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The Journal of Immunology, 1999, 162: 2008-2015.
Copyright © 1999 by The American Association of Immunologists

Dual MHC Class I and Class II Restriction of a Single T Cell Receptor: Distinct Modes of Tolerance Induction by Two Classes of Autoantigens1

Ivica Arsov and Stanislav Vukmanovic2

Michael Heidelberger Division of Immunology, Department of Pathology and Kaplan Cancer Center, New York University Medical Center, New York, NY 10016


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
In the final stages of thymic development, immature T cells undergo three distinct processes (positive selection, negative selection, and lineage commitment) that all depend on interactions of thymocyte TCRs with MHC molecules. It is currently thought that TCRs are preferentially restricted by either MHC class I or class II molecules. In this report, we present direct evidence that the TCR previously described as H-Y/H-2Db specific cross-reacts with H-2IAb if expressed in CD4+ cells. We also demonstrate an increase in thymocyte numbers in H-Y TCR-trangenic mice deficient in MHC class II, suggesting a relatively discrete form of negative selection by MHC class II compared with that induced by H-Y/H-2Db. We propose that inability to generate CD4+ T cells expressing H-Y TCR in different experimental settings may be due to tolerance to self-MHC class II. These results, therefore, support an intriguing possibility that tolerance to self may influence and/or interfere with the outcome of the lineage commitment.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
The T cell repertoire is heavily shaped by interactions with MHC-peptide ligands. T cells expressing receptors with relatively high avidities for certain MHC-peptide ligands are purged from the mature T cell pool, whereas different and generally lower avidity interactions with these ligands are required for positive selection (1, 2, 3, 4, 5). The survival of developing T cells is dependent on the balance between these two processes. In different TCR-transgenic models, with few exceptions (6, 7), mature T cells expressing transgenic receptors generally retain their original mature CD4 or CD8 phenotype and vastly outnumber T cells of the other lineage (8, 9, 10, 11). This is clearly seen when TCR-transgenic mice are bred to the backgrounds that restrict rearrangements of endogenous TCR{alpha} gene segments (10, 12, 13, 14). When allelic exclusion at the TCR{alpha} locus is inefficient, novel TCR specificities may be generated that would permit commitment of T cells to different lineages (15). However, certain differences exist among different TCR-transgenic mice with regard to the extent of positive selection of T cells with "mismatched" receptor-coreceptor combinations, as revealed by so-called "rescue" experiments in which forced expression of the coreceptors, originally expressed in the transgenic lineage, was used to drive positive selection of the opposite lineage. These experiments did not always lead to the successful liberation of T cells with mismatched receptor-coreceptor combinations. Whereas successful rescues (2, 13, 16, 17) were considered evidence that CD4+CD8+ double-positive (DP)3 thymocytes commit to either lineage stochastically and independently from their TCR specificity for either MHC class I or II molecules, unsuccessful ones (18, 19) were considered evidence that DP thymocytes were "instructed" to choose either lineage by the specificity of their TCR. Although evidence for both models exists (for reviews see Refs. 2, 3 , and 20), it is still unclear why the use of individual TCR transgenics provided different, and sometimes opposing, results.

In the H-Y TCR-transgenic model, CD8+ T cells expressing transgenic TCR are positively selected in female mice by the MHC class I H-2Db, whereas deletion of DP thymocytes occurs in male mice (8, 21, 22, 23). Although the majority of CD8+ single-positive T cells in the female thymus express the transgenic TCR, CD4+ T cells mostly express an endogenous TCR{alpha}-chain together with the transgenic ß-chain, and in SCID H-Y TCR-trangenic animals total numbers of CD4+ single-positive thymocytes are severely reduced (10). The paucity of CD4+ T cells expressing the transgenic TCR cannot be explained by structural constraints that the CD4 coreceptor might impose on TCR expression because: 1) CD4+ T cells expressing high levels of transgenic TCR are clearly present in H-Y TCR transgenic H-2d mice (21); 2) they can also be generated from DP thymocytes using Ab against the transgenic TCR{alpha}-chain (24); and 3) CD4+ hybridomas can express high levels of transgenic TCR (15).

