The FASEB Journal express article 10.1096/fj.99-0909fje. Published online July 24, 2000.

A chemical basis for recognition of nonpeptide antigens by human T cells*

Christian Belmant1,5, Eric Espinosa1, Franck Halary2, Yong Tang3, Marie-Alix Peyrat2, Hlne Sicard 1, Alan Kozikowski4, Roland Buelow4, Rmy Poupot1, Marc Bonneville2 and Jean-Jacques Fourni1
1 2

INSERM U395, Toulouse, France INSERM U463, Institut de Biologie, Nantes, France 3 Georgetown University, Washington, D.C., USA 4 Sang Stat Medical Corporation, Menlo Park, California, USA 5 Innate Pharma, Marseille Luminy, France *A study dedicated to the memory of Claude de Prval. Correspondence: Jean-Jacques Fourni, INSERM U395, CHU Purpan, BP3028, 31024 Toulouse, France. E-mail: fournie@purpan.inserm.fr Christian Belmant and Eric Espinosaoth contributed equally to this work. ABSTRACT Whereas T cell receptors (TCR) recognize processed antigenic peptides or glycolipids bound respectively to major histocompatibility complex or CD1 molecules, TCR react differently to a broad set of native antigens. A major human T cell subset is activated through a mechanism involving V 9V 2 TCR and structurally unrelated phosphorylated nonpeptide antigens (referred to as phosphoantigens). Here, the structure-function relationship of the strongest natural and synthetic phosphoantigens stimulating cells was analyzed to elucidate the molecular basis of this unconventional recognition. Besides conformational determinants, we found that chemical reactivity of antigens is critical to their bioactivity. For V 9V 2 T cell activation, both organic and phosphorylated moieties of strong ligands undergo rapid and degradative structural changes. Conversely, analogs that are resistant to degradation specifically antagonize phosphoantigen-mediated T cell activation. These data suggest a novel mode of antigen perception involving both topological recognition and ligand consumption, which confers highly specific T cell activation by structurally diverse ligands. Key Words: antigen antagonist lymphocyte gamma/delta

ntigen recognition mediated by or T cell antigen receptors (TCR) initiates specific immune responses. Whereas TCR react with antigenic peptides associated with major histocompatibility complex (MHC) molecules or glycolipids associated with CD1 molecules, TCR recognize antigens differently (1). Most T cells in human blood, which express V 9V TCR, are stimulated by diverse small non-peptide phosphoesters referred to as phosphoantigens (2-6), as well as by phosphorylated and nonphosphorylated alkylamines (7, 8). Phosphoantigens are widely distributed in nature; they were initially found and isolated in minute amounts from numerous pathogens e.g., Mycobacterium tuberculosis, the agent of human tuberculosis (3), in most mycobacteria (9, 10), or in Plasmodium falciparum, responsible for malaria. Several non-natural (synthetic) phosphoesters are also known to selectively activate V 9V 2 T cells (4, 6, 12). This highly selective but cross-reactive recognition process is mediated by the TCR (8, 13-16), requires a cell-to-cell contact, but does not involve any known MHC or MHC-like molecule (16-18). To define the molecular basis of this unusual recognition pattern, we investigated the molecular determinants contributing to the V 9V 2 T cell stimulating activity of natural and synthetic compounds. MATERIALS AND METHODS Chemical synthesis The specified pyrophosphates and analogs were synthesized according to refs 19-22). Structures were confirmed by ion trap ESI-MS n and 1H-NMR as described (19). Each product was finally purified (95-99%) by anion exchange as sodium salt in water, quantified by conductometry detection from high pH anion exchange chromatography (HPAEC), with an accuracy of 4% of each measure and detection sensitivity threshold 1 pmol (28). Cell cultures Peripheral blood lymphocytes, B lymphoblastoid cells, polyclonal V 9/V 2 T lymphocytes and CTL clones were obtained (29) and cultured in complete RPMI with 10% human serum. 3-formyl-1-butyl diphosphate was isolated from Mycobacterium tuberculosis (19), natural IPP was from Sigma, and human rIL-2 was kindly provided by Sanofi (Labge, France). Amplification of polyclonal V 9/V 2 T lymphocytes from PBL was analyzed in 10D-cultures in the presence of IL-2 (50 U/ml) (3). Where indicated, complete culture medium was supplemented with 2 mM of Levamisole, inorganic phosphate (Pi), inorganic pyrophosphate (Ppi), phenylalanine or PHA from Sigma. In vitro activation and inhibition assays Stimulation assays involved amplification of polyclonal V 9/V 2 lymphocytes among total PBL (3), cytolysis by a V 9/V 2 CTL clone and -TNF release by a V 9/V 2

