Role of TCR-restricting MHC density and thymic environment on selection and survival of cells expressing low-affinity T cell receptors

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1 Eur. J. Immunol : MHC density and thymic environment effects T cell selection and survival 1041 Role of TCR-restricting MHC density and thymic environment on selection and survival of cells expressing low-affinity T cell receptors Marianne M. Martinic 1,2,BeneditaRocha 3,KathyD.McCoy 1,HansHengartner 1 and Rolf M. Zinkernagel 1 1 Institute of Experimental Immunology, Department of Pathology, University Hospital Zurich, Zurich, Switzerland 2 Section on Immunology and Immunogenetics, Joslin Diabetes Center, Department of Medicine, Brigham and Women s Hospital, Harvard Medical School, Boston, USA 3 INSERM U591, Institut Necker, Paris, France H-Y-specific T cell receptor (TCR) transgenic mice express an MHC class I H-2D b -restricted TCR specific for a male antigen-derived peptide (H-Y). Selection and survival of cells expressing ts low-affinity TCR were analyzed under optimal and sub-optimal conditions. Optimal conditions were provided by a thymic environment with gh H-2D b density. Suboptimal conditions were provided by a thymic environment with decreased H-2D b density or by an athymic environment with either gh or low H-2D b density. Whereas negative selection was still guaranteed under sub-optimal conditions, selection and survival efficiency of cells expressing the transgenic TCR were strongly dependent on optimal conditions. These results indicated that both a thymic microenvironment and a gh density of TCR-restricting MHC molecules were needed to ensure selection and maintenance of cells expressing TCR with low affinity and hence a more diverse T cell repertoire. Key words: H-Y TCR / T cell selection and survival / MHC density / Thymic and athymic environment Received 19/1/04 Accepted 10/2/04 1 Introduction Thymocyte maturation takes place witn the thymus. After productive TCR-chain gene rearrangement, double-positive thymocytes (CD4 + CD8 + TCR g lo )interact with APC expressing self-peptide/mhc complexes. The overall avidity of ts thymocyte-apc interaction determines the fate of the thymocyte. A weak to intermediate avidity interaction transmits a survival signal to the maturing thymocyte (positive selection), whereas a strong avidity interaction transmits a signal to undergo programmed cell death (negative selection) (reviewed in [1]). Double-positive thymocytes expressing a TCR g not able to recognize self-mhc are eliminated by apoptosis (neglect) [2]. Ts selection process therefore ensures only the survival of self-mhc-restricted (potentially useful) and self-tolerant T cells, wch leave the thymus as mature T cells (reviewed in [1, 3 5]). Survival of individual T cells in the periphery, however, is only [DOI /eji ] Abbreviations: mln: Mesenteric lymph node SP: Single positive aceved if the T cell remains in continuous interaction with its TCR and TCR-restricting MHC molecules on APC, guaranteeing the maintenance of a diverse T cell repertoire [6 8]. Because continuous interaction between TCR and TCRrestricting MHC molecules is crucial for T cell selection and survival [6 12] and because it is known that cells expressing gh-affinity TCR are much less susceptible to TCR-restricting MHC density [13, 14], ts study analyzed whether changes in TCR-restricting MHC density had a direct influence on the selection and survival of cells expressing low-affinity TCR. Experiments were therefore performed using the low-affinity [15] MHC class I H-2D b -restricted H-Y-specific transgenic TCR [16] under optimal and sub-optimal conditions. Optimal conditions were provided by a thymic environment with gh TCR-restricting H-2D b density. Sub-optimal conditions were provided by a thymic environment with a decreased TCR-restricting H-2D b density or by an athymic environment with either gh or low H-2D b density. Our results indicated that, in addition to a thymic microenvironment, to obtain a diverse T cell repertoire with both gh- and low-affinity TCR, a gh density of TCR-

