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J. Biol. Chem., Vol. 281, Issue 38, 28090-28096, September 22, 2006

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CD8 Has Two Distinct Binding Modes of Interaction with Peptide-Major Histocompatibility Complex Class I^*

Hsiu-Ching Chang¹, Kemin Tan, and Yen-Ming Hsu

From the Department of Medical Oncology, Dana-Farber Cancer Institute and Department of Medicine, Harvard Medical School, Boston, Massachusetts 02115 and Molecular Discovery Department, Biogen-Idec, Inc., Cambridge, Massachusetts 02142

Received for publication, May 23, 2006 , and in revised form, July 10, 2006.

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Interaction of CD8 (CD8 or CD8) with the peptide-major histocompatibility complex (MHC) class I (pMHCI) is critical for the development and function of cytolytic T cells. Although the crystal structure of CD8·pMHCI complex revealed that two symmetric CD8 subunits interact with pMHCI asymmetrically, with one subunit engaged in more extensive interaction than the other, the details of the interaction between the CD8 heterodimer and pMHCI remained unknown. The Ig-like domains of mouse CD8 and CD8 are similar in the size, shape, and surface electrostatic potential of their pMHCI-binding regions, suggesting that their interactions with pMHCI could be very similar. Indeed, we found that the CD8 variants CD8^R8A and CD8^E27A, which were functionally inactive as homodimers, could form an active co-receptor with wild-type (WT) CD8 as a CD8^R8A or CD8^E27A heterodimer. We also identified CD8 variants that could form active receptors with WT CD8 but not with CD8^R8A. This observation is consistent with the notion that the CD8 subunit may replace either CD8 subunit in CD8·pMHCI complex. In addition, we showed that both anti-CD8 and anti-CD8 antibodies were unable to completely block the co-receptor activity of WT CD8. We propose that CD8 binds to pMHCI in at least two distinguishable orientations.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
CD8 is a co-receptor that enhances the presentation of peptide antigen complexed with MHC² class I molecule (pMHCI) to the T-cell receptor (1–3). Cell-surface CD8 is assembled as either CD8 homodimers or CD8 heterodimers (4–6). Although CD8 and CD8 are structurally similar (7), they differ in tissue distribution, ligand specificity, and efficiency of antigen presentation (8, 9). Although CD8 is expressed primarily on the surface of TCR⁺ thymocytes and peripheral T cells, CD8 has a much broader expression pattern, including the TCR⁺ and TCR⁺ intestinal intraepithelial lymphocytes, natural killer cells, and dendritic cells (9–11). CD8 has been shown to be a more efficient co-receptor than CD8 for presentation of a given antigen (12–14). However, the underlying mechanism for the enhanced efficiency is not well understood.

Both the extracellular domain and the cytoplasmic tail of the CD8 subunit have been implicated in providing increased efficiency of CD8 (12–16). We reported previously that the extracellular domain of the CD8 subunit is critical for this enhanced efficiency (14) and that introduction of the CD8 stalk region is sufficient to confer a CD8-like co-receptor efficiency to the CD8 homodimer (17). In addition, the sialylation of the O-linked glycans in the CD8 stalk region is differentiation stage-dependent and may modulate the intrinsic activity of CD8 during the transition from double-positive (CD4⁺CD8⁺) to single-positive (CD8⁺) T cells (18, 19). It was also reported that palmitoylation of a membrane-proximal cysteine residue in the cytoplasmic tail of CD8 during T-cell activation facilitates partition of CD8 heterodimers into lipid rafts, where it associates with the CD3 component of TCR complexes (20). Lastly, the kinase activity of p56^lck, which is associated only with the cytoplasmic domain of CD8, can be enhanced by CD8 (21). It is conceivable that the extracellular domain and the cytoplasmic tail of CD8 may independently contribute to the enhanced co-receptor activity of CD8.

