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REPORTS A B Proteasome OH Catalytic threonines O RTK C OH O NH2 RTKAWNRQLYPEW RTKAWNR + AWNRQLYPEW RTK + QLYPEW RTKAWNR + QLYPEW RTK + AWNRQLYPEW RTKAWNR + Ac-QLYPEW N H AWNR C N QLYPEW COOH H Proteasomes No proteasomes O NH2 AWNR C O O C O NH2 NH2 RTK 2 QLYPEW COOH 1 0 O 0 NH2 AWNR C O NH2 RTK C 100 200 0 100 200 Digestion time (min) O OH N H QLYPEW COOH Fig. 4. Mechanism of peptide splicing. (A) Model of the peptide splicing reaction. (B) Various synthetic peptides were combined in a pairwise manner and incubated with 20S proteasomes. Digests were tested for recognition by CTL 14. Mass spectrometry confirmed the presence of RTKQLYPEW in the digests recognized by the CTL and its absence in the others. Ac-QLYPEW, N-␣-acetylated peptide QLYPEW. alytic. Rather, it is catalyzed by the proteasome and therefore takes place during protein degradation. Peptide identification efforts have provided many examples of antigenic peptides that do not simply correspond to fragments of conventional proteins, but rather result from aberrant transcription, incomplete splicing, translation of alternative or cryptic open reading frames, or posttranslational modifications (11–16). Peptide splicing is another mechanism that increases the diversity of antigenic peptides presented to T cells. It represents a new aspect of the proteasome function in antigen processing. References and Notes 1. K. Hanada, J. W. Yewdell, J. C. Yang, Nature 427, 252 (2004). 2. K. L. Rock, A. L. Goldberg, Annu. Rev. Immunol. 17, 739 (1999). CD8␣␣-Mediated Survival and Differentiation of CD8 Memory T Cell Precursors Loui T. Madakamutil,1 Urs Christen,1 Christopher J. Lena,1 Yiran Wang-Zhu,1 Antoine Attinger,1 Monisha Sundarrajan,1 Wilfried Ellmeier,2* Matthias G. von Herrath,1 Peter Jensen,3 Dan R. Littman,2 Hilde Cheroutre1† Memory T cells are long-lived antigen-experienced T cells that are generally accepted to be direct descendants of proliferating primary effector cells. However, the factors that permit selective survival of these T cells are not well established. We show that homodimeric ␣ chains of the CD8 molecule (CD8␣␣) are transiently induced on a selected subset of CD8␣⫹ T cells upon antigenic stimulation. These CD8␣␣ molecules promote the survival and differentiation of activated lymphocytes into memory CD8 T cells. Thus, memory precursors can be identified among primary effector cells and are selected for survival and differentiation by CD8␣␣. The majority of T cells responding during a primary immune response subsequently undergo programmed cell death. However, a fraction of activated T cells survive and differentiate into long-lived memory T cells (1). What mechanisms mediate the 590 selective survival of these cells? To address this question, we first must identify those effector T lymphocytes that will differentiate into memory cells. The homotypic form of CD8 that uses the ␣ chain of the molecule (CD8␣␣) ap- 3. S. Morel et al., Int. J. Cancer 83, 755 (1999). 4. G. J. Adema, A. J. de Boer, A. M. Vogel, W. A. M. Loenen, C. G. Figdor, J. Biol. Chem. 269, 20126 (1994). 5. Materials and methods are available as supporting material on Science Online. 6. Single-letter abbreviations for the amino acid residues are as follows: A, Ala; C, Cys; D, Asp; E, Glu; F, Phe; G, Gly; H, His; I, Ile; K, Lys; L, Leu; M, Met; N, Asn; P, Pro; Q, Gln; R, Arg; S, Ser; T, Thr; V, Val; W, Trp; and Y, Tyr. 7. S. Kageyama, T. J. Tsomides, Y. Sykulev, H. N. Eisen, J. Immunol. 