2 1042 M. M. Martinic et al. Eur. J. Immunol : restricting MHC molecules was also required to ensure selection and maintenance of cells expressing TCR with low affinity. 2 Results 2.1 Selection of H-Y-specific TCR transgenic T cells in mice with a selecting (H-2 bb )ornonselecting (H-2 dd ) H-2 haplotype The minor stocompatibility (H) male-specific (Y) antigen (H-Y), wch is expressed in male tissues, is derived from a protein encoded by the Smcy gene located on the Y chromosome [17]. The MHC class I H-2D b -restricted H-Y-specific transgenic TCR [16] binds specifically to the male antigen-derived peptide (KCSRNRQYL) presented on H-2D b molecules [17]. T cell selection in both female and male mice expressing ts TCR transgene has been described (reviewed in [18]). Briefly, in H-Y-specific TCR transgenic females, absence of the peptide H-Y and presence of TCR-restricting H-2D b resulted in positive selection and further survival in the periphery of H-Yspecific (Fig. 1A) [16, 19]. However, only up to 40% of peripheral expressed the H-Y-specific transgenic TCR (Fig. 1A and 2). The remaining + population had recombined the endogenous TCR loci and therefore changed TCR specificity during thymocyte selection (Fig. 1A, and see also below) [20 22]. In H-Y-specific TCR transgenic males, presence of both the self-peptide H-Y and TCRrestricting H-2D b resulted in negative selection of CD4 + CD8 + DP thymocytes expressing the transgenic TCR and, therefore, an absence of H-Y-specific in the periphery (Fig. 1B) [16]. However, cells showing a down-regulation of their coreceptor and transgenic TCR, resulting in lo transgenic TCR g, were present in TCR transgenic males (Fig. 1B) [16, 19, 23]. lo + cells, where binding to self-peptide/mhc complex is most likely to be of low avidity, apparently escaped negative selection in the thymus. These cells, wch migrate into the periphery as mature self-mhc-restricted, H-Ynon-reactive T cells, do not proliferate after contact with male stimulator cells but react against non-h-y antigens [16, 19]. H-Y-specific TCR transgenic mice, wch do not express TCR-restricting MHC class I H-2D b molecules, did not show positive selection of H-Y-specific (Fig. 1C) [18, 24, 25]. These cells, however, could escape death from neglect via an endogenous rearrangement of TCR -chain genes (Fig. 1C, and see also below) [18, 24, 25]. lo transgenic TCR g were absent in both H-Y-specific TCR transgenic females and in TCR transgenic mice with a non-selecting H-2 haplotype (Fig. 1B, C). Fig. 1. Selection of the H-Y-specific transgenic TCR in mice with a selecting (H-2 bb ) or a non-selecting (H-2 dd )H-2haplotype. Blood lymphocytes of H-Y-specific TCR transgenic females [F0 (H-Y), H-2 bb ] (A) and males (B) with a selecting (H-2 bb ) or a non-selecting (H-2 dd ) H-2 haplotype [F2 (H-Y), H- 2 dd ] (C) were stained for surface expression of CD8 and CD8 g chains. Circles in dot plots represent and lo + populations. In all mice the percentage of + cells was between 7 8%, wle the percentage of CD8 cells was always below 0.5%. Blood lymphocytes of H-Yspecific TCR transgenic mice were gated on [CD8 for H-Y-specific TCR transgenic females (A) and H-Yspecific TCR transgenic mice with a non-selecting H-2 haplotype (C), CD8 lo for H-Y-specific TCR transgenic males (B)] and analyzed for surface expression of transgenic (T3.70) and transgenic g chain (V g 8.1/2) of H-Y-specific TCR. Numbers in upper right quadrants represent the percentage of expressing. Similar results were obtained with splenic lymphocytes (data not shown). At least three individual mice were tested in each group. One out of eight similar experiments is shown. 2.2 Selection of H-Y-specific transgenic TCR is independent of age The percentage of peripheral expressing the H-Y-specific transgenic TCR in females with the selecting H-2 haplotype (H-2 bb ) varied between 15 40% independent of the age of these transgenic mice (Fig. 2 and data not shown). The variability was probably because these mice were housed under conventional conditions and were on a mixed C57BL/6xSv129 background [16] rather than a pure C57BL/6 background. The percentage of peripheral expressing the transgenic TCR in females on a pure C57BL/6 background housed under SPF-conditions is 34.4%±5.3%, independent of their age. In H-Y-specific TCR transgenic males with the selecting H-2 haplotype (H-2 bb ), the percentage of lo was always above 90% (Fig. 1, and see also below) confirming earlier results [16].