Unlike other TCR transgenics, in which forced expression of "correct" coreceptors rescued some T cells of the opposite CD4/8 phenotype (13, 16, 25), the presence of the CD8 coreceptor did not allow development of CD4+ T cells expressing the H-Y-specific TCR (18, 19). Even when the intracellular domains of CD4 and CD8 were swapped, the resulting transgene still failed to rescue significant numbers of CD4+ T cells (17). It has recently been proposed that differences may lie in the context of perception of positively selecting ligand by developing thymocytes (17). Positive selection with a strong activation of p56lck might drive CD4 lineage commitment, whereas weak activation would drive CD8 commitment. Moreover, other molecules, such as Notch, have also been reported to influence the lineage choice (26). However, the inability of CD8 transgene to rescue CD4+ cells in the H-Y TCR-transgenic model might have another explanation. Overexpression of CD4 in the H-Y TCR-transgenic mice reduced the number of CD8+ T cells expressing H-Y TCR (27, 28), and the resulting peripheral DP T cells reacted with MHC class II alloantigens (27). This result raised the intriguing possibility that the H-Y TCR might be a receptor with double-MHC restriction, although this was not the original interpretation of those findings. If the cross-reactivity to MHC class II included also self-MHC class II (H-2IAb), the inability to generate significant numbers of CD4+ T cells expressing H-Y TCR may be the consequence of negative selection, as previously suggested by some investigators (29). Indeed, disruption of T cell development by CD4 transgene overexpression has recently been linked to enhanced negative selection (30). In this report, we directly demonstrate MHC class II (auto)reactivity of the H-Y TCR. We also extend this observation to H-Y TCR-transgenic mice deficient in MHC class II expression, in which we find distinct effects that are consistent with negative selection by MHC class II molecules.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals

H-Y TCR-transgenic animals were a kind gift of Dr. Janko Nikolic-Zugic (Sloan Kettering Institute, New York, NY), and were used at 6–8 wk of age. C57BL/6 (B6), as well as B6 MHC class II-deficient, B6 ß2-microglobulin (ß2m)-deficient mice, and H-Y TCR-transgenic mice on a B10.D2, RAG2-/- background were purchased from Taconic (Germantown, NY) and used at 3–5 wk of age. H-Y TCR-transgenic MHC class II-deficient animals were obtained by the backcrossing of H-Y TCR-transgenic mice to B6 MHC class II-deficient mice in the animal facility of the Skirball Institute of Biomolecular Medicine at the New York University (NYU) Medical Center, New York, NY. The second generation was obtained by mating H-Y TCR+/-, MHC class II+/- with H-Y TCR-/-, MHC class II-/- mice. Littermates were screened by immunofluorescence of peripheral blood cells.

Cell lines

P815 cells were maintained in RPMI 1640 supplemented with 10% heat-inactivated FCS, 2 mM L-glutamine, 1 mM 2-ME, and antibiotics (RP10). The CD4+ T cell line was maintained by weekly restimulations with irradiated B6 spleen cells in RP10 supplemented with 5% rat Con A supernatant. A CD4+ T cell line specific for Borrelia burgdorferi Ags (31) was provided by Dr. Alan Frey (NYU Medical Center). The generation of the CD8+ H-Y-specific transgenic cell line has been described previously (32). A T cell hybridoma expressing H-Y TCR (provided by Drs. Robert J. Hayashi and Osami Kanagawa, Departments of Pediatrics and Pathology, Washington University Medical School, St. Louis, MO, respectively) was obtained by fusing 4 x 107 Con A-stimulated lymph node cells from the H-Y TCR-transgenic, TCR{alpha}-/- mice with the same number of TCR{alpha}-ß- variants of BW 5147 thymoma (33). Cells selected with HAT-containing medium were sorted for TCR expression using Abs to either Vß8 or CD3 and cloned by limiting dilution as described previously (34).