clone (16). Titrations were repeated three to five times in different laboratories, giving similar values in the different readouts. EC50 are the lowest concentrations stimulating 50% of maximal response; IC50: lowest concentrations inhibiting 50% of maximal response to high pH anion exchange chromatography (BrHPP; 80 nM). [mt]106 nM: no response was obtained up to this concentration. Microphysiometric assays Extracellular acidification rates were monitored by Cytosensor (Molecular Devices, Crawley, UK) using polyclonal V 9/V 2 T only in the chambers (23), with 8 105 cells loaded per well, a flow (100 l/min) of low-buffered RPMI medium alone (control lanes) or same medium containing the specified molecules (arrow). The baseline acid release rates were 60-100 Vsec-1, and a data collection rate of 90 sec. Phosphoantigen samples were diluted at the indicated concentrations in low-buffered RPMI medium and adjusted at pH 7.3 before injection into chambers. HPAEC analysis of phosphoantigen degradation Cell-mediated phosphoantigen degradation was measured in Eppendorf tubes containing cells washed twice in phosphate-free buffer; prior to assay, 13 nanomoles of the specified phosphoantigens (BrHPP in Fig. 2) were incubated 1 h (37C) in 0.1 ml phosphate-free buffer with or without 2.5 10 6 polyclonal V 9/V 2 T cells. Each culture supernatant was collected and analyzed by HPAEC (28) for quantification of the introduced compound (asterisk), using an internal reference (Br, IS). The interpreted HPAEC chromatograms are shown in Fig. 2A. For BrHPP analogs differing by the pyrophosphate bond, quantification of resistance to degradation indicated in Table 2A and expressed as % was obtained by the ratio: (peak area of unaltered compound remaining in culture with cells)/(peak area of unaltered compound remaining in culture without cells). Titration of bioactivity loss in cell cultures 30 nM of BrHPP was introduced in 96-well plates containing complete medium and 2.106 cell of the indicated type, with the specified durations of incubations prior to further bioassay of the cell culture supernatant. For experiments presented in Fig. 2B, the BrHPP bioactivity remaining in these supernatants was titrated by induction of the autocytotoxicity of a V 9/V 2 T cell clone. The result (expressed as %) was obtained by the ratio: (bioactivity of the cell culture supernatant)/(bioactivity of the introduced 30 nM of BrHPP). For experiments presented in Fig. 2C, cultures were left 1 h at 37C with various cell numbers and IPP or BrHPP concentrations, and the bioactivity remaining in their respective supernatants were titrated by the -TNF release from V 9V 2 T cells. RESULTS

