In response to appropriate stimulation, T lymphocytes from systemic lupus erythematosus (SLE) patients exhibit increased and faster intracellular tyrosine phosphorylation and free calcium responses. We have explored whether the composition and dynamics of lipid rafts are responsible for the abnormal T cell responses in SLE. SLE T cells generate and possess higher amounts of ganglioside-containing lipid rafts and, unlike normal T cells, SLE T cell lipid rafts include FcRγ and activated Syk kinase. IgM anti-CD3 Ab-mediated capping of TCR complexes occurs more rapidly in SLE T cells and concomitant with dramatic acceleration of actin polymerization kinetics. The significance of these findings is evident from the observation that cross-linking of lipid rafts evokes earlier and higher calcium responses in SLE T cells. Thus, we propose that alterations in the lipid raft signaling machinery represent an important mechanism that is responsible for the heightened and accelerated T cell responses in SLE.

The TCR-mediated signals required to activate resting T cells have been well characterized; however, it is not known how TCR-coupled signals are transduced in differentiated effector T cells that coordinate ongoing immune responses. Here we demonstrate that human effector CD4 T cells up-regulate the expression of the CD3ζ-related FcRγ signaling subunit that becomes part of an altered TCR/CD3 signaling complex containing CD3ε, but not CD3ζ. The TCR/CD3/FcRγ complex in effector cells recruits and activates the Syk, but not the ZAP-70, tyrosine kinase. This physiologic switch in TCR signaling occurs exclusively in effector, and not naive or memory T cells, suggesting a potential target for manipulation of effector responses in autoimmune, malignant, and infectious diseases.

Objective. T cells from a majority of patients with systemic lupus erythematosus (SLE) display antigen receptor-mediated signaling aberrations associated with defective T cell receptor (TCR) chain, a subunit of the TCR/CD3 complex. This study was undertaken to explore the possibility that forced expression of TCR chain may reverse the known signaling abnormalities and defective interleukin-2 (IL-2) production in SLE T cells.Methods. Freshly isolated SLE T cells were transfected with TCR chain construct in a eukaryotic expression vector at high efficiency, by a recently developed nucleoporation technique. Restoration of TCR/ CD3-mediated signaling was studied in the chaintransfected cells.Results. In SLE T cells transfected with TCR chain, surface expression of TCR chain was increased and the TCR/CD3-induced increased free intracytoplasmic calcium concentration response was normalized, as was hyperphosphorylation of cellular substrates. Simultaneously, the previously noted increased expression of the Fc receptor ␥ chain was diminished in SLE T cells transfected with the chain expression vector, and the surface membrane clusters of cell signaling molecules were redistributed to a more continuous pattern. TCR chain replacement also augmented the expression of diminished TCR/CD3-mediated IL-2 production in SLE T cells, associated with increased expression of the p65 subunit of nuclear factor B in the nuclear fractions of these T cells. Conclusion. These results suggest that reconstitution of deficient TCR chain can reverse the TCR/ CD3-mediated signaling abnormalities as well as the defective IL-2 production in T cells of patients with SLE.It is well recognized that T cells from patients with systemic lupus erythematosus (SLE) display a number of signaling abnormalities (1). Many of the identified molecular aberrations explain certain established cell and cytokine defects, whereas the mechanisms of other defects have not yet been elucidated. Our group and others have demonstrated that the expression of the subunit of the T cell receptor (TCR) is decreased in a majority of SLE patients (2-4) and that this defect persists over time and is independent of disease activity (5).Despite the decreased expression of the TCR chain in SLE T cells, crosslinking of the TCR/CD3 complex leads to increased free intracytoplasmic calcium concentration ([Ca 2ϩ ] i ) response (6) and protein tyrosine phosphorylation (2,4). These events appear to occur because the Fc receptor (FcR) ␥ chain becomes a functional part of the TCR/CD3 complex (7). In support

Nitric oxide is a ubiquitous free radical that plays a key role in a broad spectrum of signaling pathways in physiological and pathophysiological processes. We have explored the transcriptional regulation of inducible nitric-oxide synthase (iNOS) by Krü ppel-like factor 6 (KLF6), an Sp1-like zinc finger transcription factor. Study of serial deletion constructs of the iNOS promoter revealed that the proximal 0.63-kb region can support a 3-6-fold reporter activity similar to that of the fulllength 16-kb promoter. Within the 0.63-kb region, we identified two CACCC sites (؊164 to ؊168 and ؊261 to ؊265) that bound KLF6 in both electrophoretic mobility shift and chromatin immunoprecipitation assays. Mutation of both these sites abrogated the KLF6-induced enhancement of the 0.63-kb iNOS promoter activity. The binding of KLF6 to the iNOS promoter was significantly increased in Jurkat cells, primary T lymphocytes, and COS-7 cells subjected to NaCN-induced hypoxia, heat shock, serum starvation, and phorbol 12-myristate 13-acetate/A23187 ionophore stimulation. Furthermore, in KLF6-transfected and NaCN-treated COS-7 cells, there was a 3-4-fold increase in the expression of the endogenous iNOS mRNA and protein that correlated with increased production of nitric oxide. These findings indicate that KLF6 is a potential transactivator of the human iNOS promoter in diverse pathophysiological conditions. Nitric-oxide synthases are key proteins that produce NO and thereby regulate many important biological processes. NO is generated during the oxidation of L-arginine to L-citrulline by at least three different isoforms of nitric-oxide synthase. Endothelial and neuronal nitric-oxide synthases are constitutively expressed, and their activity is Ca 2ϩ -and calmodulin-dependent, whereas the third isoform is transcriptionally inducible (iNOS), 1 and its activity is independent of Ca 2ϩ and calmodulin and can produce very high levels of nitric oxide over a sustained period of time (1, 2). It has been shown that iNOS is transcriptionally up-regulated in pathophysiologic conditions such as hypoxia, ischemia-reperfusion injury, and trauma and by reactive oxygen species (3, 4).