mAb and flow cytometry

F23.1, anti-mouse Vß8.1 and Vß8.2, was purified from hybridoma supernatant and conjugated to FITC (Sigma, St. Louis, MO) H57-597 (anti-mouse TCRß), F23.2 (anti-mouse Vß8.2), and MR5.2 (anti-mouse Vß8) were used as hybridoma supernatants. Ab against the transgenic TCR{alpha}-chain (T3.70) was used either conjugated to FITC or as a hybridoma supernatant. Fab fragments of T3.70 were obtained using a Fab preparation kit from Pierce (Rockford, IL), according to protocol supplied by the manufacturer. All hybridomas were kindly provided by Dr. Janko Nikolic-Zugic. Anti-mouse CD4 (H129.19) conjugated to phycoerythrin and FITC-conjugated anti-mouse CD8ß.2 (53-5.8) or CyChrome-conjugated anti-mouse CD8{alpha} (53-6.7) were purchased from PharMingen (San Diego, CA). MR 14.1 mAb is an anti-H-Y TCR anti-clonotypic Ab generated and provided by Drs. Robert J. Hayashi and Osami Kanagawa. T cells were stained using saturating concentrations of Abs and analyzed using a FACScan flow cytometer (Becton Dickinson, Mountain View, CA).

Proliferation assay and IL-2 measurement

T cells were incubated with irradiated stimulator spleen cells in round-bottom 96-well plates in RP10 without IL-2 for 48 or 96 h, as indicated. Each microculture was then pulsed with 0.5 µCi of [3H]thymidine for 12 or 24 h, as indicated, and thymidine incorporation was subsequently measured on a beta scintillation counter. For IL-2 measurements, 1 x 104 CTLL-2 cells per well (kindly provided by Dr. Alan Frey) were incubated with 50% culture supernatants for 24 h; each microculture was then pulsed with 0.5 µCi of [3H]thymidine for 24 h, and incorporation of thymidine was measured on a beta scintilation counter.

CTL assay

Targets were incubated with 51Cr-labeled sodium chromate in RP10 for 1 h at 37°C. They were then washed three times with PBS and 5 x 103, or 1 x 104 cells were transferred to a well of a round-bottom 96-well plate. CD4+ T cells were added at varying numbers to the total volume of 200 µl. Plates were incubated for 4 h at 37°C. At the end of this interval, 100 µl of supernatant was harvested from each well and counted in a gamma counter. The percentage of specific lysis was calculated as follows: 100 x ([experimental release - spontaneous release]/[maximal release - spontaneous release]). Spontaneous and maximal release were determined in the presence of RP10 or 1% Triton X-100, respectively.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Recognition of self-MHC class II by CD4+ cells expressing transgenic H-Y/H-2Db-specific TCR.

Although the number of CD4+ T cells expressing H-Y TCR in the periphery of H-Y TCR transgenic female mice is extremely low compared with CD8+ T cells bearing this receptor (<1%), the stimulation of spleen cells from these mice with T3.70 Ab in the presence of IL-2, followed by weekly restimulation with male spleen cells, unexpectedly generated a CD4+ instead of a CD8+ T cell line (Fig. 1A). This CD4+ T cell line (designated HYCD4) expressed H-Y TCR-transgenic ß-chain (Fig. 1A) and apparently low levels of transgenic {alpha}-chain (Fig. 1A). RT-PCR analysis revealed the expression of at least two TCR{alpha}-chains (data not shown), suggesting that the HYCD4 line expressed at least one endogenously rearranged TCR{alpha}-chain. To confirm surface expression of the transgenic TCR{alpha}-chain, we carried out redirected cytotoxic assays using T3.70 Ab and P815 cells as targets (Fig. 1B). These results correlated well with the FACS analysis, because targets were lysed less efficiently using T3.70 compared with F23.2 Ab recognizing transgenic ß-chain. These results confirmed low surface expression of the TCR{alpha}-chain by HYCD4 cells. Apparent weak staining with T3.70 Ab is not an artifact of in vitro culture, but rather a general feature of CD4+ H-Y TCR-transgenic cells, as shown by staining of ex vivo isolated thymocytes (Fig. 1C) and spleen cells (data not shown).