154, 567 (1995). 8. D. J. Irvine, M. A. Purbhoo, M. Krogsgaard, M. M. Davis, Nature 419, 845 (2002). 9. M. Groll, R. Huber, Int. J. Biochem. Cell Biol. 35, 606 (2003). 10. H. Paulus, Annu. Rev. Biochem. 69, 447 (2000). 11. P. G. Coulie et al., Proc. Natl. Acad. Sci. U.S.A. 92, 7976 (1995). 12. Y. Guilloux et al., J. Exp. Med. 183, 1173 (1996). 13. R.-F. Wang, M. R. Parkhurst, Y. Kawakami, P. F. Robbins, S. A. Rosenberg, J. Exp. Med. 183, 1131 (1996). 14. J. C. A. Skipper et al., J. Exp. Med. 183, 527 (1996). 15. V. H. Engelhard, A. G. Brickner, A. L. Zarling, Mol. Immunol. 39, 127 (2002). 16. N. Shastri, S. Schwab, T. Serwold, Annu. Rev. Immunol. 20, 463 (2002). 17. We thank P. Coulie for suggestions, N. Demotte for help with peptide electroporation, S. Claverol for evaluating the purity of proteasome preparations, and E. Van Schaftingen, L. Hue, D. Godelaine, J. Berthet, and E. Warren for critical reading of the manuscript. N.V. and J.C. were supported by a Télévie fellowship from the Fonds National de la Recherche Scientifique (FNRS), Belgium. This work was supported by grants from the FNRS and the Fédération Belge contre le Cancer (Belgium). Supporting Online Material www.sciencemag.org/cgi/content/full/1095522/DC1 Materials and Methods Fig. S1 References 12 January 2004; accepted 25 February 2004 Published online 4 March 2004; 10.1126/science.1095522 Include this information when citing this paper. pears to serve functions that are distinct from those of the T cell receptor (TCR) coreceptors CD4 and CD8␣ (2–4). Immature thymocytes can induce CD8␣␣ upon strong TCR stimulation (5–7), and in mice (4, 8–11) and humans (12, 13), CD8␣␣ is expressed on distinct T cell subsets that constitutively display a memory phenotype. In light of these characteristics, we hypothesized that CD8␣␣ might have functional relevance in specifying T cell memory fate. We recently showed that the thymic leukemia antigen TL, a nonclassical major histocompatibility complex (MHC) class I molecule, is a unique ligand for CD8␣␣, with TL tetramers binding specifically to 1 La Jolla Institute for Allergy and Immunology, 10355 Science Center Drive, San Diego, CA 92121, USA. 2 Howard Hughes Medical Institute and Skirball Institute of Biomedical Science, New York University School of Medicine, New York, NY 10016, USA. 3Department of Pathology and Laboratory Medicine, Emory University School of Medicine, Atlanta, GA 30322, USA. *Present address: Institute of Immunology, University of Vienna, Brunner Strasse 59, 1235 Vienna, Austria. †To whom correspondence should be addressed. Email: [email protected] 23 APRIL 2004 VOL 304 SCIENCE www.sciencemag.org REPORTS CD8␣␣ but not to CD8␣ (4). To determine whether mature T cells induce CD8␣␣ after TCR stimulation with an antibody to CD3, we stained splenocytes with TL tetramer. Although no tetramer staining could be detected on resting splenocytes, the majority of CD8␣⫹ T cells bound TL tetramer after polyclonal stimulation, and CD8␣␣ disappeared by day 5 (Fig. 1A). CD8␣␣ induction was also detected on a subset of CD8␣⫹ OT-1 TCR transgenic splenocytes stimulated with specific peptide antigen (Fig. 1B). CD8␣␣ expression was greatest on OT-1 splenocytes stimulated by antigen-presenting cells (APCs) that had been transfected with TL (APC/TL) (Fig. 1B); this result suggested that the interaction of CD8␣␣ with TL might stabilize CD8␣␣ surface expression. Binding of classical MHC class I tetramers to DP thymocytes and activated T cells is known to be influenced by modified glycosylation on CD8␣ (14, 15). To exclude the possibility that this accounted for the observed TL tetramer staining on recently activated CD8␣ T cells, we used chimeric tetramers in which the CD8binding ␣3 domain of TL was replaced by that of H2-Kb (TL/Kb). Under conditions where CD8␣␣ was readily detected by TL tetramers, TL/Kb tetramers failed to stain activated OT-1 T cells (fig. S1). Addition- ally, TL tetramer staining of activated OT-1 cells could be blocked by antibodies to CD8␣ but not by antibodies to CD8 (fig. S1), demonstrating the specificity of the TL tetramer for CD8␣␣. Previous studies have suggested that CD8␣␣ might promote thymocyte survival (2, 7). We therefore hypothesized that CD8␣␣ might also rescue mature activated CD8␣ T cells. Consistent with this idea, CD8␣␣⫹ splenic OT-1 transgenic T cells that had been activated by APC/TL retained high levels of the antiapoptotic factors BclxL (Fig. 1C) and Bcl-2 (fig. S2) (16). The high levels of survival factors were dependent on TL expression by the APC and could be blocked using antibodies to TL (Fig. 1C). Accumulation of the antiapoptotic factors also correlated with enhanced lymphocyte survival, as measured by reduced uptake of Annexin V (Fig. 1C). Activated CD8␣␣⫹ OT-1 TCR splenocytes also expressed high levels of the shared interleukin-2 (IL-2)/IL-15 receptor  (R) chain (also called CD122), but not the IL2R␣ chain (CD25) (Fig. 1D). This finding suggested that these cells expressed the IL-15 rather than the IL-2 receptor, and this was supported by the capacity of IL-15 to expand CD8␣␣⫹ OT-1 CD8⫹ splenocytes, as compared with an inhibition in the presence of IL-2 (Fig. 1D). Other cytokines, Fig. 1. CD8␣␣ is transiently induced on activated CD8 splenocytes and provides survival. (A) C57BL/6 splenocytes cultured with antibody to CD3 were analyzed for CD8␣␣ expression using TL tetramers on activated (open) and resting (filled) CD8⫹ and CD4⫹ T cells. (B) CD8⫹ OT-1 TCR splenocytes cultured with RMAS (APC) or RMAS-TL (APC/TL) with or without OVA peptide (OVAp) for 3 days were analyzed for CD8␣␣ on gated OVA/H-2Kb tetramer⫹ cells. Data in (A) and (B) are from one of five independent experiments. (C) CD8⫹ OT-1 TCR splenocytes cultured with APC or APC/TL with or without OVAp for 72 hours. Apoptotic cells were detected with Bcl-xL and Annexin-V staining; blocking of Bcl-xL accumulation was observed in the presence of antibody 18/20 to TL (thin line). (D) After OVAp stimulation for 72 hours, CD8␣␣⫹ and CD8␣␣⫺ OT-1 TCR splenocytes were analyzed for IL-2R␣ and IL-2/IL15R. Data are from one of four individual experiments. CD8 OT-1 TCR T cells were cultured with the indicated cytokines and OVAp-loaded irradiated wild-type spleen cells; the bar graphs show numbers of CD8␣␣⫹ cells on gated V␣2⫹ T cells (left) and numbers of CD8␣␣⫹ cells and total cells (right). Data are from one of three individual experiments. (E) Left panels: Total splenocytes, stimulated as above, were gated on CD11c⫹ or CD11b⫹ including IL-7, had no effect on the expansion of the CD8␣␣⫹ T cells (Fig. 1D). Costaining of polyclonal activated T cells with TL tetramers and Annexin V further demonstrated specific survival of the CD8␣␣⫹ population (Fig. 1E). To relate this apparent survival advantage to the interaction of CD8␣␣ with its ligand, we