3 cases) (Figure 7). Of note, one of the patients dis
played a CD40-L cDNA without any nucleotide change within the coding
region (207). One study on four patients showed that the CD40-L of three
of the patients' mothers had the same mutation, while a fourth patient
appeared to have had a de novo alteration (2 1 0) . Expressed mutated
CD40-L are unable to activate B lymphocytes from normal individuals
(1 36, 207), whereas the B lymphocytes from hyper-IgM patients can be
stimulated either with activated T cells (2 1 1 ) or with anti-CD40 and cyto
kines ( l 36, 207-209, 2 1 2, 2 1 3). In line with the Ig status of hyper-1gM
patients, mice develop an hyper-IgM response to the thymus-dependent
antigen DNP-Ovalbumin when CD40-CD40-L interactions are blocked in
vivo by injecting CD40-Fc chimeric molecules (D Gray, personal com
munication).
A Tentative Synthesis on Where, When, and How CD40 Is
Triggered
The key role of CD40 for T cell-dependent B cell activation in vitro was
firmly supported by the in vivo finding that mutations in CD40-L gene
result in hyper-lgM syndrome. Based on the kinetics and in vivo expression
of CD40 and CD40-L, the following model for the timing and sites of
Figure 7
Schematic representation of the coding region of CD40-Ligand and localization
of the mutations, deletions found in 12 hyper-IgM patients. Patients A.T., B.W., T.G. (209),
P I -P4 (210), P5-P7 (207) and CD., J.W. ( 1 36). IC, intracellular domain; TM, transmembrane
domain; EC, extracellular domain.
CD40 AND ITS LIGAND
90 7
CD40/CD40-L interactions during T cell-dependent immune responses is

proposed (Figure 8), based on earlier versions (21 4-2 1 8).
The entry of pathogen/antigen into the mammalian organism elicits speci
fic and nonspecific immune responses. Dendritic cells from skin or mucosa
o
mem ry
B cell

differentiation
APICAL
LIGHT ZONE
-,
lt
n into
mi gotira-primary follicle
centroblast
UGHT
ZONE
follicular d ndritic
e
cell (FOC
)
apoptosis
;o
-'-,
.o-I----s
n,
c na ex pan
somatic mutations
BASAL
-----tnaive or ,
".,
DARK ZONE
,
-
SECONDARY
LYMPHOID
QBM!'
/-0.
interdigitating
ri
I
e
II
c:
--
...
.
- -
- ,-
'- _
PARACORTICAL
.
AREA
Slillf l M!,/,!;QA
dendritic I Langerhans cell
Model for CD40/CD40-Ligand interactions in antigen dependent immune

responses (See section: A tentative synthesis on where, when and how CD40 is triggered),
Antigen, <)-<)
CD40, -0 CD40-L, shaded areas: zones of CD40-CD40-L
interactions,
Figure 8
=

908
BANCHEREAU ET AL
initiate the specific reaction by capturing antigen and migrating into the
paracortical T cell-rich areas of secondary lymphoid organs such as lymph
nodes. In these sites, these APC are called interdigitating dendritic cells
(IDC). During the antigen loading and migration phases, dendritic cells
acquire surface CD40, possibly following interaction with GM-CSF
released by various cell types (keratinocytes, neutrophils, mast cells) at the
site of antigen entry. In the T cell-rich areas, the IDCs present antigen
derived peptides bound to MHC class II antigens, to naive or memory
antigen-specific T cells initiating the extrafollicular reaction. Cross-linking
of the T cell receptor readily turns on CD40-L expression (Figure 9).
Meanwhile various sets of adhesion molecules strengthen the interactions
between T cells and the IDe. The cross-linking of CD40 on IDC by T cell
CD40-L enhances IDC cytokine production which subsequently poten
tiates T cell activation, proliferation and differentiation. In a mirror fashion
(Figure 9), the cross-linking of CD28/CTLA4 on T cells by B7 antigen on
IDC results in increased cytokine production by T cells which may then
act (i) in an autocrine fashion as T cell growth and differentiation factors,
(ii) to further activate the IDC, and (iii) to induce the proliferation and
differentiation of recruited antigen-specific B cells. It is possible that IDC
present antigen to B cells. The antigen activated B cells interact with T
cells through various surface molecules. In particular, CD40 cross-linking
boosts B cell activation and differentiation and may represent a key signal
for the migration of B cells into primary follicles composed of a network
of follicular dendritic cells (FDC) (2 19, 220). Some antigen-specific acti
vated T cells may also migrate at that time. These interactions result in
massive B cell proliferation which starts the germinal center (GC) reaction
(221 , 222). When a full germinal center is developed, proliferating centro
blasts can be identified in the dark zone. This is the anatomic site where
high-rate somatic mutations occur within the variable Ig regions (223,
224). There is no evidence, at the present time, that CD40/CD40-L inter
actions are operating at that stage. The centroblasts then mature into
nonproliferating centrocytes that compose the light zone. In the basal light
zone, the somatic mutants undergo selection based on their ability to bind
to antigen deposited on FOCs in the form of immune complexes. Antigen
receptor triggering will permit the survival of these cells while others die
from apoptosis. Subsequently, in the apical light zone, the antigen on FDC
will be processed by selected B cells and presented to antigen-specific
activated T cells that produce IL-2, IL-4, and IL- l 0 (225, 226). It is possible
that the CD40 on FDC may engage the CD40-L on activated T cells. At
that stage, C040/C040-L interactions may play a key role in (i) inducing
multiplication of the rare selected B cells, (ii) turning on the isotype switch-