3 Eur. J. Immunol : MHC density and thymic environment effects T cell selection and survival 1043 remaining + population still expressed the transgenic TCR g -chain but paired with endogenous TCR - chains (Fig. 2, and see also below). The approximately fivefold decrease in the percentage of expressing the transgenic TCR was observed exclusively in the periphery of these mice because analysis of F1 (H- Y) versus F0 (H-Y) CD8 single-positive (SP) thymocytes revealed only a twofold decrease in the percentage of CD8-SP thymocytes expressing the transgenic TCR [84.42±0.7% CD8-SP thymocytes in F0 (H-Y) (n=4) versus 44.81±1.85% in F1 (H-Y) (n=4) TCR transgenic females]. from F2 (H-Y) transgenic females with a non-selecting H-2 haplotype (H-2 dd ) did not show positive selection of H-Y-specific transgenic TCR but expressed gh levels of the transgenic TCR g -chain paired with endogenous TCR chains (Fig. 2, and see also below). Fig. 2. In females, selection and survival of cells expressing the H-Y-specific transgenic TCR is dependent on the density of TCR-restricting MHC class I H-2D b molecules. Blood lymphocytes of H-Y-specific TCR transgenic females [F0, F1, F2 (H-Y)] with different H-2 haplotypes (H-2 bb,h-2 bd,h-2 bk,h- 2 bq, H-2 dd ) were stained for surface expression of TCRrestricting H-2D b molecules. Numbers in stogram plots represent mean fluorescence intensity (MFI) of H-2D b.blood lymphocytes of transgenic females were additionally gated on cells and analyzed for surface expression of (T V g 8.1/2 + ). Numbers in upper right quadrants represent the percentage of cells expressing. Numbers beside dot plots represent ranges of percentages of cells expressing in females with the selecting (H-2 bb ), a mixed (H-2 bd,h-2 bk,h-2 bq ), or a non-selecting H-2 haplotype (H-2 dd ). At least three individual mice were tested in each group. One out of three similar experiments is shown. 2.3 Direct correlation of TCR-restricting H-2D b density with the number of H-Y-specific TCR transgenic CD8 in females To analyze the amount of CD8 expressing the transgenic TCR in mice with half the H-2D b density, H-Yspecific TCR transgenic mice [F0 (H-Y), H-2 bb ] were crossed with BALB/c (H-2 dd ), CBA (H-2 kk ) or C57B10.G (H-2 qq ) mice to obtain TCR transgenic mice with a heterozygous H-2 haplotype [F1 (H-Y), H-2 bd,h-2 bk or H-2 bq, respectively]. Additionally, F1 (H-Y), H-2 bd mice were crossedwithbalb/cmicetoobtainf2micewithanonselecting H-2 haplotype [F2 (H-Y), H-2 dd ]. All F1 (H-Y) transgenic females exbited a strong reduction in the percentage of peripheral H-Y-specific TCR transgenic, ranging only between 5 10% (Fig. 2). The In H-Y-specific TCR transgenic females housed under SPF conditions, the decrease in the percentage of TCR transgenic in females with heterozygous [F1 (H-Y) H-2 bd ] versus homozygous H-2D b density [F0 (H-Y) H-2 bb ] was always about twofold in both thymus and periphery (B. Rocha, unpublished data). The differences observed with our results (fivefold decrease in the periphery) could be due to the housing conditions of our mice. Under conventional conditions, mice can be infected with pathogens resulting in proliferation of pathogen-specific T cells, and hence an accompanying decrease in the percentage of expressing the H-Y-specific transgenic TCR. However, if selection and survival efficiency of cells expressing the H-Yspecific transgenic TCR in females were strictly dependent on H-2D b + density, absolute numbers of cells expressing the transgenic TCR, irrespective of the proliferation of other T cells, should directly correlate with H-2D b density. We therefore calculated absolute numbers of H-Y-specific TCR transgenic CD8 + T cells in both the thymus and periphery of transgenic females with homozygous [F0 (H-Y) H-2 bb ] or heterozygous [F1 (H-Y) H-2 bd ] H-2 haplotype (Fig. 3). The obtained results revealed that in H-Y-specific TCR transgenic females with heterozygous H-2D b density [F1 (H-Y) H-2 bd ], absolute numbers of TCR transgenic CD8-SP thymocytes + and of TCR transgenic splenocytes were decreased by almost half when compared to transgenic females with homozygous H-2D b density (Fig. 3). In agreement with ts, absolute numbers of peripheral expressing transgenic TCR g and endogenous V 2 chains were increased by more than twofold in F1 versus F0 H-Y-specific TCR transgenic females (data not shown). The discrepancy between a fivefold decrease in percentages (Fig. 2) and an almost twofold decrease in absolute numbers (Fig. 3) of + spleen cells expressing H-Y-specific transgenic TCR in trans-

