et al., ibid., p. 76; A. Traunecker, W. Luke, K. Karjalainen, ibid., p. 84. 15. R. E. Hussey et al., ibid., p. 78. 16. D. Capon et al., ibid. 337, 525 (1989); A. Traun- ccker et al., ibid. 339, 68 (1989). 17. D. Lamarre, T. Gregory, D. Capon, R.-P. Sekaly, Vaccines 89 (Cold Spring Harbor Laboratories, Cold Spring Harbor, NY, 1989), p. 103. 18. B. C. Cunningham, P. Jhurani, P. Ng, J. A. Wells, Science 243, 1330 (1989). 19. D. R. Littman and S. Gettner, Nature 325, 453 (1987). 20. D. Lamarre et al., unpublished. 21. J. L. Greenstein, J. Kappler, P. Marrack, S. J. Burakoff, J. Exp. Med. 159, 1213 (1984). 22. D. Lamarre et al., EMBOJ., in press. 23. E. A. Berger, T. R. Fuerst, B. Moss, Proc. Natl. Acad. Sci. U.S.A. 85, 2357 (1988). 24. T cell hybridomas expressing the mutant CD4 mole- cules were preincubated with the appropriate mu- rine antihuman CD4 monoclonal antibody (MAb) at 20 nM for 60 min at 37°C prior to addition of 105 DAP-3 DdDP cells. After a 20-hour co-culture, the culture supematants were assayed for the pres- ence of IL-2 using the IL-2-dependent cell line CTLL.2. The results are expressed as units of IL-2 per milliliter. MI, M2, M4, M7, and M8 expressed levels of the OKT4B epitope identical to the cells expressing wild-type CD4 and were treated with the OKT4B antibody. Monoclonal antibody (MAb) inhibition experiments with M3, M5, and M6 were performed in the presence of Leu3a antibody since these mutants expressed level of Leu3a comparable to those of cells expressing wild-type CD4. 25. V. D. Stacy et al., Proc. Natl. Acad. Sci. U.S.A. 81, 1799 (1984). For the stimulation of hybridomas with MAbs specific to the TcR, culture plates were coated ovemight with protein A-purified F23.1 (10 Lg/ml), a MAb specific to the variable region V,B8. CR-y& T LYMPHOCYTES CONSTITUTE a distinct T celllineage (1% to 10% of CD3+ T cells) with cytolytic activ- ity and the capacity to produce lymphokines (1). The major unresolved question con- ceming TCR-y6 T cells is the nature of the ligands they recognize. The TCRa,B reper- L. A. Matis, A. M. Fry, M. M. Cotterman, Division of Biochemistry and Biophysics, Center for Biologics Eval- uation and Research, Food and Drug Administration, Bethesda, MD 20892. R. Q. Cron, R. F. Dick, J. A. Bluestone, Ben May Institute, Department of Pathology, University of Chica- go, Chicago, IL 60637. 746 Mutants (10' cells) were incubated in these plates for 20 hours and culture supematants were tested for the presence of IL-2 with the IL-2-dependent CTLL line. Results are presented as units of IL-2 per ml of culture supematant. 26. L. K. Clayton et al., Nature 339, 548 (1989). 27. H. B. Chao et al., J. Biol. Chem. 264, 5812 (1989). 28. D. C. Diamond et al., J. Immunol. 141, 3715 (1989); K. J. Weinhold et al., ibid. 142, 3091 (1989). 29. Y. Thomas, L. Rogozinski, L. Chess, Immunol. Rev. 74, 113 (1983); Q. J. Sattentau, A. G. Dalgleish, R. A. Weiss, P. C. Beverley, Science 234, 1120 (1986). 30. M. R. Davis and P. J. Bjorkman, Nature 334, 395 (1988). 31. The dissociation constants (Kd) for rgpl2O-CD4 interaction were measured by saturation binding analysis of 251-labeled rgpl2O to intact 3DT52.5.8- transduced cells. Cells (0.5 x 106 to 1.0 x 106) were incubated with 125I-labeled rgpl20 at concentra- tions ranging from 0.1 to 20 nM (1 hour, 37C). Nonspecific binding was measured in the presence of 200-fold excess of unlabeled rgpl2O. The values reported are means ± SD of at least two indepen- dent experiments done in triplicate. 32. M. Zoller and M. Smith, Nudeic Acids Res. 10, 6487 (1982). 