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Logo of nihpaAbout Author manuscriptsSubmit a manuscriptNIH Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
J Immunol. Author manuscript; available in PMC Dec 1, 2009.
Published in final edited form as:
PMCID: PMC2586973
NIHMSID: NIHMS73494

Differential Expression and Molecular Associations of Syk in Systemic Lupus Erythematosus T cells1

Abstract

Diminished expression of TCR ζ and reciprocal up-regulation and association of FcRγ with the TCR/CD3 complex is a hallmark of SLE T cells. Here we explored if differential molecular associations of Syk kinase that preferentially binds to FcRγ contribute to pathological amplification of signals downstream of this ‘rewired TCR’ in SLE. We detected higher amounts of Syk expression and activity in SLE compared to normal T cells. Selective inhibition of the activity of Syk reduced the strength of TCR-induced calcium responses and slowed the rapid kinetics of actin polymerization exclusively in SLE T cells. Syk and ZAP-70 also associated differently with key molecules involved in cytoskeletal and calcium signaling in SLE T cells. Thus, while Vav-1 and LAT preferentially bound to Syk, PLC-γ1 bound to both Syk and ZAP-70. Our results show that differential associations of Syk family kinases contribute to the enhanced TCR-induced signaling responses in SLE T cells. Thus, we propose molecular targeting of Syk as a measure to control abnormal T cell responses in SLE.

Introduction

Signal transduction through the T cell receptor (TCR) has been shown to be augmented in T cells derived from patients with systemic lupus erythematosus (SLE) (1,2). Specifically, the kinetics of TCR-induced intracellular tyrosine phosphorylation, polymerization of actin and intracellular calcium flux are accelerated in SLE compared to normal T cells (3,4). Recent studies have identified the contribution of altered structure of TCR in mediating abnormal T cell responses. In a majority of patients with SLE T cells, TCR ζ chain undergoes extensive downregulation and instead, reciprocal upregulation and association of a homologous FcRγ chain with the TCR is observed (3,5). Signaling through this ‘rewired TCR’ is presumed to be stronger because of the association of FcRγ with Syk that is 100 times enzymatically more potent than ZAP-70, the kinase which is predominant in normal T cells and traditionally associates with TCR ζ (6,7).

Differences in the molecular associations and activities of Syk and ZAP-70 have been reported and have been ascribed to the differences in the structures of these two kinases (8,9). The recruitment for association of ZAP-70 but not Syk with Src kinase proteins for enzymatic activation is one such example (8,9). Similarly differential expression of ZAP-70 and Syk in T cell subsets might explain differential functional outcomes of TCR signaling. For example, in vitro activated effector CD4 T cells express high amounts of Syk and in these cells, Syk also demonstrates greater association with the TCR compared to ZAP-70 (10). These activated cells produce lower amounts of IL-2 compared to freshly activated naïve T cells. Similarly, human ZAP-70−/− T cells that express Syk also produce lower amounts of IL-2 compared to T cells that express ZAP-70 (11). These observations suggest a strong role for Syk/ZAP-70 kinases in shaping differential functional outcomes of TCR signaling.

However, how Syk contributes to augmentation of TCR-induced signaling in SLE remains unknown. Here, we hypothesized that Syk and ZAP-70 associate with disparate signaling molecules in normal and SLE T cells and contribute to the hyperexcitable phenotype of SLE T cells. Our assumption was based on several observations. First, the binding between Syk and FcRγ was observed in SLE but not normal T cells (5). Secondly, Syk but not ZAP-70 was found to be associated with lipid rafts in SLE T cells, and distinct composition of lipid rafts has been shown to affect the outcome of T cell responses in SLE (4,12). Thirdly, the kinetics of two TCR-induced signaling pathways involving Syk kinases, namely actin polymerization and calcium signaling are enhanced in SLE T cells (4), suggesting a possibility of differential involvement of these kinases in abnormally regulating these events. Therefore, we compared the expression and molecular associations of Syk and ZAP-70 between normal and SLE T cells. We observed that Syk was expressed in higher amounts in SLE compared to normal T cells and that SLE T cells displayed distinct binding of Syk and ZAP-70 to signaling molecules involved in actin polymerization and calcium signaling such as Vav, PLC-γ1 and LAT. These observations suggest that differential expression and association of Syk contributes to abnormal TCR signaling in SLE.