It was suggested that expression of the CD4 coreceptor might potentially introduce novel, MHC class II-restricted specificities in T cells expressing the H-Y transgenic ß-chain, including specificities to yet unidentified self-molecules (27, 35). To determine whether HYCD4 cells recognize MHC class I-associated male Ag in a coreceptor-independent manner or novel MHC class II-associated (auto)antigen, we examined the response of this line to MHC class II-deficient, ß2m-deficient (ß2m-/-), or wild-type (wt) syngeneic B6-irradiated spleen cells. As demonstrated in Fig. 2Go, HYCD4 cells mounted a proliferative response to both wt B6 and ß2m-/-, but not to MHC class II-deficient male or female spleen cells. The response to male and female ß2m-/- or wt stimulators is not significantly different, suggesting that this response is not likely to be H-Y/H-2Db-specific. This pattern of recognition, therefore, reveals that HYCD4 cells recognize self-MHC class II molecules, in agreement with previous findings (36, 37) and our (Fig. 3Go) results, demonstrating the essential role of CD8 coreceptor in H-Y TCR recognition of the H-Y/H-2Db complex.



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  		FIGURE 2. Recognition of self-MHC class II by the HYCD4 line. A proliferation assay was carried out by incubating 1 x 105 HYCD4 cells/well and different numbers of irradiated stimulator cells for 48 h in IL-2-free medium, followed by a 24-h pulse with [3H]thymidine. The results represent the mean ± SD of triplicate cultures.

 


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  		FIGURE 3. Coreceptor-dependent response of transgenic CD8+ cells to male Ag. A proliferation assay was carried out by incubating 2 x 105 H-Y TCR-transgenic CD8+ cells/well and 3 x 105 irradiated stimulator cells for 96 h in medium containing 5% Con A supernatant, followed by a 12-h pulse with [3H]thymidine. All Abs were added at 12.5 µg/ml. The results represent the mean ± SD of triplicate cultures.

 
MHC class II-specificity does not depend on endogenous TCR{alpha}-chains.

To determine if the transgenic {alpha}-chain of the H-Y TCR or endogenous {alpha}-chains, might be involved in recognition of the "self-antigen" on B6 stimulators, we first tested the effect of Fab fragments of T3.70 Ab on proliferation of HYCD4 cells. The HYCD4 cell proliferative response to B6 stimulators was inhibited by >50% by this reagent, whereas no inhibitory effect was seen on Ag-dependent proliferation of a B. burgdorferi-specific CD4+ T cell line (Fig. 4Go), arguing against nonspecific effects on T cell proliferation mediated by T3.70 Fab.



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  		FIGURE 4. T3.70 Fab partially blocks the proliferation of HYCD4 to self-MHC II. Different concentrations of T3.70 Fab were tested for the ability to block the proliferative response of HYCD4 to syngeneic B6 cells (A) or a B. burgdorferi-specific CD4+ T cell line to nominal Ag (B). Assays were carried out essentially as described in the legend to Fig. 2Go. The responses in the absence of Ags were subtracted. The results represent the mean ± SD of triplicate cultures.

