In this study, we report the characterization of mice lacking the transmembrane adapter molecule SIT. Our findings support our previous hypothesis that SIT primarily acts as a negative regulator of TCR-mediated signaling (29
). Because SIT is most highly expressed in thymocytes, we initially investigated the function of SIT during thymic selection processes. Non-TCR transgenic SIT-deficient thymocytes showed higher levels of CD5 and CD69 than wild-type animals. Upregulation of these two activation markers has been proposed to reflect the activation status of the cells within the thymus, and the expression levels of CD5 on thymocytes have been shown to directly correlate with the strength of TCR-mediated signals (6
). Therefore, the enhanced expression of CD5 on SIT-deficient thymocytes suggests that not only in Jurkat T cells (29
) but also in vivo, SIT primarily acts as a negative regulator of T-cell activation. Furthermore, it indicates that one physiologic function of SIT is to finely tune signals that emanate from the TCR during thymic development. Clearly, the possibility that SIT directly regulates the expression levels of CD5 or CD69 cannot be completely ruled out. However, the observation that the expression levels of CD5 and CD69 are normal in SIT-deficient OT-I and OT-II TCR transgenic mice (data not shown) makes this possibility unlikely.
The enhanced expression of CD5 and CD69 (together with the slightly increased thymic cellularity) in non-TCR tg mice initially suggested that loss of SIT enhances TCR-mediated signals, thereby leading to subtle alterations of the thymic selection processes. This hypothesis was confirmed in the low-affinity H-Y TCR tg model system, where we demonstrated that in female H-Y TCR tg animals, loss of SIT apparently enhances the efficiency of positive selection. This hypothesis was deduced from different observations. First, SIT-deficient CD8+
SPs show enhanced expression of CD5 (as a sign of enhanced TCR-mediated signaling during the selection processes [6
]). Second, the positively selected CD8+
SP cells in the female mice are more mature than their SIT-expressing counterparts (as assessed by lower levels of HSA expression). Third, ex vivo-isolated thymocytes show enhanced constitutive phosphorylation of the dual-specificity kinase ERK, whose activity has been reported to directly correlate with the efficiency of positive selection (1
). Fourth, loss of SIT induces a more efficient silencing of the TCRα locus that results in a decrease of non-TCR tg CD4+
SP T-cells (20
). Thus, the H-Y data are compatible with the hypothesis that loss of SIT enhances TCR-mediated signals and thereby facilitates positive selection. It is interesting that similar effects on positive selection as shown here for SIT have previously been observed in mice lacking expression of CD5 (in the H-Y and the P14 TCR tg models [5
]). Thus, it appears as if negative regulators such as SIT and CD5 serve to set the signaling thresholds for positive selection during thymic development.
In addition to enhanced positive selection, we found a decrease in DP cells in female H-Y TCR tg mice. This could suggest that in the H-Y system (and even more pronounced in the P14 system), loss of SIT not only enhances positive selection but even partially converts positive selection to negative selection. Although further experiments are required to prove this hypothesis, the idea that loss of SIT partially converts positive selection to negative selection is strongly supported by recent data that we obtained in our laboratory using female H-Y TCR tg SIT/TRIM double-knockout mice. TRIM is a second non-lipid-raft transmembrane adapter protein with unknown function that carries similar tyrosine-based signaling motifs in its cytoplasmic domain as SIT (10
Importantly, the thymi of the female H-Y TCR tg TRIM/SIT double-knockout mice are almost indistinguishable from the thymi of wild-type male H-Y TCR tg animals, in which almost all DP thymocytes are eliminated by negative selection (due to expression of the endogenous male autoantigen). Thus, in the female H-Y TCR tg mice, concomitant loss of both TRIM and SIT completely converts positive selection to negative selection (L. Simeoni et al., unpublished data). The fact that this conversion is only incomplete in SIT-single-deficient mice (note that loss of TRIM by itself has a null effect on thymic development in either male or female H-Y TCR tg mice [Simeoni et al., unpublished]) suggests that the loss of SIT is partially compensated for by other transmembrane adapter proteins, TRIM being one potential candidate (see below).
It is also possible that SIT may regulate thymocyte survival. However, two observations argue against this hypothesis. First, the thymic cellularity in the OT-I and OT-II transgenic systems is normal. Second, we did not observed any differences in the survival rate of thymocytes after treatment with various apoptotic stimuli (e.g., dexamethasone, etoposide, and anti-Fas).