Materials and Methods

Patient samples and T cell isolation

Written informed consent was obtained from 58 SLE patients (51 females, 7 males), with SLE disease activity index (SLEDAI) ranging from 0–16, and 52 healthy volunteers, and 10 rheumatoid arthritis and 9 Sjogren’s syndrome (SS) patients included in this study (Table 1). The SLEDAI scores were calculated as originally described (13). The study protocol and isolation of T cells have been described previously using Rosette Sep (StemCell Technology, Vancouver, British Columbia, Canada) following the manufacturer’s instructions (4). With this technique, we achieved a purity of >97% as assessed by CD3ε staining and analysis by FACS. The study protocol was approved by the Health Use Committees of Walter Reed Army Institute of Research, Walter Reed Army Medical Center, University of Maryland, and Medstar Research Institute.

Table 1
Details of the patients involved in the study

Western blotting, immunoprecipitation and cytochalasin release experiments

The protocols for these experiments have been described before (4,10). Briefly, the cells were lysed with a buffer containing 1% Triton X-100. The supernatants were used for western blotting and immunoprecipitation experiments. All samples were resolved on a 4–12% Bis-Tris NuPage gel (Invitrogen), proteins were transferred onto PVDF membranes and then incubated with specific antibodies against phosphotyrosine (Upstate biotechnology, Charlottesville, VA) or ZAP-70, Syk, PLC-γ1, β-actin, LAT (Santa Cruz Biotech, Santa Cruz, CA) or p-Syk (clone I122–720, Becton Dickinson, San Jose, California). Another polyclonal anti-ZAP-70 Ab was a kind gift from Ronald Wange, NIH, MD. P-coupled antibodies (Santa Cruz Biotech) were used as secondary antibodies, and detection was performed with ECL (Amersham Pharmacia Biotech). The detergent insoluble pelleted fractions after cell lysis were used for eluting actin-bound proteins by addition of 0.1 mg/ml of cytochalasin B for 1 h at 37°C as described previously (4).

Actin polymerization assay

One million normal and SLE T cells were treated with either DMSO or 2 μM R406for 1 hour at 37°C. The cells were activated with anti-CD3 IgM antibody (a kind gift from Dr. Donna Farber, UMD, Baltimore, MD) for various time points and stained with phalloidin-FITC as described before (4). The peak polymerization for each time point was calculated as the ratio between the peak at each time point and the peak at 0s. The shift in the peak between 30 s and 1 min was calculated as the ratio between peaks at 1 min and 30 s.

[Ca2+]i response analysis

These assays were performed as described previously (4), 5×106 cells were treated with 2 μM R406 or equal volumes of DMSO for 1 hr at 37°C and incubated with 1 μg/ml Indo-AM (Molecular Probes Inc., Eugene, OR) for 30 min at 37°C. Samples were run and at 30 s, either OKT3 (10 μg/ml) or the isotype control mIgG2a was added followed by goat anti-mouse cross-linker at 1 min or only PMA and 0.5 μg/ml ionomycin (Sigma) and the ratio of the fluorescence which is directly proportional to free cytosolic Ca2+, was recorded for a period of 400 s as described before using an Epics Altra, (Coulter, Hialeah, FL) flow cytometer (3,14).

Densitometry and statistical analysis

Densitometric analysis of the autoradiograms was performed with the software program GelPro (Media Cybernetics, Silver Spring, MD). Statistical analysis of the data was done by Student t test using the software MINITAB, version 14 (Minitab, State College, PA). A value of p <0.05 was considered to be significant.

Results

Increased expression and activity of Syk kinase in SLE T cells

It has been reported that in a majority of SLE patients, the peripherally circulating T cells demonstrate significantly diminished expression of TCR ζ chain and instead it is replaced by a homologous protein FcRγ that associates with Syk kinase (5). Because ZAP-70 has been shown to be the predominant kinase associated with TCR ζ signaling in normal T cells, we explored if the expression of these two kinases was altered in SLE compared to normal T cells. Western blot analysis of lysates derived from normal and SLE T cells demonstrated that while the expression of ZAP-70 remained virtually unaltered between normal and SLE T cells, the amount of Syk expression was increased by greater than 23 folds (p<0.05) in SLE T cells in more than 80% of cases (Fig. 1 A and B). Simultaneously however, we could not detect significant differences in the expression of Syk between normal and rheumatoid arthritis (RA) and Sjogren’s (SS) T cells (Fig. 1C), suggesting that this observation was limited to SLE. The level of expression of Syk in SLE T cells was unaffected by the disease activity and we did not detect differences in the expression of Syk between cells derived from patients with a wide range of SLEDAI scores ranging from 0–16.