 
Even though these results strongly imply that H-Y TCR might recognize a self-MHC class II-restricted ligand, it was still possible that the effects seen in culture could be attributed to endogenous TCR{alpha}-chains. For example, it is possible that T3.70 Ab cross-reacts with other members of the V{alpha}3.2 family. The simplest approach to exclude involvement of endogenous TCR{alpha}-chains would be to use CD4+ cells from H-Y TCR-transgenic mice bred to TCR{alpha}- or recombination-activating gene (RAG)-deficient backgrounds. However, CD4+ T cells from H-Y mice are in general not overtly reactive to self-MHC class II (data not shown), and establishment of the HYCD4 line was a relatively exceptional event. We devised two approaches to circumvent this problem. First, we used a CD4+ T cell hybridoma expressing a single-specific H-Y TCR, generated by the fusion of spleen cells from TCR{alpha}-deficient H-Y TCR-transgenic females with the TCR{alpha}-ß- BW5147 thymoma. Although this hybridoma stained well with anti-Vß8 Ab, as well as anti-H-Y TCR-specific Ab MR 14.1, it stained less efficiently with T3.70 (Fig. 5A). This hybridoma responded by secreting IL-2 to wt and ß2m-/- but not to MHC class II-deficient spleen cells (Fig. 5B), analogous to the HYCD4 line. The second approach was to allow the development of endogenous rearrangement-deficient CD4+ cells in the absence of H-2IAb. H-2d, RAG2-/-, H-Y TCR-transgenic mice had spleens of extremely reduced size (4–6 x 106 total nucleated cells) containing rare CD4+ and virtually no CD8+ cells (Fig. 6A). Consistent with the findings in two T cell lines (see Fig. 1A and 4A) and ex vivo CD4+ cells (Fig. 1C), H-2d, RAG2-/-, H-Y TCR-transgenic CD4+ cells stained relatively well with the F23.1 but poorly with T3.70 Ab (data not shown). Given that CD8+ T cells stain well with T3.70 Ab in our (32) (Fig. 1C) as well as in others’ hands, the relatively poor expression of T3.70 epitope in CD4+ cells cannot be completely explained by replacement of transgenic with endogenous TCR{alpha}-chains. Strikingly, RAG2-/- spleen cells (Fig. 6B) exhibited a pattern of functional reactivity that was identical with the HYCD4 line (see Fig. 2Go) and the TCR{alpha}-deficient hybridoma (Fig. 5B), confirming the notion that transgenic {alpha}-chain permits and/or may be actively involved in the recognition of self-MHC class II structures.

Effects of self-MHC class II on development of H-Y-specific thymocytes.

Collectively, the data presented thus far demonstrate an intrinsic affinity of the H-Y TCR to self-MHC class II. Deletional negative selection in H-Y TCR-transgenic female mice, however, has not been described up to now. Clonal deletion can be seen at different stages of thymocyte maturation, probably reflecting the differences in avidities for deletional ligands or their different distributions in the thymus (11, 23). Moreover, low affinity/avidity ligands may induce only partial deletion or nondeletional effects such as anergy or TCR/coreceptor down-regulation (38, 39, 40, 41, 42). To examine the potential effects of MHC class II expression on thymocyte development in H-Y TCR-trangenic mice, we bred H-Y TCR-transgenic mice onto an MHC class II-deficient background. H-Y TCR-transgenic mice deficient in MHC class II expression showed a significant increase in CD4 expression on DP thymocytes (Fig. 7Go). Although CD4 up-regulation occurred in nontransgenic MHC class II-deficient animals, consistent with previous reports (43, 44), we found this increase to be more pronounced in H-Y TCR-transgenic mice (1.9-fold vs on average a 1.3-fold higher expression in TCR nontransgenic animals). Given that CD4 down-modulation in DP thymocytes occurs upon TCR engagement with anti-TCR Abs (45), and that the only difference between TCR-transgenic and nontransgenic DP cells is the proportion of cells expressing the TCR and the specificity of the expressed TCR, we believe that pronounced CD4 down-modulation reflects the intrathymic H-Y-specific TCR engagement by MHC class II. In addition to these effects on CD4 expression, we have also found some evidence of deletion induced by MHC class II. In H-Y TCR-transgenic female mice deficient in MHC class II, for example, there was a twofold increase in total thymocyte numbers as compared with TCR transgenic animals expressing MHC class II, despite a severe reduction of mature CD4+ thymocytes (Fig. 8Go). Moreover, we consistently found a two- to threefold increase in relative DP thymocyte numbers in H-Y TCR-transgenic males deficient in MHC class II, as compared with control mice (Fig. 7Go), implying that the deletion of DP thymocytes in transgenic male mice is partially contributed by MHC class II. Taken together, these results suggest: 1) the in vivo reactivity to self-MHC class II of the H-Y TCR expressed by DP thymocytes; and 2) that this reactivity may induce deletion of a proportion of DP cells, albeit not as dramatic as that caused by H-Y/H-2Db.



