T cell signaling abnormalities in systemic lupus erythematosus are associated with increased mutations/polymorphisms and splice variants of T cell receptor ? chain messenger RNA

Abstract

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.

PTPN22 C1858T polymorphism in Colombian patients with autoimmune diseases

“…19 Signaling aberration and abnormalities in tyrosine phosphorylation might act as a central defect in the pathogenesis of SLE. 20,21 Our results confirm previous findings in independent SLE cohorts 12,22,23 and suggest that downregulation of T-cell activation plays an important role in T-cell-mediated response in disease.…”

Forced Expression of the Fc Receptor γ-Chain Renders Human T Cells Hyperresponsive to TCR/CD3 Stimulation

“…To determine the mechanism of TCR -chain down-regulation in Fc⑀RI␥-transfected cells, we analyzed the mRNA level by RT-PCR. Total RNA was isolated from nucleoporated cells after 18 h, reverse transcribed, and PCR amplified as described earlier (26). There was no significant difference between the level of TCR -chain mRNA in control vector or Fc⑀RI␥-transfected cells (data not shown), suggesting that the decreased expression of -chain is not the consequence of transcriptional down-regulation.…”

Abnormal expression of various molecular forms and distribution of T cell receptor ? chain in patients with systemic lupus erythematosus

“…Peripheral blood mononuclear cells were obtained by density-gradient centrifugation over Ficoll, and T cells were isolated by magnetic separation (Miltenyi Biotec, Auburn, CA) after depletion of non-T cells as described previously (36). In all cases, the percentage of T cells in the isolated subpopulation was Ͼ96% as determined by anti-CD3 staining and fluorescence-activated cell sorter analysis using an Epics Elite flow cytometer (Coulter, Hialeah, FL).…”