CD40 AND ITS LIGAND
909
ing machinery, and (iii) inducing the differentiation of selected B cells

either into memory cells which will recirculate or into plasmablasts that
will leave the germinal centers to migrate either to the bone marrow or to
the mucosal lamina propria where they become plasma cells.
The clinical and biological status of patients suffering from the hyper
IgM syndrome permits us to conclude that the role of CD40 on APC to
boost T cell responses is either of secondary importance or can be replaced
by other molecular pairs as suggested by normal T cell numbers and
lack of viral infections. However, the susceptibility of these patients to
opportunistic agents such as Pneumocystis yet indicate a partially altered
repertoire of T cell responses. The presence of normal or elevated cir
culating IgM levels of polyclonal origin indicates that primary B cell
Figure 9 CD40/CD40-Ligand in cellular interactions. T cells recognize peptide presented

by MHC Class II on antigen presenting cells (APC) (dendritic cells or B cells). Adhesion
molecules strengthen the interaction. This results in upregulation of CD40-ligand on T cells
and B7/BBI on APC. Triggering of CD40 on APC permits activation of B cells and/or
cytokine production by B cells or dendritic cells. The produced cytokines further activate T
cells and allow their proliferation. The increased B7/BB I expression on APCfB cells triggers
CD28 or CTLA-4 on T cells which then secrete cytokines which will either further activate
the proliferation/differentiation of B cells or act as autocrine T cell growth factor. Thus,
the interaction between CD40/CD40-L signals B cells and APC, while the triggering of
CD28/CTLA-4 signals T cells to produce cytokines, which results in T cell proliferation, B
cell growth and differentiation, and APC activation.

910
BANCHEREAU ET AL
reactions are not affected by lack of CD40jCD40-L interactions either

because this interaction is not involved in this process or because other
molecules can substitute at that level. In this respect, the B cell stimulatory
effect of membrane TNF on activated T cells may play a significant role
(227, 228). The lack of germinal centers in the secondary lymphoid organs
of hyper-IgM patients suggest either an altered migration of activated T
and B cells into primary follicles or an altered proliferation of centroblasts.
The mechanisms leading to the proliferation of centroblasts still remain
unclear and may be CD40-L independent, because no CD40-L + T cells
can be detected in the GC dark zone and because B cells do not appear to
undergo somatic mutations at a high rate when cultured in the CD40
system in the presence of IL-4 (229).
PERSPECTIVES
As CD40 belongs to a family of molecules that bind several ligands as
exemplified by the two TNFRs, the LNGFR, and 4-1 BB, it is plausible
that CD40 may bind to other presently uncharacterized ligands. Indeed,
as 4- l BB binds both a TNF-like molecule ( l 39a) and extracellular matrix
proteins (56), it is possible that CD40 may also bind to such a matrix. This
would be in agreement with the high levels of binding of soluble CD40
observed on murine B cells (229a). The function of CD40 on cells other
than B cells and monocytes remains to be determined. In particular the
broad expression of CD40 on CD34 + progenitors raises the possibility
that CD40 plays an important role in hematopoiesis. CD40 activation
could represent a pathway through which CD40-L + T cells may regulate
hematopoietic production levels in the bone marrow during acute
situation. Such a regulatory function would be compatible with the fact
that hyper-IgM patients have a normal output of newly formed B cells
despite alteration of their CD40-L. However, CD40 may also participate
in constitutive hematopoiesis, as might be suggested from the frequent
neutropenia, as well as the less common anemia and thrombocytopenia
observed in hyper-IgM patients. Also, the existence of an alternative ligand
for CD40, as evoked above, should be considered within the bone marrow,
as it could point to a more central function of CD40 in constitutive
hematopoiesis. Obviously, further studies are necessary to define the role
of CD40 in hematopoietic development. In this context, evaluation of mice
in which the CD40 gene has been disrupted by homologous recombination
appears of importance.
The apparently crucial role of CD40 triggering in isotype switch will
permit us to dissect the mechanisms leading to the intracellular assembly

