CD4 and CD8 molecules can physically associate with the same T-cell receptor

Transcription

1 Proc. Natl. cad. Sci. US Vol. 86, pp , December 1989 Immunology CD4 and CD8 molecules can physically associate with the same T-cell receptor (major histocompatibility complex/membrane molecules/t-lymphocyte activation) PULINE F. GLLGHER*, BRBR FZEKS DE ST. GROTHt, ND JCQUES F.. P. MILLER* *The Thymus Biology Unit, The Walter and Eliza Hall Institute of Medical Research, Post Office, Royal Melbourne Hospital, Victoria 3050, ustralia; and tdepartment of Medical Microbiology, Stanford University School of Medicine, Stanford, C Contributed by Jacques F.. P. Miller, September 3, 1989 BSTRCT The expression of the cell-surface glycoproteins CD4 and CD8 on functionally mature T cells is usually mutually exclusive and correlates with class II and class I major histocompatibility complex (MHC) restriction, respectively. CD4 and CD8 function by binding to class II and class I MHC molecules on the antigen-presenting cell (PC), thereby increasing the adhesion between the T cell and the PC. From antibody-blocking studies and from cocapping and comodulation experiments, CD4 and CD8 come into close physical contact with their appropriately restricted T-cell receptor (TCR) at the time of antigen recognition. By the use of affinity chromatography followed by two-dimensional diagonal gel electrophoresis, we have identified a Mr 43,000 disulfidebonded heterodimer copurifying with CD4. This protein was identified as the TCR by its removal after preclearing with the anti-tcr antibody F23.1 and by its generation after protease digestion of the same peptides as the TCR from this clone. When CD4 and CD8 were similarly isolated from an unusual CD4+ CD8+ class II-restricted T-cell clone, the TCR was identified as associating with either accessory molecule in the absence of activation. Therefore, CD4 and CD8 do not distinguish between class I- and class 11-restricted TCRs in their ability to form membrane complexes, indicating a need for both the TCR and its associated accessory molecule to recognize the same individual MHC molecule on the PC to optimize TCR triggering. Specific recognition of antigen in association with major histocompatibility complex (MHC) determinants by immune T lymphocytes is a function unique to the T-cell antigen receptor (TCR) (1). The interaction between a T cell and its antigen-presenting cell (PC) needs to be enhanced and stabilized by other cellular adhesion molecules before TCR triggering can occur. Two such accessory molecules are the T-cell surface glycoproteins CD8 and CD4 (Lyt-2/3 and L3T4 in the mouse), which are usually expressed in a mutually exclusive fashion on mature class I and class II MHCrestricted T cells, respectively. CD4 has been shown to bind to class II MHC molecules independently of any TCRantigen binding (2) and, similarly, isolated CD8 can bind to class I MHC (3, 4), with both reactions being of low affinity. In addition to their cytoadhesion function, a more complex role for CD4 and CD8 in T-cell activation has been indicated by several studies. First, the lymphokine production of two unusual CD4+ CD8+ class II-restricted T-cell clones, triggered by a specific antigen in association with the MHC, was inhibited by anti-cd4 monoclonal antibodies (mbs) but not anti-cd8 mbs, despite the presence of both class I and class II MHC molecules on the PCs (5). CD8 was not able to substitute for CD4 in a class II-restricted T-cell response. Second, in the absence of any MHC, T-cell activation by anti-tcr mbs could be inhibited with anti-cd4 and anti- CD8 mbs (6-8). Third, antibody-induced crosslinking of the TCR with its accessory CD4 or CD8 abrogated the inhibitory effect of anti-cd4/cd8 mbs and could even enhance T-cell activation (6, 9, 10). These observations have underpinned the hypothesis that, at the time of antigen recognition, CD4 and not CD8 must form a close physical