Fig. 1
Expression and activity of Syk are increased in SLE T cells

Next, we asked if in addition to increased expression of Syk in SLE, whether the activity of Syk kinase was increased in SLE. Because phosphorylation of Syk confers activation of this kinase, we compared the expression of pSyk in normal and SLE T cells activated with anti-CD3. As expected, we detected phosphorylation of Syk exclusively following activation of normal and SLE T cells with anti-CD3 Ab. In addition, pSyk expression was significantly higher in SLE T cells stimulated with an IgM anti-CD3 antibody (Fig. 1D). We also performed intracellular staining experiments with anti-Syk antibody using FACS to determine the pattern of Syk staining within the T cell subsets in SLE. We noted a uniform pattern of staining of Syk within entire T cell populations, and no observable differences were noted between the CD4 and CD8 T cells with regard to the expression levels of Syk (data not shown).

Associations and cellular distribution of Syk family kinases in SLE T cells

Previous studies have demonstrated qualitative differences in the TCR-induced intracellular tyrosine phosphorylation in SLE and normal T cells. As an initial step towards assessing the possibility of differential molecular associations of Syk in mediating these events, we compared the patterns of TCR-induced tyrosine phosphorylation in normal and SLE T cells treated with Syk kinase inhibitor R406. As reported previously (3), the overall kinetics of phosphorylation of signaling kinases were accelerated in SLE compared to normal T cells with peak phosphorylation noted in SLE at 60 s and in normal T cells at 120 s (Fig. 2A). Addition of R406 decreased the overall intensity of tyrosine phosphorylation in both normal and SLE T cells but more so in SLE T cells consistent with higher expression levels of Syk in SLE T cells. Peak phosphorylation of TCRζ chain (at the 21/23 kDa region) did not decrease consistently despite addition of R406 in both normal (at 120s) and SLE (at 60s) T cells (Fig. 2B) consistent with the fact kinases other than Syk such as lck and fyn are involved in the phosphorylation of TCRζ Our results with addition of another Syk inhibitor piceatannol (that also has an inhibitory effect on ZAP70 kinase) to normal and SLE T cells were similar to those observed following treatment of normal and SLE T cells with R406 but showed more extensive suppression of signals in SLE compared to normal T cells (data not shown). These results taken together, suggest an enhanced role for Syk in signaling in SLE T cells through differential molecular associations.

Fig. 2
Differential involvement of Syk in phosphorylation of cytoplasmic proteins and differential localization of Syk kinases in normal and SLE T cells

Previously, we had shown that Syk but not ZAP-70 associated with lipid rafts in SLE T cells, suggesting that these kinases associate differentially with the actin cytoskeleton. To address this possibility, we compared the pattern of localization of Syk and ZAP-70 in SLE and normal T cells in the cytoplasmic and actin-bound fractions. We observed that in SLE T cells, high amounts of Syk could be detected within both cytoplasmic and actin-bound fractions, whereas in normal T cells, very low amounts of Syk were detected within the cytoplasmic and actin fractions. Treatment with anti-CD3 antibody resulted in more significant mobilization of Syk to the actin-bound fractions in SLE compared to normal T cells (Fig. 2C). By sharp contrast, the amount of ZAP-70 that associated with the actin-bound protein fraction of SLE T cells was lower compared to that observed in normal T cells. Taken together, these observations suggest that in SLE T cells, Syk plays an important role in regulating signaling events closely associated with actin-cytoskeleton.

