Early Stages of Thymocyte Differentiation (Intrathymic Maturation/Thymocyte Subsets/Murine Thymic Lymphoma) ELLEN R

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Proc. Nati. Acad. Sci. USA Vol. 85, pp. 1174-1178, February 1988 Immunology a/fJ T-cell antigen receptor gene and protein expression occurs at early stages of thymocyte differentiation (intrathymic maturation/thymocyte subsets/murine thymic lymphoma) ELLEN R. RICHIE*t, BRENDA MCENTIRE*, NICK CRISPEt, JOHN KIMURA§, LEWIS L. LANIER¶, AND JAMES P. ALLISON§ *The University of Texas System Cancer Center, Science Park-Research Division, Smithville, TX 78957; tThe Research Institute of Scripps Clinic, La Jolla, CA 92037; §Cancer Research Laboratory, The University of California at Berkeley, Berkeley, CA 94720; and lBecton Dickinson Immunocytometry Systems, Mountain View, CA 94043 Communicated by Marian E. Koshland, October 12, 1987 (received for review May 22, 1987)

ABSTRACT Alterations in gene expression that orches- on differentiating thymocytes contribute significantly to this trate eukaryotic cellular differentiation often require appro- process (9). Ifthe antigen receptor is a critical determinant of priate interactions between differentiating cells and a special- positive and/or negative selection, one would expect to find ized microenvironment. During T-lymphocyte differentiation, antigen receptor expression on thymocytes at a relatively immature thymocytes undergo a stringent intrathymic selec- early stage in the maturation process. tion process that requires intimate contact with thymic stromal Previous reports support this general concept. KJ16 is an elements. Since this selection process generates T cells that are antibody that detects a determinant encoded by members of self-tolerant and recognize nominal antigen only within the the V/38 family of T-cell antigen receptor p-chain genes (10). context of self-major histocompatibility antigen complex mol- Approximately 20% of mature thymocytes stain brightly ecules, it is possible that thymocyte/stromal cell interactions with KJ16, while ==10% of immature thymocytes stain are mediated, in part, by antigen-specific receptors expressed weakly with this antibody in immunofluorescence assays on differentiating thymocytes. However, the developmental (11). By extrapolation, these data suggest that essentially all stage at which a/fl antigen-specific receptors are expressed of the mature subsets and =50% of immature normal thy- during T-cell maturation has been a matter of debate. To mocytes express antigen receptor molecules. Also, a recent address this issue, we have studied a/f8 T-cell antigen recep- study involving three-color flow cytometric analysis of hu- tor gene and protein expression on normal thymocyte subsets man thymocytes demonstrated that 78% of cells displaying a of AKR/J mice, as well as on a panel of AKR/J primary cortical differentiation antigen phenotype react with WT31, thymic lymphomas characterized for CD4 (L3T4) and CD8 a monoclonal antibody that detects a shared epitope on (Lyt-2) differentiation antigen expression. The data unequiv- human a/l, antigen receptors (12). Nevertheless, expression ocally demonstrate that a/fl heterodimers are expressed not of antigen receptor a/,1 heterodimer on immature thymocyte only on phenotypically mature thymocytes but also on the subsets has been a controversial issue (13, 14). majority of CD4+8+ double-positive cells that comprise the In this report, we examine antigen receptor a/,B hetero- predominant nonmature thymocyte subset. Furthermore, a dimer expression on normal thymocyte subsets of AKR/J fraction of thymocytes in the CD4-8- double-negative com- mice and a panel of AKR/J primary thymic lymphomas partment, known to contain progenitor cells, also expresses characterized for CD4 and CD8 differentiation antigen readily detectable cell-surface a/fl receptors. Therefore, dur- expression. Individual thymic lymphomas are further ana- ing the process of intrathyinic selection, interactions between lyzed for P-chain gene rearrangements as well as for a-chain nonmature thymocytes and stromal cells via