association with its class II-restricted TCR on the T-cell surface; conversely, CD8 would associate exclusively with a class I-restricted TCR (5, 9). Evidence for such complexes has relied on indirect cocapping and comodulation experiments (11-15). Up to 30% of CD4 molecules have been shown to comodulate with the TCR-CD3 complex from the cell membrane after specific antigenic stimulation (13). In the absence of T-cell triggering, such aggregates are present but at very low levels (16) and have been estimated to occur with no more than =5% of TCRs on helper T cells (13, 14). Conventional immunoprecipitation approaches have not been adequate to the demonstration of these TCR-accessory molecule associations. We have used affinity chromatography to physically isolate this tiny proportion of TCRs that are complexed with CD4 on the T-cell membrane in the absence of an activating stimulus. By the use of a CD4' CD8' cell, we show here that when CD4 and CD8 are expressed on the same cell, they both have the ability to associate with the same class II-restricted TCR. These results strongly suggest that TCR-accessory molecule complex formation is driven by the ligation of both molecules to the same MHC molecule presenting specific antigen. MTERILS ND METHODS ntibodies. The F23.1 anti-v188 (17) and 145-2C11 anti-t3 (18) antibodies were used as unpurified hybridoma culture supernatants. MR-18.5 (19) mouse anti-rat immunoglobulin mb was used as unpurified ascites fluid for immunoprecipitations. GK1.5 anti-l3t4 (20), anti-lyt-2 (21), and PC-61 anti-interleukin 2 (IL-2) receptor (22) mbs were affinity purified from ascites fluids on Sepharose 4B beads (Pharmacia) coupled with mb MR For the affinity chromatography experiments, purified antibodies (1.0 mg/ ml) were coupled to CNBr-activated Sepharose 4B (Pharmacia) according to the manufacturer's recommendation. T-Cell Clones. The derivation and characterization of the alloreactive T-cell clone 4.35F2 (8) and the hapten-specific clone (5) have been described. Briefly, 4.35F2 is a lymphokine-producing, CD4+ CD8- Vp8+ T-cell clone that responds to IEk specifically has the surface phenotype CD4+ CD8+ Vp8+ and produces lymphokines in response to azobenzenearsonate-coupled lak. Both cell lines were cultured in RPMI 1640 medium supplemented with 2 The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C solely to indicate this fact bbreviations: PC, antigen-presenting cell; IL-2, interleukin 2; mb, monoclonal antibody; MHC, major histocompatibility complex; TCR, T-cell antigen receptor.

2 mm L-glutamine, 50,M 2-mercaptoethanol, 10% fetal calf serum, and IL-2-containing supernatant from Con - stimulated rat spleen cells. Radioiodination. T cells were surface labeled with 1251 by the lactoperoxidase/h202 method (41). Labeled cells were lysed in 2 ml of 100 mm Tris HC1 (ph 7.4) containing 1 mm EDT, 0.5% Triton X-100, 1 mm phenylmethylsulfonyl fluoride, and 1 mm iodoacetamide. fter 1 hr, the lysates were centrifuged for 10 min at 15,000 x g in an Eppendorf 5141S centrifuge and pelleted debris was discarded. For the anti-t3 immunoprecipitation, the cell lysis buffer contained 1% digitonin (23) instead of 0.5% Triton X-100. Immunoprecipitation. Samples (0.5 ml) of detergent lysates were precleared three times, for 1 hr each, by the addition and removal of 50 l of a 50% suspension of Staphylococcus aureus (Commonwealth Serum Laboratories, Melbourne, ustralia), either untreated or precoated with MR-18.5 mb. The desired mbs were precomplexed with MR-18.5 and S. aureus, or with S. aureus alone before immunoprecipitation. Immunology: Gallagher et al. Unbound mb was washed from the bacteria with phosphate-buffered saline (PBS) containing 0.5% Triton X- 100 (PBS-TX), and the mb-coated bacteria were incubated with samples of precleared lysate for 4 hr. The bacteria were then washed five times in PBS-TX and precipitated proteins were eluted by boiling in Laemmli sample