examined TL expression during splenocyte stimulation. Although TL was not detected on resting splenocytes, its expression was observed on activated CD3⫺/ CD11b⫺/c⫹ dendritic cells and CD3⫺/ CD11b⫹/c⫺ monocytes (Fig. 1E), consistent with a potential CD8␣␣/TL-mediated survival process of the in vitro activated T cells. To examine CD8␣␣ induction on T lymphocytes in vivo, we infected mice with lymphocytic choriomeningitis virus (LCMV ) and analyzed them at various times for expression of CD8␣␣ on LCMVreactive T lymphocytes (16). LCMV peptide–MHC class I tetramers were used to identify virus-specific T cells in conjunction with TL tetramers. Induction of CD8␣␣ was observed on a subset of responding CD8␣⫹ LCMV-specific cells by day 7 after infection (Fig. 2A, day 7). A proportion of the CD8␣␣⫹ responder cells did not bind the LCMV-specific tetramers, most likely because they represented acti- TCR⫺ cells and analyzed for TL expression using antibody HD168 to TL. Cells were resting (filled), stimulated (open, thick line), or in the presence of biotinylated antibody to rat isotype (open, thin line). Right panel: Annexin-V and TL tetramer staining of splenocytes stimulated with antibody to CD3 for 72 hours. Data are from one of four independent experiments. Values in the horizontal bar represent percent positive cells for each staining. www.sciencemag.org SCIENCE VOL 304 23 APRIL 2004 591 REPORTS vated T cells specific for other LCMV epitopes (Fig. 2A, day 7). The expression of CD8␣␣ on the LCMV-specific CD8␣ T cells disappeared with virus clearance (Fig. 2A, days 14 and 40) (17). As was the case in vitro, activated LCMV-specific splenocytes that had induced surface CD8␣␣ expression also expressed high levels of Bcl-xL (Fig. 2B), consistent with a selective survival advantage for these cells. In contrast to primary LCMV stimulation (Fig. 2A, day 7), nearly all the long-term surviving LCMV-specific CD8⫹ T cells reinduced CD8␣␣ upon secondary stimulation (Fig. 2A, day 60). This is consistent with the possibility that the capacity to reinduce CD8␣␣ was selected specifically during the primary response. The LCMVspecific primary activated CD8␣␣⫹ T cells also displayed enhanced expression of IL7R␣ (also called CD127) and IL-15R (Fig. 2B), which are typically up-regulated on memory CD8 T cells (18, 19). Given the characteristics so far described for CD8␣␣⫹ activated lymphocytes, we reasoned that they might represent precursors of memory T cells. Naı̈ve CD8⫹ splenocytes isolated from transgenic mice expressing the P14 TCR (specific for the LCMV GP33-41 epitope presented by H-2Db) were transferred into nontransgenic naı̈ve recipient mice. These animals were then infected with LCMV and analyzed at various times after infection. At day 8, a subset of P14 transgenic donor cells had induced CD8␣␣, whereas the bulk of primary P14 responder cells remained CD8␣␣ negative (Fig. 3A, day 8). At day 10 after infection (at the beginning of the contraction phase of the response), the proportion of CD8␣␣⫹ P14 effector cells remained unchanged, whereas the CD8␣␣⫺ P14 T cells decreased markedly (Fig. 3A, day 10). Similar to wild-type LCMV-specific responder T cells, CD8␣␣⫹ P14 cells also expressed elevated levels of IL-7R (Fig. 3A). CD8␣␣ expression disappeared by day 15, however, whereas IL-7R remained high on the surviving P14 cells (Fig. 3A, day 15). The selective survival of CD8␣␣expressing primary effector T cells in these