The paradox of non-peptide ligand discrimination by -cells The two natural antigens with the strongest T cell stimulating activity are the ubiquitous isoprenod isopentenyl diphosphate [IPP (10)] and a mycobacterial aldehyde: 3-formyl-1-butyl diphosphate (19). These two acyclic C5 esters of pyrophosphate differ solely by their C3 substituent (=CH2 for the prenylic antigen vs. -CHO for the mycobacterial ligand), and have 103-fold different bioactivities on a molar basis (Table 1A). As demonstrated by inactivity of their reduced analogs (Table 1A), these C3 substituents are critical for stimulating T cells. When compared with their respective inactive analog, each of these bioactive ligand presents little topological difference brought by respectively, one and two additional hydrogen atoms. This suggests that the reactive V 9V 2 TCR is very discriminating, which is in striking paradox with its previously documented broad reactivity to structurally heterogeneous synthetic and natural compounds (4, 6, 8, 10). -cell stimulating bioactivities of natural and synthetic compounds correlate to their chemical reactivities Due to the limited availability of purified natural phosphoantigens, we performed a structure to function study on related synthetic molecules (19-22) in order to elucidate the determinants contributing to phosphoantigen bioactivity. We then compared V 9V 2 T lymphocyte responses such as proliferation, specific lysis and early activation signals (23) (Fig. 1A,B) to synthetic analogs differing from the natural phosphoantigens by the C3 substituent exclusively (Table 1B). The reasonably active carbonyl (C=O) and diol (C(OH)-CH2 OH) analogs indicated that the nature of the C3 substituent, but not unsaturation itself, is critical for bioactivity (Table 1B). This was in agreement with the [mt]50-fold higher bioactivities of yet different C3-derivatives, epoxide (-CH2-O-) and chlorohydrin (-CH2 Cl). It is interesting that all these bioactive structures and the natural ligands share polarized determinants. To assess the importance of this factor, less polarized halohydrins---e.g., (-CH2 Br) and (CH2 I)---were synthesized and their activity tested. Actually, bromohydrin and iodohydrin were respectively 10-fold and 100-fold more bioactive than chlorohydrin for activating cell responses (Table 1 and Fig. 1A). So instead of permanent polarization, polarizability of the C3 substituent matches to bioactivity. Indeed, the phosphohalohydrin Cl [lt] Br [lt] I hierarchy of bioactivity correlated with their chemical reactivity on Sn 2type nucleophilic substitution. Along this line, all natural phosphoantigens and synthetic phosphoesters or alkylamines (7, 8) with cell-stimulating bioactivities are chemically reactive compounds, via either substitution, addition or elimination (Table 1B). Furthermore, chemical reduction of the aldehyde or prenyl groups of strong natural ligands abrogated both their chemical reactivity and bioactivity (Table 1A). Similarly, the chemically reactive pamidronate and ibandronate amino bisphosphonates stimulate human cells unlike chemically unreactive clodronate and etidronate analogs (7).

The influence of topological determinants Some alkyl phosphates devoid of chemically reactive C3 substituent are weak agonists for human cells (e.g., ethyl-phosphate EC50: 30 M) (4). The partial structural homology on P-O-C1-C2 of such suboptimal ligands with the above strong agonists suggested that this restricted conformational fit may provide low-range bioactivities. This was tested by titrating the bioactivities of shorter chain homologues of the strongly bioactive compounds, whose topology differs at C2. None of these shorter chain compounds had bioactivity, despite the presence of the respective chemically reactive halohydrin groups (Table 1B). The same conclusion also applied to isomers of the highly bioactive structures bearing the C-methyl substitution on carbon 2 instead of carbon 3 (data not shown). Therefore, the presence per se of a chemically reactive group does not confer a strong bioactivity to a putative phosphoantigen, but only potentiates the topologically fit ligands. Hydrolysis of the pyrophosphate moiety is associated with potentiation of the stimulating bioactivity Some natural phosphoantigens are nucleotidic conjugates (3, 24), and may spontaneously cleave into 3-formyl-1-butyl-PP moieties (19), with a far higher bioactivity than the corresponding triphosphates, diphosphodiesters, monophosphates or unphosphorylated residues (data not shown). These results give to the unsubstituted diphosphate tail a major influence on the ligand ability to stimulate cells, reflecting either topological constraints or requirement for pyrophosphate hydrolysis. To test if pyrophosphate hydrolysis occurred on incubating phosphoantigens with T cells, we controlled the supernatants of cultures containing 13 nmoles of BrHPP with or without added cells. In the presence of V 9V 2 T cells, partial hydrolysis of the BrHPP pyrophosphate and simultaneous release of stoichiometric amounts of Pi were detected (Fig. 2A). This degradation also occurred with other cell types, and increased with time (Fig. 2B). This progressive degradation enhanced with cell numbers (Fig. 2C), and resulted in irreversible loss of phosphoantigen bioactivity, which was evidenced with compounds acting at nanomolar, but not at micromolar, ranges (Fig. 2C). Furthermore, T lymphocytes no longer responded to phosphoantigens when incubated with dephosphorylation-inhibiting drugs (Levamisole or PPi, Fig. 2C). Dephosphorylation-resistant analogs inhibit the T cell-stimulating bioactivity of phosphoantigens Together, the above data strongly suggested that phosphoantigen degradation is mandatory for efficient T cell activation. The influence of dephosphorylation on antigen bioactivity could be tested using phosphoantigens containing a pyrophosphate analog that resists to enzymatic hydrolysis. Since imidodiphosphate (P-NH-P), methylene diphosphonate (P-CH2-P) and difluoromethylenediphosphonate (P-CF2-P) are less hydrolyzed pyrophosphate isosteres (25-27), we measured both stability and bioactivity