The interaction of CD8 and pMHCI has been elucidated through x-ray analyses of the crystal structures of human CD8·HLA-A2 and mouse CD8·H2-K^b (22, 23). This structural information confirms the results of many mutational studies. The complementarity-determining region-like loops of CD8 and the MHCI 3 domain CD loop (residues 220–228) are the most critical for CD8·pMHCI interaction (24–26). In both human and mouse CD8·pMHCI complexes, two symmetric CD8 subunits clamp asymmetrically onto the relatively rigid MHCI 3 domain CD loop. One subunit, CD81, is involved in more than 70% of the total interaction surface between CD8 and the MHCI 3 domain and ₂-microglobulin (₂M). The second subunit, CD82, is less engaged and interacts only with the MHCI 3 domain (22, 23). To simplify reference to these interaction sites, we designated the site occupied by the CD81 subunit of CD8·pMHCI as site 1 and the site occupied by the CD82 subunit as site 2.

Unlike CD8·pMHCI, the structure of CD8·pMHCI is not known. Two different binding models have been proposed for the interaction of pMHCI with mouse and human CD8 (22, 23). Considering that the stalk region of CD8 is longer than that of CD8 (44 versus 35 amino acids), which allows the Ig-like domain of CD8 to extend farther from the T-cell surface, we proposed a binding mode for mouse CD8·pMHCI interaction in which CD8 and CD8 occupy site 1 and site 2, respectively (23). Consistent with this model is our observation that co-expression of WT CD8 with the CD8^R8A variant resulted in co-receptor activity indistinguishable from that of WT CD8 (17). Because mouse CD8^R8A lacks the side chain of Arg⁸ critical for engaging in extensive interaction with pMHCI (site 1 interaction), the functional CD8^R8A heterodimer must bind to pMHCI with its subunit occupying site 1 and its subunit (CD8^R8A) occupying site 2. On the other hand, based on electrostatic interactions between modeled human CD8 and pMHCI, Gao et al. (22) proposed that in the human CD8·pMHCI complex, the CD8 subunit occupies site 1, and the CD8 subunit occupies site 2. Interestingly, like mouse CD8^R8A, human CD8^R4K and CD8^L25A are not able to form functional homodimeric co-receptor (34). Yet, unlike the mouse CD8^R8A variant, neither of these human CD8 variants is able to form functional heterodimeric co-receptor with WT CD8 (34). This observation led to the conclusion that in the human CD8·pMHCI complex, the CD8 subunit occupies only site 1 (34). Thus, the interactions between CD8 and pMHCI in mouse and human systems appear to be different. The molecular basis for this difference remains unclear.

To further understand the interaction between CD8 and pMHCI and possibly resolve the apparently conflicting observations in human and mouse systems, we asked, in the context of CD8·pMHCI complex, whether mouse CD8 could also occupy site 2. Among a panel of CD8 variants that were unable to occupy site 1, we identified two CD8 variants that were capable of forming functional co-receptor with WT CD8. Therefore, the mouse CD8 subunit can also occupy site 2 in the CD8·pMHCI complex. In addition, we showed that an anti-CD8 antibody only blocked activity of co-receptors in which CD8 was occupying site 1 but not site 2. These results support the notion that CD8 interacts with pMHCI in at least two distinguishable binding orientations.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
mAbs and Flow Cytometry—All of the mAbs used in the indirect flow cytometric analyses and mAb inhibition assays are described previously (17, 23).

Constructs of Mouse CD8 and CD8 Variants and N15 Transfectants Expressing CD8 Variants—We generated alanine-substituted mouse CD8 variants, including CD8^E27A, CD8^K55A, CD8^S101A, and CD8^K103A, using the PCR-based mutagenesis (14). Cell lines expressing variant CD8^E27AE27A or CD8^E27A were generated by transfecting the CD8^E27A cDNA alone or together with WT CD8 into the N15 CD8^– cell line as described (14). For the cell lines expressing CD8^R8Avar or CD8^var heterodimers, the CD8^R8A or WT CD8 cDNA was transfected pairwise with each CD8 variant cDNA, ^K55A, ^S101A, or ^K103A, into the N15 CD8^– cell line. The resulting transfectants were tested for surface expression of TCR (detected by anti-TCR V5.2 mAb MR9.4), CD8 (by anti-CD8 mAb 53.6.72), and CD8 (by anti-CD8 mAb YTS156) proteins, and positive clones were combined (minimum of 20 independent clones) and sorted for similar levels of surface expression.

