Differential CD3z Phosphorylation Is Not Required for the Induction of T Cell Antagonism by Altered Peptide Ligands1 Haiyan Liu*† and Dario A. A. Vignali2*‡ T cells recognize foreign Ags in the form of short peptides bound to MHC molecules. Ligation of the TCR:CD3 complex gives rise to the generation of two tyrosine-phosphorylated forms of the CD3 z-chain, pp21 and pp23. Replacement of residues in MHCbound peptides that alter its recognition by the TCR can generate altered peptide ligands (APL) that antagonize T cell responses to the original agonist peptide, leading to altered T cell function and anergy. This biological process has been linked to differential CD3z phosphorylation and generation of only the pp21 phospho-species. Here, we show that T cells expressing CD3z mutants, which cannot be phosphorylated, exhibit a 5-fold reduction in IL-2 production and a 30-fold reduction in sensitivity following stimulation with an agonist peptide. However, these T cells are still strongly antagonized by APL. These data demonstrate that: 1) the threshold required for an APL to block a response is much lower than for an agonist peptide to induce a response, 2) CD3z is required for full agonist but not antagonist responses, and 3) differential CD3z phosphorylation is not a prerequisite for T cell antagonism. The Journal of Immunology, 1999, 163: 599 – 602. he TCR:CD3 complex consists of an Ag-specific ab heterodimer and six associated CD3 chains (eg, ed, and zz), which are required for correct assembly and transport to the cell surface (1, 2). Signal transduction through this complex is mediated by immunoreceptor tyrosine-based activation motifs (ITAM)3 [(D/E)X2YX2(L/I)X7YX2(L/I)], which are phosphorylated upon TCR ligation and recruit SH2-containing proteins (3– 5). While some studies have shown that all three CD3 z-chain ITAMs are required for maximal T cell function, others suggest that signal transduction proceeds normally in their absence (6 – 8). Although T cell development and function is normal in CD3z2/2 mice reconstituted with a CD3z mutant lacking ITAMs, the selection of T cells expressing certain TCR transgenes is altered and autoreactive T cells have been identified (9 –12). One of the first demonstrable biochemical events following TCR ligation by MHC:peptide complexes is the tyrosine phosphorylation of the TCR-associated CD3z homodimer (13). This gives rise to the recruitment of ZAP-70, a tyrosine kinase that has been shown to be a key component of the signaling cascade (14 – 16). Differential phosphorylation of the ITAMs on CD3z leads to the generation of two distinct m.w. species by SDS-PAGE, a lower pp21 band and an upper pp23 band; the latter is only seen following full T cell activation (17–19). Recent studies have suggested that the pp21 form contains two or three monophosphorylated ITAMs, while pp23 has all the ITAM tyrosines phosphorylated (20). The former observation is surprising given that ZAP-70 is
T
*Department of Immunology, St. Jude Children’s Research Hospital, Memphis, TN 38105; and †Graduate Program in Pathology and ‡Department of Pathology, University of Tennessee Medical Center, Memphis, TN 38163 Received for publication January 4, 1999. Accepted for publication April 26, 1999. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1
This work was supported by the National Institutes of Health Grant AI-39480, Cancer Center Support CORE Grant 5 P30 CA21765-17, and the American Lebanese Syrian Associated Charities (ALSAC). 2
Address correspondence and reprint request to Dr. Dario Vignali, Department of Immunology, St. Jude Children’s Research Hospital, 332 N. Lauderdale, Memphis, TN 38105-2794. E-mail address:
[email protected] 3 Abbreveations used in the paper: ITAM, immunoreceptor tyrosine-based activation motif; APL, altered peptide ligand; HEL, hen egg lysozyme.