of corresponding derivatives of compounds acting at nanomolar (i.e., BrHPP) and micromolar (i.e., IPP) ranges. We found (Table 2A) that the compounds most sensitive to cell-mediated degradation were the strongest cell agonists whereas the most resistant ones were not agonists. Conversely, the latter behaved as antagonists: they inhibited the activity of stimulating phosphoantigens when added simultaneously to T cells (Table 2A). Here again, introducing a chemically reactive function on the nondegradable methylene diphosphonates raised their inhibitory potency (Table 2B). As indicated by competition between increased IPP concentrations and non-hydrolyzable analogs, the stronger the stimulus, the higher the amounts of competitor for antagonizing the stimulus (Fig. 3). Therefore, pyrophosphate hydrolysis critically contributes to phosphoantigen bioactivity for T cells. Phosphoantigen degradation controls early steps of T cell activation On specific recognition of antigens by the TCR, T lymphocytes trigger early activation signals such as increased metabolic rate, which results in faster acidification of the extracellular milieu. Microphysiometric analysis by Cytosensor of T cell early activation evidenced a dose-dependent and rapid onset of signaling after exposure to phosphoantigens. Increased extracellular acidification occurred straight-away (in less than 9s., data not shown) after cell exposure to soluble phosphoantigen agonists (Fig. 1B), but not in response to the non-hydrolyzable analogs (even with up to 103 higher doses than for agonists, Fig. 4A left panel). A reduced acidification response was obtained when both compounds were added together (Fig. 4A, right panel), indicating an immediate competition between the activating phosphoantigen and its unstimulatory analog. This competition was reversible as demonstrated by reciprocal titration experiments using grading doses of either the agonist or the antagonist (Fig. 4B). The specificity of this inhibition was tested by comparing the effect of non-hydrolyzable analogs on mitogen vs. phosphoantigen-mediated activation of a V 9V 2 T cell clone. The non-hydrolyzable phosphoantigen analog only affected response to the phosphoantigen and not to the lectin mitogen (Fig. 4C). DISCUSSION The present work was undertaken to clarify several paradoxical features of nonpeptide ligand recognition by human cells. First, nonpeptide ligand discrimination by V 9V 2 T cells is surprisingly fine given their concomitant broad reactivity to structurally heterogeneous compounds (2, 6, 8, 10). Second, although recognition of such nonpeptide ligands by T cells is mediated by the V 9V 2 TCR (10, 14, 15), it occurs along a highly transient cell contact via an interaction independent of any MHC presenting molecule (16-18). The present study identified two critical factors of phosphoantigen bioactivity: the ligand susceptibility to chemical reaction and to pyrophosphate hydrolysis. Such findings may explain the paradoxical ligand discriminating properties of cells, as it is anticipated that through chemical reactions, structurally heterogeneous compounds could generate related intermediates sharing topological features recognized