the antigen-re- and 8-chain mRNA transcripts. We find that a/P antigen ceptor complex may play a pivotal role in T-cell differentiation receptor molecules are expressed not only on most pheno- and should be considered in formulating schemes for func- typically mature cells, but also on the majority of CD4+8+ tional T-cell selection. double-positive cells as well as on a fraction of cells in the CD4-8- double-negative subset. These data suggest that T-cell differentiation occurs under the influence of the thy- both phenotypically mature and nonmature thymocyte sub- mic microenvironment within which bone marrow-derived sets contain cells that are candidate populations for an progenitor cells proliferate and differentiate to generate the intrathymic selection process. small fraction of immunocompetent cells that emigrates to peripheral lymphoid tissues. Intrathymic residence is a critical step in the evolution and diversification of a functional MATERIALS AND METHODS T-cell antigen receptor repertoire. Thymocyte maturation Animals and Source of Primary Lymphomas. AKR/J mice involves a stringent selection process that generates mature were purchased from The Jackson Laboratory (Bar Harbor, T cells that are self-tolerant yet able to recognize antigen ME). Spontaneous lymphomas were obtained from 6-mo or only within the context of self-major histocompatibility older untreated mice. N-methyl-N-nitrosourea (MNU)- complex (MHC) determinants (1-5). Although the intrathy- induced thymic lymphomas were obtained from 4- to 5-mo- mic selection process is poorly understood, interactions old mice previously injected with MNU (75 mg/kg) at 7 wk between developing thymocytes and stromal elements in the of age. MNU-treated mice were sacrificed before they were thymus appear to be essential in establishing both tolerance 6 months old. and MHC restriction (6-8). In view of the specific restric- Immunofluorescence and Fluorescence-Activated Cell Sort- tions imparted to developing T cells via such interactions, er (FACS) Analysis. For single-color analyses, cells were we have suggested that T-cell antigen receptors expressed Abbreviations: MHC, major histocompatibility complex; MNU, The publication costs of this article were defrayed in part by page charge N-methyl-N-nitrosourea; FITC, fluorescein isothiocyanate; APC, payment. This article must therefore be hereby marked "advertisement" allophycocyanin; PE, phycoerythrin. in accordance with 18 U.S.C. §1734 solely to indicate this fact. tTo whom reprint requests should be addressed. 1174 Downloaded by guest on September 24, 2021 Immunology: Richie et al. Proc. Natl. Acad. Sci. USA 85 (1988) 1175 incubated with anti-CD4 (hybridoma GK1.5) (15) or anti- A. Ungated Thymocytes B. F23.1 Expression by Thymocytes u CD8 (hybridoma 53-6.7) (16), washed, and incubated with 10% 70% fluorescein isothiocyanate (FITC)-conjugated mouse anti-rat immunoglobulin Cl) (Jackson ImmunoResearch; Avondale, < 1023. PA). In the three-color immunofluorescence experiments, \Lo Hi thymocytes were stained with FITC-conjugated anti-CD8, 9.1% 3.1% biotin-conjugated anti-CD4, and arsenyl-derivatized F23.1 z monoclonal antibody (17). After washing, cells were incu- 101 E X bated with allophycocyanin (APC)-streptavidin, kindly pro- I. 2.7%l vided by Becton-Dickinson (Mountain View, CA), and phy- 10 101C 102 103 104 100 101 102 103 104 coerythrin (PE)-coupled rabbit anti-arsenyl antibody. Immu- CD8 FITC F23.1 Ars: PE a-Ars nofluorescence was analyzed on a FACS (FACS IV; Becton-Dickinson) using a 488-nm argon ion laser. Dead C. F23.1 LO Thymocytes D. F23.1 Hi Thymocytes 104 cells were excluded from analysis by forward light scatter 0 69% (18). In three-color immunofluorescence, fluorophores were 0 4% 90% 14% I 103 excited with a 488-nm argon laser (FITC and PE) and a dye _. --p 103/ ,'* laser emitting at 620 nm (APC). Three-color fluorescence 2E q. data were analyzed with Consort 30 software, which allows m.