Copyright © 1999 by The American Association of Immunologists
constitutively associated with pp21 and that ZAP-70 will only bind to doubly phosphorylated ITAMs (21–23). Presentation of altered peptide ligands (APL) has been shown to induce only the lower pp21 CD3z phospho-species, essentially no phosphorylated CD3e, and weak association of unphosphorylated ZAP-70 that lacks stable kinase activity (17–19, 24 –28). A similar phenotype has also been seen with T cells stimulated with nonmitogenic anti-CD3 mAbs (29). Thus, a correlation has been drawn between differential CD3z phosphorylation and lack of active ZAP-70, and the induction of T cell antagonism, anergy, and altered T cell function. It has recently been suggested that the lack of IL-2 production, rather than an altered pattern of TCR-mediated phosphorylation, is the crucial factor controlling anergy induction (24). While an altered pattern of CD3e and CD3z phosphorylation was observed in T cells anergized by exposure to APL, this was not observed when anergy was induced by a lack of costimulation. However, it is not clear whether the biochemical events that lead to reduced IL-2 production are the same in the two systems. In addition, several groups have observed a correlation between differential CD3z phosphorylation and antagonism induced by APL in murine T cell hybridomas, despite their lack of autocrine dependency on IL-2 (28, 30). Thus, the relative importance of differential CD3z phosphorylation remains unclear, and there has been no direct molecular examination of this issue. Resolution of this question is important given current interest in the use of APL as a strategy for treating autoimmune disease. In this study, we have utilized CD3z-loss variants of the hen egg lysozyme (HEL) 48-62-specific, H-2Ak-restricted murine T cell hybridoma 3A9 to determine whether differential CD3z phosphorylation is a prerequisite for, or a consequence of, T cell antagonism.
Materials and Methods
Hybridomas and CD3z mutants CD3z-loss variants of 3A9 were cloned by using FACS of cells stained with Abs against CD3 and CD4 (PharMingen, San Diego, CA), as previously described (17, 31). CD3z mutants were made by recombinant PCR, using a murine CD3z cDNA as a template (gift from Larry Samelson, National Institutes of Health, Bethesda, MD), as previously described (32, 33). Details of the oligos used are available on request. Mutants were 0022-1767/99/$02.00
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ROLE OF CD3z IN T CELL ANTAGONISM
verified by sequencing and cloned into one of two eukaryotic expression vectors, pCIneo (Promega, Madison, WI) or pHbApr-1neo (33). CD3z loss variants were transfected by electroporation, selected with G418, and either cloned or bulk sorted by FACS (32, 33).
Ag presentation and antagonism assays Ag presentation assays were performed essentially as described elsewhere (32, 34). Briefly, T cell hybridomas were stimulated with synthetic peptides (Center for Biotechnology CORE facility at St. Jude Children’s Research Hospital, Memphis, TN) at the concentrations indicated, together with LK35.2 as APC (murine B cell lymphoma, H-2Akd). After 24 h, supernatants were removed for the estimation of IL-2 secretion against a recombinant murine IL-2 standard (Genzyme, Cambridge, MA) by culturing with the IL-2-dependent T cell line CTLL-2. Antagonism assays were set up in the same way, except that the APC were first prepulsed with the agonist for 6 h, washed three times, pulsed with the antagonist peptide for 2 h, and then T cell hybridomas added (28).
CD3z tyrosine phosphorylation analysis. CD3z tyrosine phosphorylation analysis was determined as previously described (17). Briefly, LK35.2 cells pulsed with 3 mM peptide were mixed with T cell hybridomas and incubated at 37°C for 5 min. The cell pellet was lysed in 1% Brij 97 (polyoxyethylene 10 oleyl ether; Sigma, St. Louis, MO) at room temperature for 1 h, and immunoprecipitated with a rabbit anti-CD3z antisera (2 ml, No. 551; gift from David Weist, Fox Chase Cancer Center, Philadelphia, PA) for 2 h at room temperature followed by incubation with 25 ml protein A-Sepharose (Pharmacia, Piscataway, NJ) for 1 h at room temperature (unlike other commonly used detergents, Brij97 precipitates at 4°C and so has to be used at room temperature). Eluted proteins were resolved by SDS-PAGE and transferred onto polyvinylidene difluoride membrane (Schleicher & Schuell, Keene, NH). Blots were blocked with 5% BSA (Boehringer Mannheim, Indianapolis, IN) in TBST, and tyrosine phosphorylation was detected with biotinylated 4G10 (0.1 mg/ml; Upstate Biotechnology, Lake Placid, NY) (90 min at room temperature), followed by 1:12,000 dilution of streptavidin-HRP (Amersham, Arlington Heights, IL) (60 min at room temperature). Blots were developed using ECLplus (Amersham). To detect the original protein, blots were stripped in 100 mM 2-ME (Bio-Rad, Hercules, CA), 2% SDS, 62.5 mM Tris-HCl (pH 6.7) for 30 min at 50°C, washed three times and blocked with 5% nonfat dry milk in TBST at 4°C overnight. Blots were probed with an anti-CD3z mAb, H146 (1:4 for 60 min at room temperature; gift from Ralph Kubo, Cytel, San Diego, CA), followed by protein A-HRP (1:12,000; Amersham) (60 min at room temperature). Blots were developed as described above.