by the TCR. Ligands fulfilling minimal topological requirements illustrate the critical contribution to bioactivity of chemical but not topological features located in critical positions (e.g., C3 substituent, Table 1B). The chemically reactive groups of antigens may undergo changes driven by Sn2-type substitution, nucleophilic additions or eliminations with various substituents at the cell surface, yielding either cell surfacebound or soluble but altered phosphoantigens. Cell cultures progressively release partially dephosphorylated phosphoantigens in supernatants (Fig. 2A), but this is not proper to the reactive lymphocytes. As highly reactive ligands such as BrHPP present a higher level of dephosphorylation than less reactive homologues (e.g., IPP, Table 2A), the chemically reactive group might favor pyrophosphate hydrolysis, thereby positively influencing phosphoantigen bioactivity. Although this dephosphorylation process occurs on cells of diverse origin including the reactive cells, it is nevertheless mandatory to their activation by phosphoantigens (Fig. 2C). Indeed, phosphoantigen degradation by lymphocytes had not been observed before, but most likely because it could only be evidenced with highly bioactive (nanomolar) ligands that so far had not been available. Based on BrHPP data (~70% of 13 nanomoles per hour correspond to 3.6 nanomole per million cell per hour, see Fig. 2A and Table 2A), the cell-mediated degradation rates may be estimated of 600,000 molecules per cell sec-1, which is undetectable by bioassays with micromolar bioactive ligands. Accordingly, analogs of stimulating ligands that did not permit pyrophosphate hydrolysis remained unaltered in cell culture supernatants, and antagonized activation by phosphoantigens following classical mass action laws. Furthermore, their selectivity and reversibility imply a true competitive antagonism between hydrolyzable and nonhydrolyzable compounds, without generation of a broadly inhibitory signal to the effector cells. So, chemically reactive moieties and hydrolyzable pyrophosphate greatly enhance (by up to 104-fold) the bioactivities of topologically suited ligands. Along the same line, milli-to-micromolar concentrations of the chemically reactive pamidronate and ibandronate amino bisphosphonates were recently described as stimulatory for human cells while their chemically unreactive homologues clodronate and etidronate were not (7). Degradative recognition of antigens most likely accounts for the great structural heterogeneity of human V 9V 2 T cells stimuli: the best ligands are known to be dephosphorylation-prone compounds (3, 4, 6, 8, 10, 24). Further, this rapid and irreversible degradation of the stimulus explains the need for a cell-to-cell contact to activate T cells by phosphoantigens, but also the lack of target sensitization by a phosphoantigen pulse (16, 18). At the border of innate and adaptive immunity, this recognition allows T cell activation by various stimuli (12) while focusing the responses to the very site of antigen production. Beyond this, given the broad spectrum of physiopathological contexts where V 9V 2 T cells have been involved, these new molecules available in gram amounts and acting in the 108-1010 M may prove valuable therapeutic tools for in vivo evaluation of T cell responses in various infectious and tumor pathologies.

ACKNOWLEDGMENTS We thank F. Pont (IFR 30, Toulouse, France) for mass spectrometry facility and Sanofi (Labge, France) for generous gifts of recombinant human IL-2. This study was supported by institutional grants from INSERM, program APEX, lAssociation pour la Recherche sur le Cancer, la Rgion Midi-Pyrnes, La Ligue Nationale pour la Recherche Contre le Cancer, Sang Stat. Med. Corp., and Innate Pharma. REFERENCES 1. Chien, Y. H., Jores, R., and Crowley, M. P. (1996) Recognition by gamma/delta T cells. Annu. Rev. Immunol. 14, 511-532 2. Fourni, J. J., and Bonneville, M. (1996) Stimulation of T cells by phosphoantigens. Res. Immunol. 147, 338-346 3. Constant, P., Davodeau, F., Peyrat, M. A., Poquet, Y., Puzo, G., Bonneville, M., and Fournie, J. J. (1994) Stimulation of human gamma delta T cells by nonpeptidic mycobacterial ligands. Science 264, 267-270 4. Tanaka, Y., Sano, S., Nieves, E., De Libero, G., Rosa, D., Modlin, R. L., Brenner, M. B., Bloom, B. R., and Morita, C. T. (1994) Nonpeptide ligands for human gamma delta T cells. Proc. Natl. Acad. Sci. USA 91, 8175-8179 5. Schoel, B., Sprenger, S., and Kaufmann, S. H. (1994) Phosphate is essential for stimulation of V gamma 9V delta 2 T lymphocytes by mycobacterial low molecular weight ligand. Eur. J. Immunol. 24, 1886-1892 6. Burk, M. R., Mori, L., and De Libero, G. (1995) Human V gamma 9-V delta 2 cells are stimulated in a cross-reactive fashion by a variety of phosphorylated metabolites. Eur. J. Immunol. 25, 2052-2058 7. Kunzmann, V., Bauer, E., and Wilhelm, M. (1999) (gamma)/(delta) T-cell stimulation by pamidronate. N. Engl. J. Med. 340, 737-738 8. Bukowski, J. F., Morita, C. T., and Brenner, M. B. (1999) Human T cells recognize alkylamines derived from microbes, edible plants and tea: implication for innate immunity. Immunity 11, 57-65 9. Constant, P., Poquet, Y., Peyrat, M. A., Davodeau, F., Bonneville, M., and Fournie, J. J. (1995) The antituberculous Mycobacterium bovis BCG vaccine is an attenuated mycobacterial producer of phosphorylated nonpeptidic antigens for human gamma delta T cells. Infect. Immun. 63, 4628-4633

