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Fas Modulates both Positive and Negative Selection of Thymocytes
Cellular Immunology 194, 127–135 (1999) Article ID cimm.1999.1502, available online at http://www.idealibrary.com on
Fas Modulates both Positive and Negative Selection of Thymocytes Kazuhiro Kurasawa, Yoshiko Hashimoto, and Itsuo Iwamoto Department of Internal Medicine II, Chiba University School of Medicine, Chiba 260, Japan Received July 15, 1998; accepted March 31, 1999
We studied the functional role of Fas (CD95) in thymic T cell development using the TCR transgenic mice homozygous for the lpr mutation, DO10 lpr/lpr mice. In DO10 lpr/lpr mice, the differentiation of CD4 1CD8 1 double-positive (DP) thymocytes to CD4 1 single-positive (SP) thymocytes was markedly impaired, as indicated by decreased generation of CD4 1 SP thymocytes and reduced ratio of CD4 1 SP thymocytes to DP thymocytes in lpr/lpr mice compared with those of 1/1 mice. Activation of DP thymocytes in the process of positive selection was also significantly inhibited in DO10 lpr/lpr mice, as shown by the lower levels of CD69 expression on DP thymocytes in lpr/lpr mice compared to 1/1 mice. Furthermore, the deletion of DP thymocytes induced by in vivo administration of OVA peptide (up to 150 mg) and anti-TCR clonotype mAb did not occur in DO10 lpr/lpr mice, whereas these treatments significantly decreased DP thymocytes in DO10 1/1 mice. On the other hand, no significant difference in DO10 transgenic TCR expression on DP thymocytes was found between DO10 lpr/lpr and 1/1 mice. Together, these results indicate that Fas is importantly involved in both positive and negative selection of thymocytes. © 1999 Academic Press
INTRODUCTION The fate of T lymphocytes in the thymus is determined by the interaction between their T cell receptors (TCR) and peptide/MHC complexes on thymic stroma cells (1–3). Thymocytes bearing TCR with low avidity to the peptide/MHC complex are positively selected and rescued from apoptosis, being allowed to differentiate to mature T cells (positive selection). On the other hand, thymocytes bearing TCR with high avidity to the peptide/MHC complex are induced to undergo apoptosis (negative selection). Thymocytes with TCR that cannot interact with the peptide/MHC complex are also led to apoptosis, termed “death by neglect.” During these processes, more than 95% of T cells generated in the thymus die there; the remaining 5% migrate to the peripheral lymphoid organs as mature T cells (4, 5). However, little is known about the roles of cell-surface
molecules other than TCR in inducing positive and negative selection of thymocytes. The Fas antigen (Fas/CD95) is a 45-kDa cell-surface protein that belongs to the tumor necrosis factor (TNF)/nerve growth factor receptor family (6 – 8). When Fas ligand (FasL) or agonistic anti-Fas antibody binds to Fas, apoptosis is induced in sensitive cells (9 –13). Among thymocytes, CD4 1 CD8 1 doublepositive (DP) 1 cells and CD4 1 CD8 2 or CD4 2 CD8 1 single-positive (SP) cells abundantly express Fas (14 –16). In addition, DP thymocytes selectively undergo apoptosis when exposed to agonistic anti-Fas antibody in vitro and in vivo, whereas SP thymocytes were resistant to Fas-induced apoptosis despite their abundant expression of Fas (15, 16). However, the functional importance of Fas in thymocyte development has not been fully elucidated, although recent studies have shown that the Fas/FasL system is involved in peripheral clonal deletion of mature T cells (17–22). Therefore, we decided to determine whether Fas is involved in thymocyte development, especially in positive and negative selection of thymocytes. For this purpose, we introduced the lpr mutation, a defective Fas gene (23, 24), into a TCRab transgenic mouse (DO10 mouse) (25) and generated the TCR transgenic mice homozygous for the lpr mutation, DO10 lpr/lpr mice, which express no detectable Fas on the cell surface. Using DO10 lpr/lpr mice, we studied the role of Fas in positive selection of thymocytes by analyzing the generation of CD4 1 SP thymocytes from CD4 1 CD8 1 DP thymocytes and the expression levels of CD69 and TCR on thymocytes in the Fas-mutated mice. We also studied the role of Fas in negative selection of thymocytes by administrating antigenic peptide and anti-TCR clonotype antibody to DO10 lpr/lpr mice. Our results indicate that Fas is involved in both positive and negative selection of thymocytes.
1 Abbreviations used: DP, CD4 1CD8 1 double-positive; OVA, ovalbumin; SP, CD4 1 or CD8 1 single-positive.
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0008-8749/99 $30.00 Copyright © 1999 by Academic Press All rights of reproduction in any form reserved.
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MATERIALS AND METHODS Generation of DO10 TCR Transgenic Mice Carrying the lpr/lpr Mutation (DO10 lpr/lpr Mice) DO10 TCR transgenic mice, which express a transgenic TCRab specific for ovalbumin peptide 323–339 (OVA 323–339)/I-A d complex (25), were kindly provided by Dr. D. Loh (Washington University, Seattle, WA) and were maintained in our university animal facility. MRL-lpr/lpr mice were purchased from Clea Japan (Tokyo, Japan). DO10 TCR transgenic mice (H-2 d) were crossed with MRL-lpr/lpr mice (H-2 k). F1 mice were analyzed for DO10 transgenic TCR expression as described below. The F1 mice bearing the transgenic TCR (H-2 d/k, lpr/1) were then mated with the F1 mice without transgenic TCR (H-2 d/k, lpr/1) to generate two genotypes of F2 mice bearing the transgenic TCR, DO10 1/1 mice (H-2 d/d) and DO10 lpr/lpr mice (H-2 d/d). F2 mice were analyzed for DO10 transgenic TCR expression, lpr genotype, and H-2 haplotype as described below. All animals were housed under specific pathogen-free conditions. DO10 transgenic TCR expression was detected by PCR analysis of tail DNA using Va13.1-5 and Ja primers (sense: 59-CAGGAGGGATCCAGTGCCAGC and anti-sense: 59-TGGCTCTACAGTGAGTTTGGT) (the primer sequences were provided by Dr. D. Loh). Tail DNA was prepared by protease K digestion, phenol/ chloroform extraction, and ethanol precipitation. The lpr genotype was determined by PCR analysis of intron 2 of the Fas gene, where an early transposable element is inserted in the mutation (24). The PCR analysis was performed with a sense primer for intron 2 (59-GTAAATAATTGTGCTTCGTCAG) (18) and two antisense primers; one is for intron 2 (59-CAGGGAAATGTAGCAAGATG) and the other is for ETn (59-GTTGCGACACCAGTTATGAA). The PCR products from lpr/1 were 215 bp (intron 2–intron 2) and 282 bp (intron 2–Etn) in size, whereas only the 215-bp product was amplified from lpr/lpr mice. The PCR products were distinguished by electrophoresis on 2% agarose gel. The expression of Fas on thymocytes was analyzed by flow cytometry with anti-murine Fas mAb (Jo2) plus FITC-conjugated anti-hamster IgG mAb (Pharmingen, San Diego, CA) to confirm the results of PCR analysis of lpr genotype, when mice were sacrificed for further analysis. The H-2 haplotype was examined by flow cytometry analysis with FITC-conjugated anti-H-2 d mAb (Meiji Nyugyo, Tokyo, Japan) and FITC-conjugated anti-H-2 k mAb (Pharmingen). Flow Cytometry Cells from thymus and spleens (1 3 10 6) were first incubated with anti-Fc receptor mAb 2.4G2 to prevent nonspecific staining and were stained with fluorescence- or biotin-conjugated antibodies in phosphate-