33. We wish to thank S. Marsters, G. Croteau, and V. Foster for their expert technical assistance and N. Guay for her kind assistance in typing this manu- script. We are indebted to R. Ward for her critical comments on the manuscript. Supported by grants from the Medical Research Council of Canada (MRC-10.055 and MRC 10116) from the National Cancer Institute of Canada, by a fellowship to D.L. from the Cancer Research Society Inc. and by Genentech Inc. 13 June 1989; accepted 11 July 1989 toire displays specificity for both class I and class II MHC (Ia) antigens. Several develop- mental parallels between TCRy& and TCRoa3 suggest that some TCRy& might also recognize MHC antigens. For example, the TCRB locus is situated with the TCRa locus and V,,. gene elements can rearrange productively to DJb (2-4). In this light, several murine and human alloreactive TCRyb T cell lines and clones specific for class I MHC antigens have been identified (3, 5). Evidence for different anti- gen-processing pathways for presentation of peptides by class I versus class II MHC molecules (6) suggests that class I and class II MHC-restricted T cells may recognize distinct sets of foreign protein antigens. Therefore, in order to better characterize the potential diversity of the MHC-specific TCR-yb repertoire, it was important to es- tablish whether TCR-yb T cells have the capacity for class II MHC molecule recogni- tion. Alloreactive T cell lines were derived from the peripheral lymph nodes of athymic B10 (H-2b) nu/nu mice by in vitro stimulation of T cells with H-2-congenic B10.BR (H-2k) splenic antigen-presenting cells (APCs) (7). The structure of the receptor proteins ex- pressed by these lines was examined by performing immunoprecipitations with a monoclonal antibody specific for the CD3e chain of the TCR complex (8). One CD3+CD4+CD8- line, LBK4, expressed a CD3-associated heterodimer with a molecu- lar mass of 41 to 43 kD, which appeared to be an a,B receptor (Fig. IA and Fig. 1B, lane 1). That LBK4 was an o,B T cell line was confirmed by showing that an antiserum to Cpl (9) precipitated an identical 41- to 43- kD heterodimer (Fig. 1B, lane 2). A second independently derived line, LBK5, with a CD3+CD4-CD8- surface phenotype, expressed a markedly different CD3-associated heterodimer consisting of proteins of 31 and 45 kD (Fig. 1C). A clone derived from this line, G11, expressed an identical heterodimer (Fig. ID, lane 1). After reduction and alkylation of the immu- noprecipitate of the G 1 clone obtained with an antibody to CD3, the 31-kD and 45-kD proteins were immunoprecipitated by an antiserum to the CG,l/Cy2 peptide (9) (Fig. 1D, lane 3) and an antiserum to TCRb (10) (Fig. 1D, lane 2), respectively. The specificities of the TCRao-bearing LBK4 line and the TCR-yb-expressing LBK5 line were tested in cell-mediated cyto- toxicity assays with target cells from various H-2 recombinant mouse strains (Fig. 2A). Because TCRY8 T cells cultured in the pres- ence of exogenous growth factors may de- velop nonspecific cytolytic activity (5), the assays of cytolytic T lymphocytes (CTLs) were performed with cells that had been cultured in the absence of interleukin-2 (IL- 2) for 24 to 48 hours. First, the MHC- linked specificity of both lines was shown by the fact that they lysed H-2 congenic B10.BR (H-2k) (not shown) and B10.A (H-25), but not syngeneic B10 (H-2b) or allogeneic B10.D2 (H-2d) target cells (Fig. 2A, Exp. 1). The LBK5 but not the LBK4 T cells also killed B10.A(5R) (H-2'5), BlO.S(9R) (H-2t4), and B10.RIII (H-2r) targets (Fig. 2A, Exp. 2). More detailed analysis of the specificity of the -yb receptor expressed by LBK5 was SCIENCE, VOL. 245 Structure and Specificity of a Class II MHC Alloreactive y8 T Cell Receptor Heterodimer Louis A. MATIs, ALiCIA M. FRY, RANDY Q. CRON, MELISSA M. COTrERMAN, ROBERT F. DIcK, JEFFREY A. BLUESTONE Two distinct CD3-associated T cell receptors (TCRac and TCR-y8) are expressed in a mutually exclusive fashion on separate subsets of T lymphocytes. While the specificity of the TCRa3 repertoire for major histocompatibility complex (MHC) antigens is well established, the diversity of expressed 'yS receptors and the ligands they recognize are less well understood. An alloreactive CD3+CD4-CD8- T cell line specific for murine class II MHC (Ia) antigens encoded in the I-E subregion of the H-2 gene complex was identified, and the primary structure of its y8 receptor heterodimer was characterized. In contrast to a TCRac-expressing alloreactive T cell line selected for similar specificity, the TCRy8 line displayed broad cross-reactivity for multiple distinct I-Eencoded allogeneic Ia molecules. on March 26, 2016 Downloaded from on March 26, 2016 Downloaded from on March 26, 2016 Downloaded from on March 26, 2016 Downloaded from