Results
Significant contribution of the CD3z ITAMs in IL-2 production Two CD3z-loss variants of 3A9 (3A9z-.4 and 3A9z-.7) were transfected with either wild-type CD3z (CD3z.WT), a CD3z mutant lacking functional ITAM motifs (CD3z.DITAM), or a truncated form of CD3z lacking most of the cytoplasmic domain (CD3z.DCY) (Fig. 1A). All three molecules restored TCR:CD3 expression to levels comparable to the parental TCR 1 3A9 T cell hybridoma (Fig. 1B, and data not shown). Western blot analysis demonstrated that the amount of CD3z.WT, CD3z.DCY, and CD3z.DITAM expressed by the transfectants was similar (Fig. 1C). Furthermore, no parental wild-type CD3z could be detected in the CD3z.DCY transfectants or the loss mutants. The CD3z.WT T cell transfectants were phenotypically and functionally indistinguishable from the original 3A9 T cell hybridoma in all assays performed in this study (data not shown). The CD3z.DCY and CD3z.DITAM T cell transfectants, however, produced 5 times less IL-2 than the CD3z.WT control, and required 30 times more peptide to induce IL-2 production (Fig. 2A). Thus, the loss of CD3z ITAMs reduces T cell sensitivity and strength of response. Some clones of CD3z.WT.3A9z-.4 were isolated that produced lower levels of IL-2 comparable to the T cells expressing mutant CD3z (Fig. 2A). These served as useful controls for subsequent experiments. As expected, stimulation of the CD3z.WT, but not the CD3z.DCY and CD3z.DITAM T cell transfectants with HEL 48 –
FIGURE 1. Characteristics of T cell hybridomas expressing mutant and wild-type CD3z. A, Schematic depicting the CD3z molecules expressed by the T cell hybridomas. The extracellular domain is on the left, and the transmembrane region is in black. In CD3z.DITAM, the tyrosine (Y) residues in the ITAMs have been substituted with phenylalanine (F). In CD3z.DCY, residues 46 –129 have been deleted. CY, cytoplasmic tail. B, Flow cytometric analysis of TCR expression by the 3A9 CD3z loss mutants and their transfectants, as indicated by surface staining with an antiCD3e Ab. C, Western blot analysis of T cell hybridomas expressing mutant and wild-type CD3z. The untransfected CD3z loss mutants are included as controls.