buffered saline (PBS) containing 1% fetal calf serum (FCS) for 30 min at 4°C. The following FITC-, PE-, or biotin-conjugated antibodies were used: CD4 (YTS191.1), CD8a (YTS169.4) (Caltag, South San Francisco, CA), CD3 (2C11), TCRab (H57–597), CD69 (H1.2F3), Thy1.2 (53-2.1), B220 (RA3-6B2), and antimouse IgM antibody (Pharmingen). DO10 transgenic TCR clonotype mAb KJ1-26 (25) (a kind gift from Dr. D. Loh) was purified from the hybridoma supernatant by a protein G affinity column and was then biotinylated. Cells stained with biotinylated mAb were then incubated with streptavidin–PE or –Tricolor (Caltag). Stained cells were resuspended in PBS containing 1% FCS and analyzed by FACScan (Becton–Dickinson, Mountain View, CA) using the Cell Quest program. In Vivo Administration of Antigenic Peptide and Anti-TCR Antibody Mice were injected intraperitoneally with PBS or OVA peptide 323–339 (ISQAVHAAHAEINEAGR) (10, 25, 50, 100, and 250 mg) three times every 24 h. Seventy-two hours after the first injection, the cell number of thymocytes was counted and thymocyte subpopulations were analyzed by flow cytometry. Mice were also injected intraperitoneally with PBS or anti-DO10 transgenic TCR mAb (KJ1-26; 100 mg). Seventy-two hours after injection, the cell number of thymocytes was counted and thymocyte subpopulations were analyzed by flow cytometry. Data Analysis Data are summarized as means 6 SD. Statistical analysis of the results was performed by analysis of variance using Fisher’s least significant difference test for multiple comparisons. P values ,0.05 were considered significant. RESULTS Cellularity of Lymphoid Organs of DO10 lpr/lpr Mice In order to study the role of Fas in thymic T cell development, we generated Fas-deficient TCRab transgenic mice (DO10 lpr/lpr mice). DO10 lpr/lpr mice developed in an indistinguishable manner from DO10 1/1 mice and showed no gross abnormality. The cell number of thymocytes was not significantly different between DO10 lpr/lpr and 1/1 mice (16.1 6 5.2 3 10 7 vs 20.3 6 6.5 3 10 7, mean 6 SD, n 5 12 mice). On the other hand, splenocytes were increased in DO10 lpr/lpr mice (8 to 10 weeks old) compared with those of 1/1 mice (17.4 6 5.1 3 10 7 vs 11.5 6 3.1 3 10 7, n 5 12, P , 0.005). However, no gross splenomegaly, lymphadenopathy, or hepatomegaly, which developed in lpr/lpr mice without the DO10 transgenic TCR after the age of 12 weeks, developed even in 20-week-old
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FIG. 1. Thymocyte subpopulations in DO10 1/1 and lpr/lpr mice. Thymocyte subpopulations in DO10 1/1 and lpr/lpr mice (8 to 10 weeks old) were analyzed by flow cytometry using anti-CD4 – FITC mAb, anti-CD8 –PE mAb, biotinylated anti-DO10 transgenic TCR clonotype mAb KJ1-26, and streptoavidin–Tricolor. CD4/CD8 profiles of DO10 transgenic TCR 1 thymocytes (KJ1-26 1) are depicted. The data shown are representative of 12 mice in each group, and the figures indicate the percentages of the corresponding populations.
DO10 lpr/lpr mice. B220 1 Thy1 1 CD4 2CD8 2 abnormal lpr T cells (26) were not detected in peripheral lymphoid organs of DO10 lpr/lpr mice either (data not shown). Effect of Fas on Positive Selection It has been shown that only the CD4 1CD8 1 doublepositive thymocytes that are positively selected are able to survive and differentiate to CD4 1 or CD8 1 single-positive thymocytes (1, 2). To determine the role of Fas in positive selection of thymocytes, we first examined the generation of CD4 1 SP thymocytes, a major mature thymocyte population in DO10 TCR transgenic mice, and the efficiency of differentiation of DP thymocytes to CD4 1 SP thymocytes in DO10 lpr/lpr mice. As shown in Fig. 1 and Table 1, CD4 1 SP thymocytes bearing DO10 transgenic TCR were significantly decreased in lpr/lpr mice compared with those of 1/1 mice. CD8 1 SP thymocytes that were not primarily selected in DO10 TCR transgenic mice were increased in DO10 lpr/lpr mice compared with 1/1 mice. Fur-
thermore, the ratio of CD4 1 SP thymocytes to DP thymocytes, which reflects the efficiency of the differentiation from DP to SP stage and positive selection, was also significantly lower in lpr/lpr mice than that in 1/1 mice (Table 1). However, in thymocytes that did not express DO10 transgenic TCR, no significant difference was found in the efficiency of the differentiation from DP to SP stage between 1/1 and lpr/lpr mice (CD4 SP/DP: 0.165 6 0.064 vs 0.134 6 0.043, n 5 12). Therefore, these results indicate that the differentiation of DP thymocytes to CD4 1 SP thymocytes is impaired in DO10 lpr/lpr mice, suggesting that Fas is involved in the maturation of thymocytes in positive selection. Because CD69 has been reported to be a marker of thymocytes that are in the process of positive selection (27–29), we then examined the expression of CD69 on thymocytes in DO10 lpr/lpr mice. The CD69 high DP thymocytes were significantly decreased in lpr/lpr mice compared with those of 1/1 mice (4.0 6 1.3% vs 12.8 6 2.8%, n 5 8, P , 0.001) (Fig. 2). The mean fluorescence intensity of CD69 expression on DP thymocytes was also significantly lower in lpr/lpr mice than that in 1/1 mice (4.1 6 1.0 vs 10.9 6 2.4, n 5 8, P , 0.001). On the other hand, CD69 high CD4 1 SP thymocytes were similarly observed between 1/1 and lpr/lpr mice (27.8 6 5.6% vs 28.8 6 7.1%, n 5 8) (Fig. 2). These findings of the decreased expression of CD69 on DP thymocytes from lpr/lpr mice also suggest that Fas is involved in positive selection of thymocytes. To further study the role of Fas in thymic T cell development, we examined the expression of TCR on thymocytes in DO10 lpr/lpr mice. DP thymocytes from 1/1 and lpr/lpr mice expressed intermediate levels of DO10 transgenic TCR (Fig. 3). Similarly, DP thymocytes from 1/1 and lpr/lpr mice expressed intermediate levels of total TCR detected with anti-TCRab mAb (H57-597) (data not shown). On the other hand, CD4 1 SP thymocytes from 1/1 and lpr/lpr mice expressed high level of DO10 transgenic TCR (Fig. 3) and of total TCR (data not shown). Thus, no significant differences in DO10 transgenic and total TCR expressions on DP
TABLE 1 Subpopulations of DO10 Transgenic TCR 1 (KJ1-26 1) Thymocytes in DO10 1/1 and lpr/lpr Mice
1/1 lpr/lpr
DN (%)
DP (%)
CD4 SP (%)
CD8 SP (%)
CD4 SP/DP
6.41 6 2.87 7.90 6 3.06
64.68 6 9.50 75.19 6 8.56*
26.86 6 6.02 13.17 6 4.87**
1.68 6 0.59 3.36 6 1.13**
0.435 6 0.151 0.186 6 0.071**
Note. Thymocytes from DO10 1/1 and lpr/lpr mice (8 to 10 weeks old) were analyzed by flow cytometry using anti-CD4 –FITC mAb, anti-CD8 –PE mAb, biotinylated anti-DO10 transgenic TCR clonotype mAb KJ1-26, and streptoavidin–Tricolor. DN, CD4 2CD8 2 doublenegative thymocytes; DP, CD4 1CD8 1 double-positive thymocytes; CD4 or CD8 SP, CD4 1 or CD8 1 single-positive thymocytes. Data are means 6 SD for 12 mice in each group. *, ** Significantly different from the corresponding mean value of DO10 1/1 mice. * P , 0.02. ** P , 0.001.