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  		FIGURE 7. Attenuation of negative selection in H-Y TCR-transgenic mice deficient in MHC class II expression. Thymocytes (1 x 106 cells per sample) were analyzed for CD4/CD8 expression using flow cytometry and directly labeled Abs. The numbers inside the quadrants represent the percentage of cells in each population. The right-end histograms represent the comparisons of CD4 expression on gated DP thymocyte populations for each experimental group (plain lines, MHC class II+/-; bold lines, MHC class II-/-). Data are representative of three experiments.

 


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  		FIGURE 8. MHC class II deficiency increases total thymocyte numbers in H-Y TCR-transgenic female mice. Total thymocyte numbers are given as average values with SDs in parentheses. Three pairs of age-matched littermates were tested. *, Differences statistically significant at p < 0.01 (Student’s t test).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
We demonstrate in this report that the TCR initially described as H-Y/H-2Db specific (reviewed in Ref. 46) cross-reacts to the self-MHC class II when expressed in CD4+ cells, and that the presence of MHC class II during thymic development leads to a deletion of a proportion of TCR-transgenic T cells. The exact MHC class II-associated ligand for the H-Y TCR still remains unknown. Theoretically, it could be H-2IAb itself, superantigen, or a self-peptide bound to H-2IAb. The fact that spleen cells from mice with an almost identical background but a different MHC haplotype (B10.BR) are not stimulatory for HYCD4 cells or for endogenous TCR{alpha}-deficient hybridoma cells (data not shown), and that superantigens in general bind well to H-2k class II molecules (47, 48) argues against the superantigens as stimulatory ligands. The true nature of the ligand remains to be established.

CD4 coreceptor expression has been shown in different models to introduce novel MHC class II specificities in CD8+ T cells initially recognizing MHC class I-restricted Ags (49, 50). Because CD8+ T cells from MHC class II-deficient mice readily respond to self-MHC class II, it has been suggested that a number of T cells may be capable of recognizing both classes of MHC molecules (51). It has also been shown that CD8+ T cells in H-Y TCR-transgenic mice forced to express CD4 might also contain cells capable of recognizing MHC class II (27). In all these cases, however, due to an inefficient allelic exclusion at the TCR{alpha} locus, the possibility that distinct TCRs were carriers of MHC class I- or class II-restricted specificities could not be ruled out. In contrast, through the use of T3.70 Fab fragments and endogenous TCR{alpha}- or RAG2-deficient cells we were able to demonstrate for the first time dual MHC-restricted specificity of the single TCR. In addition, in light of the fact that the transgenic mouse carrying this TCR has been extensively used by immunologists during the last 10 years as a model to study diverse issues in immunology (such as thymic selections, lineage commitment, tolerance induction, graft rejection, mature T cell homeostasis, and T cell memory generation), the discovery of its additional (auto)specificity may bear important implications on each of these processes.

