The present study shows that replacement of ZAP-70 tyrosines 315 and 319 with alanines results in attenuated TCR signaling, leading to compromised development in CD4+ and CD8+ thymocytes, as well as defective positive and negative selection in the context of certain fixed transgenic TCR systems. This results in impaired development of normal numbers of T cells and in an apparent shift in the TCR repertoire. Because of the apparent similarities between YYAA and SKG mice, with regard to T cell development, in this study we compared these two mice and explored the mechanisms that contribute to or protect from the development of autoimmune arthritis in the YYAA mice.
The YYAA mice have diminished TCR signaling, and consequently, perturbed thymocyte development. One explanation for the reduced level of TCR signaling may be the decrease in YYAA ZAP-70 protein expression levels. The most severe reduction is seen in DP thymocytes. However, in the CD4 SP thymocytes, the level of the YYAA protein is comparable to the level of the wild-type protein. Yet, signaling is still substantially reduced in the CD4 SP population as indicated by diminished calcium flux and Erk phosphorylation in response to TCR stimulation. Although in the periphery the level of the YYAA ZAP-70 is reduced to ~50% wild-type levels, we and others have not seen a substantial reduction in signaling in heterozygous ZAP-70 KO mice that express half the normal level of protein. The basis for the reduction of the YYAA protein is not clear, but could reflect a less stable protein or the influence of compromised ZAP-70–dependent TCR signaling. We favor the latter hypothesis, as we have not noted difficulties in expressing the YYAA mutant in cell lines. Moreover, at similar levels of wild-type or YYAA ZAP-70 protein in reconstitution studies of a deficient Jurkat line, we still note decreased TCR signaling as indicated by diminished total tyrosine phosphorylation as well as that of specific substrates (T. Kadlecek, personal communication; unpublished data). The reduction of ZAP-70 protein in the SKG mice is much more profound in all subsets than the YYAA and, yet, these mice have sufficient T cell functional activity in the periphery to mediate T cell-dependent responses that result in autoimmune arthritis. Thus, it appears that the scaffolding function mediated by the phospho-Y315 and -Y319 to recruit critical effector molecules is, indeed, important.
The YYAA and SKG mice have very different ZAP-70 mutations; compromised scaffolding function and decreased binding to phosphorylated ITAMs, respectively. Because of the similar developmental defects in YYAA and SKG mice, we compared TCR signaling events in wild-type, YYAA, and SKG mice and found quantitative decreases in TCR signaling, as indicated by calcium responses, phosphorylation of Erk, and surface expression of CD5 and CD69 at different stages of T cell development. Consistent with the TCR signaling strength, the patterns of CFSE dilution and IL-2 production in response to TCR stimulation in vitro were greatly reduced in SKG T cells and more moderately decreased in YYAA T cells. Thus, the SKG mutation has a greater negative impact on TCR signaling than the YYAA mutation. Despite the greater impact on TCR signaling in the SKG mice, the absolute numbers of CD4 SP and CD8 SP thymocytes are very similar in SKG and YYAA mice. Our results with the YYAA and SKG mice contrast with another more severe hypomorphic allelic series of ZAP-70 in which the absolute numbers of CD4 SP and CD8 SP thymocytes correlate with the signaling strength of different ZAP-70 alleles (Siggs et al., 2007
). The discrepancy might be caused by different efficiencies in positive and negative selection in various ZAP-70 alleles, or in quantitatively or qualitatively different signals based on the specific functions of ZAP-70 impacted by the individual mutations.
The reduced number of single positive thymocytes in the YYAA mice suggested that the impairment in TCR signaling results in defective positive selection. This is supported by the reduced numbers of TCRhi
DP thymocytes, indicative of cells that have recently been positively selected. By crossing the YYAA mutation with two TCR transgenic mice, we have demonstrated that, like the SKG mice, the efficiency of positive selection in YYAA mice is reduced in both transgenic systems. As compared with the reported positive selection defects in SKG mice in both HY TCR and DO 11.10 TCR systems (Sakaguchi et al., 2003
), the efficiency of positive selection in YYAA mice appears to be very similar.