Analyses of Co-receptor Activity by IL-2 Production and Inhibition of Co-receptor Activity by Anti-CD8 mAbs—IL-2 production by various N15 CD8⁺ transfectants triggered by antigen stimulation was quantified by a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium assay described previously (17). In brief, 1 x 10⁵ R8 antigen-presenting cells loaded with 10^–9 – 10^–4 M vesicular stomatitis virus octapeptide was incubated with an equal number of N15 transfectants expressing CD8 variants plus 10 ng/ml phorbol myristate acetate for 24 h. Inhibition of co-receptor activity by anti-CD8 mAb was analyzed by the IL-2 assay. Briefly, 10 µg of affinity-purified anti-CD8 mAb H59, anti-CD8 mAb YTS156, or control mAb 2H11 was added to transfectants at 4 °C for 30 min before incubation with peptide-loaded antigen-presenting cells. IL-2 induced in the supernatant by antigen stimulation was quantified by IL-2-dependent growth of CTLL20 cells. N15 transfectants treated with anti-CD3 mAb 145.2C11 were used as a positive control for T-cell activation. Recombinant human IL-2 (Chiron, Emeryville, CA) was used as an IL-2 standard.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Structural Basis for CD8 Occupancy of Both Site 1 and Site 2—The overall protein architecture of CD8 is similar to that of CD8 (22, 23) and resembles the Ig-variable domain fragment of an antibody (Fig. 1A). Despite the low degree of protein sequence identity between CD8 and CD8 (Fig. 1C), superimposing their structures reveals only limited differences in the loop regions, including the CDR1- and CDR2-like loops (Fig. 1B). The CDR-like loops are involved in ligand binding and are expected to be more variable than other regions. However, the nine residues that are identical in human and mouse CD8 and CD8 are important for maintaining an Ig-fold structure and dimeric interface (7). This conservation ensures similarity in the overall structure of and subunits (Fig. 1B). The apparent structural similarity between CD8 and CD8 raises the possibility that CD8·pMHCI interactions may be very similar to those of CD8·pMHCI, with the subunit replacing either one of the subunits in CD8·pMHCI complex.

CD8 Subunit of Heterodimeric CD8^E27A Occupies Site 1—We recently showed that CD8^R8A is functionally active (17, 28). This suggests that Arg⁸ residue of the CD8 subunit is dispensable for the co-receptor function of CD8^R8A. The Arg⁸ is not only critical for the ability of CD8 to occupy site 1 but is also the antigenic epitope recognized by mAb H59 (17). Hence, it is possible that mAb H59 blocks the interaction of pMHCI and CD81 but not that of pMHCI and CD82. If so, mAb H59 would be expected to block the co-receptor activity of CD8 but not that of CD8^R8A. However, as the binding epitope of mAb H59 is lost in CD8^R8A (17), this variant is not suitable for performing the antibody blocking experiment. Because both Arg⁸ and Glu²⁷ form hydrogen bonds with Lys⁵⁸ of ₂M in the crystal complex of CD8·H-2K^b (Fig. 1, D and E), we engineered a substitute variant, CD8^E27A, and tested whether, like CD8^R8A, CD8^E27A is functionally active. Fig. 2A shows that despite comparable surface expression levels of CD8 and TCR among the CD8 transfectants, cells expressing CD8^E27AE27A exhibit co-receptor activity about 1000-fold less efficiently than cells expressing WT CD8 (Fig. 2B). Thus, like CD8^R8AR8A, CD8^E27AE27A is incapable of interacting productively with pMHCI. In addition, the co-receptor activity of CD8^E27A is indistinguishable from that of WT CD8 (Fig. 2C). Therefore, similar to CD8^R8A, CD8^E27A can productively engage with pMHCI. mAb H59 binds to cells expressing WT CD8 and CD8^E27A variants (Fig. 2A) indicating that the binding epitope of mAb H59 is retained. Fig. 2E shows that mAb H59 completely inhibits the co-receptor activity of CD8, yet it does not affect that of CD8^E27A (Fig. 2D). We also tested the co-receptor activity of CD8^E27A in the presence of an anti-CD8 mAb YTS156. In contrast to mAb H59, mAb YTS156 completely blocks the activity of CD8^E27A (Fig. 2D).