4 1044 M. M. Martinic et al. Eur. J. Immunol : genic females with heterozygous (H-2 bd )comparedto homozygous (H-2 bb ) H-2 haplotype could be explained by a threefold gher number of total + spleen cells in the former [1.14± spleen cells in F0 (H-Y) (n=4) versus 3.44± spleen cells in F1 (H-Y) (n=4) TCR transgenic females]. In summary, in H-Y-specific TCR transgenic females a reduction in H-2D b density by 50% [F1 (H-Y) H-2 bd ] resulted in an almost 50% decrease in absolute numbers of both thymic and peripheral CD8 expressing the transgenic TCR (Fig. 3). Therefore, efficiency of positive selection and maintenance of the H-Y-specific transgenic TCR appeared to depend directly on TCRrestricting H-2D b density. 2.4 H-2D b density determines the degree of CD8 > I co-receptor down-regulation in H-Yspecific TCR transgenic males Fig. 3. Direct correlation in females of absolute numbers of H-Y-specific TCR transgenic CD8 with TCR-restricting H-2D b density. Total numbers of H-Y-specific TCR transgenic CD8-SP thymocytes and of TCR transgenic + spleen cells were calculated in H-Y-specific TCR transgenic females with the homozygous [F0 (H-Y) H-2 bb ]orheterozygous [F1 (H-Y) H-2 bd ] H-2 haplotype. The ratio of H-Y-specific TCR transgenic CD8 in females with heterozygous to homozygous H-2 haplotype is about 0.6 for both thymocytes and spleen cells. Four individual mice were tested in each group. One out of two similar experiments is shown. Fig. 4. Direct correlation of expression levels of coreceptor in H-Y-specific TCR transgenic males with TCRrestricting H-2D b density. Blood lymphocytes of H-Y-specific TCR transgenic males [F0, F1, F2 (H-Y)] with different H-2 haplotypes (H-2 bb,h-2 bd,h-2 bk,h-2 bq,h-2 dd )werestained for surface expression of H-2D b, and CD8 and CD8 g chains. Numbers in stogram plots represent MFI of H-2D b, CD8 and CD8 g, as indicated. Numbers beside stogram plots represent percentages of expressing in males with the selecting (H-2 bb ), a mixed (H-2 bd,h-2 bk,h-2 bq ), or a non-selecting H-2 haplotype (H-2 dd ). At least three individual mice were tested in each group. One out of three similar experiments is shown. In H-Y-specific TCR transgenic males with a homozygous or heterozygous H-2 haplotype the percentage of lo cells expressing the transgenic TCR was comparable in the thymus (data not shown) and in the periphery (Fig. 4). from H-Y-specific TCR transgenic males with a non-selecting H-2 haplotype did not express the H-Y-specific transgenic TCR (Fig. 4), but expressed transgenic TCR g chains paired with endogenous TCR chains, confirming our previous observations (Fig. 1C). Surprisingly, although the percentage of lo cells expressing the transgenic TCR was comparableinf0(h-y)andf1(h-y)transgenicmales,a drastic difference was seen in CD8 and CD8 g chain expression levels on both thymic (data not shown) and peripheral CD8 (Fig. 4). from F0 (H-Y) transgenic males with homozygous H-2D b density showed the strongest down-regulation for both CD8 and CD8 g chains (Fig. 4). As expected, from F2 (H-Y) transgenic males with a non-selecting H-2 haplotype exbited the same -expression levels as their female counterparts (data not shown). Interestingly, from F1 (H-Y) transgenic males with half the H-2D b density had less down-regulated CD8 and CD8 g chains than transgenic males with homozygous H-2D b density (Fig. 4). In ts case, due to the lower overall avidity interaction between the transgenic TCR and H-Y/H-2D b complexes in males with heterozygous H-2D b density, weaker down-regulation of the CD8 and CD8 g chains appeared to be sufficient to escape from negative selection. Additionally, transgenic males with heterozygous H-2D b density appeared to display a gher escape from negative selection when compared to transgenic males with homozygous H-2D b density, as determined by a 1.7-fold increase in total numbers of CD8 lo -SP thymocytes expressing the transgenic TCR [4.49± CD8 lo -SP thymocytes in F1 (H-Y) (n=3)