Based on analyses of male HY TCR transgenic mice, negative selection in YYAA mice bearing the HY-TCR is similarly defective as in the SKG mice bearing this transgene (Sakaguchi et al., 2003
). Given the defects in both positive and negative selection in YYAA mice, we anticipated a breakdown of normal self-tolerance and the appearance of autoimmunity in the YYAA mice. Surprisingly, YYAA mice failed to develop spontaneous autoimmunity. Nor did they develop the autoimmune arthritis seen in the SKG mice after zymosan challenge. However, the YYAA mice, unlike wild-type mice, did develop serum rheumatoid factor. Thus, activation of the innate immune system in YYAA mice, as in SKG mice, led to some evidence of autoimmunity but, unlike SKG, did not lead to autoimmune disease. Further studies of autoantibodies and histological examination failed to reveal any evidence of other autoimmune manifestations in YYAA mice. However, we did find evidence for peripheral T cell activation and this correlated with the signaling strength of both ZAP-70 alleles. But only SKG mice, which showed the highest increase in CD44hi
, and CD69+
cells in the periphery, develop autoimmune arthritis. These findings raise three possibilities. First, differences in peripheral TCR signaling caused by the YYAA and SKG mutations may determine whether autoimmune arthritis develops because of differences in the quality of the immune response generated. Second, peripheral tolerance is relatively intact in YYAA but defective in SKG mice. Third, the skewed repertoires selected in the thymus of the SKG and the YYAA mice substantially differ, leading to induction of autoimmune arthritis only in the SKG mice after zymosan challenge.
T cells are responsible for autoimmune arthritis in the SKG mouse model, we reasoned that altered cytokine production would likely be important in the evolution of T cell–mediated disease. We specifically examined IL-17 production because it has been implicated in the pathogenesis of autoimmune arthritis (Hirota et al., 2007
). However, when challenged with zymosan, an elevation of IL-17 production was seen in both YYAA and SKG T cells. The number of IL-17–producing cells increased somewhat in both YYAA and SKG mice after treatment with zymosan, although the induced increase in IL-17–producing cells in the YYAA mouse was somewhat lower than that seen in T cells from the SKG mouse. One interpretation of these data are that YYAA CD4+
T cells are less able to differentiate into Th17 cells, although the change compared with the SKG T cells is rather modest. Another alternative possibility is that some cytokines such as IFN-γ in the YYAA mice suppress Th17 development. We found that IFN-γ–deficient YYAA mice did not show enhanced development of Th17 T cells (unpublished data). Collectively, these data suggest that the failure to develop arthritis in the YYAA mice is not likely to be caused by a selective difference in the ability of the YYAA and SKG mice to generate increased numbers of IL-17–secreting effector cells. However, it is possible that other differences in cytokine production or local production might influence responses leading to arthritis.
Phenotypic and functional characterization of T reg cells, which are essential for peripheral self-tolerance, indicated that much lower percentages and absolute numbers of thymic T reg cell were found in the YYAA and SKG mice. This marked decrease is probably the result of their severely impaired abilities to produce IL-2, partial TCR signaling defect, or both. Interestingly, unlike another partial T cell deficiency mouse mutant, LATY136F
in which T reg cells are nearly absent in the periphery (Koonpaew et al., 2006
), the percentages of Foxp3+
T reg cell in the YYAA and SKG mice were essentially normal or increased, respectively. Whereas YYAA T reg cells can develop in the periphery despite fewer numbers, we demonstrated that the suppressive activity of YYAA T reg cells consistently was not as efficient as WT controls. Given that SKG T reg cells have relatively normal suppressive function in vitro, the lower suppressive activity of YYAA T reg cell, which was consistently observed, is unexpected. Whether YYAA T reg cells are less functional in vivo remains to be determined. However, significant decreases in both thymic and peripheral T reg cell numbers in the YYAA mice suggest that YYAA T reg cells are impaired to some extent. Despite the observation that T reg cell mediated–function appears to be defective in the YYAA mice in contrast to the SKG mice, very limited autoimmunity was observed in the YYAA mice. Thus, the development of arthritis in SKG mice does not reflect a greater impairment in T reg cell numbers or function than in the YYAA mice.
