Stochastic component to development of class I major histocompatibility complex-specific T cells

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1 Proc. Natl. Acad. Sci. USA Vol. 91, pp , January 1994 Immunology Stochastic component to development of class I major histocompatibility complex-specific T cells ANDREA ITANO*, DIMITIUS KIOUSSISt, AND ELLEN ROBEY*t *Department of Molecular and Cell Biology, University of California, Berkeley, CA 94720; and tdivision of Molecular Immunology, National Institute of Medical Research, Mill Hill, London, United Kingdom Communicated by Marian Koshland, September 21, 1993 ABSTRACT The mechanism by which an initially uncommitted cell chooses between alternative fates is a central Issue In developmental biology. In the mammalian thymus, CD4 helper T cells and CDB cytotoxic T cells arise from a common precursor that expreses both CD4 and CD8. The choice between the CD4 and CD8 lineage is linked to the specificity of the T-cell antigen receptor expressed by a thymocyte, but whether lineage commitment is stochastic or instructed has not been definitively resolved. We present evidence that expression ofa constitutive CD8 transgene during thymic selection permits development of mature CD4 cells bearing the class I-restricted FS T-ceUl antigen receptor. These results suggest that there is a stocastic component to the development of class I major histocompatibility complex-restricted T cells. Mature T cells expressing either the CD4 or CD8 coreceptors arise from a thymic precursor that expresses both CD4 and CD8. The mechanism by which this precursor chooses between these alternative fates has not been established. Studies with transgenic mice bearing rearranged a,b T-cell antigen receptor (TCR) genes have demonstrated a link between the specificity of the TCR for class I or class II major histocompatibility complex (MHC) and the expression of CD4 or CD8 by mature T cells. Mice bearing class I-specific TCR transgenes often have increased numbers of mature CD8 T cells, and transgenic class I-specific TCRs are selectively expressed on mature CD8 T cells. In contrast, mice bearing class II-specific TCR transgenes generally have more mature CD4 cells, and transgenic class II-specific TCRs are expressed primarily on mature CD4 cells (1-5). One model to explain this finding proposes that the recognition of MHC class I during positive selection "instructs" a CD4+CD8+ precursor to become a CD8 cell, whereas recognition of class II MHC directs a precursor to become a CD4 T cell (6). According to an alternative "stochastic/selection" model, the commitment to the CD4 or CD8 lineage precedes positive selection and is made independently of the specificity of the TCR (7, 8). Because recognition of class I MHC requires the coordinate binding of a class I-specific TCR and the CD8 co-receptor, thymocytes that bear class I-specific TCRs and choose the CD4 pathway of development cannot undergo positive selection and thus cannot mature. Thymocytes bearing class II-specific TCRs that choose the CD8 lineage cannot undergo positive selection because they lack the CD4 coreceptor, which is required for class II MHC recognition. Either instruction or stochastic/selection models are adequate to explain the bias in development of CD4 and CD8 T cells, which is observed in TCR transgenic mice. The stochastic/selection model predicts a developmental intermediate that expresses a class I-restricted TCR and is committed to a CD4 lineage but that cannot complete maturation because it has low or no CD8 expression. Expression 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. 220 of a constitutive CD8 transgene should allow this intermediate to proceed through development. We have tested this notion by asking whether coexpression of a CD8 transgene along with a class I-specific TCR transgene would "rescue" mature CD4 T cells bearing a class I-specific TCR transgene. Using CD8.1 a cdna and CD8 f3 genomic clones under the control of the human CD2 regulatory regions, we generated a transgenic line in which CD8.1 is expressed on virtually all T cells at levels close to that of endogenous CD8 (CD8.2). When this CD8.1 transgene is coexpressed with the anti-h-y TCR transgene, improved positive selection in the CD8 lineage was observed with no apparent rescue of mature CD4 cells (9). Two other groups have obtained similar results (10, 11). However, because all three studies used the same transgenic TCR, we felt it was important to reexamine this question by using a transgenic TCR of a different specificity. Here we describe the effect of constitutive CD8 expression on the selection of a TCR from a CD8+ class I-specific T-cell clone called F5. MATERIALS AND METHODS Transgenic mice expressing the F5TCR (12) and constitutive CD8.1 a and p transgenes (9) (H-2b) were mated to each other. Transgenic offspring were identified by Southern blot hybridization and sacrificed at 1-2 months of age. For analysis by flow cytometry, thymus and lymph node were teased apart in cold M199 medium (GIBCO) supplemented with 10%o fetal bovine serum, and cells were passed through nylon mesh. Lymph node cells were depleted of B cells by passage over plates coated with anti-mouse immunoglobulin as described (13). Three-color analysis was performed with the following reagents: anti-cd8.2 (2.43), anti-cd4 (H129.19), fluorescein isothiocyanate-labeled anti-rat immunoglobulin (Caltag, South San Francisco, CA), biotinylated anti-v1ll (KT11; V, variable region), biotinylated anti- CD8.2 (2.43), phycoerythrin-labeled anti-cd4 antibody (GK1.5; Becton Dickinson), biotinylated anti-interleukin 2 (IL-2) receptor, biotinylated anti-v,a2 (PharMingen), and rat immunoglobulin (CalBiochem). Data were collected with a FACS IV (Becton Dickinson). For IL-2 receptor expression measurements, splenocytes or thymocytes (2 x 106 cells per ml) from F5 TCR and F5 TCR/CD8.1 transgenic mice were incubated in 24-well plates with 0.5 pg of nucleoprotein (NP)-( ) peptide per ml in complete RPMI 1640 medium with 50 units of recombinant IL-2 per ml (Amgen, Thousand Oaks, CA). After 48 hr, cells were purified over Ficoll gradients and stained for flow cytometry as described above. Abbreviations: TCR, T-cell antigen receptor; MHC, major histocompatibility complex; IL-2, interleukin 2; V, variable region; NP, nucleoprotein. *To whom reprint requests should be addressed at: Department of Molecular and Cell Biology, 471 Life Sciences Addition, University of California, Berkeley, CA

2 Immunology: Itano et al. RESULTS The F5 TCR recognizes a NP peptide bound to Db and, like the anti-h-y TCR, permits development of mature CD8 cells in H-2b mice (12). To determine whether expression of the CD8.1 transgene could permit development of mature CD4 cells bearing the F5 TCR, we bred mice bearing the F5 TCR transgene to mice bearing the CD8.1 transgene. Both transgenic parents were H-2b. In addition, both transgenic parents carry an endogenous CD8.2 allele, allowing the transgenic CD8 (CD8.1) to be distinguished from the endogenous allele by monoclonal antibodies. We then analyzed T-cell development in the offspring of this cross by flow cytometry. The results of this analysis are presented in Table 1 and Fig. 1. The thymus of nontransgenic mice contains mostly CD4+CD8+ cells, which consist of immature cells, as well as dead-end cells, which are destined to die in the thymus. There are also smaller numbers of more mature single-positive cells, which are either CD4+CD8- or CD4-CD8+. In F5 TCR transgenic mice (ref. 12; Table 1) there is a large proportion of mature CD8 single-positive cells as a consequence of positive selection by class I MHC. Very few CD4 single-positive cells are found in F5 TCR transgenic mice. Interestingly, in mice that carry both the F5 TCR transgene and the CD8.1 transgene, a significant population of CD4 single-positive cells is observed. We also examined the expression of transgeneencoded TCR (V911) by three-parameter flow cytometry (Fig. 1; Table 1). Nontransgenic and CD8.1 transgenic mice contain very few thymocytes with high levels of V911, whereas a large proportion of the thymocytes from F5 TCR transgenic mice and F5 TCR/CD8.1 transgenic (double transgenic) mice are V911+. We find that there is a significant population of V911+ cells in F5 TCR/CD8.1 transgenic mice, whereas very few V 1+ cells are present in F5 TCR transgenic mice. Taking into account the 3-fold reduction in the size of the thymus in F5 TCR/ CD8.1 transgenic mice, there is still an :3-fold increase in the total number of Vll+ cells in these mice. We also examined the lymph node T-cell populations in F5 TCR/CD8.1 transgenic mice to determine whether these V.11+ cells could emigrate and populate the periphery, as would be expected