5 Eur. J. Immunol : MHC density and thymic environment effects T cell selection and survival 1045 versus 2.70± CD8 lo -SPthymocytesinF0(n=3) TCR transgenic males]. In the periphery, however, absolute numbers of lo in both F0 (H-Y) and F1 (H-Y) TCR transgenic males were comparable (data not shown). Ts was due to the fact that, despite having a 2-fold decrease in the percentage of total + spleen cells, F1 (H-Y) TCR transgenic males had a 2-fold increase in total spleen cell numbers when compared to F0 (H-Y) TCR transgenic males. Ts lo resulted in comparable absolute numbers of + spleen cells (data not shown). Importantly, H-Y-specific were never detected in TCR transgenic males irrespective of their TCR-restricting H-2D b density (Fig. 4). Taken together, these results indicated that the density of TCR-restricting MHC class I H-2D b molecules was an important parameter in determining the fate of the H-Yspecific TCR transgenic T cells. 2.5 Selection of H-Y-specific transgenic TCR in athymicmicewithdifferenth-2haplotypes We next analyzed the fate of the low-affinity H-Y-specific transgenic TCR in an athymic environment. For ts purpose, H-Y-specific TCR transgenic mice were crossed with C57BL/6- or BALB/c-nudes for two generations, yielding euthymic and athymic H-Y-specific TCR transgenicmice(tcrh-y +/ ) expressing different H-2 haplotypes (H-2 bb, H-2 bd or H-2 dd ). Because of the strong decrease in CD8 + cell numbers in athymic compared to euthymic TCR transgenic mice, mesenteric lymph node (mln) and spleen cells of athymic mice were first enriched for Thy1.2 via MACS purification before analysis of transgenic TCR expression. The amount of CD8 + mln and spleen cells in athymic mice was reduced by fold when compared to + populations (data not shown). As already observed in euthymic females (Fig. 2, 3), selection and survival efficiency of cells expressing the transgenic TCR in athymic females appeared to depend directly on the TCRrestricting H-2D b density (Fig. 5). The percentage of expressing the transgenic TCR in athymic females with half the H-2D b density (TCR H-Y +/,H-2 bd ) was decreased by half when compared to athymic females with homozygous H-2D b density (TCR H-Y +/,H- 2 bb ) (Fig. 5). In contrast, as already observed in euthymic males, in athymic TCR transgenic males with selecting H-2 haplotypes (TCR H-Y +/,H-2 bb,h-2 bd ) H-Y-specific were never detected (Fig. 6). In addition, escape from negative selection was also observed in athymic males via down-regulation of both co-receptor and transgenic TCR (Fig. 6). The degree of down-regulation of and transgenic Fig. 5. Selection of the H-Y-specific transgenic TCR in athymic mice with different H-2 haplotypes. Thy1.2 + MACSpurified mln cells of athymic H-Y-specific TCR transgenic (TCR H-Y +/ ) females and males with different H-2 haplotypes (H-2 bb,h-2 bd,h-2 dd ) were gated on and analyzed for surface expression of (T V g 8.1/2 + ). Athymic H-Y-specific TCR transgenic males with selecting H-2 haplotypes (TCR H-Y +/,H-2 bb,h- 2 bd ) were devoid of cells and therefore were gated on lo cells. Transgenic negative littermates (TCR H- Y /,H-2 bb ) were used as negative controls. Numbers in dot plots represent percentage of + mln cells expressing. Similar results were obtained with splenic lymphocytes (data not shown). At least four mice were tested in each group. One out of three similar experimentsisshown. TCR directly correlated with the H-2D b density; the gher the H-2D b density, the stronger the downregulation of both and transgenic TCR in athymic males (Fig. 6). Surprisingly, athymic TCR transgenic mice with a nonselecting H-2 haplotype (TCR H-Y +/,H-2 dd ) expressed + + Tcells(Fig.5)incontrastto their euthymic counterparts (Fig. 1C). With increasing age, the percentage of TCR transgenic + T cells decreased but never disappeared completely (data not shown). Furthermore, in athymic TCR transgenic females with a non-selecting H-2 haplotype (TCR H-Y +/,H-2 dd ) around one-fourth of expressed the transgenic TCR,butnotthetransgenicTCRg, chain (Fig. 5). With increasing age, the percentage of these cells increased and they also appeared in athymic males with a non-selecting H-2 haplotype (data not shown). In young athymic TCR transgenic mice with a non-