Compensatory mechanisms might also explain why SIT deficiency does not enhance positive selection (or convert positive selection to negative selection) in the OT-I and OT-II TCR transgenic models. Alternatively, it simply might suggest that the fine-tuning function of SIT becomes negligible beyond a certain strength of the TCR-mediated input signal. In line with a gatekeeper function of SIT only in low-strength signaling systems might be the finding that the loss of SIT apparently does not influence negative selection processes in any model that we investigated (i.e., the strong TCR-mediated signals that induce negative selection apparently can no longer be influenced by SIT).
Besides regulating thymic development, SIT seems to partially control the size of the peripheral T-cell pool. Thus, the loss of SIT leads to an expansion of γδ T cells in peripheral lymphoid organs without enhancing the production of γδ T cells within the thymus or impairing their homing to the gut. In addition, SIT-deficient mice show diminished numbers of αβ T cells, selectively in lymph nodes. The latter finding could be due either to a reduced production of αβ T cells within the thymus or to a defect in peripheral T-cell homeostasis or to both. The mild reduction in the numbers of SP thymocytes in nontransgenic SIT−/−
mice could in fact be partially responsible for the reduced numbers of mature peripheral T cells. However, it is also known that similarly to immature thymocytes, naive T cells require continuous contact with self-peptide/MHC molecules (low-affinity signals) to prevent death by neglect (15
). Thus, low-affinity ligands dictate positive selection in immature thymocytes and survival in naive peripheral T cells. Loss of a negative regulatory molecule such as SIT could enhance the strength of TCR-mediated signaling in the presence of low-affinity peptides, thereby altering the survival/death rate of peripheral T cells.
Independently of the mechanism that underlies altered homeostasis of the peripheral T-cell pool, the absence of SIT results in hyperreactivity of peripheral T cells towards TCR-mediated stimuli, with the consequence of enhanced production of the TH1 cytokines tumor necrosis factor alpha and gamma interferon in vitro. In line with this, loss of SIT significantly enhances the clinical course of EAE, a TH1-mediated autoimmune disease of the central nervous system.
How SIT regulates peripheral T-cell functions and how it balances TH1 versus TH2 T-helper cells in the periphery is not known at present. Indeed, despite considerable effort, we could not reveal significant alterations of membrane-proximal signaling events in SIT-deficient peripheral T cells. One possibility to explain the hyperreactivity of peripheral T cells was based on our previous observation that in Jurkat T cells, SIT exerts its negative regulatory function via the tyrosine-based signaling motif YASV (29
). Moreover, in the Jurkat system, it was shown that upon pervanadate stimulation, the tyrosine kinase Csk, the major negative regulator of Src protein tyrosine kinases, is capable of binding to this motif. Therefore, Csk represented an attractive candidate for mediating the inhibitory function of SIT on TCR signaling. However, so far, we could not show an association between SIT and Csk in human or mouse T cells under more physiological conditions of stimulation (e.g., CD3 MAb instead of pervanadate [data not shown]). Similarly, we did not observe an upregulation of the enzymatic activity of Lck, a crucial substrate of Csk in peripheral T cells of SIT−/−
mice. Furthermore, CD3-mediated phosphorylation of the TCR-ζ chain is not impaired in SIT-deficient thymocytes. Collectively, these data suggest that SIT is not involved in the inhibition of Src kinases (either directly or indirectly by recruiting Csk).
Another possibility to explain the negative regulatory role of SIT was based on our previous observation that SIT is capable of recruiting cytosolic protein tyrosine phosphatases (e.g., SHP2) to the plasma membrane (29
). However, extensive biochemical analysis of SIT-deficient T cells did not reveal alteration of TCR-mediated membrane targeting of SHP1 or SHP2 (data not shown). This largely rules out the possibility that impaired membrane targeting of SHP1 or SHP2 underlies the enhanced signaling capacity of SIT−/−
T cells. Nevertheless, the fact that SIT-deficient T cells react normally after stimulation with PMA and ionomycin suggests (and confirms our previous data) that SIT exerts its negative regulatory role upstream of activation of protein kinase C and production of IP3.