° 102 0 102 two parameters (forward light scatter and/or one or two u I fluorescence signals) to be used for selective gating. r001 010 7.1% 9.1% Radiolabeling, Immunoprecipitation, and Electrophoresis. I. 1003.6% 2.1% *° Cells were surface-labeled with 1251 by lactoperoxidase- catalyzed iodination and extracts were subjected to immu- 100 101 102 103 104 100 101 102 103 104 CD8 FITC CD8 FITC noprecipitation as described, using antiserum 8177 (19). Two-dimensional NaDodSO4/PAGE was performed in FIG. 1. Expression of a/8 antigen receptor by murine thy- which the first dimension was run on 7.5% gels under mocyte subpopulations. Normal AKR/J thymocytes were stained nonreducing conditions followed by NaDodSO4/PAGE on with FITC-conjugated anti-CD8, biotin-conjugated anti-CD4, ar- 10% gels under reducing conditions in the second dimension. senyl (a-Ars)-derivatized anti-V,38 (F23.1), streptavidin-APC Nucleic Acid Extraction and Hybridization Analysis. High (AvAPC), and PE-conjugated rabbit anti-arsenyl antibody. The molecular weight DNA was prepared from 108 cells by percentages given indicate the percentage of gated cells falling within the indicated quadrant of the two-dimensional contour plots proteinase K digestion followed by spermine extraction (20). (A, C, and D) or within the indicated gates of the histogram (B). (C DNA was precipitated with ethanol, redissolved in appro- and D) Two-dimensional contour plots of cells exhibiting low (9.1% priate buffers (21), digested with restriction endonucleases of total) or high (3.1% of total) levels of staining, respectively, with (New England BioLabs), and electrophoresed. Southern the F23.1 antibody. blots were hybridized with a nick-translated probe using dextran sulfate and formamide and analyzed by autoradiog- CD4 + or CD8 + single-positive immunocompetent thy- raphy using Kodak X-Omat AR film and an intensifying mocytes. These results are also consistent with a previous screen at - 70°C (22-24). Total cellular RNA was isolated report showing that staining of immature thymocytes with an from approximately 108 cells using vanadylribonuclease anti-V,88 antibody is less intense than that observed for complex and electrophoresed in 1.1% agarose containing 2.2 mature cells (11). The thymocyte sample analyzed also M formaldehyde (21). After transfer to nitrocellulose, the showed a disparity between F23.1 expression on CD4+ blots were hybridized as described above. The C,31, J.31, and single-positive and CD8+ single-positive thymocytes. This J2 probes were gifts from Leroy Hood (25). The Ca probe disparity was particularly evident with regard to the F23.1 was provided by Astar Winoto (26). bright cells; CD8+ single-positive cells expressed a high level of F23.1 on 10.3% of cells, whereas CD4+ single- RESULTS positive cells were 21.2% F23.1 high. T-Cell Antigen Receptor Expression on Thymic Lym- T-Cell Antigen Receptor Expression on Normal Thymocyte phomas. Further evidence supporting antigen receptor Subsets. The expression of antigen receptor molecules on expression on phenotypically nonmature T cells was ob- thymocyte subsets defined by CD8 and CD4 differentiation tained from analyses of primary murine thymic lymphomas, antigen phenotypes was examined by multicolor immuno- which we regard as monoclonal proliferations representative fluorescence. This technique allows simultaneous detection of normal thymocyte subsets. Indirect immunofluorescence of three distinct cell-surface antigens. To detect antigen analyses of CD4 and CD8 antigens on a series of thymic receptor expression, thymocytes were stained with arso- lymphomas identified tumors expressing double-negative, nate-conjugated F23.1 monoclonal antibody, which detects double-positive, or single-positive phenotypes. To investi- an allotypic determinant on antigen receptor 3 chains of gate the relationship between antigenic phenotype and anti- 20-25% of peripheral T-cells of both CD4 and CD8 single- gen positive subsets (17, 27, 28). As shown in Fig. 1, -9.1% of receptor expression, lymphomas were radioiodinated AKR thymocytes displayed positive but dull staining, while Table 1. Expression of F23.1 by thymocyte subpopulations =3.1% displayed bright staining for the F23.1 determinant. Electronic gates were set on the dull- and bright-staining %4-,8- %4+,8+ %4+,8- %4-,8+ subsets for further analysis of CD4 and CD8 antigen expres- F23.1 low (120-169) 1.9 11.6 3.8 7.0 sion. As illustrated in Fig. 1 and summarized in Table 1, the F23.1 high (169-235) 1.3 0.6 21.2 10.3 F23.1 bright cells are localized predominantly to the pheno- F23.1 total (120-235) 3.0 12.2 24.9 17.2 typically mature CD4+ or CD8+ single-positive subsets. In Calculated expression contrast, the vast majority of F23.1 dull cells is present in the of ( chain* 12 49 100 69 CD4 +8 + double-positive thymocyte subset, while a fraction Thymus was from 16-week-old AKR/J. Distribution of pheno- of F23.1 dull-staining cells is localized to the double-negative types was as follows: 4-,8-, 17%; 4+,8+, 70.1%; 4+,8-, 10%; subset. These data clearly demonstrate that antigen receptor 4-,8+, 2.7%. expression is not restricted to the mature subsets containing *Based on expression in periphery and 4+,8- of 25%. Downloaded by guest on September 24, 2021 1176 Immunology: Richie et al. Proc. Natl. Acad. Sci. USA 85 (1988) and subjected to immunoprecipitation with antiserum 8177, Table 2. Summary of T-cell antigen receptor a- and (3-chain which is specific for framework epitopes borne by murine transcripts and heterodimer expression on phenotypically T-cell antigen receptors (19). The immunoprecipitates were characterized thymic lymphomas subjected to diagonal gel electrophoresis. Representative 1.5-kb 1.3-kb results are shown in Fig. 2 and the compiled data are a-chain (-chain Cell-surface T-cell summarized in Table 2. As in the above analyses of normal Lymphoma CD4 CD8 mRNA mRNA antigen receptor thymocyte subsets, antigen receptors are expressed on lymphomas from each phenotypic group defined by CD4 and 705 - - _ + CD8 antigen profiles. Seven of nine tumors that displayed a 1019 - - - + cortical phenotype (i.e., CD4+8+) expressed readily detect- 621 - - + + + able a/(, antigen receptor molecules, again indicating that 629 - - + + receptor expression is not restricted to cells at a relatively 728M + + + + mature stage of differentiation. Furthermore, one lymphoma 812M + + + + 522 + + + + + in the CD4-8- group, a phenotype that corresponds to an 524 + + + + + immature precursor stage in T-cell development (29, 30), 830 + + + + + expressed readily detectable a/(3 receptors. Among the 1009M + + + + + CD8 + single-positive lymphomas, most but not all ex- 814 + + + + + pressed a/P antigen receptors, while each of the CD4' 920 + + + + + single-positive lymphomas was a/(3 receptor positive. These 1010 + + + + + results precisely parallel the findings from the three-color 722M - + + + immunofluorescence analysis of normal AKR thymocytes. 903M - + + + Rearrangement and Expression of T-Cell Antigen Receptor 720M - + + + + a- and f-Chain Genes in Thymic Lymphomas. Southern blots 918M - + + + + of restriction endonuclease digested tumor DNA were hy- 1018M - + + + + bridized with probes to the J131 and J,32 gene segments (25). 829M - + + + + All of the lymphomas in each phenotypic group contained 727 + - + + + one or more antigen receptor (-chain gene rearrangements 904 + - + + + (data not shown). To determine whether transcription of antigen receptor a- or 8-chain genes correlated with cell- M, MNU-induced lymphoma. surface protein expression, RNA blot analyses were performed. As illustrated in Fig. 3 and summarized in Table 2, 3-chain transcripts. However, a-chain transcripts were not each of the antigen receptor-positive lymphomas studied detected in two antigen receptor-negative lymphomas, both contained full-length 1.3-kilobase (kb) and truncated 1.0-kb of which displayed a CD4-8- double-negative phenotype, (3-chain transcripts as well as 1.6-kb a-chain transcripts. All corresponding to the most primitive thymocyte subset (29, of the contained 30). The remaining antigen receptor-negative lymphomas, of receptor-negative lymphomas full-length both CD4+8+ and CD4-8' surface phenotypes, contained NR _ a- as well as (3-chain transcripts, indicating that full-length a- REDI and (-chain mRNA transcripts are necessary but insufficient for expression of antigen receptor protein at the cell surface.