experiments was consistent with their possible role as memory precursors (19). We next examined the long-term survival of the CD8␣␣⫹ CD8␣⫹ T cells and their potential to mature toward memory T cells (16). P14 T cells transferred to recipient mice were isolated on day 8 after LCMV infection and sorted into CD8␣␣⫹ and CD8␣␣⫺ fractions. The two subsets were then retransferred to naı̈ve recipient animals. To control for potential migration differences between the two cell subsets, we analyzed a set of recipient mice in each case 2 days after cell transfer for the pres- 592 Fig. 2. CD8␣␣ is selectively induced on effector CD8 T cells in vivo. (A) Wild-type mice infected with LCMV were bled on the indicated days after infection (day 60 mice were rechallenged 3 days before analysis) and gated CD8⫹ cells were stained with GP276-286/Db and TL tetramers. (B) Responder cells isolated at day 7 after infection were analyzed for intracellular Bcl-xL and surface expression of IL-2/IL-15R and IL7R␣. Filled and open histograms denote CD8␣␣⫺ and CD8␣␣⫹ effector cells, respectively. Data are representative of two individual experiments in (A) and (B) with 30 mice in each experiment. Values shown in each quadrant of (A) are percentages of tetramer⫹ cells on gated CD8⫹ T cells; percentages of tetramer⫹ cells of total splenocytes are in parentheses. Fig. 3. Direct progeny of CD8␣␣⫹ effector cells survive and differentiate into memory T cells. (A) Splenocytes from GP33-41–specific TCR transgenic mice (P14; 106 cells per mouse) were transferred into C57BL/6 mice, which were infected with LCMV 1 day later. Splenocytes from these animals were analyzed on days 8, 10, and 15 after infection. After gating on cells stained for V␣2⫹/V8.1⫹, T cells were analyzed for CD8␣␣ and IL7R␣ expression. Data represent one of four mice per time point. (B) GP33tet⫹/ CD8␣␣⫹DP and GP33tet⫹SP sorted primary effector P14 cells (Ly5.2⫹) were transferred a second time into naı̈ve wild-type (Ly5.1) mice (106 cells per mouse). Secondary recipient mice were rested for 40 days and 3 days after rechallenge with LCMV (⬃105 plaque-forming units per mouse) and analyzed for the recovery and function of Ly5.2⫹ splenocytes. Upper panels: recovered Ly5.2⫹ CD8 cells from one recipient mouse in each case. Middle panels: IFN-␥ secretion of these cells stimulated with GP33-41 peptide in vitro. Bar graphs: numbers of recovered P14 cells and IFN-␥–secreting secondary P14 cells from three recipient mice. Data in (B) are from one of two independent experiments. ence of the donor cells in various tissues. Comparable numbers of both cell types were isolated from each tissue analyzed, indicating that CD8␣␣⫹ and CD8␣␣⫺ effector cells migrated similarly. Remaining recipients were rested for 40 days and analyzed after rechallenge with the virus. Substantial numbers of P14 T cells were recovered from mice that had received CD8␣␣⫹ primary P14 effector cells, but not from those that had received the CD8␣␣⫺ fraction (Fig. 3B). To test for functional differentiation of the surviving donor cells, we measured cytokine responses after in vitro stimulation with GP33-41 peptide. Most progeny of CD8␣␣⫹ P14 effector cells displayed a potent secondary response, as measured by intracellular interferon-␥ (IFN-␥) staining, whereas CD8␣␣⫺ progeny did not (Fig. 3B). As a direct test for a role of CD8␣␣ in memory formation, mice carrying an enhancer deletion within the murine cd8 locus (E8I⫺/⫺) (20) were