6 1046 M. M. Martinic et al. Eur. J. Immunol : Fig. 6. Direct correlation of expression levels of coreceptor and inathymich-y-specific TCR transgenic males with TCR-restricting H-2D b density. Thy1.2 + MACS-purified mln cells of athymic H-Y-specific TCR transgenic mice (TCR H-Y +/ )withdifferenth-2haplotypes (H-2 bb,h-2 bd,h-2 dd ) were stained for surface expression of CD8 and CD8 g chains. + mln cells were further analyzed for surface expression of transgenic TCR (T3.70) and transgenic TCR g (V g 8.1/2) chains. Numbers in stogram plots represent MFI of CD8 g,cd8g,transgenic TCR, and transgenic TCR g, as indicated. Athymic H-Yspecific TCR transgenic females (TCR H-Y +/,H-2 bd )(bottom panels) were used as positive controls. Similar results were obtained with splenic lymphocytes. Three mice were used in each group. One out of three similar experiments is shown. selecting H-2 haplotype we could only detect endogenous V 2 (Fig. 7B) and V 8 rearrangements (data not shown) in males but not in females. However, extrathymic differentiation is much less efficient than thymus differentiation and, therefore, generation of T cells is rare [26]. In addition, there are considerable variations in the TCR chains expressed from mouse to mouse [27]. For these reasons, one cannot predict the frequency of individual V expression in nudes. Furthermore, in athymic females with a non-selecting H-2 haplotype two-trds of expressed the transgenic TCR g chain but not the transgenic TCR chain (Fig. 5). It is therefore very likely that the transgenic TCR g chain is associated with endogenous TCR chains. Finally, when older TCR transgenic mice with non-selecting H-2 haplotype were analyzed, endogenous V chain gene rearrangements were detected in both males and females (data not shown). Taken together, the results obtained with athymic TCR transgenic mice with a non-selecting H-2 haplotype (Fig. 5, 7B) differed from the ones obtained with their euthymic counterparts (Fig. 1C, 7A). Perhaps in the absence of a thymic microenvironment, H-Yspecific TCR transgenic cells used different mechanisms to escape death from neglect, for example by rearranging not only endogenous V but also V g chains (explaining the presence of expressing exclusively Fig. 7. Endogenous V chain gene rearrangement in euthymic (A) and athymic (B) H-Y-specific TCR transgenic mice. Blood lymphocytes of euthymic (A) and Thy1.2 + MACS-purified mln cells of athymic (B) H-Y-specific TCR transgenic (TCR H-Y +/ ) females and males with different H-2 haplotypes (H-2 bb,h-2 bd,h-2 dd ) were gated on and analyzed for surface expression of endogenous V 2 and transgenic TCR g chains (V 2 + V g 8.1/2 + ). Transgenic negative littermates (TCR H-Y / ) were used as negative controls. Numbers in upper left and upper right quadrants represent the percentage of being V 2 V g 8.1/2 + and V 2 + V g 8.1/2 +, respectively. Similar results were obtained with splenic lymphocytes. At least four individual mice were tested in each group. One out of four similar experimentsisshown. transgenic TCR, but not transgenic TCR g,chainsin athymic females with a non-selecting H-2 haplotype) or by expressing dual TCR (explaining the presence of

7 Eur. J. Immunol : MHC density and thymic environment effects T cell selection and survival 1047 expressing in athymic mice with a non-selecting H-2 haplotype). These questions will be addressed in detail in a future study using different ages of athymic TCR transgenic mice with a non-selecting H-2 haplotype. Finally, endogenous V chain gene rearrangements in euthymic (Fig. 7A) and athymic (Fig. 7B) TCR transgenic mice with selecting H-2 haplotype (TCR H-Y +/,H-2 bb,h- 2 bd ) were analyzed. Whereas in euthymic TCR transgenic females with homozygous (H-2 bb ) and heterozygous (H- 2 bd ) H-2 haplotypes expression of endogenous V 2 (Fig. 7A) and V 8 chains (data not shown) could be detected, only athymic transgenic females with heterozygous H-2 haplotype showed expression of endogenous V 2 chains (Fig. 7B). Perhaps due to the less efficient selection and survival of H-Y-specific transgenic T cells in an environment with decreased H-2D b density, had gher chances