Role of Syk in TCR-induced signaling in SLE T cells

In order to test the possible role of Syk in enhancement of TCR-induced signaling in SLE, we studied the effect of specific inhibition of Syk using a Syk-selective inhibitor R406 (15) on two cytoskeleton regulated events namely, polymerization of β-actin and TCR-induced calcium response. We chose this compound over piceatannol because it is more selective for Syk kinase and therefore provided greater flexibility with dosing ranges compared to piceatannol which exerts different effects on Syk and ZAP-70 at different doses. We had previously shown that the peak β-actin polymerization following treatment with anti-CD3 IgM Ab occurred at 30 s in SLE T cells, whereas it occurred at 1 min in normal T cells (4). Here, we observed that when SLE T cells were treated with DMSO, the peak actin polymerization was noted at 30 s, consistent with our previous observations (Fig. 3 and Table 2). However, inhibition of Syk following treatment with R406 resulted in retardation of the kinetics of actin polymerization in SLE T cells, with the peak actin polymerization observed at 60 s. This difference in the shift of peaks between DMSO and R406 treatments was statistically significant (Fig. 3 and Table 2, p=0.011). However, the kinetics of actin polymerization in normal T cells remained minimally altered with in fact some statistically insignificant acceleration of kinetics of actin polymerization with peak actin polymerization occurring at 30 s in the presence of R406 (p=0.092). T cells derived from patients with RA and Sjogren’s syndrome showed kinetics of actin polymerization similar to that observed in normal T cells (Fig. 3 and data not shown). This retardation of the kinetics of β-actin polymerization exclusively in SLE T cells suggests that Syk plays a role in regulating actin-polymerization events SLE but not normal T cells. In this experiment, we used T cells derived from SLE patients with SLEDAI scores ranging from 0–16 but observed consistent results, suggesting that these results are not affected by differences in the disease activity.

Fig. 3
Inhibition of Syk kinase by R406 retards actin-polymerization
Table 2
F-actin polymerization in normal, SLE T and RA cells treated with R406

Similarly, culturing SLE and normal T cells in the presence of R406 resulted in profound suppression of intracellular calcium signaling in SLE (65% reduction) but not normal T cells (13% reduction) the difference being statistically significant (p=0.005). This finding demonstrates the important role of Syk in the calcium signaling pathway exclusively in SLE (Fig. 4A). Although the magnitude of calcium responses were lower in RA T cells compared to normal T cells, the kinetics of calcium induction observed in RA T cells were similar to that observed in normal T cells (Fig. 4A). Although these results suggest Syk as the predominant kinase in calcium signaling in SLE, the lack of complete suppression of calcium signaling in the presence of R406 in SLE T cells suggested that possibly other kinases including ZAP-70 have an overlapping role in mediating TCR-induced calcium responses in SLE T cells. These results were also consistently observed in T cells derived from patients with varying disease activity (SLEDAI ranging from 0–16). We observed that while R406 treatment suppressed TCR/CD3 complex-induced calcium responses in SLE, bypassing the TCR by PMA-ionomycin treatment resulted in high intracellular calcium responses suggesting that the R406-induced suppression of calcium responses observed in SLE T cells was not due to depletion of intracellular calcium by R406 (Fig. 4C).

Fig. 4
Inhibition of Syk kinase by R406 dampens TCR-induced calcium response in SLE T cells

Differential associations of Syk and ZAP-70 in SLE T cells

In order to understand the mechanism by which Syk contributes to heightened kinetics of cytoskeletal events, we compared the molecular associations of Syk and ZAP-70 in SLE and normal T cells. Specifically, we studied the association between Syk and three substrates of ZAP-70/Syk kinases namely Vav, LAT and PLC-γ1. Vav is an important protein required in the F-actin polymerization and TCR capping. In addition, Vav is also required for optimal calcium mobilization via its role in production of PIP2, a substrate of PLC-γ1 (16,17). LAT is critical to the calcium responses via coupling the TCR complex to PLC-γ1-calcium signaling pathway (18). Immunoprecipitation studies demonstrated that the association between Syk and Vav was about 10 folds higher in SLE compared to normal T cells (Fig. 5A, p=0.006). Stripping and reprobing the blots with anti-pVav Ab revealed that Vav was inducibly phosphorylated in both normal and SLE T cells with modestly more intense phosphorylation bands noted in SLE compared to normal T cells following anti-CD3 Ab treatment. The association between ZAP-70 and Vav was not found to be significantly different between normal and SLE (Fig. 5A).

Fig. 5
Differential association of Syk with Vav, LAT and PLC γ1 in SLE and normal T cells

Next, we evaluated the association between Syk/ZAP-70 and LAT in normal and SLE T cells. Immunuprecipitation of LAT resulted in co-precipitation of similar amounts of Syk with LAT in normal and SLE T cells (Fig. 5B). However, while normal T cells demonstrated high amounts of LAT binding with ZAP-70, the amount of LAT that associated with ZAP-70 in SLE T cells was significantly reduced in a majority of SLE T cells (Fig. 5B, p=0.012). Again, we did not notice differences in the pattern of binding of ZAP-70 with LAT between patients with different disease activity (SLEDAI ranging from 0–8). Because LAT is an important resident of lipid rafts, these findings are consistent with our previous observations that Syk alone but not ZAP-70 associated with lipid rafts in SLE T cells (4) and provide a plausible mechanism by which Syk augments signaling through lipid rafts.