buffer without reducing agents for the two-dimensional analyses or with 50 mm dithiothreitol for one-dimensional SDS/PGE. ffinity Chromatography of Detergent Lysates. Detergent lysates (0.5-ml vol) were precleared three to six times, for 60 min each, using S. aureus precoated with the relevant mbs. The precleared samples were- passed slowly through mb- Sepharose columns and were washed with 40 ml of PBS-TX. Bound proteins were eluted in 0.5-ml fractions with 0.1 M diethylamine (ph 11) containing 0.5% Triton X-100. The ph of each fraction was immediately adjusted to ph 7.5 with 100 l of 1.0 M Tris-HCl. Fractions were assayed and those samples containing peak radioactivity were precipitated overnight at -20 C with 90% methanol. The precipitated proteins were pelleted by centrifugation at 15,000 x g for 30 min and then dissolved in 50 l of Laemmli sample buffer without any reducing agent. Samples were analyzed by two-dimensional nonreducing/reducing PGE as described (24) followed by autoradiography. Peptide Mapping. The radioactive proteins of interest were cut out of the dried gels and digested as described (25) for 30 min with 50,ug of S. aureus V8 protease per ml (Sigma). The resulting peptides were resolved on a 15% polyacrylamide gel and autoradiographed. RESULTS Initial experiments were performed on the CD4+ CD8- T-cell 4.35F2, whose TCR could be immunoprecipitated with mb F23.1. The TCR a/3 heterodimer of 4.35F2 in nonreducing/ reducing two-dimensional PGE migrates at Mr 43,000 and below the diagonal, as typical for a disulfide-bonded dimeric protein (Fig. 1). To detect the tiny proportion of CD4-TCR complexes that presumably would exist in a low-affinity association on resting T cells, we required a gentle purification method that was capable of isolating all the CD4 molecules from a detergent lysate of- cells. We therefore used GK1.5 mb covalently bound to Sepharose in an affinity column, with a capacity well in excess of antigen loaded, as a separation procedure that also permitted gentle washing of bound molecules to minimize disruption of molecular complexes. When CD4 was isolated in this way from the 4.35F2 cell lysate, a small amount of a disulfide-bonded dimer from the same column fraction was also observed migrating in the same position as the TCR (Fig. 1B). This protein was not seen in the fractions eluted from the PC-61-Sepharose column Proc. Natl. cad. Sci. US 86 (1989) a FIG. 1. Comparison of the TCR with membrane molecules of "25t-labeled 4.35F2 detergent lysates separated by an anti-cd4- Sepharose affinity column and two-dimensional nonreducing/ reducing SDS/PGE. Electrophoresis proceeded from left to right and then from top to bottom. () TCR af8 heterodimer immunoprecipitated with F23.1 mb. Dotted line marks the diagonal formed by non-disulfide-bonded proteins. (B) Surface proteins of 4.35F2, which bound to anti-cd4-sepharose, including the low abundance TCRlike dimer (arrow). (C) Proteins isolated by anti-il-2 receptor column; the TCR-like dimer was not copurified (position shown by arrow). (D) Proteins separating on the anti-cd4-sepharose column after repeated preclearing of the detergent lysate with S. aureus coated with F23.1; the TCR-like dimer has been removed (arrow shows position). Mr markers x 10-3 are indicated for each gel. utoradiographs were exposed for 1 day () or 2 weeks (B-D). used as a specificity control (Fig. 1C). Repeated preclearing of the detergent lysate with F23.1 mb before affinity chromatography removed this protein from the gel (Fig. 1D), indicating that it was indeed the TCR. Identification was further established when the protein was cut out of the gel shown in Fig. 1B and, together with the immunoprecipitated TCR (Fig. l), was digested with V8 protease. Fig. 2 shows that, despite their difference in radioactive intensity due to the vastly different amounts of protein analyzed, the two proteins generated the same resolvable peptides and therefore were homologous. Thus, the disulfide-bonded dimer coeluting with CD4 is the small proportion of TCRs that are physically associated with CD4 on the resting T-cell membrane. In our published model of antigen recognition (5), we proposed that CD4 could aggregate with the class IIrestricted TCR, but CD8 would be excluded from such a complex: CD8 could only associate with a class I-restricted TCR. We were able to address the capability of a given TCR B C D