-88K DISCUSSION I This investigation demonstrates that a/P antigen receptor -43K expression occurs on thymocytes within each of the four major subsets of thymocytes defined by CD4 and CD8 differentiation antigen expression. Earlier studies estab- -29K lished the presence of antigen receptor-bearing thymocytes in phenotypically and functionally mature thymocyte subsets (11-14). However, whether antigen receptors are ex- A. 8177 antiserumn pressed on nonmature cortical-derived thymocytes has been NFR a controversial issue. The present data demonstrate that a major fraction of CD4+8 + normal thymocytes express anti- RED gen receptors, although the relative level of expression is I lower than that observed on single-positive cells. This is in substantial agreement with the recent demonstration by -68K Bluestone et al. that murine CD4+8' cells express low levels of CD3 (31). Even in the CD4-8- subset, a small but -43K significant fraction of cells expresses detectable a/(3 receptors. The thymic lymphoma data not only agree precisely with the observations on normal thymocytes but also pro- -29K vide further information on antigen receptor gene and protein expression in monoclonal populations. Seven ofthe nine CD4+8+ lymphomas expressed a/(3 antigen receptors, and all lymphomas in this phenotypic group contained appar- B. Normal rabbit serum ently full-length a-chain and (-chain transcripts. This obser- demon- FIG. 2. Diagonal NaDodSO4/PAGE analysis of "2I surface- vation agrees with the report of Kinnon et al. (32) labeled proteins from T-lymphoma 115M. (A) T-cell antigen receptor strating that cells of the major cortical lineage express high heterodimer isolated by immunoprecipitation with antiserum 8177 levels of these transcripts. The presence of the a- and that recognizes framework determinants on a/,8 receptor molecules. (3-chain transcripts is necessary, but it does not ensure (B) Normal rabbit serum control. Numbers on right represent kDa. antigen receptor expression. Possibly, the lymphomas lack- Downloaded by guest on September 24, 2021 Immunology: Richie et A Proc. Natl. Acad. Sci. USA 85 (1988) 1177 A only the CD8 single-positive subset was found to contain a functionally incompetent subset defined by expression of the TCR NegDative TCR Positive In cortical thymocyte marker Jl1d (39). -10: Previous studies have concluded that CD4-8- double- 1* negative thymocytes express mRNA encoding X-chain but not a-chain message, although recent work suggests that a subset of these cells expresses both types of transcripts (32, 40, 41). The double-negative lymphomas analyzed in this report are consistent with these findings in that two tumors did not contain a-chain transcripts while two other lym- ww116kb phomas contained both a- and p-chain transcripts. Interest- ingly, one of the latter lymphomas expressed cell-surface a/13 receptor and may represent an intermediate stage in maturation prior to acquisition of CD4 or CD8 molecules. Thus, as other investigators have suggested, we speculate 1 23 4 5 6 7 8 that the double-negative population may be heterogeneous B with respect to differentiation stage and possibly lineage TCR Negative TCR Positive commitment (30, 32). Since the double-negative lymphomas Fr...... ------.-, that lacked a-chain transcripts failed to express a cell-