Comparison of the expression of PLC-γ1 in normal and SLE T cells revealed that from similar amounts of protein lysates derived from normal and SLE T cells, similar amounts of PLC-γ1 could be immunoprecipitated from SLE T cells (Fig. 5C). However, PLC-γ1 showed much higher binding to Syk (Fig. 5C) in SLE compared to normal T cells that were activated with anti-CD3. Association between PLC-γ1 and β-actin remained unchanged between normal and SLE T cells. Similar to Syk, the association between PLC-γ1 and ZAP-70 was also enhanced in SLE compared to normal T cells (5 out of 8 patients, Fig. 5C and 5D). These results suggests that both Syk and ZAP-70 are involved in the regulation of TCR-induced calcium flux in SLE and is consistent with our earlier observation that inhibition of Syk alone could not completely inhibit the calcium pathway in SLE T cells (Fig. 4A).

Discussion

Mechanisms that contribute to pathological amplification of TCR-induced signaling remain unclear. We and others have reported that the structure of TCR is altered in a majority of SLE T cells wherein, TCR ζ is replaced by FcRγ (reviewed in 19). The finding that Syk associated with FcRγ and that Syk but not ZAP-70 associated with lipid-rafts exclusively in SLE T cells raised the possibility that Syk plays an important role in altering the outcome of signals emanating from the TCR in SLE (4). In this study, we provide several lines of evidence that support this hypothesis: 1) Expression of Syk but not ZAP-70 is enhanced in SLE. 2) Compared to ZAP-70, Syk demonstrated greater association with actin-cytoskeleton in SLE T cells. 3) Inhibition of Syk ‘normalized’ the kinetics of actin polymerization and suppressed signaling events demonstrated by cytoskeleton such as TCR-induced calcium signaling. 4) Syk and ZAP-70 displayed differences in their patterns of association with key signaling molecules that shape the outcome of TCR-induced signaling.

Previously, we had shown that following ligation of TCR/CD3 complex, SLE T cells displayed overall increases in intracellular tyrosine phosphorylation events (3). The observations in our present study that inhibition of Syk kinases reduced the overall level of tyrosine phosphorylation in SLE T cells (Fig. 2A) suggest that enhanced activity of Syk kinases contributes to increased tyrosine phosphorylation of key signaling elements in SLE.

The integrity of actin-cytoskeleton is essential to efficient formation and dissolution of immunological synapse. And mobilization of lipid rafts and proteins bound to actin-cytoskeleton have been shown to play a major role in regulating signaling through lipid rafts. Thus, the increased association of Syk with actin compared to association between ZAP-70 and actin (Fig. 2B) might explain in part why lipid rafts from SLE T cells exclusively contain Syk but not ZAP-70. The finding that selective inhibition of Syk resulted in reversal of the pathologically accelerated kinetics of F-actin polymerization also demonstrates the functional significance of increased association of Syk with the cytoskeleton (Fig. 3). One plausible explanation for the accelerated kinetics of F-actin polymerization is greater enzymatic potency of Syk compared to ZAP-70 (6). Another mechanism could be via the association of Syk with Vav (Fig. 5A) which results in activation of Vav. Phosphorylation-induced activation of Vav is critical to its function and Vav has been shown to play an important role in the kinetics of TCR/CD3 receptor capping (16,17,20). The observation that in normal T cells ZAP-70 is the predominant kinase that binds to Vav but in SLE both Syk and ZAP-70 bind in high amounts to Vav (Fig. 5A top and bottom panels) with possible further enhancement of its activity, lends another rationale for increased kinetics of TCR-induced actin polymerization observed in SLE T cells.