10. Tanaka, Y., Morita, C. T., Nieves, E., Brenner, M. B., and Bloom, B. R. (1995) Natural and synthetic non-peptide antigens recognized by human gamma delta T cells. Nature (London) 375, 155-158 11. Behr, C., Poupot, R., Peyrat, M. A., Poquet, Y., Constant, P., Dubois, P., Bonneville, M., and Fournie, J. J. (1996) Plasmodium falciparum stimuli for human gamma-delta T cells are related to phosphorylated antigens of mycobacteria. Infect. Immun. 64, 28922896 12. De Libero, G. (1997) Sentinel function of broadly reactive human T cells. Immunol. Today 18, 22-26 13. Davodeau, F., Peyrat, M. A., Hallet, M. M., Houde, I., Vie, H., and Bonneville, M. (1993) Peripheral selection of antigen receptor junctional features in a major human gamma delta subset. Eur. J. Immunol. 23, 804-808 14. Bukowski, J. F., Morita, C. T., Tanaka, Y., Bloom, B. R., Brenner, M. B., and Band, H. (1995) V gamma 2V delta 2 TCR-dependent recognition of non-peptide antigens and Daudi cells analyzed by TCR gene transfer. J. Immunol. 154, 998-1006 15. Bukowski, J. F., Morita, C. T., Band, H., and Brenner, M. B. (1998) Crucial role of TCR gamma chain junctional region in prenyl pyrophosphate antigen recognition by gamma delta T cells. J. Immunol. 161, 286-293 16. Lang, F., Peyrat, M. A., Constant, P., Davodeau, F., David-Ameline, J., Poquet, Y., Vie, H., Fournie, J. J., and Bonneville, M. (1995) Early activation of human V gamma 9V delta 2 T cell broad cytotoxicity and TNF production by nonpeptidic mycobacterial ligands. J. Immunol. 154, 5986-5994 17. Holoshitz, J., Romzek, N. C., Jia, Y., Wagner, L., Vila, L. M., Chen, S. J., Wilson, J. M., and Karp, D. R. (1993) MHC-independent presentation of mycobacteria to human gamma delta T cells. Int. Immunol. 5, 1437-1443 18. Morita, C. T., Beckman, E. M., Bukowski, J. F., Tanaka, Y., Band, H., Bloom, B. R., Golan, D. E., and Brenner, M. B. (1995) Direct presentation of nonpeptide prenyl pyrophosphate antigens to human gamma delta T cells. Immunity 3, 495-507 19. Belmant, C., Espinosa, E., Poupot, R., Peyrat, M. A., Guiraud, M., Poquet, Y., M., B., and Fourni, J. J. (1999) 3-formyl-1-butyl diphosphate, a novel mycobacterial metabolite stimulating human T cells. J. Biol. Chem. 274, 32079-32084 20. Belmant, C., Bonneville, M., Fourni, J. J., and Peyrat, M. A. (1998) In nouveaux composs phosphohalohydrines, procd de fabrication et applications (INSERM patent PCT/FR 99/02058/INPI, Paris, France)