63-pulsed B cells induced the tyrosine phosphorylation of CD3z (Fig. 2B). These data confirmed that the mutant CD3z molecules could not be phosphorylated and that these T cell transfectants had not re-expressed wild-type CD3z. T cell antagonism in the absence of the CD3z cytoplasmic domain Using T cells from 3A9.TCR transgenic mice, we have shown that HEL 48-Q57A-63 is a strong peptide antagonist, HEL 48-L56A-63 is a weak peptide antagonist, and HEL 48-L56A-61AA is a null peptide (R. T. Carson and D. A. A. Vignali, unpublished observations). These substitutions do not affect peptide affinity (17, 35, 36). Stimulation of the CD3z.WT T cell transfectants with HEL 48-63 induced the tyrosine phosphorylation of CD3z and generation of both the pp21 and pp23 forms (Fig. 3A). The strong antagonist, HEL 48-Q57A-63, induced only the lower pp21 phosphorylated form of CD3z. This was consistent with previous studies that had demonstrated a correlation between differential CD3z phosphorylation and the induction of anergy (18, 19). However, no CD3z phosphorylation above basal levels was observed with HEL 48-L56A-63 and 48-L56A-61AA in the CD3z.WT transfectants. The ability of these three APL to antagonize T cell responses to the agonist, HEL 48-63, was assessed. A 30-fold molar excess of the HEL 48-Q57A-63 APL over agonist resulted in a substantial reduction in IL-2 secretion by the CD3z.WT T cell transfectants (3A9z-.7, 97%; 3A9z-.4, 86%: Fig. 3B). Both HEL 48-L56A-63 and HEL 48-L56A-61AA manifested reduced levels of antagonism. Three conclusions can be drawn from these data. 1) The
The Journal of Immunology
FIGURE 2. T cell function in cells expressing mutant CD3z. A, IL-2 production by T cell hybridomas following stimulation with HEL 48-63 presented by LK35.2 B cells. IL-2 concentration was determined using CTLL-2 cells. Data are representative of three experiments. B, Only wildtype CD3z is tyrosine phosphorylated following T cell stimulation. T cell hybridomas transfected with wild-type and mutant CD3z, and the CD3z2 control, were incubated with or without HEL 48 – 63 and the degree of CD3z phosphorylation determined. 3A9z-.4 transfectants gave identical results.
ability of APL to modulate responses of 3A9 T cell hybridomas and T cells from 3A9.TCR transgenic mice was comparable. 2) A low level of T cell antagonism could be obtained with only basal levels of CD3z phosphorylation. 3) The ability of HEL 48Q57A-63 and 48-L56A-63 to antagonize CD3z.WT transfectants of 3A9z-.7 and 3A9z-.4 was comparable, suggesting that differences in sensitivity and IL-2 production in response to the agonist has little effect on the ability of T cells to be antagonized by APL. To our surprise, the CD3z.DCY and CD3z.DITAM T cell transfectants were also antagonized with HEL 48-Q57A-63 and 48L56A-63 to a level comparable to the CD3z.WT T cell transfectant (Fig. 3B). Furthermore, antagonism was observed despite the low IL-2 production by, and reduced sensitivity of, T cell transfectants expressing mutant CD3z in response to the agonist HEL 46-63. This phenotype was comparable to the 3A9z-.4 CD3z.WT transfectant, which was also less sensitive to the agonist (Fig. 2A). The ability of HEL 48-Q57A-63 and 48-L56A-63 to antagonize T cell responses was consistent and highly reproducible among all the clones and bulk transfectants tested. Although it is not clear why results with the HEL 48-L56A-61AA peptide were variable, the results do show that mutation of the CD3 z-chain has no effect on the ability of T cells to be antagonized.
Discussion Our data highlight two important observations. First, differential phosphorylation of CD3z is not a prerequisite for the induction of T cell antagonism. Indeed the complete loss of signal transduction through CD3z has no effect, implying that the critical biochemical events that underlie T cell antagonism can be efficiently mediated via CD3e, CD3g, and/or CD3d. It remains to be determined whether T cell antagonism could occur in a TCR:CD3 complex in which CD3z was the only component possessing an intact cytoplasmic tail. Second, despite a significant reduction in sensitivity
601
FIGURE 3. The absence of a functional CD3 z-chain has no effect on the ability of T cells to be antagonized. A, Differential CD3z phosphorylation in response to peptide antagonists. 3A9z-.7 CD3z.WT T cells were stimulated with LK35.2 cells pulsed with 3 mM agonist (48-63) and APL (48-Q57A-63, 48-L56A-63, 48-L56A-61AA) as indicated, and tyrosine phosphorylation of CD3z determined. Blots were stripped and reprobed with anti-CD3z to demonstrate comparable loading. 3A9z-.4 CD3z. WT transfectants gave comparable results. B, The ability of three APL to antagonize the response of T cell hybridomas expressing mutant and wildtype CD3z to the agonist, HEL 48-63, was tested. Percentages on the righthand side represent the reduction in IL-2 release induced by the agonist following addition of 10 mM of the APL indicated. Data are representative of nine experiments.