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FIG. 2. CD69 expression on thymocyte subpopulations in DO10 1/1 and lpr/lpr mice. Thymocytes from DO10 1/1 and lpr/lpr mice were stained with biotinylated anti-CD69, anti-CD4 –FITC, anti-CD8-PE, and streptavidin–Tricolor, and cells were analyzed by flow cytometry. CD69 expression of CD4 1CD8 1 double-positive (DP) thymocytes and CD4 1 single-positive (SP) thymocytes are depicted. Solid lines and dotted lines indicate cells stained with anti-CD 69 mAb and control IgG, respectively. The data shown are representative of eight mice in each group, and the figures indicate the percentages of CD69 high populations.
thymocytes were found between DO10 lpr/lpr and 1/1 mice. Effect of Fas on Negative Selection To determine the role of Fas in negative selection of thymocytes, we examined the effect of in vivo administration of antigenic peptide on the deletion of DP thymocytes in DO10 lpr/lpr mice. Administration of OVA 323–339 (10 –250 mg, three times every 24 h) significantly decreased DP thymocytes at 3 days after the first injection in DO10 1/1 mice in a dose-dependent manner (Figs. 4A and 4B), which was consistent with the results described by Murphy et al. (25). In contrast to 1/1 mice, the administration of up to 150 mg (in total) of OVA 323–339 failed to decrease DP thymocytes in lpr/lpr mice (Figs. 4A and 4B). The reduction of DP thymocytes by the administration of 150 mg of OVA 323–339 was also observed at 5 and 7 days after the first injection in 1/1 mice, but not in lpr/lpr
mice (data not shown). Thus, DO10 lpr/lpr mice required an approximately 10-fold higher concentration of OVA 323–339 to induce the deletion of DP thymocytes than DO10 1/1 mice. These results indicate that Fas is involved in negative selection of thymocytes. In addition, it is also important to note that the administration of extremely high doses of OVA 323–339, such as 750 mg, was able to decrease DP thymocytes even in the lpr/lpr mice (Figs. 4A and 4B). Effect of in Vivo Administration of Anti-TCR mAb To further determine whether Fas is involved in negative selection of thymocytes, we administered anti-TCR clonotype mAb KJ1-26 (100 mg) into DO10 lpr/lpr mice, and the deletion of DP thymocytes was analyzed 72 h after the administration of the mAb. Administration of KJ1-26 mAb significantly decreased DP thymocytes in 1/1 mice, whereas this treatment failed to decrease DP thymocytes in lpr/lpr mice (Fig.
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FIG. 3. DO10 transgenic TCR expression on thymocyte subpopulations in DO10 1/1 and lpr/lpr mice. Thymocytes from DO10 1/1 and lpr/lpr mice were stained with biotinylated KJ1-26 mAb, anti-CD4 –FITC, anti-CD8 –PE, and streptavidin–Tricolor, and cells were analyzed by flow cytometry. DO10 transgenic TCR (KJ1-26) expression of DP thymocytes and CD4 1 SP thymocytes are depicted. The data shown are representative of 12 mice in each group.
5). These results also suggest that Fas modulates apoptosis of DP thymocytes induced by TCR stimulation in vivo. DISCUSSION In this study, we show that Fas is importantly involved in both positive and negative selection of thymocytes. We found that the differentiation of DP thymocytes to CD4 1 SP thymocytes was markedly impaired in DO10 lpr/lpr mice, as indicated by decreased generation of CD4 1 SP thymocytes and reduced ratio of CD4 1 SP thymocytes to DP thymocytes in lpr/lpr mice compared with those of 1/1 mice (Fig. 1 and Table 1) and that the activation of DP thymocytes in the process of positive selection was significantly inhibited in DO10 lpr/lpr mice, as indicated by the lower levels of CD69 expression on DP thymocytes in lpr/lpr mice compared to 1/1 mice (Fig. 2). Furthermore, we also
found that the deletion of DP thymocytes induced by in vivo administration of OVA peptide (up to 150 mg) and anti-TCR clonotype mAb did not occur in DO10 lpr/lpr mice, whereas these treatments significantly decreased DP thymocytes in DO10 1/1 mice (Figs. 4 and 5). On the other hand, no significant difference in DO10 transgenic TCR expression on DP thymocytes was found between DO10 lpr/lpr and 1/1 mice (Fig. 3). Taken together, these results indicate that Fas is required for inducing efficient positive and negative selection of thymocytes. We show that Fas plays an important role in the positive selection of thymocytes. Fas could modulate thymic T cell activation and could enhance positive selection that is an activation process (1–3, 27). This is suggested by the decreased CD69 levels on DP thymocytes in DO10 lpr/lpr mice compared with 1/1 mice, since CD69 expression on DP thymocytes was induced by stimulation through TCR (27–29). Alternatively,
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FIG. 5. Effect of in vivo administration of anti-DO10 transgenic TCR mAb on the deletion of CD4 1CD8 1 double-positive thymocytes in DO10 1/1 and lpr/lpr mice. DO10 1/1 and lpr/lpr mice were injected intraperitoneally with PBS or anti-DO10 transgenic TCR mAb (KJ1-26; 100 mg). Seventy-two hours after the injection, the cell number of thymocytes was counted and thymocyte subpopulations were analyzed by flow cytometry using anti-CD4 –FITC and antiCD8 –PE mAb. The data shown are representative of six mice in each group, and the figures indicate the cell numbers of total thymocytes and the percentages of the corresponding populations.
FIG. 4. Effect of in vivo administration of antigenic peptide on the deletion of CD4 1CD8 1 double-positive thymocytes in DO10 1/1 and lpr/lpr mice. DO10 1/1 and lpr/lpr mice were injected intraperitoneally with PBS or OVA peptide 323–339 (10, 25, 50, 100, and 250 mg) three times every 24 h. Seventy-two hours after the first injection, the cell number of thymocytes was counted and thymocyte subpopulations were analyzed by flow cytometry using anti-CD4 – FITC and anti-CD8 –PE mAb. A shows representative CD4/CD8 profiles of thymocytes in DO10 1/1 and lpr/lpr mice given OVA 323–339 (0, 150, and 750 mg in total) (n 5 8 mice in each group). The figures indicate the cell numbers of total thymocytes and the percentages of DP thymocytes. B shows the cell number of DP thymocytes in DO10 1/1 (E) and lpr/lpr mice (■) given OVA 323–339 (30 to 750 mg in total). Data are means 6 SD for eight mice at each dose. *, **Significantly different from the mean value of the corresponding control response (PBS), *P , 0.01, **P , 0.001.