Human effector T cells have been difficult to isolate and characterize due to their phenotypic and functional similarity to the memory subset. In this study, a biochemical approach was used to analyze human effector CD4 T cells generated in vitro by activation with anti-CD3 and autologous monocytes for 3 to 5 days. The resultant effector cells expressed the appropriate activation/differentiation markers and secreted high levels of interferon ␥ (IFN-␥) when restimulated. Biochemically, effector CD4 T cells exhibited increases in total intracellular tyrosine phosphorylation and effector-associated phosphorylated species. Paradoxically, these alterations in tyrosine phosphorylation were concomitant with greatly reduced expression of CD3 and CD3⑀ signaling subunits coincident with a reduction in surface T-cell receptor (TCR) expression. Because loss of CD3 has also been detected in T cells isolated ex vivo from individuals with cancer, chronic viral infection, and autoimmune diseases, the requirements and kinetics of CD3 down-regulation were examined. The loss of CD3 expression persisted throughout the course of effector T-cell differentiation, was reversible on removal from the activating stimulus, and was modulated by activation conditions. These biochemical changes occurred in effector T cells generated from naive or memory CD4 T-cell precursors and distinguished effector from memory T cells. The results suggest that human effector T-cell differentiation is accompanied by alterations in the TCR signal transduction and that loss of CD3 expression may be a feature of chronic T-cell activation and effector gen- IntroductionThe initiation of adaptive immune responses depends on the activation and differentiation of naive T cells into effector cells that migrate to antigenic sites to orchestrate immune-mediated antigen clearance. Effector T cells can also predominate in diseases characterized by chronic immune activation, such as the autoimmune diseases systemic lupus erythematosus (SLE) 1 and rheumatoid arthritis, 2 and chronic viral infections, 3 and during acute rejection of transplanted tissue. 4 Thus, characterization of the effector T-cell subset at the mechanistic level is essential to understand its role in normal and pathologic immune states.Effector T-cell differentiation involves a series of profound cellular and molecular changes, including an increase in size, up-regulation of activation/differentiation markers such as CD69, CD25, and CD45RO, 5 transcription of effector cytokine genes such as interferon ␥ (IFN-␥) and interleukin-4 (IL-4), 6 and reconfiguring of chromatin structure in the regions of these differentially expressed genes. 7 In contrast to naive CD4 T-cell precursors, the resultant effector CD4 T cells produce high levels of effector cytokines, can be reactivated in the absence of costimulation, 8,9 and exhibit an increased susceptibility to apoptosis. 8 The disparate functions and activation requirements of effector and naive CD4 T cells suggest that distinct signals are being transduced th...

Objective T cells from patients with systemic lupus erythematosus (SLE) display antigen receptor–mediated signaling aberrations associated with defective T cell receptor (TCR) ζ chain protein and messenger RNA (mRNA) expression. This study was undertaken to explore the possibility that coding‐region mutations/polymorphisms of the TCR ζ chain could account for its decreased expression and altered signaling in SLE T cells. Methods TCR ζ chain mRNA from 48 SLE patients, 18 disease controls, and 21 healthy volunteers was reverse transcribed, amplified by polymerase chain reaction, and cloned, and complementary DNA (cDNA) was sequenced. DNA sequences from multiple clones were analyzed for silent single‐nucleotide polymorphisms, mutations, and splice variations, to promote the identification of heterozygosity. Results DNA sequence analysis revealed several widely distributed missense mutations and silent polymorphisms in the coding region of the TCR ζ chain, which were more frequent in SLE patients than in patients with other rheumatic diseases or healthy controls (P < 0.0001). Several of the missense mutations were located in the 3 immunoreceptor tyrosine activation motifs or the GTP binding domain, and this could lead to functional alterations in the TCR ζ chain. A splice variant of the TCR ζ chain with a codon CAG (glutamine) insertion between exons IV and V was found in half of the SLE and control samples. Two larger spliced isoforms of the TCR ζ chain, with an insertion of 145 bases and 93 bases between exons I and II, were found only in SLE T cells. We also identified various alternatively spliced forms of the TCR ζ chain resulting from the deletion of individual exons II, VI, or VII, or a combined deletion of exons V and VI; VI and VII; II, III, and IV; or V, VI, and VII in SLE T cells. The frequency of the deletion splice variants was significantly higher in SLE than in control samples (P = 0.004). These variations were observed in cDNA and may not reflect the status of the genomic DNA. Conclusion These findings demonstrate that heterogeneous mutations/polymorphisms and alternative splicing of TCR ζ chain cDNA are more frequent in SLE T cells than in T cells from non‐SLE subjects and may underlie the molecular basis of known T cell signaling abnormalities in this disease.

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