to Ag and quantity of IL-2 released, T cells expressing TCRs that have lost 6 of 10 ITAMs can still be potently antagonized. This suggests that the threshold required for an antagonist to block a response is much lower than for an agonist to induce a response. This is synonymous with the observation that the absence of CD4 has a significant effect on the response of a T cell hybridoma to agonist peptides, but has little effect on the ability of APL to induce T cell antagonism (37). Although exogenous IL-2 has been shown to break anergy, the biochemical basis for the lack of IL-2 production by antagonized and anergic T cells remains unresolved. It has been argued that the lack of IL-2 production is the primary factor controlling anergy induction, rather than an altered pattern of TCR-mediated phosphorylation (24). This conclusion was based on the observation that anergy induced by a lack of costimulation failed to give the altered pattern of CD3e and CD3z phosphorylation that was observed in T cells anergized by exposure to APL. However, in the former, there was still a clear difference in the amount of ZAP-70 phosphorylation, and this could have given rise to the same biochemical phenotype as in the APL-stimulated T cells (24) (Fig. 2C). Furthermore, no indication was provided for the relative amount of ZAP-70 kinase activity in the two systems. In addition, we and others have demonstrated strong T cell antagonism in T
602 cell hybridomas despite their lack of autocrine dependency on IL-2 (28, 30, and the present study). Several groups have shown a correlation between the induction of T cell antagonism and anergy and a novel CD3z and CD3e tyrosine phosphorylation pattern (18, 19, 24 –28). This leads to a transient association of unphosphorylated ZAP-70, which lacks stable kinase activity. This has lead to the suggestion that differential CD3z phosphorylation may be a key biochemical event in the induction of T cell anergy (19, 20). Our data suggest that while this may be a phenotype of peptide antagonism (or anergy), it may not be a prerequisite. Indeed, the loss of a functional CD3z cytoplasmic tail clearly has a significant effect on IL-2 production but no effect on the ability of APL to induce antagonism. Recent studies have suggested that successive, ordered CD3z phosphorylation occurs following TCR ligation of MHC:peptide complexes (20). The authors suggest that this process sets thresholds that determine whether interaction with a TCR ligand is sufficient to result in T cell activation. APL give rise to incomplete ITAM phosphorylation, thus allowing for the recruitment of single SH2 domain-containing signaling molecules, but not ZAP-70. Given that the T cells used in our study can be potently antagonized in the complete absence of CD3z phosphorylation, it is probable that such multistep CD3z phosphorylation plays no role in the induction of T cell antagonism. While we cannot rule out the possibility that this could be mediated by phosphorylation of only one tyrosine in CD3e, CD3g, and/or CD3d, it is important to point out that such differential phosphorylation has not been directly demonstrated for these CD3 molecules. Although the loss of CD3e phosphorylation has also been shown as a hallmark of APL-induced TCR signaling (18, 24, 25), it is unclear if this represents partial or no phosphorylation. This could be determined using phosphopeptide-specific Abs against each of the CD3e, CD3g, and CD3d tyrosine motifs, as was recently used to analyze CD3z phosphorylation (20). While it is likely that the controlled genetic manipulation of all the CD3 components will be required to elucidate the biochemical events that lead to T cell antagonism, it is clear that the differential phosphorylation of CD3z is not a prerequisite.
Acknowledgments We thank Kate Vignali for assistance with phosphorylation analysis, David Wiest for the anti-CD3z antisera 551, Ralph Kubo for the H146 anti-CD3z mAb, and Larry Samelson for the CD3z cDNA. We also thank Paula Arnold, Dharmesh Desai, David Woodland, and Creg Workman for their critical review of the manuscript.