for a mature population. The results of this analysis are shown in Fig. 2 and Table 2. Indeed, there are significantly increased numbers of V911+ CD4+ T cells in F5 TCR/CD8.1 transgenic mice as compared to mice expressing the F5 TCR transgene alone. The observation that increased numbers of mature VP11+ T cells develop in F5 TCR/CD8.1 transgenic mice suggests that the CD8.1 transgene permits maturation of cells ofthe CD4 lineage with class I-specific TCRs. However, the possibility must be considered that the CD4 cells in F5 TCR/CD8.1 transgenic mice express endogenous TCR a, rather than transgenic TCR a (VA4), which could permit positive selection by class II MHC recognition to occur. To address this question, we examined the expression of an endogenous Va (Va2) in the lymph node T cells of these mice. The results of these analyses are shown in Fig. 2. While Table 1. Proc. Natl. Acad. Sci. USA 91 (1994) 221 Thymocytes from F5 TCR transgenic and F5 TCR/CD8.1 transgenic (double transgenic) mice % of total thymocytes CD4+ No. of thymocytes CD4+ CD4- CD8.2- CD8.2+ X 10-7 CD8.2- CD8.2+ VPll+ VP11+ n Nontransgenic 12 (4.6) 5.8 (1.1) 3.5 (1.4) <0.8 <0.4 4 CD8.1 transgenic 7.0 (1.5) 8.1 (0.6) 3.6 (0.4) <0.8 <0.4 2 F5 TCR transgenic 15 (3.5) 2.2 (0.2) 18 (7.6) 1.6 (0.3) 16 (8.3) 4 F5 TCR and CD8.1 transgenic 5.5 (2.0) 15 (3.3) 16 (3.6) 12 (3.7) 10 (2.2) 6 Numbers represent average values from several individuals. SDs are given in parentheses; n represents number of individuals examined. 415% of CD4 and 9%o of CD8 T cells from nontransgenic and CD8.1 transgenic mice express Va2 (ref. 14; Fig. 2), in F5 TCR transgenic mice only 1% of CD8+ T cells express V.2. This suppression of endogenous a chains could be the result of the shutoff of rearrangement as a result of positive selection and subsequent maturation (15), or it may reflect competition between transgenic and endogenous a chain for pairing with the transgenic chain (16, 17). In contrast, 19% of CD4 cells of F5 TCR transgenic mice express Va2. This is consistent with previous observations in anti-h-y TCR transgenic mice, in which the expression of endogenous TCR a permits mature T cells of the CD4 lineage to develop (18). If the lymph node CD4 cells in F5 TCR/CD8.1 transgenic mice had been selected with endogenous TCRs, we would expect their Va2 usage to resemble the CD4 population in F5 TCR transgenic mice. In contrast only 6.5% of the lymph node CD4 cells in F5 TCR/CD8.1 transgenic mice express Va2. We also examined the expression of Vail and found a similar trend (data not shown). These observations, taken together with the fact that a majority of the lymph node CD4 cells in F5 TCR/CD8.1 transgenic mice express transgene (3, imply that a significant number of CD4 cells in F5 TCR/CD8.1 transgenic mice express the F5 TCR. A more direct test of whether the CD4 cells in F5 TCR/ CD8.1 transgenic mice express transgenic TCR is their ability to respond to the NP peptide. To examine this question, we incubated splenocytes and thymocytes from F5 TCR and FS TCR/CD8.1 transgenic mice with NP peptide and H-2b splenocytes for 48 hr. We then examined expression of the IL-2 receptor, which is induced on mature T cells upon activation through the TCR. The results are shown in Fig. 3. While a vast majority of CD8 single-positive cells from either F5 TCR and F5 TCR/CD8.1 transgenic mice express IL-2 receptor after activation by NP peptide, only a small proportion of the CD4 single-positive cells from F5 TCR transgenic mice are IL-2 receptor positive. In contrast, 68% of peripheral CD4 cells and 74% of CD4 single-positive thymocytes from F5 TCR/CD8.1 transgenic mice express IL-2 receptor. This indicates that a significant proportion of mature CD4 cells in FS TCR/CD8.1 transgenic mice express the F5 TCR and can respond to the NP peptide in vitro. This conclusion is also confirmed by the observation that equivalent numbers of mature CD4 cells develop in F5 TCR/CD8.1 transgenic mice, whether or not the mice are wild type or mutant for RAG-1 (D.K. and E.R., unpublished data). We and others have previously observed that coexpression of a CD8 transgene with the 2C TCR can lead to negative selection (19, 20). The 3-fold reduction in the size of the thymus of F5 TCR/CD8.1 transgenic mice relative to F5 TCR transgene-only mice (Table 1), and the slight reduction in the level of endogenous CD8 expression (Fig. 1), suggests that some negative selection also occurs in F5 TCR/CD8.1 transgenic mice. Nevertheless, there is still a 3-fold increase in the absolute number of thymocytes in F5 TCR/ CD8.1 transgenic mice compared to F5 TCR transgenic mice. Moreover, negative selection cannot account for the appear- CD4-