1,q (ID "P lb 0 surface heterodimer component, it is unlikely that they rl, , 4V NN ". 'o 0,(.) ,.D t.v "'Oe IFJ Cb et, represent the y/8 receptor-bearing double-negative thymocyte subset (42). In light of the events involved in the process of elaborating functional T cells, expression of antigen receptor by immature thymocytes is not surprising. Differentiating thymocytes appear to require interaction with thymic stromal cells to acquire self-tolerance and MHC restriction. It seems unlikely that these selective events would occur in the absence of antigen receptor expression. Interactions between antigen receptor molecules expressed on immature thymocytes and ligands on thymic stroma might generate 1 2 3 4 5 6 7 8 9 10 appropriate signals for further T-cell maturation. Indeed, immunohistochemical localization studies with KJ16 anti- FIG. 3. RNA blot analysis of T-cell antigen receptor (TCR) a- and p-chain RNA transcripts. (A) Lanes: 1-3, a-chain RNA tran- body revealed that antigen receptors expressed on cortical scripts from TCR-negative lymphomas; 4-7, TCR-positive lym- thymocytes were frequently in intimate contact with pro- phomas; 8, normal thymus. A C. probe was used to reveal a-chain cesses of thymic epithelial cells (43). The accessory mole- gene transcription. (B) Lanes: 1-4, (3-chain transcripts from TCR- cules CD8 and CD4 are thought to stabilize interactions negative lymphomas; 5-9, TCR-positive lymphomas; 10, normal between low-affinity T-cell receptors and antigen-presenting thymus. A Cal probe was used to reveal 3-chain gene transcription. cells, perhaps as a result of recognition by CD8 and CD4 of monomorphic epitopes on class I or class II MHC mole- ing cell-surface a/18 heterodimer contained sterile transcripts cules, respectively (44-46). Similarly, these accessory mol- or defects in synthesis or processing of CD3-like compo- ecules may stabilize antigen receptor-mediated thymocyte- nents, which are thought to be required for cell-surface stromal cell interactions, particularly in view of our findings expression of the antigen-receptor complex (33-36). that the majority of thymocytes express low levels of a//3 One consequence of intrathymic differentiation is the receptor. If cells in the double-positive compartment partic- somatic rearrangement of T-cell antigen receptor gene seg- ipate in intrathymic selection, via a/(8 T-cell receptors, it ments (25, 26). Since rearrangement is an imprecise process, seems reasonable that the expression of both CD8 and CD4 only one in three rearrangements is expected to yield in- molecules would facilitate the selection process. Since at frame junctions. Thus, it seems likely that the frequency of least 90% of thymocytes die in situ (47, 48), those few cells productive rearrangements would be in the minority and well that are positively selected might then undergo final matu- below the frequency of antigen receptor-positive thymocytes ration, including loss ofCD8 or CD4 molecules depending on noted in this and a previous study (12). One explanation for whether selection for class I or class II restriction has the high frequency observed is that the double-positive occurred. cortical thymocytes are under selective pressure for expres- In related studies, we have recently determined that the sion of antigen receptor protein because this compartment antigen receptor expressed by CD4+ 8 + thymocytes is capa- may be of central importance in the selection offunctional T ble of mediating the proximal events of transmembrane cells during thymocyte differentiation. However, contro- signaling associated with activation in mature T cells, al- versy exists as to whether cells in the double-positive subset though the CD4+8+ cells do not respond to this signal by give rise to more