examined. This enhancer is required for expression of CD8␣␣, and although cells from these mice express normal levels of CD8␣ (20), no CD8␣␣ could be detected after stimulation of E8I⫺/⫺ T cells in vitro (fig. S3). These observations indicated that the induction of CD8␣␣ on activated CD8␣⫹ splenocytes is controlled by the E8I enhancer. Consis- 23 APRIL 2004 VOL 304 SCIENCE www.sciencemag.org REPORTS Fig. 4. CD8␣ enhancer– deficient mice (E8I⫺/⫺) are compromised in generating memory CD8 T cells. Wild-type (WT) and E8I⫺/⫺ mice infected with LCMV were analyzed for primary, memory, and secondary response on days 7, 50, and 56 after rechallenge, respectively. (A) Gated NP396 tet⫹, GP276 tet⫹, or GP33 tet⫹ CD8 primary effector T cells analyzed for CD8␣␣. Data are from one of four mice in each group. (B) Splenocytes stained for GP33/Db tetramer on gated CD8⫹ cells were assessed for LCMV-specific primary and secondary responses. (C) Dot plots of IFN-␥ production from primary, memory, and secondary responder cells measured after in vitro restimulation with GP33-41 peptide. Values indicate percentage of IFN-␥⫹ cells in total splenocytes. Bar graphs show percentages of IFN-␥ ⫹ primary, memory, or secondary LCMV effector cells from WT or E8I⫺/⫺ mice and stimulated in vitro with GP33-41 or NP396-404 peptide. Data in (B) and (C) are from 9 to 12 mice each. (D) LCMV-specific primary effector cells gated on GP33/Db tetramer⫹ cells were stained for CD8␣␣ and IL-7R␣ expression. Data are from one of five mice. tent with this idea, LCMV-infected E8I⫺/⫺ mice generated substantial numbers of primary LCMV-specific CD8␣⫹ effector cells (Fig. 4, A and B), none of which expressed CD8␣␣ (Fig. 4A). Analysis of E8I⫺/⫺ and wild-type mice for primary responses revealed comparable amounts of IFN-␥–producing primary T cells from both mice (Fig. 4C). In contrast, the low numbers of E8I⫺/⫺ LCMV-specific T cells isolated 50 days after the initial infection, and the poor response of these cells upon restimulation in vitro, indicated a severe defect of the E8I⫺/⫺ lymphocytes in terms of survival and memory cell differentiation (Fig. 4, B and C). Additionally, IL-7Rhigh cells were also absent among primary E8I⫺/⫺ effector cells, indicating that CD8␣␣ is required for the initial upregulation of IL-7R on CD8 memory T cell precursors (Fig. 4D). Unique features of CD8␣␣ distinguish this molecule from the conventional TCR coreceptor CD8␣. CD8 facilitates translocation of the CD8 coreceptor into lipid rafts and localization of the CD8␣-associated p56lck in proximity to the TCR activation complex (21). CD8␣␣ is largely excluded from these rafts (21) and could thus sequester p56lck away from the TCR. Dual expression of CD8␣␣ with CD8␣ also appears to mediate raft disruption and an overall decrease of TCR activation signals (22). However, the effect of CD8␣␣ may not simply be inhibitory, as CD8␣␣ might redirect the p56lck toward other immune receptors outside rafts, including IL-7R and IL-15R. The impaired CD8 T cell memory observed in E8I⫺/⫺ mice provides direct evi- dence that CD8␣␣ is a key player in the initial survival of primary effector cells as well as ultimately in memory differentiation itself. The activation-induced expression of TL on APCs at the same time as CD8␣␣ on T cells suggests that CD8␣␣ is likely to mediate its function through interaction with TL. TL is not a typical peptide-binding MHC molecule (23), and the preferential