3 10046 Immunology: Gallagher et al. FIG. 2. Peptide maps of the TCR (lane ) and the CD4-associated TCR-like protein (lane B) from the T-cell 4.35F2. The appropriate radioactive spots were cut out of the gels (Fig. 1 and B) and subjected to digestion by S. aureus V8 protease. utoradiograph exposures were for 2 days (lane ) and 4 weeks (lane B). to distinguish between CD4 and CD8 molecules by means of the functional CD4' CD8' T-cell clone s shown in Fig. 3 -C, TCR-accessory molecule complexes were not usually evident in immunoprecipitates of TCR/CD3, CD4, and CD8 from When the affinity column isolation method was used, TCRs again were not seen with the IL-2 receptor (Fig. 3D). Surprisingly, TCR molecules copurified with both CD4 and CD8, as seen particularly well in Fig. 3 E and F. Peptide analyses of these Mr 43,000 dimers found them to be indistinguishable from the CD3-immunoprecipitated TCR of (data not shown). The amount of associated TCR from varied in repeated experiments between the level shown in Fig. 3 and that shown above for 4.35F ,. - %.TCR B _ i~~f;cd4.~~~~~~~~~~~~~~~.: Proc. Natl. cad. Sci. US 86 (1989) (Fig. 1B), but it was always present in the eluates from both anti-cd4 and anti-cd8 columns. The relative intensities of the TCR and CD8 spots (Fig. 3F) are not indicative of the relative amounts of protein separated but rather of their relative efficiencies of radioiodination. CD4, for example, is notoriously poorly labeled with 125I (26) and is not distinguished from other aggregating proteins on the diagonal of the two-dimensional gel (Fig. 3E). Occasionally, albeit unreliably, the TCR protein would be coprecipitated in immunoprecipitations of the accessory molecules, indicating that the complexes we have identified were not simply aggregations forming in the affinity columns. Fig. 4 shows a one-dimensional PGE separation of CD4 and CD8 immunoprecipitates from In this experiment, the Mr 43,000 TCR protein remained associated with CD4 but not CD8. In other immunoprecipitation experiments (data not shown), CD8 coprecipitated the Mr 43,000 protein. Veillette et al. (38) have published CD4 immunoprecipitations with coprecipitated molecules of the same molecular weight as the TCR, although they did not show further evidence to identify the TCR as such. The inconsistent coisolation of TCRs with CD4 and CD8 by this technique may be partly due to the choice of detergent used for solubilizing the cells. We have not been able to identify these complexes by using, for example, 1% digitonin, which was used to show the TCR- CD3 association (23). In addition, the vigorous washing procedure used for immunoprecipitations may disaggregate the small number of TCR-accessory molecule associations, which, as indicated by their low frequency on resting T cells, are of low affinity. From the gentler method of affinity chromatography, therefore, CD4 and CD8 clearly do not distinguish between class I- and class II-restricted TCRs; either accessory molecule is capable of complexing with a given TCR. DISCUSSION Most TCRs need the help of an accessory molecule to be triggered. This has been most definitively demonstrated in experiments involving the transfer of class I-restricted TCR CDS C i 94 - ~L - - _sllq D F FIG. 3. Two-dimensional PGE analysis of 1251-labeled surface proteins from the T-cell clone Immunoprecipitations of TCR-CD3 with mb 145-2C11 (), CD4 with mbs GK1.5 and MR-18.5 (B), CD8 with mb and MR-18.5 (C), in comparison with affinity column separations of IL-2 receptor (IL-2R) (D) (arrow marks the position of the TCR that was not copurified), CD4, which migrates on the diagonal among other proteins (E), and CD8, which is close to but distinct from the TCR (F). rrows in E and F point to the TCR. Mr standards x 10-3 are indicated. utoradiographs were exposed for 1 day (), 3 days (B and C), and 2 weeks (D-F)..4