View larger version (42K): [in this window] [in a new window]   FIGURE 1. Structural comparison of mouse CD8 and CD8. A, ribbon diagram of the CD8 heterodimer. The CD8 subunit is blue, and CD8 is purple. B, superimposition of CD8 and CD8. The -carbon trace of each CD8 subunit is color-coded as in A. C, protein sequence alignment of CD8 and CD8. The strand assignments are based on the crystal structure of mouse CD8. Residues identical in and subunits are highlighted in yellow, and CDRs are boxed. Residues Arg8 and Glu27 in mouse CD8 and their counterparts in CD8 are highlighted with black background. D, ribbon diagram of the crystal structure of mCD8·H-2Kb. The heavy chain of H-2Kb is dark green, and 2M is light green. Within CD8, CD81is light blue, and CD82is dark blue. Side chains of residues Arg8 and Glu27, in ball-and-stick form, are indicated on both CD8 subunits. E, local interactions between CD81 and 2M are detailed. Hydrogen bonds between CD8 Glu6 and CD8 Arg8, CD8 Arg8 and2M Lys58, as well as CD8 Glu27 and2M Lys58, are shown as dashed pink lines. A, D, and E were created with MOLSCRIPT (35) and B with GRASP (36).

View larger version (26K): [in this window] [in a new window]   FIGURE 2. CD8 subunit of heterodimeric CD8E27A occupies site 1. A, fluorescence-activated cell sorter analysis of the CD8var transfectants. B and C, CD8 co-receptor activity in cell lines expressing CD8E27AE27A (B) or CD8E27A (C) measured by the amounts of IL-2 produced upon stimulation with various concentrations of peptide antigen. D and E, effects of anti-CD8 mAb H59 and anti-CD8 mAb YTS156 on the co-receptor activity associated with cell lines expressing CD8E27A (D) and WT CD8 (E). VSV8, vesicular stomatitis virus octapeptide.

View larger version (31K): [in this window] [in a new window]   FIGURE 3. CD8 subunit of CD8var occupies site 2. A, CD8 co-receptor activity of N15 transfectants expressing CD8 variants and WT CD8 measured by the amounts of IL-2 produced upon stimulation with various concentrations of peptide antigen. B–D, effects of anti-CD8 mAb H59 and anti-CD8 mAb YTS156 on the co-receptor activity of N15 transfectants expressing the CD8 variants, CD8K55A (B), CD8K103A (C), and CD8S101A (D). VSV8, vesicular stomatitis virus octapeptide.

CD8 Subunit of Heterodimeric CD8^var Occupies Site 2—Because CD8^R8A and CD8^E27A interacted with pMHCI in only one orientation, the CD8^R8A co-transfection system also provided a measure to identify residues of CD8 that were critical for CD8 being able to occupy site 1. We recently reported that the Lys⁵⁵ in the CDR2-like loop and also Ser¹⁰¹ and Lys¹⁰³ in the CD8 CDR3-like loop is critical for the co-receptor activity of CD8^R8A, as co-expression of each of these CD8 variants with CD8^R8A does not lead to detectable co-receptor activity (28). In light of the observation that heterodimeric CD8^R8A is functionally active (17), we questioned whether each of these CD8 variants can form a functional co-receptor with WT CD8. The co-receptor activity of the CD8^var would indicate that each of these CD8 variants could occupy site 2 and form active co-receptors with site 1-occupying WT CD8. We found that cell lines expressing similar levels of CD8 and CD8, CD8^K55A, CD8^S101A, or CD8^K103A proteins on the cell surface (data not shown) exhibited co-receptor activity (Fig. 3A). These results suggest that the heterodimeric CD8^var can productively interact with pMHCI.