Although the molecular mechanism(s) underlying SIT-mediated inhibition of signaling is still unknown, it appears as if SIT would negatively regulate TCR-mediated activation of ERK in thymocytes. Indeed, ex vivo-isolated H-Y transgenic SIT-deficient thymocytes show enhanced phosphorylation of ERK1/2, and SIT−/−
DP thymocytes display higher expression levels of CD5 and CD69, both molecules whose expression is regulated via the ERK-dependent transcription factors Ets-1 (14
) and AP-1 (11
). In light of these observations, it is important that we have previously demonstrated that SIT can bind the cytosolic adapter protein Grb2, an upstream activator of Erk, via a membrane-proximal YGNL motif. However, when overexpressed in Jurkat T cells, a CD8/CD8/SIT chimera (extracellular domain/transmembrane domain/intracellular domain) that carries an isolated YGNL motif does not inhibit T-cell functions (35
). Rather, when this chimera is co-cross-linked with the TCR, it strongly upregulates TCR-mediated activation of the transcription factor NFAT. This suggests a positive rather than a negative regulatory role of the YGNL motif. However, further experiments are required to assess the role of the YGNL motif for SIT-mediated T-cell function.
Another possibility to explain enhanced TCR-mediated signaling in the absence of SIT would be to assume that SIT exerts its negative regulatory role in normal cells through sequestration of key signaling molecules from the lipid raft to the nonraft fraction, thereby limiting the numbers of signaling molecules in the lipid rafts. Loss of SIT within the nonraft fraction could then result in a redistribution of these signaling molecules to the lipid rafts. This would then permit enhanced signaling. A redistribution of molecules from nonrafts to rafts has most recently been proposed to cause the augmented Fc
RIII-mediated signaling in NTAL/LAB-deficient mast cells (46
However, another recent report suggested that when present in the nonraft fraction of chicken DT-40 cells, Grb2 inhibits B-cell receptor (BCR)-mediated signals via an unknown mechanism (38a
). Targeting of Grb2 to the lipid rafts (e.g., by ectopic expression of the lipid raft-associated transmembrane adapter NTAL) rescues the BCR from the Grb2-mediated inhibitory signal, thus facilitating BCR-mediated responses. If this model would also apply for T cells, then it would be reasonable to assume that SIT inhibits TCR-mediated signaling by enhancing the inhibitory pool Grb2 in the nonraft fraction. The fact that we did not observe major alterations of biochemical signaling events following CD3 stimulation of SIT-deficient peripheral T cells does not necessarily contradict this hypothesis, simply because the subtle changes that are induced by loss of SIT might be too weak to be detected by standard biochemical techniques. In addition, loss of SIT might be to a large extent compensated by other non-raft-associated transmembrane adapter proteins exerting similar functions, such as TRIM and LAX.
Although the molecular mechanism(s) underlying SIT function is still elusive, it is obvious that SIT controls signaling pathways at different levels within the immune system. First, SIT-mediated signals are required to maintain a proper T-cell repertoire; second, they are mandatory to maintain the composition of the peripheral T-cell pools; third, they regulate the activation thresholds of peripheral T cells; and fourth, they control cytokine production/expansion of particular peripheral T-cell populations. In all cases, SIT seems to act as a fine tuner of TCR-mediated signaling processes.
The only mild alterations of thymic selection processes as well as the rather moderate hyperreactivity of peripheral T cells in SIT-deficient mice might be due to a functional redundancy among those transmembrane adapter proteins that serve as negative regulators during TCR-mediated signaling. In this regard, other nonraft transmembrane adapter proteins, for example, TRIM and LAX, might compensate for the loss of SIT. TRIM shares not only structural properties with SIT (both molecules are disulfide-linked homodimers) but also two tyrosine-based signaling motifs (YGNL and YASV, which is YASL in TRIM) (18
). Similarly, LAX is also capable of binding, e.g., Grb2, and has been reported to negatively regulate TCR-mediated signals via an unknown mechanism (50
). Therefore, the analysis of the SIT/LAX or SIT/TRIM double knockout or the SIT/LAX/TRIM triple knockout will possibly help to elucidate the biochemical pathways that are controlled by these transmembrane adapter proteins. Nevertheless, it is tempting to speculate that SIT as well as other transmembrane adapter proteins play important roles in priming the organism for the development of autoimmune diseases either by regulating peripheral tolerance or by altering the generation of an appropriate T-cell repertoire. In this regard, it will be important to assess the genetic status and the phosphorylation status of SIT and other negative regulatory transmembrane adapter proteins in patients suffering from autoimmune disease.