3 222 Immunology: Itano et al. Proc. Natl. Acad. Sci. USA 91 (1994) non tg a Total thymus CD8.1 tg b CU *,X ' *v _ ) 3.9 anti-cd8.2 _4.9 10' Co. c,5- U) anti-vp i j "I'll 6, 'Of I t.$4 I..,,. v.., -..."J" "I I Vp11 high anti-cd X 5.8i.. IJ ; e Vd -- FIG. 1. Expression of CD4, endogenous CD8, and Vs11 in thymocytes from F5 TCR or F5 TCR/CD8.1 transgenic (tg) mice. Thymocytes from the offspring of a cross between F5 TCR and CD8.1 transgenic mice were analyzed with fluorescent antibodies as described in Table 1. The anti-cd8.2 antibody recognizes endogenous but not transgenic CD8. Numbers inside quadrants represent percentage of cells in each population. ance of F5 TCR+ cells. Selective deletion of CD8 cells might change the relative proportion of CD4 and CD8 T cells but would not be expected to increase the proportion of CD4 cells expressing the F5 TCR. DISCUSSION According to a stochastic/selection model for the development of CD4 and CD8 T cells, there exist developmental intermediates that are committed to a CD4 lineage and bear class I-specific TCRs, which cannot mature because they lack CD8, which is required for class I MHC recognition and positive selection. A prediction of this model is that constitutive expression of CD8 should allow class I-specific CD4+ T cells to mature. The first attempts to test this prediction used a constitutive CD8 transgene and the class I-specific anti-h-y TCR (9-11). In these studies, it was found that, although the CD8 transgene dramatically enhanced the selection of CD8+ T cells, no rescue of CD4+ T cells was observed, arguing against the stochastic/selection model. In contrast, when an analogous experiment was performed with a constitutive CD4 transgene and a class II-specific transgene, CD8+ class II-specific T cells were observed, implying that stochastic commitment to the CD4 or CD8 lineage could occur, at least for class II-specific T cells (5, 21). In addition, examination of thymic subsets in class II MHC mutant mice also supports a stochastic model (22). Although very few mature CD4 single-positive T cells develop in mice lacking class II MHC, there is a significant population of CD4+CD8lOW cells. These cells may represent developmental intermediates whose TCRs recognize class I MHC and have t 9.' 2.9. initiated positive selection but cannot complete a second phase of positive selection because their CD8 levels are too low to permit continued engagement of class I MHC. In the current study, we reexamine the question of whether there is a stochastic comment to the development of class I-specific T cells using a different class I-specific TCR, the F5 TCR. Here we show that constitutive CD8 expression does permit maturation of CD4 cells expressing the F5 TCR, implying that class I-specific T cells can choose the CD4 lineage, as predicted by the stochastic/selection model. The fact that the F5 TCR can be selected into the CD4 lineage, whereas the anti-h-y TCR cannot, may be related to the observation that, in cases in which rescue of T cells expressing the inappropriate co-receptor is observed, it is invariably inefficient. For example, in 632-microglobulin mutant mice, which express a CD4 transgene at normal levels, only 1% of lymph node cells are CD8+ (21). This implies that the rescue of CD8+ class II-specific T cells by constitutive CD4 expression is a less efficient process than the generation of single-positive cells of the appropriate CD4/CD8 lineage. In the current study, the levels of transgenic CD8 were close to normal and the thymus of F5 TCR/CD8.1 transgenic mice contains almost equivalent numbers of CD4 and CD8 singlepositive cells. However, in the periphery of F5 TCR/CD8.1 transgenic mice there is still a predominance of CD8+ cells relative to CD4+ cells. This is reflected in the ratio of mature CD4+ cells to CD8+ cells in the lymph node, which in nontransgenic mice is -2:1. Although the CD4/CD8 ratio in F5 TCR/CD8.1 transgenic mice is closer to normal than F5 TCR transgene-only mice (1:4 compared to 1:10), there is still a bias toward the development of CD8+ T cells.