4 Immunology: Gallagher et al. a 94-1 b 43-4 FIG. 4. Immunoprecipitations of CD4 and CD8 from 125I-labeled cells. SDS/PGE analysis of CD8 (Mr, 38,000) precipitated with mb (lane a) and CD4 (Mr, 55,000) with GK1.5 mb (lane b). rrowhead indicates the TCR-like protein. Mr standards x 10-3 are shown. The autoradiograph was exposed for 3 days. a and genes into CD8- cells; reconstitution of the full antigen specificity of the donor T cells in the recipients required the cotransfection of the CD8 gene (27, 28). CD4 has even been shown to increase the sensitivity of a given TCR enough to confer additional specificity on a T-helper cell (29). That the engagement of the TCR artificially crosslinked with their appropriate accessory molecules facilitates T-cell activation is now well documented (9, 10, 12, 40). Yet in the absence of an activating stimulus, the TCR aggregates with the accessory molecule at an extremely low level. In this report, we have described the isolation and identification of a small proportion of TCRs that occur naturally on the resting T-cell membrane in a close physical association with CD4. This result confirms the antibody-induced cocapping and comodulation experiments mentioned above and provides an immunochemical basis for TCR-accessory molecule interactions. Furthermore, we have provided evidence that CD4 and CD8 do not distinguish between class I- and class 11-restricted TCRs, by virtue of the ability of TCR molecules from a CD4' CD8' class II-restricted T cell to be copurified with both CD4 and CD8. This result is not surprising in terms of the current accumulated data on TCR gene usage, which does not allow for the two classes of TCRs differing in their ability to interact with CD4 or CD8. lthough a strong correlation exists between the expression of certain Vp gene sequences and recognition of a given MHC molecule-for example, V1317a and IE reactivity-the pool of receptor chains utilized for class I and class II MHC recognition is overlapping (reviewed in ref. 30). Our observation, nevertheless, was unexpected in light of antibody-blocking experiments performed on this T-cell clone, among others, where the inability of CD8 to function interchangeably with CD4 in a given class II-restricted response pointed to a TCR interaction preference for CD4 at the time of antigen recognition (5, 31). Rather, our observation indicates a requirement for the TCR and the accessory molecule to bind the same antigen-presenting MHC molecule in a three-way interaction, as proposed by Eichmann and coworkers (9). Janeway and colleagues (12) have estimated that CD4 aggregated with a class 11-restricted TCR can increase the effectiveness of T-cell triggering by up to 100-fold. Our estimates of enhancement go even higher (5). With a lowaffinity TCR, or low levels of antigen-mhc, such augmen- Proc. Natl. cad. Sci. US 86 (1989) tation might very well determine whether or not the T cell is activated. The atypical examples of the CD41 CD8' class II-restricted T-cell responses illustrate the effects of affinity enhancement by accessory molecules. The CD4-TCR complexes on would have a higher affinity for antigen in association with class II MHC than the CD8-TCR complexes or monovalent TCRs. ctivation was not inhibited by anti- CD8 mbs because triggering would occur through the CD4-TCR complexes. Conversely, when anti-cd4 mbs were used to block function, the CD8-TCR complexes and unassociated TCRs presumably did not have sufficient affinity for antigen-class II MHC to trigger a response. For CD4 and CD8, the key