In this study, we show that SLAP deficiency increased TCR complex signaling and prevented development of chronic auto-immune arthritis in SKG mice treated with zymosan. SLAP deficiency partially rescues positive selection and has a minimal effect on negative selection of thymocytes in naive SKG mice; the most dramatic effect was the increased number of Tregs in both thymus and spleen of naive DSSKO mice. After zymosan treatment, DSSKO mice had a further expansion of peripheral Tregs but did not have the increase in effector Th17 cells seen in SKG mice, thus preventing arthritis development (). Depletion and/or functional blockade of Tregs in zymosan-treated mice unleashed the autoreactive T cells resulting in development of arthritis in the DSSKO mice. In vitro, DSSKO Tregs displayed enhanced suppressive capacity upon TCR activation. Taken together, these data demonstrate that increasing TCR signal strength can enhance development and function of Tregs and prevent development of autoimmunity.
SLAP is a negative regulator of TCR signaling that adapts the E3 ubiquitin ligase c-Cbl to the ζ-chain of the TCR (TCRζ), targeting it for degradation (6
). SLAP is highly expressed in DP thymocytes (6
). Thus, our initial predictions on how SLAP deficiency would affect selection and development of T cells were based primarily on the thymus. The original prediction was that SLAP deficiency would enhance negative selection of autoreactive thymocytes thus eliminating arthritogenic thymocytes in SKG mice. Surprisingly, the data show a minimal effect of SLAP deficiency in negative selection and support a more significant role for SLAP in agonist selection of Tregs in the thymus. However, SLAP expression is dynamic in peripheral T cells with upregulation occurring upon activation (6
) (). In addition, a recent report demonstrated that signals delivered hours after T cell activation are capable of influencing T cell function. Upregulation of Crtam (an MHC class I-restricted T cell-associated molecule) on a subset of peripheral CD4+
T cells more than 6 h after TCR activation was shown to control polarity, proliferation, and cytokine secretion, thus affecting adaptive immunity to infection (23
). Thus, we examined the role of SLAP deficiency in peripheral T cells upon activation. In vivo SLAP deficiency altered the differentiation and/or proliferation of both Th17 cells and Tregs, skewing the ratio between the two in favor of Tregs. In vitro SLAP deficiency enhanced both the proliferation of CD4+
T cells and the suppressive capacity of CD4+
Tregs after stimulation through the TCR complex. Our data provide the first evidence that SLAP plays a role in autoimmune disease pathogenesis and that regulated expression of SLAP upon TCR activation in peripheral T cells plays a role in determining T cell responses. These responses include effector differentiation of Th17 cells, differentiation and/or proliferation of Tregs, the suppressive capacity of Tregs, and the balance between Tregs and effector Th17 cells.
ZAP70 plays a critical role in TCR signaling during both thymocyte selection and in mature T cell responses (24
). Hypomorphic mutations of ZAP70 have been generated that span the spectrum of immune dysregulation from autoantibody production to immunodeficiency (3
). A recent study on one of these mutants suggested that reduction in the number and function of Tregs was not sufficient for arthritis to develop (26
). Mice with the YYAA mutation of ZAP70 had fewer Tregs with diminished suppressive capacity compared with that of SKG Tregs but failed to develop arthritis after zymosan injection. However, there were significant differences in the repertoire of T cells between these mice, with the likely elimination of many arthritogenic cells as well, thus preventing the development of arthritis. The effects of changing the copy number and/or functional capacity of ZAP70 was examined in a recent report in which the intensity of signaling through the TCR complex was altered by using different combinations of WT, SKG, or null alleles of ZAP70 (21
). This allelic series of ZAP70 mutations demonstrated that alterations in TCR signaling can affect thymic selection, effector function of peripheral T cells, and development or function of Tregs. These studies have extended our understanding, but it remains to be determined whether the affected processes cause or prevent autoimmunity independently or only in combination. Although differences in number and/or function of Tregs were observed, prevention of autoimmune arthritis in ZAP70 mutants was always associated with a shift in the T cell repertoire compared with that of SKG, as was development of spontaneous arthritis in the SKG/null mice (26
Intellectually, the logic behind the aforementioned experiments and those presented in this study was the same with the prediction that increasing signaling through the TCR complex would lead to enhanced negative selection of arthritogenic T cells with a potential increase in Tregs resulting in prevention of arthritis in SKG mice. Yet, strikingly different mechanisms were responsible for prevention of arthritis in these studies. Both mice heterozygous for the SKG allele of ZAP70 and mice homozygous for the YYAA allele of ZAP70 have a different repertoire of T cells containing fewer arthritogenic T cells thus preventing the development of arthritis, regardless of the increased or decreased number and/or function of Tregs, respectively. In contrast, instead of preventing arthritis by eliminating the arthritogenic T cells, DSSKO mice possess a TCR repertoire similar to that of SKG mice that is held in check by more Tregs with enhanced suppressive capacity. Collectively, these studies demonstrate that the balance between induction of and protection from autoimmune disease can be affected by altering the sensitivity of autoreactive thymocytes to selection, by changing their effector function, and by altering the development and function of Tregs (21
). In addition, these studies demonstrate that altering TCR signaling not only changes the repertoire and function of peripheral effector T cells and Tregs but also the balance between the two. Preventing autoimmunity by altering thymic selection, as was likely the case in YYAA and SKG heterozygous mice, is not possible in a patient with overt autoimmunity. However, increasing the number or function of Tregs, as in DSSKO mice, might be.
Several non-mutually exclusive mechanisms could explain how SLAP deficiency in the context of SKG affects Treg numbers and function, preventing or ameliorating arthritis. First, enhanced signaling in thymocytes could lead to more nTregs. Second, enhanced signaling in activated peripheral T cells could improve their capacity to convert into iTregs. Third, altered signaling upon zymosan exposure could enhance the suppressive capacity of Tregs. Fourth, increasing the stability of Tregs could affect both their number and function, as it has been shown that Tregs can lose Foxp3 expression, loss of which eliminated their suppressive capacity (27
). In addition, alterations in TCR signaling could affect survival and/or expansion of Tregs. Further experiments are under way to examine the effects of SLAP deficiency on these processes in nTregs, as well as whether SLAP deficiency also affects these processes in iTregs, as most of the data on Tregs in this report focuses on nTregs.
Enhancement of both number and function of Tregs as a consequence of increased TCR complex-mediated signaling has important implications for the treatment of autoimmunity. Accumulating evidence implicates Tregs as a therapeutic target for autoimmunity, but the lack of good manufacturing procedures for Ag-specific Tregs limits the translation of this approach to the clinic (28
). Technical hurdles to the manufacturing process include the identification of suitable target Ags, purity of Tregs versus potentially autoreactive T cells as Foxp3 is intracellular, and that individualized generation of Ag-specific Tregs may be cost prohibitive (29
). Thus, stimulating endogenous Tregs to increase in number and/or efficacy by qualitatively enhancing TCR complex-mediated signaling in a manner similar to SLAP deficiency represents a more practical and useful alternative. Greater understanding of the effects of SLAP deficiency on the generation and function of Tregs will provide insight into the pathogenesis of autoimmunity, as well as information on how Tregs might be generated and/or manipulated to enhance their function in the treatment of autoimmunity.