to survive if they expressed another TCR with gher affinity for selfpeptide/mhc. Endogenous V chain gene rearrangements in TCR transgenic males with selecting H-2 haplotype, however, were undetectable for both euthymic (Fig. 7A) and athymic (Fig. 7B) males. Taken together, these results showed that protection against self-reactive H-Y-specific TCR transgenic T cells in males was guaranteed, even under sub-optimal conditions. The efficiency of selection and maintenance of cells expressing the H-Y-specific transgenic TCR in females, however, corresponded to the TCR-restricting H-2D b density. Optimal conditions such as a thymic microenvironment and gh TCR-restricting H-2D b density resulted in gh numbers of H-Y-specific transgenic T cells wle sub-optimal conditions resulted in loss of H- Y-specific transgenic T cell numbers. 3 Discussion Ts study showed that positive selection and subsequent survival of cells expressing the low-affinity H-Yspecific transgenic TCR was less efficient with reduced TCR-restricting MHC density and/or an athymic environment. These findings contrast with data obtained with rather gher affinity TCR, such as the 318 or 2C transgenic TCR [28, 29]. In 318 TCR transgenic mice with exclusively selecting (H-2 bb ) or heterozygous H-2 haplotype (H-2 bd,h-2 bk or H-2 bq ), the percentage of peripheral CD8 expressing transgenic TCR always remained above 50% independent of reduced TCR-restricting H- 2D b density and independent of the presence of a thymic environment ([14] and unpublished data). Moreover, Zerrahn et al. [13] showed that only 2C TCR, but not H-Yspecific TCR, transgenic cells are positively selected and survive in the periphery when hematopoietic cells exclusively express TCR-restricting MHC molecules. Furthermore, in Oncostatin M (OM) transgenic mice, wch show a profound thymic atrophy but massive extrathymic T cell development particularly in mln [30, 31], positive selection and survival of the H-Y-specific but not of the 2C transgenic TCR is drastically reduced [32]. Taken together, these results indicate that as soon as conditions are sub-optimal (reduced TCR-restricting MHC density, unfavorable microenvironment and cytokine milieu as opposed to a thymic microenvironment) the efficiency of positive selection and survival of cells expressing low-affinity TCR is decreased. In contrast, even under sub-optimal conditions the intrinsic affinity of the gh-affinity TCR for self-peptide/mhc complexes was gh enough to provide sufficient survival signals. Negative selection of gh-avidity self-reactive T cells, however, is always complete [33, 34]. Even under suboptimal conditions, as already shown in H-Y/LckOM and 2C TCR/LckOM double transgenic mice, all self-reactive T cells are absent [32]. The latter observation was confirmed here by showing the absence of all H-Y-specific cells ( + ) in euthymic and athymic TCR transgenic males with homozygous or with decreased H-2D b density (Fig. 1B, 4, 6). Finally, escape from negative selection is probably only possible with low-affinity TCR, as seen with the H-Yspecific transgenic TCR (Fig. 1, 4 6).Apparently,asmall down-modulation of the co-receptor was sufficient to fall beneath the overall avidity threshold for negative selection. In contrast, TCR with an intrinsically gh affinity have usually no chance to escape negative selection under physiological conditions. In summary, under sub-optimal conditions, negative selection remains tight, therefore ensuring absence of potential auto-reactivity. In contrast, the efficiency of positive selection and survival of cells expressing TCR with low affinity is decreased, leading to a less diverse T cell repertoire. Depending on the severity of sub-optimal conditions, the decrease in T cell repertoire diversity may sft from an impaired immunocompetence to a general immunodeficiency as seen in thymus-deficient nude mice or humans. Therefore, selection and survival of cells expressing TCR with low affinity, and hence of a more diverse T cell repertoire, is efficient only under optimal conditions when restricting MHC antigens are optimally present both in the thymus, for initial positive selection, and in the periphery, to ensure further survival of all TCR-expressing cells.