4 Immunology: Itano et al. non tg Proc. Natl. Acad. Sci. USA 91 (1994) 223 CD8.1 tg a 3.7 d , 1., (9.2%) 437 antl-cd dx -.,.j Nb _. anti-cd4 Expression of CD4, endogenous CD8, V.ll (transgenic TCR,), and V.2 (endogenous V.) in lymph node T cells from F5 TCR or FIG. 2. F5 TCR/CD8.1 transgenic (tg) mice. Numbers inside quadrants represent percentage of cells in each population. Numbers in parentheses represent frequency of Va2+ cells as a percentage of CD8.2+ T cells (e-h) or as a percentage of CD4+ T cells (m-p). T cells (B-depleted lymph node cells) from the offspring of a cross between F5 TCR and CD8.1 transgenic mice were analyzed with fluorescent antibodies as described in Table 1. Why is the development of CD4+ class I-specific T cells in CD8.1 transgenic mice less efficient than the formation of CD8+ class I-specific T cells? One possibility is that the majority of T cells receive instructive signals during positive selection and that the stochastic pathway represents a minor pathway for development. It is also possible that the decision to become a CD4 cell or a CD8 cell is made completely randomly but that the rescue of class I-specific CD4 cells is Table 2. Lymph node T cells from F5 TCR transgenic and F5 TCR/CD8.1 transgenic mice % of T cells CD4+ CD4- CD4+ CD4- CD8.2- CD8.2+ CD8.2- CD8.2+ Vpsll+ Vpll+ n Nontransgenic 52 (0) 37 (5.7) 3.2 (0.2) 2.5 (0.1) 2 CD8.1 transgenic F5 TCR transgenic 11 (0.6) 83 (2.6) 5.8 (2.2) 69 (11) 3 F5 TCR and CD8.1 transgenic 20 (1.5) 67 (1.5) 11 (2.2) 51 (14) 3 Numbers represent average values from several individuals. SDs are given in parentheses; n represents number of individuals examined. inefficient because restoring co-receptor expression is not sufficient to restore complete co-receptor function. For example, restoring CD8 expression in CD4 single-positive thymocytes might not completely restore co-receptor function if the activity of CD8 is inhibited by the presence of CD4. Alternatively, it is conceivable that when a thymocyte chooses the CD8 or CD4 lineage it turns off not only the unused co-receptor but also unidentified signaling molecules that transduce a signal through that co-receptor. If this were the case, introducing a co-receptor transgene would not be sufficient to restore co-receptor function because the coreceptor would be "uncoupled" to intracellular signaling machinery. The F5 TCR recognizes antigen bound to the class I MHC molecule Db, and therefore it is expected that mature T cells bearing the F5 TCR would be selected in the thymus by using class I MHC. However, it has been observed that both class I and class II MHCs can participate in development oft cells bearing a class II MHC-restricted TCR (B. J. Fowlkes, personal communication), raising the possibility that both class I and class II MHCs might also participate in the development of the CD4+ T cells in F5 TCR/CD8.1 transgenic mice. In fact, evidence from radiation chimeras in which bone marrow from F5 TCR/CD8.1 transgenic mice