Fas could delete thymocytes that do not undergo normal positive selection (30), since CD8 1 SP thymocytes were increased in lpr/lpr mice compared with 1/1 mice (Table 1). We also demonstrate that Fas promotes negative selection of thymocytes, as indicated by the findings that more antigenic peptides were required for the induction of negative selection in DO10 lpr/lpr mice than those in DO10 1/1 mice. Our results are consistent with the observation by Zhou et al. (31) that decreased clonal deletion of thymocytes to an endogenously expressed male antigen was found in H-Y TCR transgenic lpr/lpr mice. Castro et al. (32) also recently showed that Fas enhanced apoptosis of thymocytes at 12 h after the injection of OVA peptide in DO10 TCR transgenic mice. In contrast to our results, most of the previous studies have shown no significant involvement of Fas in negative selection of thymocytes in lpr/lpr mice. Some studies have shown normal clonal deletion of thymocytes to endogenous superantigens in lpr/lpr mice (33, 34) and to the endogenous male antigen in TCR transgenic lpr/lpr mice (35). Other studies have also shown that peptide antigens induce normal thymocyte deletion in TCR transgenic lpr/lpr mice (18, 36). Singer and Abbas (18) investigated thymocyte deletion in 2B4
MODULATION OF THYMIC SELECTION BY FAS
TCR transgenic lpr/lpr mice after three injections of antigenic peptide of pigeon cytochrome c (100 mg, each) and found normal negative selection of thymocytes in the lpr/lpr mice. Sytwu et al. (36) also analyzed antigen-induced thymocyte deletion in the HNT TCR transgenic lpr/lpr mice and found comparable deletion by the injection of antigenic peptide of influenza hemagglutinin (750 mg) in lpr/lpr and 1/1 mice. A major difference between our experiments and previous studies using peptide antigens (18, 36) is the dose of antigenic peptides given to mice. In studies of Singer and Abbas (18) and Sytwu et al. (36), they injected 300 and 750 mg (in total) of antigenic peptides, respectively, to induce negative selection, whereas we administered much lower doses (30 –150 mg) of antigenic peptide to mice. We found that low doses of antigenic peptide (up to 150 mg) induced clonal deletion of thymocytes in DO10 1/1 mice in a dose-dependent manner, but not in DO10 lpr/lpr mice, indicating that Fas promotes negative selection of thymocytes and that the involvement of Fas is obvious when TCR is stimulated by small doses of antigens. In contrast, their previous studies examined the effects of only a high dose of antigenic peptide on negative selection in their transgenic TCR lpr/lpr mice and failed to detect the effects of Fas on negative selection (18, 36). In accordance with their results, we also found that high doses of antigenic peptide, such as 750 mg, induced comparable deletion of thymocytes in DO10 lpr/lpr and 1/1 mice, indicating that TCR stimulation without Fas signals is able to induce negative selection of thymocytes when TCR is stimulated by high doses of antigens. It has been shown that in vivo administration of antigenic peptide activates peripheral T cells and induces apoptosis of thymocytes mediated by TNF (37). Martin et al. (37) showed that adoptively transferred transgenic TCR T cells were able to delete nontransgenic thymocytes in vivo when transgenic T cells were activated by antigen injection. One might speculate that thymocyte death by OVA peptide injection in our in vivo model for negative selection might be caused by peripheral T cell activation and the difference of thymocyte death might reflect the susceptibility of thymocytes to TNF. However, we found that thymocytes from DO10 lpr/lpr mice were induced into apoptosis by TNF equally to those from 1/1 mice (our unpublished data). The mechanism by which signals through Fas enhance both positive and negative selection of thymocytes can be explained in several ways. One possible explanation is that Fas may modulate TCR-mediated activation of thymocytes, thereby enhancing both positive and negative selection. Recent studies suggest that Fas may mediate activation signals other than induction of apoptosis (38 – 41). Alderson et al. (38) reported that some of the immobilized anti-Fas mAb stimulated human peripheral T cell proliferation in the
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presence of immobilized anti-CD3 mAb, suggesting that Fas transduces activation signals for TCR stimulation in human T cells. Aggarwal et al. (39) reported that ligation of Fas induced proliferation of fibroblasts rather than apoptosis. Furthermore, it has recently been shown that T cells lacking FADD (Fas-associating protein with death domain), an essential molecule for Fas-mediated apoptosis (40), show impaired proliferation in response to anti-CD3 mAb stimulation (41). Alternatively, it is also possible that Fas transduces the death signal that eliminates misselected thymocytes and facilitates positive selection (30) and that, on the other hand, the death signal enhances apoptosis of activated thymocytes in negative selection. This notion is also supported by the finding that Fas stimulation directly induced apoptosis of thymocytes, particularly of DP thymocytes (15, 16). The differential effects of Fas on thymocytes may be explained by multiple signaling pathways through Fas. Recent studies have shown that Fas interacts with FADD, which is recruited to Fas upon its activation (42). FADD then interacts with caspase-8 and induces activation of caspase-8 that triggers ICE (interleukin-1b converting enzyme) protease cascade leading to apoptosis (40, 43). In addition to the interaction with FADD, it has been shown that Fas also interacts with RIP (receptor interacting protein) (43). RIP interacts with TRAF2 (TNF receptor-associated factor 2) that induces activation of NF-kB (44 – 46) and thereby induces the expression of survival genes. Thus, Fas activates not only an apoptotic pathway, the Fas–FADD– caspase-8 pathway, but also the Fas–RIP–TRAF2 pathway that results in NF-kB activation. In addition, Fas has recently been shown to interact with a novel protein Daxx (47), which activates JNK kinase that plays important roles not only in apoptosis, but also in T cell activation (48). Furthermore, it has recently been shown that Fas is associated with the tyrosine kinase p59fyn (49), which is also an important tyrosine kinase for TCR-mediated activation of T cells (50), suggesting the possibility that Fas-generated signals can merge to TCR-mediated signals in thymocytes. Our findings that Fas modulates thymic selection imply that a defect in Fas affects the generation of the T cell repertoire by increasing the threshold of thymic selection. Thus, in the mice with defects in Fas such as lpr mice, thymocytes that should be positively selected in normal mice would fail to differentiate to mature T cells, and thymocytes that should be deleted would escape from negative selection, and then these abnormal thymocyte developments would generate autoreactive T cells and cause autoimmunity. On the other hand, the accepted explanation for autoimmunity in lpr mice is that deficient Fas causes the impairment in deletion of activated peripheral mature T and B cells (17–22), resulting in autoimmune diseases. This proposed mechanism of autoimmunity predicts that mice
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with defects in ICE, a crucial molecule in Fas-mediated apoptosis (51, 52), would develop autoimmune diseases as lpr mice. On the contrary to the prediction, no autoimmune features in ICE-deficient mice have been reported (53, 54), suggesting a mechanism of autoimmunity other than the defect of peripheral lymphocyte deletion in lpr mice. Therefore, the abnormal thymic selection as we have shown might contribute to autoimmunity in Fas-deficient lpr mice. In summary, we have shown that Fas is importantly involved in both positive and negative selection of thymocytes. It is thus suggested that abnormal thymic T cell selection by the defect of Fas may cause the generation of autoreactive T cells and autoimmunity.
19.
ACKNOWLEDGMENTS
26.