However, WT CD8 can form functional CD8 homodimers in these transfectants (17) and contribute to the observed co-receptor activity (Fig. 3A). We reported previously that the co-receptor activity of WT CD8 and CD8 can be triggered by peptide antigen at concentrations of 10^–9 and 10^–7 M, respectively (14). Thus, the CD8^var heterodimers are likely to be functionally active if cells expressing CD8 and CD8^var are responsive to peptide antigen at a concentration lower than 10^–7 M. Fig. 3A shows that cells expressing CD8 and CD8^K55A or CD8^K103A is responsive to peptide antigen at a concentration of 10^–8 M. This result suggests that heterodimeric CD8^K55A and CD8^K103A are functionally active. Because cells expressing CD8 and CD8^S101A exhibited a CD8-like antigen sensitivity of 10^–7 M, it appears that CD8^S101A was not functionally active.

To further verify that CD8^K55A and CD8^K103A are indeed functionally active, we also examined their co-receptor activity in the presence of anti-CD8 mAb YTS156 or anti-CD8 mAb H59. Because mAb YTS156 is chain-specific, it is expected to block the activity of the CD8^var heterodimeric co-receptor. Conversely, mAb H59, which is specific for the chain, would block the co-receptor activity associated with CD8 homodimers and CD8^var heterodimers in which CD8 subunits occupy site 1. Fig. 3B shows that mAb YTS156 was able to block some co-receptor activity associated with cells transfected with WT CD8 and CD8^K55A, indicating that CD8^K55A is indeed a contributor to that activity. Similar results were obtained from the cell line expressing WT CD8 and CD8^K103A (Fig. 3C). Thus, although CD8^K55A and CD8^K103A are unable to occupy site 1 (7), they are capable of forming functional co-receptors with WT CD8 by occupying site 2. Surprisingly, very little, if any, co-receptor activity associated with cells expressing CD8 and CD8^S101A is inhibited by mAb YTS156 (Fig. 3D), indicating that CD8^S101A does not contribute to the co-receptor activity shown in A. This is consistent with the observation that these cells exhibit CD8-like antigen sensitivity (10^–7 M). In contrast to mAb YTS156, mAb H59 completely blocks the co-receptor activity in all three cell lines (Fig. 3, B–D) suggesting that all functional co-receptors expressed must interact with pMHCI with a CD8 subunit occupying site 1 and that the portion of co-receptor activity that cannot be blocked by mAb YTS156 is contributed by CD8. These results show that the subunit of heterodimeric CD8^K55A or CD8^K103A occupies site 2.

View larger version (21K): [in this window] [in a new window]   FIGURE 4. Wild-type CD8 binds to pMHCI in two distinct orientations. A, schematic representation of CD8 or CD8 interactions with pMHCI in N15 TCR transfectants. B, CD8 co-receptor activity of WT CD8 in N15 transfectant in the presence of control mAb 2H11, anti-CD8 mAb H59, or anti-CD8 mAb YTS156 measured by the amounts of IL-2 produced upon stimulation with various concentrations of peptide antigen. VSV8, vesicular stomatitis virus octapeptide.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
The structure of the CD8·pMHCI complex reveals that the two subunits of the CD8 homodimer bind to the peptide-MHCI complex asymmetrically, with one subunit interacting extensively with pMHCI (site 1) and the other subunit interacting only with the 3 domain of pMHCI (site 2). Based on this information, we identified residues Arg⁸ and Glu²⁷ as being critical to the ability of CD8 to occupy site 1. Using CD8^R8A and CD8^E27A, we found that CD8 could form a functional co-receptor with either variant. Therefore, in the context of CD8·pMHCI interaction, CD8 can occupy site 1. We also used the CD8^R8A variant to identify three CD8 variants incapable of occupying site 1. Two of these three CD8 variants could form a functionally active co-receptor with WT CD8 indicating that although, CD8^var could not occupy site 1, it could still occupy site 2. The concept that CD8 and pMHCI can form a complex in different orientations is also supported by the observations that anti-CD8 mAb H59 completely blocks the co-receptor activity of CD8^var, yet it does not affect that of CD8^var and only partially blocks that of WT CD8. Our results support a two-binding-mode model in which heterodimeric CD8 can interact with pMHCI with two distinct binding orientations, one sensitive to mAb H59 and the other not.