8 1048 M. M. Martinic et al. Eur. J. Immunol : Materials and methods 4.1 Mice Breeding stocks of H-Y-specific TCR transgenic mice [F0 (H-Y), H-2 bb ] were a generous gift from Dr. J. Kirberg (Max Planck Institute for Immunobiology, Freiburg, Germany). C57BL/6 (H-2 bb ), BALB/c (H-2 dd ), CBA (H-2 kk ), C57B10.G (H- 2 qq ) and BALB/c-nudes (H-2 dd )wereobtainedfromtheinstitute of Laboratory Animal Science, University of Zurich, Switzerland. C57BL/6-nudes (H-2 bb ) were purchased from RCC Biotech (Fullinsdorf, Switzerland). H-Y-specific TCR transgenic mice were bred with BALB/c, CBA or C57B10.G mice yielding H-Y-specific TCR transgenic mice [F1 (H-Y)] with a heterozygous H-2 haplotype (H-2 bd,h-2 bk or H-2 bq, respectively). To obtain transgenic mice with a non-selecting H-2 haplotype (H-2 dd ), H-Y-specific TCR transgenic mice were bred twice with BALB/c [F2 (H-Y)] and tested with PCR and FACS analysis for expression of transgenic TCR, presence of H-2 dd and absence of H-2 bb MHC haplotype. To obtain athymic transgenic mice, H-Y-specific TCR transgenicmicewerebredwithc57bl/6-orbalb/c-nudesfor two generations, yielding euthymic and athymic H-Yspecific TCR transgenic mice (TCR H-Y +/ ) expressing different H-2 haplotypes (H-2 bb,h-2 bd or H-2 dd ). The H-2 haplotype of these mice and expression of transgenic TCR were tested using PCR and FACS analysis. All mice were housed under conventional conditions. All animal experiments were performed in compliance with guidelines of the Swiss law on animal protection and the Institute of Experimental Immunology. 4.2 PCR DNA for typing was prepared from mouse-tail biopsy specimens [35]. Typing was performed using primers specific for the H-Y-specific transgenic TCR (V g 8.2-fwd: 5 -ACA AGG TGG CAG TAA CAG GA-3 and J g 2.3-rev: 5 -ACA GTC AGT CTG GTT CCT GA-3 ), for both H-2 bb and H-2 dd MHC haplotypes(ea5 -fwd:5 -AGTCTTCCCAGCCTTCACACTCAG AGG TAC-3 and Ea3 -rev: 5 -CAT AGC CCC AAA TGT CTG ACC TCT GGA GAG-3 ) and for the H-2 dd MHC haplotype (K5 -fwd:5 -CATGGGCATAGAAAGGGCAGTCTTTGA ACT-3 and Ea3 -rev primer). A PCR product of 302 base pairs (bp) indicated the presence of H-Y-specific transgenic TCR, 155 bp and 700 bp indicated the presence of H-2 b and H-2 d MHC haplotype, respectively, and 210 bp indicated the presence of H-2 d MHC haplotype. 4.3 MACS purification Spleen and mln cells from athymicmicewereincubated with Thy1.2 (CD90) MicroBeads (Miltenyi Biotec) at 1:10 dilution for 30 min on ice. After wasng, Thy1.2 were purified using an automacs according to standard protocol (Miltenyi Biotec). 4.4 Flow cytometric analysis Single-cell suspensions from peripheral blood, thymus and spleen from euthymic mice, and from Thy1.2 + MACSpurified spleen and mln cells from athymic mice were stained with the following antibodies: anti-cd8 -APC (53 6.7), anti-cd8 g.2-pe (53 5.8), T3.70-Biotin (specific for transgenic TCR chain of H-Y-specific TCR) (ebioscience and generous gift from Dr. B. Rocha), anti-v 2-biotin (B20.1), anti-v g 8.1/2-FITC (specific for transgenic TCR g chain of H-Y-specific TCR) (MR5 2) and anti-h-2d b -biotin (KH95). Except for T3.70-biotin, all antibodies were purchased from PharMingen. Streptavidin-PerCP and streptavidin-tricolor were purchased from PharMingen and Caltag Laboratories, respectively. All samples were acquired on a FACSCalibur and analyzed using CellQuest software (Becton Dickinson). Acknowledgements: The authors wish to thank V. 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