5 224 Immunology:,Itano et al Spleen Proc. Natl. Acad. Sci. USA 91 (1994) Thymus 6, u U, D4+C8I % 74% h I lx Anti-IL-2 receptor I >YI FIG. 3. Expression of IL-2 receptor after in vitro stimulation with NP peptide. Splenocytes (a-d) and thymocytes (e-h) from F5 TCR or F5 TCR/CD8.1 transgenic (tg) mice were incubated with NP peptide and H-2b splenocytes for 48 hr and analyzed for expression ofcd4, endogenous CD8 (CD8.2), and IL-2 receptor. develops in class II MHC mutant hosts indicates that, although class II MHC is not absolutely required for the generation of rescued CD4+ T cells, it does contribute to their efficient maturation (B. J. Fowlkes, A.I., and E.R., unpublished data). Although cross-reactivity of mature T cells with different MHCs is relatively rare, cross-reactivity may occur more often in positive selection, given that positive selection is believed to be mediated by very low-affmity interactions (19, 20, 23). Cross-reactivity of TCR for class I and class II MHCs could account for some of the rescue of transgeneencoded TCR described here and in previous reports (21). Nevertheless, cross-reactivity cannot account for the recent observation that mature CD4+ T cells develop in class II-deficient, CD8.1 transgenic mice (B. J. Fowlkes, A.I., and E.R., unpublished data), in which all selection presumably takes place using class I MHC molecules. In summary, we have shown that mature CD4+ T cells bearing the class I-specific F5 TCR are present in CD8.1 transgenic mice, indicating that there is a stochastic component to the development of class I MHC-restricted T cells. However, the inefficiency of generating rescued CD4 cells leaves open the possibility that instructive signals may also be important in development of CD4+ and CD8+ T cells. The authors thank B. J. Fowlkes, David Raulet, and James Allison for helpful discussions and for comments on the manuscript and Keith Volk for typing transgenic mice. This work was supported by National Institutes of Health Grant R01 AI to E.R. 1. Pircher, H., Burki, K., Lang, R., Hengartner, H. & Zinkernagel, R. M. (1989) Nature (London) 342, Sha, W. C., Nelson, C. A., Newberry, R. D., Kranz, D. M., Russell, J. H. & Loh, D. Y. (1988) Nature (London) 335, Kisielow, P., Teh, H. S., Bluthmann, H. & von Boehmer, H. (1988) Nature (London) 335, Berg, L. J., Pullen, A. & Davis, M. M. (1989) Cell 58, Kaye, J., Hsu, M. L., Sauron, M. E., Jameson, S. C., Gascoigne, N. R. & Hedrick, S. M. (1989) Nature (London) 341, von Boehmer, H. (1986) Immunol. Today 7, Janeway, C. A. (1988) Nature (London) 335, Robey, E. A., Fowlkes, B. J. & Pardoll, D. M. (1990) Semin. Immunol. 2, Robey, E. A., Fowlkes, B. J., Gordon, J. W., Kioussis, D., von Boehmer, H., Ramsdell, F. & Axel, R. (1991) Cell 64, Borgulya, P., Kishi, H., Muller, U., Kirberg, J. & von Boehmer, H. (1991) EMBO J. 10, Seong, R. H., Chamberlain, J. W. & Parnes, J. R. (1992) Nature (London) 356, Mamalaki, C., Elliott, J., Norton, T., Yannoutsos, N., Townsend, A. R., Chandler, P., Simpson, E. & Kioussis, D. (1993) Dev. Immunol. 3, Mage, M. (1984) Methods Immunol. 108, Pircher, H., Rebai, N., Groettrup, M., Gregoire, C., Speiser, D. E., Happ, M. P., Palmer, E., Zinkernagel, R. M., Hengartner, H. & Malissen, B. (1992) Eur. J. Immunol. 22, Turka, L. A., Schatz, D. G., Oettinger, M. A., Chun, J. J. M., Gorka, C., Lee, K., McCormack, W. T. & Thompson, C. B. (1991) Science 253, Gabert, J., Langlet, C., Zamoyska, R., Parnes, J. R., Schmitt, V. A. M. & Malissen, B. (1987) Cell 50, Saito, T. & Germain, R. N. (1989) J. Immunol. 143, von Boehmer, H. (1990) Annu. Rev. Immunol. 8, Robey, E. A., Ramsdell, F., Kioussis, D., Sha, W., Loh, D., Axel, R. A. & Fowlkes, B. J. (1992) Cell 69, Lee, N. A., Loh, D. Y. & Lacy, E. (1992) J. Exp. Med. 175, Davis, C. B., Killeen, N., Crooks, M. E. C., Raulet, D. & Littman, D. R. (1993) Cell 73, Chan, S. H., Cosgrove, D., Waltzinger, C., Benoist, C. & Mathis, D. (1993) Cell 73, Sprent, J., Lo, D., Gao, E. K. & Ron, Y. (1988) Immunol. Rev. 101,