To explore the third possibility that TCR repertoire skewing may differ in the YYAA and SKG mice, we examined how signaling strength affects TCR repertoire usage. The YYAA mice exhibited defects in positive selection in the HY-TCR or DO11.10-TCR systems that were similar to those reported for the SKG mice (Sakaguchi et al., 2003
). However, the difference in TCR signaling could lead to differences in negative selection in a polyclonal repertoire that were not revealed by the model HY TCR transgenic systems. Therefore, we used a more direct and quantitative assessment of polyclonal TCR Vβ deletion in response to endogenous MMTV superantigens. Indeed, the magnitude of bias in Vβ deletion to endogenous superantigens correlates with the signaling strength in these different ZAP-70 allelic variants. The skewing in repertoires is not associated with a monoclonal or pauciclonal repertoire in either YYAA or SKG mice, as assessed by immunoscope analysis (Fig. S6
). These findings suggest that the window of thymic selection has been shifted to different levels in these ZAP-70 mutants. This could well result in differences in the diverse T cell repertoires that result in distinct predispositions for the induction of autoimmune disease such as arthritis. These findings support the idea that development of autoimmune disease requires breakdowns of multiple tolerance checkpoints (Goodnow, 2007
We attempted to determine whether decreasing the TCR signaling level from the YYAA T cell level to one closer to the SKG level might result in autoimmune arthritis. Therefore, we examined whether compound heterozygous mice sharing one SKG and one YYAA allele (SKG/YYAA) might develop arthritis. However, it appears that CD4+
T cells developing in YYAA mice or SKG/YYAA mice never reach the selection threshold or altered peripheral immune responsiveness of those T cells in the SKG mice for development of autoimmune arthritis. This could reflect a dominant ability of the YYAA allele to interact with phospho-ITAMs over the SKG allele. It is interesting that in contrast to another hypomorphic allelic series recently reported that developed anti-DNA autoantibodies (Siggs et al., 2007
), the YYAA, SKG, and the SKG/YYAA mice did not develop anti-DNA autoantibodies. Whereas genetic background can play a role in the development of autoimmunity, YYAA did not develop anti-dsDNA antibodies in either C57BL/6 or BALB/c backgrounds. By comparing the numbers of T cells developing on the B6 background in YYAA mice to those described in the studies by Siggs, et al., it is likely that neither the YYAA nor SKG mutations impair TCR signaling as severely. Therefore, we suspect it is likely that differences in TCR signaling that are responsible for differences in TCR repertoire explain the failure to develop arthritis or anti-dsDNA antibodies in the YYAA mice.
In conclusion, our studies demonstrate that in addition to the recently reported autoinhibitory function of Y315 and Y319 in ZAP-70, these phosphorylation sites play other important roles in TCR signaling, presumably by recruiting SH2 domain effector molecules to activated ZAP-70. These sites are important for the generation of a normal T cell repertoire and a normally functioning peripheral T cell compartment. Although reduced T reg cell development could also contribute to the development of arthritis in SKG mice, it is clearly insufficient as indicated by the failure of YYAA mice to develop frank arthritis. Our studies suggest that it is likely that distinct T cell repertoires containing potentially autoreactive cells are selected in YYAA and SKG mice based on differences of Vβ deletion to endogenous superantigens. Even in the presence of induced RF production, the YYAA mice fail to develop arthritis, whereas SKG mice do. These findings strongly suggest rather narrow windows of repertoire selection may dictate whether autoimmunity or autoimmune disease develops to the same challenge of the immune system.