We thank Dr. D. Loh for providing DO10 TCR transgenic mice and KJ1-26 mAb. We also thank Dr. Y. Saito for helpful discussion. This work was supported in part by grants from the Ministry of Education, Science, and Culture, Japan.
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Fas Modulates both Positive and Negative Selection of Thymocytes Kazuhiro Kurasawa, Yoshiko Hashimoto, and Itsuo Iwamoto Department of Internal Medicine II, Chiba University School of Medicine, Chiba 260, Japan Received July 15, 1998; accepted March 31, 1999
We studied the functional role of Fas (CD95) in thymic T cell development using the TCR transgenic mice homozygous for the lpr mutation, DO10 lpr/lpr mice. In DO10 lpr/lpr mice, the differentiation of CD4 1CD8 1 double-positive (DP) thymocytes to CD4 1 single-positive (SP) thymocytes was markedly impaired, as indicated by decreased generation of CD4 1 SP thymocytes and reduced ratio of CD4 1 SP thymocytes to DP thymocytes in lpr/lpr mice compared with those of 1/1 mice. Activation of DP thymocytes in the process of positive selection was also significantly inhibited in DO10 lpr/lpr mice, as shown by the lower levels of CD69 expression on DP thymocytes in lpr/lpr mice compared to 1/1 mice. Furthermore, the deletion of DP thymocytes induced by in vivo administration of OVA peptide (up to 150 mg) and anti-TCR clonotype mAb did not occur in DO10 lpr/lpr mice, whereas these treatments significantly decreased DP thymocytes in DO10 1/1 mice. On the other hand, no significant difference in DO10 transgenic TCR expression on DP thymocytes was found between DO10 lpr/lpr and 1/1 mice. Together, these results indicate that Fas is importantly involved in both positive and negative selection of thymocytes. © 1999 Academic Press
INTRODUCTION The fate of T lymphocytes in the thymus is determined by the interaction between their T cell receptors (TCR) and peptide/MHC complexes on thymic stroma cells (1–3). Thymocytes bearing TCR with low avidity to the peptide/MHC complex are positively selected and rescued from apoptosis, being allowed to differentiate to mature T cells (positive selection). On the other hand, thymocytes bearing TCR with high avidity to the peptide/MHC complex are induced to undergo apoptosis (negative selection). Thymocytes with TCR that cannot interact with the peptide/MHC complex are also led to apoptosis, termed “death by neglect.” During these processes, more than 95% of T cells generated in the thymus die there; the remaining 5% migrate to the peripheral lymphoid organs as mature T cells (4, 5). However, little is known about the roles of cell-surface
molecules other than TCR in inducing positive and negative selection of thymocytes. The Fas antigen (Fas/CD95) is a 45-kDa cell-surface protein that belongs to the tumor necrosis factor (TNF)/nerve growth factor receptor family (6 – 8). When Fas ligand (FasL) or agonistic anti-Fas antibody binds to Fas, apoptosis is induced in sensitive cells (9 –13). Among thymocytes, CD4 1 CD8 1 doublepositive (DP) 1 cells and CD4 1 CD8 2 or CD4 2 CD8 1 single-positive (SP) cells abundantly express Fas (14 –16). In addition, DP thymocytes selectively undergo apoptosis when exposed to agonistic anti-Fas antibody in vitro and in vivo, whereas SP thymocytes were resistant to Fas-induced apoptosis despite their abundant expression of Fas (15, 16). However, the functional importance of Fas in thymocyte development has not been fully elucidated, although recent studies have shown that the Fas/FasL system is involved in peripheral clonal deletion of mature T cells (17–22). Therefore, we decided to determine whether Fas is involved in thymocyte development, especially in positive and negative selection of thymocytes. For this purpose, we introduced the lpr mutation, a defective Fas gene (23, 24), into a TCRab transgenic mouse (DO10 mouse) (25) and generated the TCR transgenic mice homozygous for the lpr mutation, DO10 lpr/lpr mice, which express no detectable Fas on the cell surface. Using DO10 lpr/lpr mice, we studied the role of Fas in positive selection of thymocytes by analyzing the generation of CD4 1 SP thymocytes from CD4 1 CD8 1 DP thymocytes and the expression levels of CD69 and TCR on thymocytes in the Fas-mutated mice. We also studied the role of Fas in negative selection of thymocytes by administrating antigenic peptide and anti-TCR clonotype antibody to DO10 lpr/lpr mice. Our results indicate that Fas is involved in both positive and negative selection of thymocytes.
1 Abbreviations used: DP, CD4 1CD8 1 double-positive; OVA, ovalbumin; SP, CD4 1 or CD8 1 single-positive.
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MATERIALS AND METHODS Generation of DO10 TCR Transgenic Mice Carrying the lpr/lpr Mutation (DO10 lpr/lpr Mice) DO10 TCR transgenic mice, which express a transgenic TCRab specific for ovalbumin peptide 323–339 (OVA 323–339)/I-A d complex (25), were kindly provided by Dr. D. Loh (Washington University, Seattle, WA) and were maintained in our university animal facility. MRL-lpr/lpr mice were purchased from Clea Japan (Tokyo, Japan). DO10 TCR transgenic mice (H-2 d) were crossed with MRL-lpr/lpr mice (H-2 k). F1 mice were analyzed for DO10 transgenic TCR expression as described below. The F1 mice bearing the transgenic TCR (H-2 d/k, lpr/1) were then mated with the F1 mice without transgenic TCR (H-2 d/k, lpr/1) to generate two genotypes of F2 mice bearing the transgenic TCR, DO10 1/1 mice (H-2 d/d) and DO10 lpr/lpr mice (H-2 d/d). F2 mice were analyzed for DO10 transgenic TCR expression, lpr genotype, and H-2 haplotype as described below. All animals were housed under specific pathogen-free conditions. DO10 transgenic TCR expression was detected by PCR analysis of tail DNA using Va13.1-5 and Ja primers (sense: 59-CAGGAGGGATCCAGTGCCAGC and anti-sense: 59-TGGCTCTACAGTGAGTTTGGT) (the primer sequences were provided by Dr. D. Loh). Tail DNA was prepared by protease K digestion, phenol/ chloroform extraction, and ethanol precipitation. The lpr genotype was determined by PCR analysis of intron 2 of the Fas gene, where an early transposable element is inserted in the mutation (24). The PCR analysis was performed with a sense primer for intron 2 (59-GTAAATAATTGTGCTTCGTCAG) (18) and two antisense primers; one is for intron 2 (59-CAGGGAAATGTAGCAAGATG) and the other is for ETn (59-GTTGCGACACCAGTTATGAA). The PCR products from lpr/1 were 215 bp (intron 2–intron 2) and 282 bp (intron 2–Etn) in size, whereas only the 215-bp product was amplified from lpr/lpr mice. The PCR products were distinguished by electrophoresis on 2% agarose gel. The expression of Fas on thymocytes was analyzed by flow cytometry with anti-murine Fas mAb (Jo2) plus FITC-conjugated anti-hamster IgG mAb (Pharmingen, San Diego, CA) to confirm the results of PCR analysis of lpr genotype, when mice were sacrificed for further analysis. The H-2 haplotype was examined by flow cytometry analysis with FITC-conjugated anti-H-2 d mAb (Meiji Nyugyo, Tokyo, Japan) and FITC-conjugated anti-H-2 k mAb (Pharmingen). Flow Cytometry Cells from thymus and spleens (1 3 10 6) were first incubated with anti-Fc receptor mAb 2.4G2 to prevent nonspecific staining and were stained with fluorescence- or biotin-conjugated antibodies in phosphate-