CD40 AND ITS LIGAND
911
of a functional isotype switch machinery. The elucidation of these intra

cellular pathways may ultimately allow the construction of antagonists
blocking isotype switch. Finally, the lack of memory cells in patients
suffering from CD40-L mutations suggests that pharmacological targeting
of the CD40/CD40-L interactions may make it possible to down-regulate
undesired humoral responses such as antibody-mediated autoimmune dis
eases. The administration of anti-CD40-L antibody to animals was
recently found to interfere with the development of primary and secondary
humoral immune responses (230). Furthermore, such antibody treatment
also prevented the arthritis induced in mice by immunization with type II
collagen (23 1). The available structural information on CD40 and CD40L will be refined in the near future and will facilitate the construction of
mutants acting as antagonists.
ACKNOWLEDGMENTS
The authors wish to thank Nicole Courbiere for her invaluable editorial
assistance and Drs. R. Armitage, R. Geha, J. Gordon, D. Gray and M .
Howard for communicating preprints before publication.
Any Annual Review chapter, as well as any article cited in an Annual Review chapter,
may be purchased from the Annual Reviews Preprints and Reprints service.
1-800-347-8007; 415-259-5017; email: arpr@class.org
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THE CD40 ANTIGEN AND ITS

Annu. Rev. Immunol. 1994.12:881-926. Downloaded from www.annualreviews.org

J. Banchereau, F. Bazan', D. Blanchard, F. Briere,

Schering-Plough, Laboratory for Immunological Research, Dardilly,

CD40, CD40-ligand, lymphocyte activation, B cell(T cell inter

actions, hyper IgM syndrome

CD40 is an integral membrane protein found on the surface of B lym

Annu. Rev. Immunol. 1994.12:881-926. Downloaded from www.annualreviews.org

Modular Structural Design and Evolutionary Relationship of

The CD40 antigen is a phosphorylated glycoprotein which migrates in

Annu. Rev. Immunol. 1994.12:881-926. Downloaded from www.annualreviews.org

CD40 AND ITS LIGAND

both reducing and nonreducing conditions. It is a hydrophobic mo lecule

Annu. Rev. Immunol. 1994.12:881-926. Downloaded from www.annualreviews.org

isolated from a human chromosome 12-specific library of heterogeneous

CD40 AND ITS LIGAND

' ' GIEI"' 'Y

Annu. Rev. Immunol. 1994.12:881-926. Downloaded from www.annualreviews.org

TNFR-like proteins appear to encode species homologs of superfamily

Annu. Rev. Immunol. 1994.12:881-926. Downloaded from www.annualreviews.org

Figure 2_ Schematic organization of receptor and cytokine superfamilies: The re

Annu. Rev. Immunol. 1994.12:881-926. Downloaded from www.annualreviews.org

Chain length (aa)

399; 396; 397 (h; r; c)

233; 235 (h;

single viral orfs

Annu. Rev. Immunol. 1994.12:881-926. Downloaded from www.annualreviews.org

CD40 AND ITS LIGAND

Annu. Rev. Immunol. 1994.12:881-926. Downloaded from www.annualreviews.org

A notable superstructural motif in the TNFRI rods is a spiralling

CD40 AND ITS LIGAND

Cells Expressing CD40

C D40 was early considered as a pan B cell antigen (68).

Annu. Rev. Immunol. 1994.12:881-926. Downloaded from www.annualreviews.org

Annu. Rev. Immunol. 1994.12:881-926. Downloaded from www.annualreviews.org

In human fetal and adult bone marrow,

Annu. Rev. Immunol. 1994.12:881-926. Downloaded from www.annualreviews.org

CD40 AND ITS LIGAND

is acquired early in B cell ontogeny, as it is present on BCP expressing the

DENDRITIC CELLS AND MONOCYTES

CD40 is expressed in the CD45-negative stromal

OTHER CELL TYPES

CD40-L in NK cells and purifie d m on ocytes (1 27). Northern blot analysis

The initial characterizati on of the m ouse CD40-L did n ot easily yield i ts

Annu. Rev. Immunol. 1994.12:881-926. Downloaded from www.annualreviews.org

Annu. Rev. Immunol. 1994.12:881-926. Downloaded from www.annualreviews.org

Figure 4. Expression of CD40 antigen on frozen sections of human spleen, tonsil

Annu. Rev. Immunol. 1994.12:881-926. Downloaded from www.annualreviews.org

Signal Transduction Through CD40

The TNFR superfamily is primarily defined by common structural motifs

Annu. Rev. Immunol. 1994.12:881-926. Downloaded from www.annualreviews.org

CD40 AND ITS LIGAND

the T cell antigen receptor complex (9S) or the hematopoietic superfamily

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Activation of protein tyrosine kinases (PTK) appears important for the

Expression of CD40-Ligand on Activated T Cells

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CD40 AND ITS LIGAND

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