References 1. Samelson, L. E., J. B. Harford, and R. D. Klausner. 1985. Identification of the components of the murine T cell antigen receptor complex. Cell 43:223. 2. Weissman, A.M., S. J. Frank, D. G. Orloff, M. Mercep, J. D. Ashwell, and R. D. Klausner. 1989. Role of the z chain in the expression of the T cell antigen receptor: genetic reconstitution studies. EMBO J. 8:3651. 3. Reth, M. 1987. Antigen receptor tail clue. Nature 38:383. 4. Wange, R. L., and L. E. Samelson. 1996. Complex complexes: signaling at the TCR. Immunity 5:197. 5. Shaw, A. S., and M. L. Dustin. 1997. Making the T cell receptor go the distance: a topological view of T cell activation. Immunity 6:361. 6. Frank, S. J., C. Cenciarelli, B. B. Niklinska, F. Letourneur, J. D. Ashwell, and A. M. Weissman. 1992. Mutagenesis of T cell antigen receptor z chain tyrosine residues: effects on tyrosine phosphorylation and lymphokine production. J. Biol. Chem. 267:13656. 7. Irving, B. A., A. C. Chan, and A. Weiss. 1993. Functional characterization of a signal transducing motif present in the T cell antigen receptor z chain. J. Exp. Med. 177:1093. 8. van Oers, N. S., P. E. Love, E. W. Shores, and A. Weiss. 1998. Regulation of TCR signal transduction in murine thymocytes by multiple TCR z-chain signaling motifs. J. Immunol. 160:163. 9. Shores, E. W., K. Huang, T. Tran, E. Lee, A. Grinberg, and P. E. Love. 1994. Role of TCR z chain in T cell development and selection. Science 266:1047.
ROLE OF CD3z IN T CELL ANTAGONISM 10. Shores, E. W., T. Tran, A. Grinberg, C. L. Sommers, H. Shen, and P. E. Love. 1997. Role of the multiple T cell receptor (TCR)-z chain signaling motifs in selection of the T cell repertoire. J. Exp. Med. 185:893. 11. Lin, S. Y., L. Ardouin, A. Gillet, M. Malissen, and B. Malissen. 1997. The single positive T cells found in CD3-zeta/eta2/2 mice overtly react with self-major histocompatibility complex molecules upon restoration of normal surface density of T cell receptor-CD3 complex. J. Exp. Med. 185:707. 12. Yamazaki, T., H. Arase, S. Ono, H. Ohno, H. Watanabe, and T. Saito. 1997. A shift from negative to positive selection of autoreactive T cells by the reduced level of TCR signal in TCR-transgenic CD3 z-deficient mice. J. Immunol. 158: 1634. 13. Samelson, L. E., M. D. Patel, A. M. Weissman, J. B. Harford, and R. D. Klausner. 1986. Antigen activation of murine T cells induces tyrosine phosphorylation of a polypeptide associated with the T cell antigen receptor. Cell 46:1083. 14. Chan, A. C., M. Iwashima, C. W. Turck, and A. Weiss. 1992. ZAP-70: a 70 kd protein-tyrosine kinase that associates with the TCR z chain. Cell 71:649. 15. Negishi, I., N. Motoyama, K. Nakayama, S. Senju, S. Hatakeyama, Q. Zhang, A. C. Chan, and D. Y. Loh. 1995. Essential role for ZAP-70 in both positive and negative selection of thymocytes. Nature 376:435. 16. Cheng, A. M., I. Negishi, S. J. Anderson, A. C. Chan, J. Bolen, D. Y. Loh, and T. Pawson. 1997. The Syk and ZAP-70 SH2-containing tyrosine kinases are implicated in pre-T cell receptor signaling. Proc. Natl. Acad. Sci. USA 94:9797. 17. Vignali, D. A. A., and J. L. Strominger. 1994. Amino acid residues that flank core peptide epitopes and the extracellular domains of CD4 modulate differential signaling through the T cell receptor. J. Exp. Med. 179:1945. 18. Madrenas, J., R. L. Wange, J. L. Wang, N. Isakov, L. E. Samelson, and R. N. Germain. 1995. z phosphorylation without ZAP-70 activation induced by TCR antagonists or partial agonists. Science 267:515. 19. Sloan-Lancaster, J., A. S. Shaw, J. B. Rothbard, and P. M. Allen. 1994. Partial T cell signaling: altered phospho-z and lack of ZAP-70 recruitment in APL-induced T cell anergy. Cell 79:913. 