21. Belmant, C., Bonneville, M., Fourni, J. J., and Peyrat, M. A. (1998) In nouveaux composs phosphopoxydes, procd de fabrication et applications (INSERM patent PCT/FR 99/02057/INPI, Paris, France) 22. Belmant, C., Bonneville, M., Fourni, J. J., Kozikowski, A., and Peyrat, M. A. (1999) In Composs inhibant slectivement les lymphocytes T 92 et leurs applications (INSERM patent 99.04263 INPI, Paris, France) 23. Rabinowitz, J. D., Beeson, C., Wlfing, K., Tate, C. K., Allen, P. M., Davis, M. M., and McConnell, H. M. (1996) Altered T cell receptor ligands trigger a subset of early T cell signals. Immunity 125-135 24. Poquet, Y., Constant, P., Halary, F., Peyrat, M. A., Gilleron, M., Davodeau, F., Bonneville, M., and Fourni, J. J. (1996) A novel nucleotide-containing antigen for human blood gamma delta T lymphocytes. Eur. J. Immunol. 26, 2344-2349 25. Yount, R. G., Babcock, D., Ballantyne, W., and Ojala, D. (1971) Adenylyl imidophosphate, an adenosine triphosphate analog containing a P-N-P linkage. Biochemistry 10, 2484-2489 26. Yount, R. G., Babcock, D., and Ojala, D. (1971) Interaction of P-N-P and P-C-P analogs of adenosine triphosphate with heavy meromyosin, myosin, and actomyosin. Biochemistry 10, 2490-2496 27. Chambers, R. D., OHagan, D., Brian Lamont, R., and Jain, S. C. (1990) The difluoromethylenephosphonate moiety as a phosphate mimic: X-ray structure of 2-amino1,1-difluoroethylphosphonic acid. J. Chem. Soc. Chem. Commun. 1053-1054 28. Poquet, Y., Constant, P., Peyrat, M. A., Poupot, R., Halary, F., Bonneville, M., and Fourni, J. J. (1996) High-pH anion-exchange chromatographic analysis of phosphorylated compounds: application to isolation and characterization of nonpeptide mycobacterial antigens. Anal. Biochem. 243, 119-126 29. Halary, F., Peyrat, M. A., Champagne, E., Lopez-Botet, M., Moretta, A., Moretta, L., Vi, H., Fourni, J. J., and Bonneville, M. (1997) Control of self-reactive cytotoxic T lymphocytes expressing gamma delta T cell receptors by natural killer inhibitory receptors. Eur. J. Immunol. 27, 2812-2821 FIGURE LEGENDS Figure 1. In vitro titration of V9V2 T cell-stimulating bioactivity by phosphoantigens A: Left) Amplification of V 9/V 2 T cells among PBL cultured 8 days in complete medium with IL-2 containing the specified concentrations (M) of iodohydrin (filled