Despite the reactivity of the H-Y TCR for self-MHC class II, negative selection has not been reported thus far in H-Y TCR-transgenic mice, other than clonal deletion induced by H-Y/H-2Db. This is, we believe, likely due to relatively subtle effects of the presence of H-2IAb on thymic development. Our findings are compatible with the view that some DP thymocytes expressing high levels of CD4 coreceptor and H-Y TCR are deleted due to cross-reactivity to self-MHC class II. We observed a two- to threefold increase in the percentage of DP thymocytes in male H-Y TCR-transgenic mice devoid of MHC class II expression as well as a twofold increase in the total number of thymocytes in female mice. Partial deletion of DP thymocytes due to tolerance was observed previously in different experimental models (38, 52, 53). It would appear that only thymocytes expressing high levels of CD4 were deleted by MHC class II in female H-Y mice. Deletion was probably compensated well by the generation of sufficient numbers of DP cells with lower CD4 expression, so that deletion could not be observed as a reduced percentage of DP cells. However, additional selection pressure from the H-Y Ag clearly unmasked the contribution of the MHC class II in clonal deletion. Taken together, compared with the male Ag, MHC class II would therefore have to qualify as a relatively weak tolerogen for the H-Y TCR. It remains to be established whether this is due to different levels or cell-type expression of the two Ags in the thymus, or some other unknown factor.

The ability of HYCD4 cells to grow in culture when stimulated with syngeneic B6 cells raises the issue of the control of the reactivity of CD4+ cells in the mouse, as H-Y female or male mice apparently do not suffer from autoimmunity. The overt autoreactivity of the HYCD4 cell line in vitro can probably be ascribed to exogenous IL-2 supply, which was shown to be essential for the tolerance breakdown in other cases of autoreactive CD4+ T cells (54). Interestingly, a CD4+ T cell population was found in H-Y TCR-transgenic SCID mice which predominantly (but not exclusively) expressed endogenous TCR{alpha}-chain, and which induced inflammatory bowel disease after transfer into nontransgenic, syngeneic SCID mice (35). Because this cell population failed to induce inflammatory bowel disease when transferred into H-Y TCR-transgenic hosts, it was suggested that these cells are held in check in vivo, presumably by some other T cell populations. Similar mechanisms might be responsible for the control over in vivo equivalents of HYCD4 cells.

The paucity of CD4+ T cells expressing H-Y TCR in the H-Y TCR-transgenic mice could result from a failure of DP thymocytes to commit to the CD4+ lineage or to an inability of already committed CD4+ T cells to be positively selected by self-MHC II ligands. Initial observations of H-Y TCR-transgenic mice devoid of MHC class II molecules revealed a population of CD4+CD8low H-Y TCRhigh, which was scarce in mice expressing MHC class II (55). Because the CD4+CD8low phenotype was thought to be transient, leading to the mature CD4+CD8- phenotype, the existence of this population in MHC class I-deficient mice but not in class I, class II double-deficient mice, was proposed as an argument for the stochastic model of lineage commitment. Recent data revealed, however, that this transient population of thymocytes might contain a significant proportion of CD4-CD8+ precursors (56, 57). In elegant sets of experiments using the coreceptor reexpression assay, this population of cells from H-Y TCR-transgenic animals primarily appeared to be committed to the CD8+ lineage (12). Because this was achieved only if positively selecting ligand H-2Db was present, it was concluded that commitment to the CD4 lineage might be preempted by MHC class I-restricted signals. Indeed, successful commitment to the CD4 lineage occurred if H-Y TCR nonselecting, H-2d thymocytes were used. Inasmuch as all these reports focused on the CD4+CD8low TCRhigh thymocyte progeny argue against the stochastic interpretation for the lineage commitment, they do not explain the virtual absence of CD4+ T cells expressing H-Y TCR in CD8 transgenic mice. Even though this can be explained by the instructive model for lineage commitment, it still remains puzzling that CD8 transgene expression resulted in the generation of CD4+ T cells in other, MHC class I-restricted, TCR-transgenic mice.

Collectively, our results suggest that mature T cells expressing CD4 and high levels of H-Y TCR may be deleted due to the reactivity of the TCR to self-MHC class II. This may explain why mature CD4+ T cells expressing H-Y TCR are rare in H-Y TCR-transgenic mice, even after CD8 coreceptor overexpression. More importantly, these findings also raise an intriguing possibility that tolerance could control the decision process of lineage commitment or might interfere with the program of commitment after the decision is made. In either case, the effects of tolerance may mask the influence of other determinants of lineage commitment.