to successful function in an immune response must lie in their ability not only to associate physically with the TCR but also to recognize the same MHC molecule on the PCs. If the ligands for the accessory molecule and the TCR are on separate molecules but in close physical contact on the antigen-presenting membrane, a TCR-accessory complex might still be able to provide some enhancement, particularly if the TCR stimulus is suboptimal. Experiments by Jones et al. (31) on other class II-restricted T-cell clones expressing both CD4 and CD8 have shown a minor contribution by CD8 when TCR triggering was not optimal; inhibition of responsiveness by anti-cd8 mb was much less effective than by anti-cd4. Similarly, some CD4 involvement in activation was demonstrable in the class I-restricted response of a CD4' CD8' T-cell hybridoma when class II MHC molecules were present in large amounts on the PCs (32). Both these examples, however, emphasize the importance of a tripartite binding event to optimize the response of a normal T cell. feature of the accessory molecule affinity purifications from both cell lines used in this study was the large number of other radioactive proteins that coeluted from the columns and migrated both on and off the diagonal of the twodimensional gels. This apparent "background" was not due to proteins nonspecifically sticking to the mb-sepharose columns since it was not observed in the control IL-2 receptor purifications. n increasing number of cell-surface molecules have been documented as associating or cocapping together-for instance, TCR af3 and CD3 (23), CD8 and class I MHC (33, 34), CD4 and LF-1 (35), and CD45 and a variety of independent cell-surface antigens (36, 37). When the T cell binds to the specific PC, some of these molecules are reorganized with the cytoskeletal components into the site of membrane contact (11), thereby demonstrating their connection, either direct or indirect, with the cytoskeleton and with each other. Functionally, mb-induced coaggregation of a number of cell-surface molecules with the TCR has been shown to enhance T-cell activation (10) and may reflect an intercellular binding mechanism dependent on multimolecular associations on the T-cell membrane synergizing to maintain cellular adhesion. No evidence exists that the IL-2 receptor participates in these aggregations, hence the reason we chose it as the negative control molecule. If the nature of the CD4 and CD8 glycoproteins is to aggregate easily with other cell-surface components, then the large number of associated proteins we observed on the gels is not surprising. further signaling role for the accessory molecules has been mooted as a consequence of the observed coupling of CD4 and CD8 to a protein kinase p561ck (38, 39) that is capable of phosphorylating the CD3 complex. Whether CD4 and CD8 are capable of transmitting an independent signal to the T cell or whether they act through CD3 remains to be determined. The fact that TCR-accessory complexes appear to be so critical to T-cell activation implicates the latter option as being more likely in mature T cells. Yet the contribution of the accessory molecules to thymocyte development may hinge on their transmission of a signal separate from that of CD3, leading to CD4 being expressed with a class II-

5 10048 Immunology: Gallagher et al. restricted TCR and CD8 being expressed with class I- restricted TCRs as differentiation progresses. In conclusion, we have provided immunochemical evidence for the association of the class II-restricted TCR with CD4 and shown that in the absence of antigen-mhc, a given class II-restricted TCR is capable of associating with either CD4 or CD8. These results emphasize the need for a tripartite binding event between the TCR-accessory molecule complex and antigen-mhc and directly implicate the MHC molecule as the driving stimulus for increased interaction of the TCR with its appropriate accessory molecule. We are grateful to Prof. J. W. Goding and Dr.. W. Boyd for critical appraisal of the manuscript. This study was supported by grants from The National Health and Medical Research Council of ustralia, the Utah Foundation, the Buckland Foundation, the Jack Brockhoff Foundation, the Sunshine and H. B. McKay Charitable Trust, and the Multiple Sclerosis Foundation of ustralia. 