circles). BrHPP (empty circles) or IPP (triangles); right) % of cytolytic response of a V 9/V 2 T cell clone on B-LCL targets cultured with iodohydrin(filled circles):BrHPP (empty circles) or IPP (triangles). B: Cytosensor microphysiometric detection of early response of a V9V 2 CTL clone to the specified concentrations of BrHPP (underlined: BrHPP in culture medium). Figure 2. Phosphoantigen degradation in cell culture A: Comparative HPAEC analysis of 13 nmoles of BrHPP in culture supernatants (<zref>28<zrefx>). IS: internal standard for quantification; asterisk indicates the BrHPP peak., Pi: inorganic phosphate. B: % of bioactivity remaining in supernatants from 30 nM BrHPP introduced in cell cultures (2.106 cell per well) with the specified conditions. C: T cell numbers causing the biologically measured level of degradation. Bioactivity (V 9V 2 T cell-mediated -TNF release) in supernatant of primary cultures (1 h, 37C) that contained BrHPP (left panel) or IPP (middle panel) concentrations and various cell numbers: no cell per well (black diamonds), 2.106 cell per well (empty squares), 5.106 cell per well (empty triangles) and 107 cell per well (empty circles). D: Response (-TNF release) of polyclonal V 9V 2 T cells stimulated in culture media with or without BrHPP (25 nM) and the following drugs (2 mM each): Leva, levamisole; Ppi, inorganic pyrophosphate; Phe, phenylalanine; Pi, inorganic phosphate. Figure 3. Antagonism of phosphoantigen activation by methylene diphosphonate analogs Relative response of V 9V 2 T cells (-TNF secretion) to phosphoantigens in the presence of methylene diphosphonates. Results are expressed as percent of the maximal response in cultures with BrHPP alone. They were obtained by dividing the -TNF secretion induced by BrHPP and the methylene diphosphonates of iodohydrin (IHPCP, filled triangles), bromohydrin (BrHPCP, empty triangles) or isopentenyl (IPCP, filled squares) by the -TNF secretion of the V 9V 2 T cell clone in culture with BrHPP alone (106 cells/well). Empty dots: background -TNF secretion by V 9V 2 T cells in medium alone (0% of response corresponding to 0-5 pg TNF/ml), filled dots: maximal TNF secretion by V 9V 2 T cells in culture medium with BrHPP alone (100% response corresponding to 50-400 pg TNF/ml). Figure 4. Phosphoantigen antagonism by bromohydrin methylene diphosphonate (BrHPCP) A: Comparative cytosensor response of 106 polyclonal V 9V 2 T cells exposed (left panel, arrow) to 10 nM iodohydrinpyrophosphate (filled circles), or to BrHPCP alone at 0 M (empty triangles),10 M (empty squares), 100 M (empty circles); right panel: cytosensor response of 106 polyclonal V 9V 2 T cells exposed to 10 M IPP alone (filled circles), 0 M (empty circle) or 10 M (empty squares) of BrHPCP, and 10 M IPP+ 10 M BrHPCP (empty triangles). B: Reversible responses (specific target lysis in

upper panels, -TNF release in lower panels) of a V 9V 2 CTL clone in complete culture medium containing various BrHPCP concentrations (left panels) with (black circles) or without 9 nM BrHPP (empty circles) or reciprocally, in complete culture medium containing various BrHPP concentrations (right panels) with (black circles) or without 60 M BrHPCP (empty circles). C: BrHPCP (70 M, black bars) specifically antagonizes phosphoantigen stimulation but not mitogenic lectin-dependent P815 target cell lysis by a V 9V 2 CTL clone (white bars, stimulus alone).

Table 1:
) natural phosphoantigens and their chemically reduced analogues:
HO HO O P O O

-O

R
CH 3

R:
CH 2 C CHO

(nM)

R:
CH 3

(nM)

P O

natural isopentenyl -pyrophosphate:

10 000

CH CH2OH

>106

mycobacterial 3-formyl-1-butyl-pyrophosphate:

CH

10

CH

>106

) synthetic phosphoantigens :
-O
R:

HO P O O

HO P O O

R
CH 3

HO

HO O P O O

-O

P O

R
CH 3

Chem.reac. type:

EC50 , (nM) :

EC50 , (nM) :

OH C O C HO C O C HO C HO C HO C CH2 I CH2 Br CH2 Cl CH2 CH2 OH

weak An E-1,E-2,Sn-2 Sn-2 ++ Sn-2 + Sn-2 ++ Sn-2 +++

>106 50 000 5 000 20 100 10 1

nt nt nt >106 >106 >106 >106

Table 2:
HO HO

-O

R2
O CH 3

P O

R1 R1 :

P O

R2:
HO

bioactivity for T cells Resistance to degradation by T cells antagonist agonist (%) IC 50 (M) EC 50 (nM) 28 39 10 3 000 12 000 -a -a 15 700 150 1000a

CH 2 Br C CH 2 C

-O -

HO

-NH(Imidodiphosphates)

CH 2 Br C CH 2 C

61 75
CH 2 Br

HO

-CH2 (Diphosphonates)
HO

C CH 2 C CH 2 Br C CH 2 C

92 92 >99 >99

-CF2 (Difluorodiphosphonates)

B
-O

HO P O CH 2

HO P O O

R
CH3

R:
HO C CH 2 C O C HO C HO C CH 2 I CH 2 CH 3

IC 50 (M) 1000 700 30

CH 2 Br

15 10

a: unspecific inhibitory activity above the specified concentration -: none