20. Neumeister Kersh, E., A. S. Shaw, and P. M. Allen. 1998. Fidelity of T cell activation through multistep T cell receptor z phosphorylation. Science 281:572. 21. Wiest, D. L., J. M. Ashe, R. Abe, J. B. Bolen, and A. Singer. 1996. TCR activation of ZAP70 is impaired in CD41 CD81 thymocytes as a consequence of intrathymic interactions that diminish available p56lck. Immunity 4:495. 22. van Oers, N. S., N. Killeen, and A. Weiss. 1994. ZAP-70 is constitutively associated with tyrosine-phosphorylated TCR zeta in murine thymocytes and lymph node T cells. Immunity 1:675. 23. Isakov, N., R. L. Wange, J. D. Watts, R. Aebersold, and L. E. Samelson. 1996. Purification and characterization of human ZAP-70 protein-tyrosine kinase from a baculovirus expression system. J. Biol. Chem. 271:15753. 24. Madrenas, J., R. H. Schwartz, and R. N. Germain. 1996. Interleukin 2 production, not the pattern of early T-cell antigen receptor-dependent tyrosine phosphorylation, controls anergy induction by both agonists and partial agonists. Proc. Natl. Acad. Sci. USA 93:9736. 25. Reis, E. H. Levine, and R. N. Germain. 1996. Partial signaling by CD81 T cells in response to antagonist ligands. J. Exp. Med. 184:149. 26. La Face, D. M., C. Couture, K. Anderson, G. Shih, J. Alexander, A. Sette, T. Mustelin, A. Altman, and H. M. Grey. 1997. Differential T cell signaling induced by antagonist peptide-MHC complexes and the associated phenotypic responses. J. Immunol. 158:2057. 27. Hemmer, B., I. Stefanova, M. Vergelli, R. N. Germain, and R. Martin. 1998. Relationships among TCR ligand potency, thresholds for effector function elicitation, and the quality of early signaling events in human T cells. J. Immunol. 160:5807. 28. Carson, R. T., D. D. Desai, K. M. Vignali, and D. A. A. Vignali. 1999. Immunoregulation of T helper cells by naturally processed peptide antagonists. J. Immunol. 162:1. 29. Smith, J. A., J. T. Tso, M. R. Clark, M. S. Cole, and J. A. Bluestone. 1997. Nonmitogenic anti-CD3 monoclonal antibodies deliver a partial T cell receptor signal and induce clonal anergy. J. Exp. Med. 185:1413. 30. Evavold, B. D., J. Sloan-Lancaster, and P. M. Allen. 1994. Antagonism of superantigen-stimulated helper T-cell clones and hybridomas by altered peptide ligand. Proc. Natl. Acad. Sci. USA 91:2300. 31. Vignali, D. A. A., J. Moreno, D. Schiller, and G. J. Hammerling. 1992. Does CD4 help to maintain the fidelity of T cell receptor specificity? Int. Immunol. 4:621. 32. Vignali, D. A. A., R. T. Carson, B. Chang, R. S. Mittler, and J. L. Strominger. 1996. The two membrane proximal domains of CD4 interact with the T cell receptor. J. Exp. Med. 183:2097. 33. Vignali, D. A. A., and K. M. Vignali. 1999. Profound enhancement of T cell activation mediated by the interaction between the T cell receptor and the D3 domain of CD4. J. Immunol. In press. 34. Carson, R. T., K. M. Vignali, D. L. Woodland, and D. A. A. Vignali. 1997. T cell receptor recognition of MHC class II-bound peptide flanking residues enhances immunogenicity and results in altered TCR V region usage. Immunity 7:387. 35. Allen, P. M., G. R. Matsueda, R. J. Evans, J. B. Dunbar, G. R. Marshall, and E. R. Unanue. 1987. Identification of the T-cell and Ia contact residues of a T-cell antigenic epitope. Nature 327:713. 36. Nelson, C. A., N. J. Viner, S. P. Young, S. J. Petzold, and E. R. Unanue. 1996. A negatively charged anchor residue promotes high affinity binding to the MHC class II molecule I-Ak. J. Immunol. 157:755. 37. Hampl, J., Y. H. Chien, and M. M. Davis. 1997. CD4 augments the response of a T cell to agonist but not to antagonist ligands. Immunity 7:379.