interaction of TL with CD8␣␣ (4) could potentially have the effect of further reinforcing the sequestration of p56lck away from the TCR. Although at this stage the CD8␣␣dependent mechanism for memory formation described here appears to be confined to CD8␣⫹ T cells, the previous observations that under certain activation conditions CD4 T cells can induce CD8␣␣ (24, 25) leave open the possibility that a similar process might also operate in CD4 T cell memory. Human CD8␣⫹ TCR␣⫹ peripheral blood lymphocytes also express CD8␣␣, and human CD8␣␣⫹ T cells with a memory phenotype have recently been identified (13), suggesting that CD8␣␣-dependent memory formation may be preserved across species. In the design of vaccines, much attention has been focused on the magnitude of the initial immune effector cell burst in terms of establishing robust immune memory (26). Our results indicate that this is not the sole determining factor and that selective survival and differentiation of CD8␣␣⫹ precursors, leading to long-lived CD8 memory T cells, also plays an important role. References and Notes 1. J. Sprent, D. F. Tough, Science 293, 245 (2001). 2. M. J. Barnden, W. R. Heath, F. R. Carbone, Cell. Immunol. 175, 111 (1997). 3. R. Bosselut et al., Immunity 12, 409 (2000). 4. A. J. Leishman et al., Science 294, 1936 (2001). 5. U. Moebius, G. Kober, A. L. Griscelli, T. Hercend, S. C. Meuer, Eur. J. Immunol. 21, 1793 (1991). 6. N. S. van Oers et al., J. Immunol. 151, 777 (1993). 7. A. Chidgey, R. Boyd, Int. Immunol. 9, 1527 (1997). 8. B. Rocha, P. Vassalli, D. Guy-Grand, J. Exp. Med. 173, 483 (1991). 9. B. Rocha, H. von Boehmer, D. Guy-Grand, Proc. Natl. Acad. Sci. U.S.A. 89, 5336 (1992). 10. D. Cruz et al., J. Exp. Med. 188, 255 (1998). 11. A. J. Leishman et al., Immunity 16, 355 (2002). 12. L. A. Terry, J. P. DiSanto, T. N. Small, N. Flomenberg, Tissue Antigens 35, 82 (1990). 13. A. Konno et al., Blood 100, 4090 (2002). 14. A. M. Moody et al., J. Biol. Chem. 278, 7240 (2003). 15. M. A. Daniels et al., Immunity 15, 1051 (2001). 16. See supporting data on Science Online. 17. K. Murali-Krishna et al., Science 286, 1377 (1999). 18. K. S. Schluns, L. Lefrancois, Nature Rev. Immunol. 3, 269 (2003). 19. S. M. Kaech et al., Nature Immunol. 4, 1191 (2003). 20. W. Ellmeier, M. J. Sunshine, K. Losos, D. R. Littman, Immunity 9, 485 (1998). 21. A. Arcaro et al., J. Immunol. 165, 2068 (2000). 22. A. G. Cawthon, H. Lu, M. A. Alexander-Miller, J. Immunol. 167, 2577 (2001). 23. Y. Liu et al., Immunity 18, 205 (2003). 24. X. Paliard, R. W. Malefijt, J. E. de Vries, H. Spits, Nature 335, 642 (1988). 25. R. Aranda et al., J. Immunol. 158, 3464 (1997). 26. S. M. Kaech, E. J. Wherry, R. Ahmed, Nature Rev. Immunol. 2, 251 (2002). 27. We thank M. Cheroutre and O. Turovskaya for their contributions; M. Kronenberg, J. Braun, E. Reinherz and E. Sercarz for helpful discussions; and C. Miceli, J. Altman, R. Ahmed, N. Shastri, and W. Heath for constructs, reagents, cell lines, and mice. Supported by NIH grants DK54451 and AI50263 (H.C., L.T.M.), AI33614 (P.J.), and AI51973 (M.G.V.) and by Juvenile Diabetes Research Foundation grant 3-2000-510 (U.C.). This is manuscript number 512 of the La Jolla Institute for Allergy and Immunology. Supporting Online Material www.sciencemag.org/cgi/content/full/304/5670/590/DC1 Materials and Methods Figs. S1 to S3 References 7 October 2003; accepted 18 February 2004 www.sciencemag.org SCIENCE VOL 304 23 APRIL 2004 593