buffered saline (PBS) containing 1% fetal calf serum (FCS) for 30 min at 4°C. The following FITC-, PE-, or biotin-conjugated antibodies were used: CD4 (YTS191.1), CD8a (YTS169.4) (Caltag, South San Francisco, CA), CD3 (2C11), TCRab (H57–597), CD69 (H1.2F3), Thy1.2 (53-2.1), B220 (RA3-6B2), and antimouse IgM antibody (Pharmingen). DO10 transgenic TCR clonotype mAb KJ1-26 (25) (a kind gift from Dr. D. Loh) was purified from the hybridoma supernatant by a protein G affinity column and was then biotinylated. Cells stained with biotinylated mAb were then incubated with streptavidin–PE or –Tricolor (Caltag). Stained cells were resuspended in PBS containing 1% FCS and analyzed by FACScan (Becton–Dickinson, Mountain View, CA) using the Cell Quest program. In Vivo Administration of Antigenic Peptide and Anti-TCR Antibody Mice were injected intraperitoneally with PBS or OVA peptide 323–339 (ISQAVHAAHAEINEAGR) (10, 25, 50, 100, and 250 mg) three times every 24 h. Seventy-two hours after the first injection, the cell number of thymocytes was counted and thymocyte subpopulations were analyzed by flow cytometry. Mice were also injected intraperitoneally with PBS or anti-DO10 transgenic TCR mAb (KJ1-26; 100 mg). Seventy-two hours after injection, the cell number of thymocytes was counted and thymocyte subpopulations were analyzed by flow cytometry. Data Analysis Data are summarized as means 6 SD. Statistical analysis of the results was performed by analysis of variance using Fisher’s least significant difference test for multiple comparisons. P values ,0.05 were considered significant. RESULTS Cellularity of Lymphoid Organs of DO10 lpr/lpr Mice In order to study the role of Fas in thymic T cell development, we generated Fas-deficient TCRab transgenic mice (DO10 lpr/lpr mice). DO10 lpr/lpr mice developed in an indistinguishable manner from DO10 1/1 mice and showed no gross abnormality. The cell number of thymocytes was not significantly different between DO10 lpr/lpr and 1/1 mice (16.1 6 5.2 3 10 7 vs 20.3 6 6.5 3 10 7, mean 6 SD, n 5 12 mice). On the other hand, splenocytes were increased in DO10 lpr/lpr mice (8 to 10 weeks old) compared with those of 1/1 mice (17.4 6 5.1 3 10 7 vs 11.5 6 3.1 3 10 7, n 5 12, P , 0.005). However, no gross splenomegaly, lymphadenopathy, or hepatomegaly, which developed in lpr/lpr mice without the DO10 transgenic TCR after the age of 12 weeks, developed even in 20-week-old
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FIG. 1. Thymocyte subpopulations in DO10 1/1 and lpr/lpr mice. Thymocyte subpopulations in DO10 1/1 and lpr/lpr mice (8 to 10 weeks old) were analyzed by flow cytometry using anti-CD4 – FITC mAb, anti-CD8 –PE mAb, biotinylated anti-DO10 transgenic TCR clonotype mAb KJ1-26, and streptoavidin–Tricolor. CD4/CD8 profiles of DO10 transgenic TCR 1 thymocytes (KJ1-26 1) are depicted. The data shown are representative of 12 mice in each group, and the figures indicate the percentages of the corresponding populations.
DO10 lpr/lpr mice. B220 1 Thy1 1 CD4 2CD8 2 abnormal lpr T cells (26) were not detected in peripheral lymphoid organs of DO10 lpr/lpr mice either (data not shown). Effect of Fas on Positive Selection It has been shown that only the CD4 1CD8 1 doublepositive thymocytes that are positively selected are able to survive and differentiate to CD4 1 or CD8 1 single-positive thymocytes (1, 2). To determine the role of Fas in positive selection of thymocytes, we first examined the generation of CD4 1 SP thymocytes, a major mature thymocyte population in DO10 TCR transgenic mice, and the efficiency of differentiation of DP thymocytes to CD4 1 SP thymocytes in DO10 lpr/lpr mice. As shown in Fig. 1 and Table 1, CD4 1 SP thymocytes bearing DO10 transgenic TCR were significantly decreased in lpr/lpr mice compared with those of 1/1 mice. CD8 1 SP thymocytes that were not primarily selected in DO10 TCR transgenic mice were increased in DO10 lpr/lpr mice compared with 1/1 mice. Fur-
thermore, the ratio of CD4 1 SP thymocytes to DP thymocytes, which reflects the efficiency of the differentiation from DP to SP stage and positive selection, was also significantly lower in lpr/lpr mice than that in 1/1 mice (Table 1). However, in thymocytes that did not express DO10 transgenic TCR, no significant difference was found in the efficiency of the differentiation from DP to SP stage between 1/1 and lpr/lpr mice (CD4 SP/DP: 0.165 6 0.064 vs 0.134 6 0.043, n 5 12). Therefore, these results indicate that the differentiation of DP thymocytes to CD4 1 SP thymocytes is impaired in DO10 lpr/lpr mice, suggesting that Fas is involved in the maturation of thymocytes in positive selection. Because CD69 has been reported to be a marker of thymocytes that are in the process of positive selection (27–29), we then examined the expression of CD69 on thymocytes in DO10 lpr/lpr mice. The CD69 high DP thymocytes were significantly decreased in lpr/lpr mice compared with those of 1/1 mice (4.0 6 1.3% vs 12.8 6 2.8%, n 5 8, P , 0.001) (Fig. 2). The mean fluorescence intensity of CD69 expression on DP thymocytes was also significantly lower in lpr/lpr mice than that in 1/1 mice (4.1 6 1.0 vs 10.9 6 2.4, n 5 8, P , 0.001). On the other hand, CD69 high CD4 1 SP thymocytes were similarly observed between 1/1 and lpr/lpr mice (27.8 6 5.6% vs 28.8 6 7.1%, n 5 8) (Fig. 2). These findings of the decreased expression of CD69 on DP thymocytes from lpr/lpr mice also suggest that Fas is involved in positive selection of thymocytes. To further study the role of Fas in thymic T cell development, we examined the expression of TCR on thymocytes in DO10 lpr/lpr mice. DP thymocytes from 1/1 and lpr/lpr mice expressed intermediate levels of DO10 transgenic TCR (Fig. 3). Similarly, DP thymocytes from 1/1 and lpr/lpr mice expressed intermediate levels of total TCR detected with anti-TCRab mAb (H57-597) (data not shown). On the other hand, CD4 1 SP thymocytes from 1/1 and lpr/lpr mice expressed high level of DO10 transgenic TCR (Fig. 3) and of total TCR (data not shown). Thus, no significant differences in DO10 transgenic and total TCR expressions on DP
TABLE 1 Subpopulations of DO10 Transgenic TCR 1 (KJ1-26 1) Thymocytes in DO10 1/1 and lpr/lpr Mice
1/1 lpr/lpr
DN (%)
DP (%)
CD4 SP (%)
CD8 SP (%)
CD4 SP/DP
6.41 6 2.87 7.90 6 3.06
64.68 6 9.50 75.19 6 8.56*
26.86 6 6.02 13.17 6 4.87**
1.68 6 0.59 3.36 6 1.13**
0.435 6 0.151 0.186 6 0.071**
Note. Thymocytes from DO10 1/1 and lpr/lpr mice (8 to 10 weeks old) were analyzed by flow cytometry using anti-CD4 –FITC mAb, anti-CD8 –PE mAb, biotinylated anti-DO10 transgenic TCR clonotype mAb KJ1-26, and streptoavidin–Tricolor. DN, CD4 2CD8 2 doublenegative thymocytes; DP, CD4 1CD8 1 double-positive thymocytes; CD4 or CD8 SP, CD4 1 or CD8 1 single-positive thymocytes. Data are means 6 SD for 12 mice in each group. *, ** Significantly different from the corresponding mean value of DO10 1/1 mice. * P , 0.02. ** P , 0.001.