differentiated progeny or represent a proliferation (49). Any putative transition of CD4+8+ cells terminally differentiated state (37, 38). expressing antigen receptor at low density to high-density Among the CD8+ single-positive lymphomas, as among mature cells must therefore be accompanied by a maturation normal thymocytes of the same phenotype, there were cells event related to the manner in which the cells perceive the that lacked detectable a/,8 receptors on the surface, al- receptor-mediated signal, and not merely by acquisition of though they expressed full-length a- and P-chain mRNA. antigen receptor capable of signaling. On the other hand, it is However, the majority of the CD8+ lymphomas, and all of also possible that the inability ofCD4+8+ cells to respond to the CD4' lymphomas, expressed surface a/13 receptors as antigen receptor-mediated stimuli by proliferation renders well as full-length mRNA. The disparity in receptor expres- the cells incapable of further maturation. It is clear that an sion between CD8 single-positive and CD4 single-positive understanding of the events in thymocyte maturation will thymocytes has a precedent in functional studies in which require further studies of the response of thymic subpopula- Downloaded by guest on September 24, 2021 1178 Immunology: Richie et al. Proc. Natl. Acad. Sci. USA 85 (1988)

tions to ligand-receptor interactions and the response of the tory, Cold Spring Harbor, NY). cells to signals generated by these interactions. 22. Southern, E. M. (1975) J. Mol. Biol. 98, 503-517. The findings reported here do not, of course, demonstrate 23. Wahl, G. M., Stern, M. & Stark, G. R. (1979) Proc. Natl. lineage relationships between phenotypically distinct thy- Acad. Sci. USA 76, 3683-3687. 24. Smith, G. E. & Summers, M. D. (1980) Anal. Biochem. 109, mocyte subpopulations. However, these results do indicate 123-129. that antigen receptor expression is not an exclusive property 25. Malissen, M., Minard, K., Mjolsness, S., Kronenberg, M., of mature functional T-cells, and that early expression of Goverman, J., Hunkapiller, T., Prystowsky, M. B., Yoshikai, a// antigen receptors must be taken into account in the Y., Fitch, F., Mak, T. W. & Hood, L. (1984) Cell 37, 1101- formulation of schemes for functional T-cell selection during 1110. 26. Winoto, A., Mjolsness, S. & Hood, L. (1985) Nature (London) thymic differentiation. 316, 832-836. 27. Crispe, I. N., Bevan, M. J. & Staerz, U. D. (1985) Nature We thank Drs. Leroy Hood and Astar Winoto for providing the (London) 317, 627-629. DNA probes. We also thank Dawn Walker for excellent technical 28. Epstein, R., Roehm, N., Marrack, P., Kappler, J., Davis, M., assistance and Joyce Mayhugh and Nancy Caplan for help in Hedrick, S. & Cohn, M. (1985) J. Exp. Med. 161, 1219-1224. manuscript preparation. This work was supported by Grants CA- 29. Fowlkes, B. J., Edison, L., Mathieson, B. J. & Chused, T. M. 37912 (to E.R.R.) and CA40041 (to J.P.A.) from the National (1985) J. Exp. Med. 162, 802-822. Institutes of Health (Bethesda, MD) and by a grant from the Bruce 30. Scollay, R. & Shortman, K. (1985) J. Immunol. 134, 3632- McMillan, Jr., Foundation to E.R.R. 3642. 31. Bluestone, J. A., Pardoll, D., Sharrow, S. 0. & Fowlkes, B. J. 1. Zinkernagel, R. M. (1978) Immunol. Rev. 42, 224-236. (1987) Nature (London) 326, 82-84. 2. Fink, P. J. & Bevan, M. J. (1978) J. Exp. Med. 148, 766-773. 32. Kinnon, C., Diamond, R. A. & Rothenberg, E. V. (1986) J. 3. Von Boehmer, H., Haas, W. & Jerne, N. K. (1978) Proc. Natl. Immunol. 