1. Marrack, P. & Kappler, J. (1986) dv. Immunol. 38, Doyle, C. & Strominger, J. L. (1987) Nature (London) 330, Norment,. M., Salter, R. D., Parham, P., Engelhard, V. H. & Littman, D. R. (1988) Nature (London) 336, Rosenstein, Y., Ratnofsky, S., Burakoff, S. J. & Herrmann, S. H. (1989) J. Exp. Med. 169, Fazekas de St. Groth, B., Gallagher, P. F. & Miller, J. F.. P. (1986) Proc. Natl. cad. Sci. US 83, Emmrich, F., Kanz, L. & Eichmann, K. (1987) Eur. J. Immunol. 17, Janeway, C.., Haque, S., Smith, L.. & Saizawa, K. (1987) J. Mol. Cell. Immunol. 3, Owens, T. & Fazekas de St. Groth, B. (1987) J. Immunol. 138, Emmrich, F., Strittmatter, U. & Eichmann, K. (1986) Proc. Natl. cad. Sci. US 83, Owens, T., Fazekas de St. Groth, B. & Miller, J. F.. P. (1987) Proc. Natl. cad. Sci. US 84, Kupfer,., Singer, S. J., Janeway, C.. & Swain, S. L. (1987) Proc. Natl. cad. Sci. US 84, Saizawa, K., Rojo, J. & Janeway, C.. (1987) Nature (London) 328, Rivas,., Takada, S., Koide, J., Sonderstrup-McDevitt, G. & Engelman, E. G. (1988) J. Immunol. 140, nderson, P., Blue, M.-L. & Schlossman, S. F. (1988) J. Immunol. 140, Rojo, J. M., Saizawa, K. & Janeway, C.. (1989) Proc. Natl. cad. Sci. US 86, O'Neill, H. C., McGrath, M. S., llison, J. P. & Weissman, I. L. (1987) Cell 49, Proc. Natl. cad. Sci. US 86 (1989) 17. Staerz, U. D., Rammensee, H.-G., Benedetto, J. D. & Bevan, M. J. (1985) J. Immunol. 134, Leo, O., Foo, M., Sachs, D. H., Samelson, L. E. & Bluestone, J.. (1987) Proc. Natl. cad. Sci. US 84, Lanier, L. L., Gutman, G.., Lewis, D. E., Griswold, S. T. & Warner, N. L. (1982) Hybridoma 1, Dialynas, D. P., Wilde, D. B., Marrack, P., Pierres,., Wall, K.., Havran, W., Otten, G., Loken, M. R., Pierres, M., Kappler, J. & Fitch, F. W. (1983) Immunol. Rev. 74, Ledbetter, J.. & Herzenberg, L.. (1979) Immunol. Rev. 47, Ceredig, R., Lowenthal, J. W., Nabholz, M. & MacDonald, H. R. (1985) Nature (London) 314, Oettgen, H. C., Pettey, C. L., Maloy, W. L. & Terhorst, C. (1986) Nature (London) 320, Goding, J. W. & Harris,. W. (1981) Proc. Natl. cad. Sci. US 78, Gallagher, P. F., Fazekas de St. Groth, B. & Miller, J. F.. P. (1986) Eur. J. Immunol. 16, Dialynas, D. P., Quan, Z. S., Wall, K.., Pierres,., Quintans, J., Loken, M. R., Pierres, M. & Fitch, F. W. (1983) J. Immunol. 131, Gabert, J., Langlet, C., Zamoyska, R., Parnes, J., Schmitt- Verhulst,.-M. & Malissen, B. (1987) Cell 50, Dembic, Z., Haas, W., Zamoyska, R., Parnes, J., Steinmetz, M. & von Boehmer, H. (1987) Nature (London) 326, Ballhausen. W. G., Reske-Kunz,. B., Tourvielle, B., Ohashi, P. S., Parnes, J. R. & Mak, T. W. (1988) J. Exp. Med. 167, Blackman, M.., Kappler, J. W. & Marrack, P. (1988) Immunol. Rev. 101, Jones, B., Khavari, P.., Conrad, P. J. & Janeway, C.. (1987) J. Immunol. 139, Greenstein, J. L., Kappler, J., Marrack, P. & Burakoff, S. J. (1984) J. Exp. Med. 159, Blue, M.-L., Craig, K.., nderson, P., Branton, K. R. & Schlossman, S. F. (1988) Cell 54, Bushkin, Y., Demaria, S., Le, J. & Schwab, R. (1988) Proc. Natl. cad. Sci. US 85, Kupfer,. & Singer, S. J. (1988) Proc. Natl. cad. Sci. US 85, Bourguignon, L. Y. W., Hyman, R., Trowbridge, I. & Singer, S. J. (1978) Proc. Natl. cad. Sci. US 75, Ledbetter, J.., Tonks, N. K., Fischer, E. H. & Clark, E.. (1988) Proc. Natl. cad. Sci. US 85, Veillette,., Bookman, M.., Horak, E. M. & Bolen, J. B. (1988) Cell 55, Barber, E. K., Dasgupta, J. D., Schlossman, S. F., Trevillyan, J. M. & Rudd, C. E. (1989) Proc. Natl. cad. Sci. US 86, Boyse, N. W., Jonsson, J. I., Emmrich, F. & Eichmann, K. (1988) J. Immunol. 141, Goding, J. W. (1980) J. Immunol. 124,