The idea that CD8 can interact with pMHCI in two orientations prompted questions regarding whether the antigen presentations mediated by these two distinct CD8·pMHCI complexes are qualitatively and/or quantitatively different. Note that the p56^lck kinase is only associated with the cytoplasmic domain of CD8 but not with that of CD8 (21). Thus, the distance between p56^lck and the chains in a TCR complex that has CD8 engaged in one orientation is expected to be different from that in another TCR complex that has CD8 engaged in the opposite orientation. In addition, a specific pMHCIa or pMHCIb, such as H-2K, H-2D, H-2L, or Qa-1, Qa-2 plus H2-M3 (31), might favor the interaction with CD8 in one orientation over the other. It is also possible that one specific binding mode might be better accommodated because of the particular position at which a specific TCR docks with pMHCI (29–31). Furthermore, the differentiation stage-related sialylation of the O-glycans associated with the stalk regions of CD8 proteins (18, 19) may be compatible with one, but not another, binding orientation. Therefore, a given TCR·pMHCI complex may preferentially or exclusively interact with CD8 in one binding mode but not the other. Such an asymmetric binding mode might facilitate a better engagement between a given TCR complex and the heterodimeric co-receptor that is not possible for its homodimeric counterpart.

Both CD8 and Ly49A NK cell receptors are shown to interact with pMHCI on the same cell (cis interaction) (32, 33). Specifically, the inhibitory Ly49A NK cell receptor can bind to its pMHCI ligand, H-2D^d, expressed on either the target cells (trans interaction) or the same NK cell (cis interaction) (33). Cis interaction of Ly49A with pMHCI reduces the number of Ly49A available for binding of pMHCI ligand on the target cells. By attenuating the inhibition on NK cells, Ly49A can fine-tune an optimal sensitivity for NK cells to discriminate normal versus abnormal host cells. Similarly, interaction of CD8 to pMHCI in cis may be compatible with one, but not another, binding mode. Future studies are needed to better understand the potential regulatory role of heterodimeric CD8 on antigen presentation.

The structural comparison of human and mouse CD8 shows that the corresponding residue of mouse Arg⁸ is human Arg⁴. Similar to the mouse CD8^R8AR8A, human CD8^R4KR4K homodimer cannot productively interact with pMHCI (34). However, unlike its mouse counterpart, human CD8^R4K cannot form a functional co-receptor with WT human CD8 (34). Judging from the overall structural similarities between human and mouse CD8 as well as pMHCI proteins, it is difficult to understand the molecular basis for such a discrepancy. However, close examination reveals some differences that may explain this discrepancy. For example, human CD8 can be expressed on the cell surface as functionally inactive homodimers CD8, whereas mouse CD8 cannot (27). This may drastically reduce the surface abundance of heterodimeric CD8 and may therefore explain why no apparent co-receptor activity was detected in the human experimental system. In addition, transient expression cultures, rather than established cell lines, were used for experimentation in the human system. The background noise inherently associated with transient expression systems may compromise the detection of any functional activity of heterodimers in a cell adhesion assay. Thus, it is conceivable that the observed discrepancy was because of differences in experimentation systems and that human CD8, like mouse CD8, also interacts with pMHCI with two distinct binding orientations.

Our results may also explain the apparent difficulty in obtaining protein crystals for structure determination of the CD8·pMHCI complex, as WT protein mixtures may contain two types of complexes preventing formation of ordered crystals. In this regard, our CD8 heterodimeric variants capable of only one binding mode may provide an alternative to obtain the protein crystal of CD8·pMHCI complexes and facilitate future efforts to elucidate their structures by crystallographic or other biophysical approaches. By generating transgenic mice expressing one heterodimeric variant, it will be possible to investigate the physiological significance of one binding mode without the complications associated with the second binding mode.

	FOOTNOTES

 
^* This work was supported by National Institutes of Health Grant AI45789 and by the Claudia Adams Barr investigator award of Dana-Farber Cancer Institute (to H.-C. C.). 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 To whom correspondence should be addressed: Dana-Farber Cancer Inst., Harvard Medical School, 77 Ave. Louis Pasteur, HIM-442, Boston, MA 02115. Tel.: 617-632-4497; Fax: 617-632-3668; E-mail: hsiu-ching_chang{at}dfci.harvard.edu.