137, 4010-4015. Acad. Sci. USA 75, 2439-2443. 33. Ohaski, P. S., Mak, T. W., van den Elsen, P., Yanagi, Y., 4. Kappler, J. W. & Marrack, P. (1976) Nature (London) 262, Yoshikai, Y., Calman, A. F., Terhorst, C., Stobo, J. D. & 727-728. Weiss, A. (1985) Nature (London) 316, 606-609. 5. Klein, J. & Nagy, Z. A. (1982) Adv. Cancer Res. 37, 234-317. 34. Allison, J. P. & Lanier, L. L. (1985) Nature (London) 314, 6. Jordan, R. K., Robinson, J. H., Hopkinson, N. A., House, 107-109. K. C. & Bentley, A. L. (1985) Nature (London) 314, 454-456. 35. Samelson, L. E., Harford, J. B. & Klausner, R. D. (1985) Cell 7. Lo, D. & Sprent, J. (1986) Nature (London) 319, 672-675. 43, 223-231. 8. Von Boehmer, H. & Hafen, K. (1986) Nature (London) 320, 36. Oettgen, H. C., Pettey, C. L., Maloy, V. L. & Terhorst, C. 626-628. (1986) Nature (London) 320, 272-275. 9. Allison, J. P., Lanier, L. L., Guyden, J. & Richie, E. (1986) 37. Lugo, J. P., Krishnan, S. N., Sailor, R. D. & Rothenberg, Fed. Proc. Fed. Am. Soc. Exp. Biol. 45, 376 (abstr.). E. V. (1986) Proc. Natl. Acad. Sci. USA 83, 1862-1866. 10. Haskins, K., Hannum, C., White, J., Roehm, N., Kubo, K., 38. Penit, C. (1986) J. Immunol. 137, 2115-2121. Kappler, J. & Marrack, P. (1984) J. Exp. Med. 160, 452-471. 39. Crispe, I. N. & Bevan, M. J. (1987) J. Immunol. 138, 2013- 11. Roehm, N. L., Herron, J., Cambier, D., DiGuisto, K., Has- 2018. kins, J., Kappler, J. & Marrack, P. (1984) Cell 38, 577-584. 40. Raulet, D. H., Garman, R. D., Saito, H. & Tonegawa, S. 12. Lanier, L. L., Allison, J. P. & Phillips, J. H. (1986) J. Immu- (1985) Nature (London) 314, 103-107. nol. 137, 2501-2507. 41. Samelson, L. E., Lindsten, T., Fowlkes, B. J., van den Elsen, 13. Acuto, O., Hussey, R. E., Fitzgerald, K. A., Protentis, J. P., P., Terhorst, C., Davis, M. M., Germain, R. N. & Schwartz, Meuer, S. C., Schlossman, S. F. & Reinherz, E. L. (1983) Cell R. H. (1985) Nature (London) 315, 765-768. 34, 717-726. 42. Pardoll, D. M., Fowlkes, B. J., Bluestone, J. A., Kruisbeck, 14. Snodgrass, H. R., Kisielow, P., Kiefer, M., Steinmetz, M. & A., Maloy, W. L., Coligan, J. E. & Schwartz, R. H. (1987) von Boehmer, H. (1985) Nature (London) 313, 592-596. Nature (London) 326, 79-81. 15. Dialynas, D. P., Quan, Z. S., Wall, K. A., Pierres, A., Quin- 43. Farr, A. G., Anderson, S. P., Marrack, P. & Kappler, J. (1985) tans, J., Loken, M. R., Pierres, M. & Fitch, F. W. (1983) J. Cell 43, 543-550. Immunol. 131, 2445-2451. 44. Meuer, S. C., Schlossman, S. F. & Reinherz, E. R. (1982) 16. Ledbetter, J. A. & Herzenberg, L. A. (1979) Immunol. Rev. Proc. Natl. Acad. Sci. USA 79, 4395-4399. 47, 63-90. 45. Marrack, P., Endres, R., Schimonkevitz, R., Szlotnik, A., 17. Staerz, U. D., Rammensee, H.-G., Benedetto, J. & Bevan, Dialynas, D., Fitch, F. & Kappler, J. (1983) J. Exp. Med. 158, M. J. (1985) J. Immunol. 34, 3994-4000. 1077-1091. 18. Loken, M. R. & Herzenberg, L. A. (1975) Ann. N.Y. Acad. 46. Swain, S. L. (1981) Proc. Natl. Acad. Sci. USA 78, 1101-1105. Sci. 254, 163-171. 47. McPhee, D., Pye, J. & Shortman, K. (1979) Thymus 1, 19. McIntyre, B. W. & Allison, J. P. (1984) Cell 38, 659-699. 151-162. 20. Hoopes, B. C. & McClure, W. R. (1981) Nucleic Acids Res. 9, 48. Scollay, R., Butcher, E. C. & Weismann, I. L. (1980) Eur. J. 5493-5504. Immunol. 10, 210-218. 21. Maniatis, T., Fritsch, E. F. & Sambrook, J. (1982) Molecular 49. Havran, W. L., Poenie, M., Kimura, J., Tsien, R., Weiss, A. Cloning: A Laboratory Manual (Cold Spring Harbor Labora- & Allison, J. P. (1987) Nature (London) 330, 170-173. Downloaded by guest on September 24, 2021