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FIG. 2. CD69 expression on thymocyte subpopulations in DO10 1/1 and lpr/lpr mice. Thymocytes from DO10 1/1 and lpr/lpr mice were stained with biotinylated anti-CD69, anti-CD4 –FITC, anti-CD8-PE, and streptavidin–Tricolor, and cells were analyzed by flow cytometry. CD69 expression of CD4 1CD8 1 double-positive (DP) thymocytes and CD4 1 single-positive (SP) thymocytes are depicted. Solid lines and dotted lines indicate cells stained with anti-CD 69 mAb and control IgG, respectively. The data shown are representative of eight mice in each group, and the figures indicate the percentages of CD69 high populations.
thymocytes were found between DO10 lpr/lpr and 1/1 mice. Effect of Fas on Negative Selection To determine the role of Fas in negative selection of thymocytes, we examined the effect of in vivo administration of antigenic peptide on the deletion of DP thymocytes in DO10 lpr/lpr mice. Administration of OVA 323–339 (10 –250 mg, three times every 24 h) significantly decreased DP thymocytes at 3 days after the first injection in DO10 1/1 mice in a dose-dependent manner (Figs. 4A and 4B), which was consistent with the results described by Murphy et al. (25). In contrast to 1/1 mice, the administration of up to 150 mg (in total) of OVA 323–339 failed to decrease DP thymocytes in lpr/lpr mice (Figs. 4A and 4B). The reduction of DP thymocytes by the administration of 150 mg of OVA 323–339 was also observed at 5 and 7 days after the first injection in 1/1 mice, but not in lpr/lpr
mice (data not shown). Thus, DO10 lpr/lpr mice required an approximately 10-fold higher concentration of OVA 323–339 to induce the deletion of DP thymocytes than DO10 1/1 mice. These results indicate that Fas is involved in negative selection of thymocytes. In addition, it is also important to note that the administration of extremely high doses of OVA 323–339, such as 750 mg, was able to decrease DP thymocytes even in the lpr/lpr mice (Figs. 4A and 4B). Effect of in Vivo Administration of Anti-TCR mAb To further determine whether Fas is involved in negative selection of thymocytes, we administered anti-TCR clonotype mAb KJ1-26 (100 mg) into DO10 lpr/lpr mice, and the deletion of DP thymocytes was analyzed 72 h after the administration of the mAb. Administration of KJ1-26 mAb significantly decreased DP thymocytes in 1/1 mice, whereas this treatment failed to decrease DP thymocytes in lpr/lpr mice (Fig.
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FIG. 3. DO10 transgenic TCR expression on thymocyte subpopulations in DO10 1/1 and lpr/lpr mice. Thymocytes from DO10 1/1 and lpr/lpr mice were stained with biotinylated KJ1-26 mAb, anti-CD4 –FITC, anti-CD8 –PE, and streptavidin–Tricolor, and cells were analyzed by flow cytometry. DO10 transgenic TCR (KJ1-26) expression of DP thymocytes and CD4 1 SP thymocytes are depicted. The data shown are representative of 12 mice in each group.
5). These results also suggest that Fas modulates apoptosis of DP thymocytes induced by TCR stimulation in vivo. DISCUSSION In this study, we show that Fas is importantly involved in both positive and negative selection of thymocytes. We found that the differentiation of DP thymocytes to CD4 1 SP thymocytes was markedly impaired in DO10 lpr/lpr mice, as indicated by decreased generation of CD4 1 SP thymocytes and reduced ratio of CD4 1 SP thymocytes to DP thymocytes in lpr/lpr mice compared with those of 1/1 mice (Fig. 1 and Table 1) and that the activation of DP thymocytes in the process of positive selection was significantly inhibited in DO10 lpr/lpr mice, as indicated by the lower levels of CD69 expression on DP thymocytes in lpr/lpr mice compared to 1/1 mice (Fig. 2). Furthermore, we also
found that the deletion of DP thymocytes induced by in vivo administration of OVA peptide (up to 150 mg) and anti-TCR clonotype mAb did not occur in DO10 lpr/lpr mice, whereas these treatments significantly decreased DP thymocytes in DO10 1/1 mice (Figs. 4 and 5). On the other hand, no significant difference in DO10 transgenic TCR expression on DP thymocytes was found between DO10 lpr/lpr and 1/1 mice (Fig. 3). Taken together, these results indicate that Fas is required for inducing efficient positive and negative selection of thymocytes. We show that Fas plays an important role in the positive selection of thymocytes. Fas could modulate thymic T cell activation and could enhance positive selection that is an activation process (1–3, 27). This is suggested by the decreased CD69 levels on DP thymocytes in DO10 lpr/lpr mice compared with 1/1 mice, since CD69 expression on DP thymocytes was induced by stimulation through TCR (27–29). Alternatively,
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FIG. 5. Effect of in vivo administration of anti-DO10 transgenic TCR mAb on the deletion of CD4 1CD8 1 double-positive thymocytes in DO10 1/1 and lpr/lpr mice. DO10 1/1 and lpr/lpr mice were injected intraperitoneally with PBS or anti-DO10 transgenic TCR mAb (KJ1-26; 100 mg). Seventy-two hours after the injection, the cell number of thymocytes was counted and thymocyte subpopulations were analyzed by flow cytometry using anti-CD4 –FITC and antiCD8 –PE mAb. The data shown are representative of six mice in each group, and the figures indicate the cell numbers of total thymocytes and the percentages of the corresponding populations.
FIG. 4. Effect of in vivo administration of antigenic peptide on the deletion of CD4 1CD8 1 double-positive thymocytes in DO10 1/1 and lpr/lpr mice. DO10 1/1 and lpr/lpr mice were injected intraperitoneally with PBS or OVA peptide 323–339 (10, 25, 50, 100, and 250 mg) three times every 24 h. Seventy-two hours after the first injection, the cell number of thymocytes was counted and thymocyte subpopulations were analyzed by flow cytometry using anti-CD4 – FITC and anti-CD8 –PE mAb. A shows representative CD4/CD8 profiles of thymocytes in DO10 1/1 and lpr/lpr mice given OVA 323–339 (0, 150, and 750 mg in total) (n 5 8 mice in each group). The figures indicate the cell numbers of total thymocytes and the percentages of DP thymocytes. B shows the cell number of DP thymocytes in DO10 1/1 (E) and lpr/lpr mice (■) given OVA 323–339 (30 to 750 mg in total). Data are means 6 SD for eight mice at each dose. *, **Significantly different from the mean value of the corresponding control response (PBS), *P , 0.01, **P , 0.001.