² The abbreviations used are: MHC, major histocompatibility complex; pMHCI, peptide-MHC class I; WT, wild type; ₂M, ₂-microglobulin; mAb, monoclonal antibody; IL-2, interleukin 2; CDR, complementarity-determining region; TCR, T-cell receptor; NK, natural killer.

	ACKNOWLEDGMENTS

 
We are grateful to Drs. Raymond M. Welsh, Linda K. Clayton, Robert J. Mallis, Ann-Marie Moody, and Kyoung-Joon Oh for their critical reading of this manuscript.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 

1. Cantor, H., and Boyse, E. A. (1975) J. Exp. Med. 141, 1376–1389[Abstract/Free Full Text]
2. Reinherz, E. L., and Schlossman, S. F. (1980) Cell 19, 821–827[CrossRef][Medline] [Order article via Infotrieve]
3. Zuniga-Pflucker, J. C., Jones, L. A., Chin, L. T., and Kruisbeek, A. M. (1991) Semin. Immunol. 3, 167–175[Medline] [Order article via Infotrieve]
4. Moebius, U., Kober, G., Griscelli, M. L., Hercend, T., and Meuer, S. C. (1991) Eur. J. Immunol. 21, 1793–1800[Medline] [Order article via Infotrieve]
5. de Totero, D., Tazzari, P. L., DiSanto, J. P., di Celle, P. F., Raspadori, D., Conte, R., Gobbi, M., Ferrara, G. B., Flomenberg, N., and Lauria, F. (1992) Blood 80, 1765–1773[Abstract/Free Full Text]
6. Terry, L. A., DiSanto, J. P., Small, T. N., and Flomenberg, N. (1990) Tissue Antigens 35, 82–91[Medline] [Order article via Infotrieve]
7. Chang, H. C., Tan, K., Ouyang J., Parisini, E., Liu, J. H., Le, Y., Wang, X., Reinherz, E. L., and Wang, J. H. (2005) Immunity 23, 661–671[CrossRef][Medline] [Order article via Infotrieve]
8. Gao, G. F., Rao, Z., and Bell, J. I. (2002) Trends Immunol. 23, 408–413[CrossRef][Medline] [Order article via Infotrieve]
9. Konig, R. (2002) Curr. Opin. Immunol. 14, 75–83[CrossRef][Medline] [Order article via Infotrieve]
10. Hayday, A., Theodoridis, E., Ramsburg, E., and Shires, J. (2001) Nat. Immunol. 2, 997–1003[CrossRef][Medline] [Order article via Infotrieve]
11. Gangadharan, D., and Cheroutre, H. (2004) Curr. Opin. Immunol. 16, 264–270[CrossRef][Medline] [Order article via Infotrieve]
12. Renard, V., Romero, P., Vivier, E., Malissen, B., and Luescher, I. F. (1996) J. Exp. Med. 184, 2439–2444[Abstract/Free Full Text]
13. Wheeler, C. J., Chen, Y. F., Potter, T. A., and Parnes, J. R. (1998) J. Immunol. 160, 4199–4207[Abstract/Free Full Text]
14. Witte, T., Spoerl, R., and Chang, H. C. (1999) Cell. Immunol. 191, 90–96[CrossRef][Medline] [Order article via Infotrieve]
15. Luescher, I. F., Vivier, E., Layer, A., Mahiou, J., Godeau, F., Malissen, B., and Romero, P. (1995) Nature 373, 353–356[CrossRef][Medline] [Order article via Infotrieve]
16. Bosselut, R., Kubo, S., Guinter, T., Kopacz, J. L., Altman, J. D., Feigenbaum, L., and Singer, A. (2000) Immunity 12, 409–418[CrossRef][Medline] [Order article via Infotrieve]
17. Wong, J. S., Wang, X., Witte, T., Nie, L., Carvou, N., Kern, P., and Chang, H. C. (2003) J. Immunol. 171, 867–874[Abstract/Free Full Text]