TCR-induced calcium signaling is another intra-cytoplasmic event that is augmented in SLE. Our observation that suppression of Syk resulted in significant reduction in the calcium response suggests a critical role of Syk in the pathological augmentation of TCR-induced calcium flux in SLE (Fig. 4). Detection of association between Syk and PLC-γ1 exclusively in SLE T cells is significant in this regard (Fig. 5C). Similarly, LAT has been shown to couple the TCR to PLC-γ1 and thus participate in the calcium signaling (18). Compared to normal T cells, SLE T cells demonstrate decreased association between ZAP-70 and LAT, whereas Syk associates with LAT in high amounts (Fig. 5B), suggesting that Syk is the predominant kinase that regulates signaling events involving LAT such as calcium responses in SLE T cells.

It is not clear how precisely distal signaling leading upto IL-2 production is affected because of the differences in the utilization of Syk and ZAP-70 in the proximal signaling pathways in SLE. Differential regulation of the calcium regulated pathways which are critical to IL-2 gene expression is a possible explanation (21,22). Further studies are necessary to address this issue. Also, it remains unclear why Syk expression is increased in SLE T cells. Previously, we had drawn comparisons between SLE T cells and effector T cells which share many common features including downregulation of TCR ζ and upregulation of FcRγ, demonstration of membrane clustering of TCR/CD3 complex at baseline, and even demonstration of increased expression of Syk by effector cells and association of active form of Syk to lipid rafts in SLE T cells (4). We had argued that conceivably, the autoimmune process of SLE largely accounts for these observations by driving the T cells into a perpetual state of activation. Our studies herein suggest that in this context, controlling signaling pathways involving Syk would be an important means of controlling the abnormal T cell activity in SLE.

More than a decade ago it was proposed that Syk and ZAP-70 might play different roles in different subsets of T cells (23). Subsequently, the flexibility of TCR-signaling in various immunological contexts has been observed by a simple switch in the utilization of Syk and ZAP-70 by the TCR. For example, in the thymus, Syk is expressed more in early thymocytes and subsequently undergoes relative downmodulation in response to pre-TCR-induced signals, whereas ZAP-70 appears later during the transition of CD4+CD8+ to single positive (SP) cells, a stage at which TCR ζ signaling is established (23). In periphery, the circulating naïve and memory T cells in both humans and mice express high amounts of ZAP-70 and low amounts of Syk. As observed above, in vitro generated effector CD4 T cells preferentially utilized Syk instead of ZAP-70 (10). More recently, the double negative regulatory T cells (DN Treg) that display a TCRαβCD4CD8 and are involved in suppressing host defenses against a transplanted organ, were reported to signal through the FcRγ-Syk pair (24). The observation that FcRγ binds to Syk in SLE T cells suggests that utilization of Syk by the TCR-signaling machinery might be significant in certain autoimmune contexts as well. It is unclear why ZAP-70 is the predominant kinase that participates in TCR signaling in normal T cells despite the presence and activation of Syk kinase albeit at lower levels. Our observation that Syk is excluded from the lipid rafts in normal T cells might be significant in this regard and might provide a mechanism by which the activity of Syk is restricted by normal T cells.

In summary, we show here that in SLE T cells, there is higher expression of Syk kinase compared to normal T cells. This observation is also associated with alterations in the associations of Syk and ZAP-70 in SLE T cells with greater association of Syk with key signaling molecules that shape T cell responses. While the precise outcome of these changes remain unknown, they might be involved in abnormal T cell responses such as defective production of IL-2 or sustained activation of T cells (10,11). Therefore, we propose that Syk could serve as an important molecular target for controlling abnormal T cell activation in SLE.

Acknowledgments

We gratefully acknowledge Doug Smoot (Naval Medical Research Center, Silver Spring, MD) and James Robertson, Leah Duke and Drs. Michael Zidanic, Ashima Saxena and Ramachandra Naik (Walter Reed Army Institute of Research, Silver Spring, MD) for their help with various experiments and Dr. Donna Farber (University of Maryland, Baltimore, MD) for the kind gift of antibodies.

Footnotes

1This work was supported by National Institutes of Health Grants R01 AI42269.

4Abbreviations used in this paper: SLE, systemic lupus erythematosus; Syk, Spleen tyrosine kinase; ZAP-70, zeta associated protein 70; PLC-γ1, phospholipase C-γ1; LAT, linker for activation of T cells, [Ca2+]i, intracellular Ca2+ concentration; DMSO, Dimethyl Sulphoxide; SLEDAI, SLE disease activity index

Publisher's Disclaimer: Disclaimer: The opinions expressed herein are the private ones of the authors and they do not represent those of the Department of Defense or the Department of the Army.

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