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  		FIGURE 1. HYCD4 line expresses low levels of H-Y TCR. A, The expression of CD4/CD8 was analyzed by flow cytometry using 1 x 106 cells and fluorochrome-conjugated Abs. TCR expression was analyzed using F23.1 or T3.70 hybridoma supernatants followed by goat anti-mouse FITC (bold lines) or secondary Ab alone (plain lines). B, A redirected cytotoxic assay was performed using 25% T3.70 or F23.2 hybridoma supernatant. C, Transgenic TCR expression on ex vivo cells. Transgenic thymocytes were triple stained with phycoerythrin-conjugated anti-CD4, CyChrome-conjugated anti-CD8, and FITC-conjugated F23.1 or T3.70. Gates were placed on CD4+CD8+ or CD4-CD8+ single-positive (SP) cells as shown and staining with anti-TCR reagents was analyzed.

 


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  		FIGURE 5. Phenotypic and functional analysis of a T cell hybridoma expressing H-Y TCR. The surface expression of CD4 was analyzed by flow cytometry using 1 x 106 cells. H-Y TCR expression was analyzed using directly conjugated F23.1 Ab (anti-TCR Vß8.2), MR 14.1 (anti-clonotypic H-Y TCR Ab), and T3.70 (anti-H-Y TCR{alpha}-chain) (see Fig. 4A). IL-2 production by a T cell hybridoma in response to different numbers of stimulator cells was determined using IL-2-dependent CTLL-2 cells (see Fig. 4B). The results represent means ± SD of triplicate cultures. Data are representative of three independent experiments.

 


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  		FIGURE 6. H-2d, RAG2-/-, H-Y TCR-transgenic spleen cells respond to H-2IAb. Spleen cells (1 x 106) from B10.D2 RAG2-/- H-Y TCR-transgenic mice were stained for CD4 and CD8 expression (A). The same cells were tested for their proliferative response to irradiated stimulator cells (4 x 105/well) of wt, ß2m-, or MHC class II-deficient B6 haplotype or to anti-TCR Ab (H597, 25% hybridoma supernatant). The results represent the mean ± SD of triplicate cultures (B).

 

   Acknowledgments
 
We thank Janko Nikolic-Zugic (Sloan Kettering Memorial Cancer Center, New York, NY) for providing the H-Y breeder mouse; Dan Littman and members of his laboratory (NYU Medical Center, New York, NY) for the help with rederivation of the transgenic line; T. Darma Rao (NYU Medical Center) for performing the experiment using Borrelia-specific T cells; Robert Hayashi and Osami Kanagawa (Washington University School of Medicine, St. Louis, MO) for providing the TCR{alpha}-deficient H-Y hybridoma, MR14.1 mAb, and FACS profiles shown in Fig. 5A; and Alan Frey (NYU Medical Center) and Craig Davis (NYU Medical Center) for reading the manuscript.


   Footnotes
 
1 This work was supported in part by the Markey Charitable Trust Junior Investigator Award, National Institutes of Health Grant AI041573, and National Cancer Institute Core Support Grant 5P30 CA16087. I.A. is a Jeannette Greenspan Fellow in Cancer Research of the Kaplan Cancer Center. Back

2 Address correspondence and reprint requests to Dr. Stanislav Vukmanovic, Michael Heidelberger Division of Immunology, Department of Pathology, NYU Medical Center, 550 First Avenue, New York, NY 10016. E-mail address: Back

3 Abbreviations used in this paper: DP, CD4+CD8+ double-positive thymocytes; B6, C57BL/6; ß2m, ß2-microglobulin; wt, wild type. Back

Received for publication August 4, 1998. Accepted for publication November 5, 1998.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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