18. Priatel, J. J., Chui, D., Hiraoka, N., Simmons, C. J., Richardson, K. B., Page, D. M., Fukuda, M., Varki, N. M., and Marth, J. D. (2000) Immunity 12, 273–283[CrossRef][Medline] [Order article via Infotrieve]
19. Moody, A. M., Chui, D., Reche, P. A., Priatel, J. J., Marth, J. D., and Reinherz, E. L. (2001) Cell 107, 501–512[CrossRef][Medline] [Order article via Infotrieve]
20. Arcaro, A., Gregoire, C., Bakker, T. R., Baldi, L., Jordan, M., Goffin, L., Boucheron, N., Wurm, F., van der Merwe, P. A., Malissen, B., and Luescher, I. F. (2001) J. Exp. Med. 194, 1485–1495[Abstract/Free Full Text]
21. Irie, H. Y., Mong, M., Itano, A., Crooks, C., Littman, D., Burakoff, B., and Robey, E. (1998) J. Immunol. 161, 183–191[Abstract/Free Full Text]
22. Gao, G. F., Tormo, J., Gerth, U. C., Wyer, J. R., McMichael, A. J., Stuart, D. I., Bell, J. I., Jones, E. Y., and Jakobsen, B. K. (1997) Nature 387, 630–634[CrossRef][Medline] [Order article via Infotrieve]
23. Kern, P. S., Teng, M. K., Smolyar, A., Liu, J. H., Liu, J., Hussey, R. E., Spoerl, R., Chang, H. C., Reinherz, E. L., and Wang, J. H. (1998) Immunity 9, 519–530[CrossRef][Medline] [Order article via Infotrieve]
24. Sun, J., and Kavathas, P. B. (1997) J. Immunol. 159, 6077–6082[Abstract]
25. Salter, R. D., Benjamin, R. J., Wesley, P. K., Buxton, S. E., Garrett, T. P., Clayberger, C., Krensky, A. M., Norment, A. M., Littman, D. R., and Parham, P. (1990) Nature 345, 41–46[CrossRef][Medline] [Order article via Infotrieve]
26. Connolly, J. M., Hansen, T. H., Ingold, A. L., and Potter, T. A. (1990) Proc. Natl. Acad. Sci. U. S. A. 87, 2137–2141[Abstract/Free Full Text]
27. Devine, L., Kieffer, L. J., Aitken, V., and Kavathas, P. B. (2000) J. Immunol. 164, 833–838[Abstract/Free Full Text]
28. Chang, H. C., Smolyar, A., Spoerl, R., Witte, T., Yao, Y., Goyarts, E. C., Nathenson, S. G., and Reinherz, E. L. (1997) J. Mol. Biol. 271, 278–293[CrossRef][Medline] [Order article via Infotrieve]
29. Rudolph, M. G., and Wilson, I. A. (2002) Curr. Opin. Immunol. 14, 52–65[CrossRef][Medline] [Order article via Infotrieve]
30. Buslepp, J., Wang, H., Biddison, W. E., Appella, E., and Collins, E. J. (2003) Immunity 19, 595–606[CrossRef][Medline] [Order article via Infotrieve]
31. Stewart-Jones, G. B., McMichael, A. J., Bell, J. I., Stuart, D. I., and Jones, E. Y. (2003) Nat. Immunol. 4, 657–663[CrossRef][Medline] [Order article via Infotrieve]
32. Auphan, N., Boyer, C., Andre, P., Bongrand, P., and Schmitt-Verhulst, A. M. (1991) Mol. Immunol. 28, 827–837[CrossRef][Medline] [Order article via Infotrieve]
33. Doucey, M. A., Scarpellino, L., Zimmer, J., Guillaume, P., Luescher, I. F., Bron, C., and Held, W. (2004) Nat. Immunol. 5, 328–336[CrossRef][Medline] [Order article via Infotrieve]
34. Devine, L., Sun, J., Barr, M. R., and Kavathas, P. B. (1999) J. Immunol. 162, 846–851[Abstract/Free Full Text]
35. Kraulis, P. J. (1991) J. Appl. Crystallogr. 24, 946–950[CrossRef]
36. Nicholls, A., Sharp, K. A., and Honig, B. (1991) Proteins 11, 281–296[CrossRef][Medline] [Order article via Infotrieve]

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