Fas could delete thymocytes that do not undergo normal positive selection (30), since CD8 1 SP thymocytes were increased in lpr/lpr mice compared with 1/1 mice (Table 1). We also demonstrate that Fas promotes negative selection of thymocytes, as indicated by the findings that more antigenic peptides were required for the induction of negative selection in DO10 lpr/lpr mice than those in DO10 1/1 mice. Our results are consistent with the observation by Zhou et al. (31) that decreased clonal deletion of thymocytes to an endogenously expressed male antigen was found in H-Y TCR transgenic lpr/lpr mice. Castro et al. (32) also recently showed that Fas enhanced apoptosis of thymocytes at 12 h after the injection of OVA peptide in DO10 TCR transgenic mice. In contrast to our results, most of the previous studies have shown no significant involvement of Fas in negative selection of thymocytes in lpr/lpr mice. Some studies have shown normal clonal deletion of thymocytes to endogenous superantigens in lpr/lpr mice (33, 34) and to the endogenous male antigen in TCR transgenic lpr/lpr mice (35). Other studies have also shown that peptide antigens induce normal thymocyte deletion in TCR transgenic lpr/lpr mice (18, 36). Singer and Abbas (18) investigated thymocyte deletion in 2B4
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TCR transgenic lpr/lpr mice after three injections of antigenic peptide of pigeon cytochrome c (100 mg, each) and found normal negative selection of thymocytes in the lpr/lpr mice. Sytwu et al. (36) also analyzed antigen-induced thymocyte deletion in the HNT TCR transgenic lpr/lpr mice and found comparable deletion by the injection of antigenic peptide of influenza hemagglutinin (750 mg) in lpr/lpr and 1/1 mice. A major difference between our experiments and previous studies using peptide antigens (18, 36) is the dose of antigenic peptides given to mice. In studies of Singer and Abbas (18) and Sytwu et al. (36), they injected 300 and 750 mg (in total) of antigenic peptides, respectively, to induce negative selection, whereas we administered much lower doses (30 –150 mg) of antigenic peptide to mice. We found that low doses of antigenic peptide (up to 150 mg) induced clonal deletion of thymocytes in DO10 1/1 mice in a dose-dependent manner, but not in DO10 lpr/lpr mice, indicating that Fas promotes negative selection of thymocytes and that the involvement of Fas is obvious when TCR is stimulated by small doses of antigens. In contrast, their previous studies examined the effects of only a high dose of antigenic peptide on negative selection in their transgenic TCR lpr/lpr mice and failed to detect the effects of Fas on negative selection (18, 36). In accordance with their results, we also found that high doses of antigenic peptide, such as 750 mg, induced comparable deletion of thymocytes in DO10 lpr/lpr and 1/1 mice, indicating that TCR stimulation without Fas signals is able to induce negative selection of thymocytes when TCR is stimulated by high doses of antigens. It has been shown that in vivo administration of antigenic peptide activates peripheral T cells and induces apoptosis of thymocytes mediated by TNF (37). Martin et al. (37) showed that adoptively transferred transgenic TCR T cells were able to delete nontransgenic thymocytes in vivo when transgenic T cells were activated by antigen injection. One might speculate that thymocyte death by OVA peptide injection in our in vivo model for negative selection might be caused by peripheral T cell activation and the difference of thymocyte death might reflect the susceptibility of thymocytes to TNF. However, we found that thymocytes from DO10 lpr/lpr mice were induced into apoptosis by TNF equally to those from 1/1 mice (our unpublished data). The mechanism by which signals through Fas enhance both positive and negative selection of thymocytes can be explained in several ways. One possible explanation is that Fas may modulate TCR-mediated activation of thymocytes, thereby enhancing both positive and negative selection. Recent studies suggest that Fas may mediate activation signals other than induction of apoptosis (38 – 41). Alderson et al. (38) reported that some of the immobilized anti-Fas mAb stimulated human peripheral T cell proliferation in the
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presence of immobilized anti-CD3 mAb, suggesting that Fas transduces activation signals for TCR stimulation in human T cells. Aggarwal et al. (39) reported that ligation of Fas induced proliferation of fibroblasts rather than apoptosis. Furthermore, it has recently been shown that T cells lacking FADD (Fas-associating protein with death domain), an essential molecule for Fas-mediated apoptosis (40), show impaired proliferation in response to anti-CD3 mAb stimulation (41). Alternatively, it is also possible that Fas transduces the death signal that eliminates misselected thymocytes and facilitates positive selection (30) and that, on the other hand, the death signal enhances apoptosis of activated thymocytes in negative selection. This notion is also supported by the finding that Fas stimulation directly induced apoptosis of thymocytes, particularly of DP thymocytes (15, 16). The differential effects of Fas on thymocytes may be explained by multiple signaling pathways through Fas. Recent studies have shown that Fas interacts with FADD, which is recruited to Fas upon its activation (42). FADD then interacts with caspase-8 and induces activation of caspase-8 that triggers ICE (interleukin-1b converting enzyme) protease cascade leading to apoptosis (40, 43). In addition to the interaction with FADD, it has been shown that Fas also interacts with RIP (receptor interacting protein) (43). RIP interacts with TRAF2 (TNF receptor-associated factor 2) that induces activation of NF-kB (44 – 46) and thereby induces the expression of survival genes. Thus, Fas activates not only an apoptotic pathway, the Fas–FADD– caspase-8 pathway, but also the Fas–RIP–TRAF2 pathway that results in NF-kB activation. In addition, Fas has recently been shown to interact with a novel protein Daxx (47), which activates JNK kinase that plays important roles not only in apoptosis, but also in T cell activation (48). Furthermore, it has recently been shown that Fas is associated with the tyrosine kinase p59fyn (49), which is also an important tyrosine kinase for TCR-mediated activation of T cells (50), suggesting the possibility that Fas-generated signals can merge to TCR-mediated signals in thymocytes. Our findings that Fas modulates thymic selection imply that a defect in Fas affects the generation of the T cell repertoire by increasing the threshold of thymic selection. Thus, in the mice with defects in Fas such as lpr mice, thymocytes that should be positively selected in normal mice would fail to differentiate to mature T cells, and thymocytes that should be deleted would escape from negative selection, and then these abnormal thymocyte developments would generate autoreactive T cells and cause autoimmunity. On the other hand, the accepted explanation for autoimmunity in lpr mice is that deficient Fas causes the impairment in deletion of activated peripheral mature T and B cells (17–22), resulting in autoimmune diseases. This proposed mechanism of autoimmunity predicts that mice
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with defects in ICE, a crucial molecule in Fas-mediated apoptosis (51, 52), would develop autoimmune diseases as lpr mice. On the contrary to the prediction, no autoimmune features in ICE-deficient mice have been reported (53, 54), suggesting a mechanism of autoimmunity other than the defect of peripheral lymphocyte deletion in lpr mice. Therefore, the abnormal thymic selection as we have shown might contribute to autoimmunity in Fas-deficient lpr mice. In summary, we have shown that Fas is importantly involved in both positive and negative selection of thymocytes. It is thus suggested that abnormal thymic T cell selection by the defect of Fas may cause the generation of autoreactive T cells and autoimmunity.
19.
ACKNOWLEDGMENTS
26.
We thank Dr. D. Loh for providing DO10 TCR transgenic mice and KJ1-26 mAb. We also thank Dr. Y. Saito for helpful discussion. This work was supported in part by grants from the Ministry of Education, Science, and Culture, Japan.
27.
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