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DNA Microarray Gene Expression Profile of T Cells with the Splice Variants of TCR mRNA Observed in Systemic Lupus Erythematosus¹

Kensei Tsuzaka^2,*,, Kyoko Nozaki, Chika Kumazawa, Kiyono Shiraishi, Yumiko Setoyama^*, Keiko Yoshimoto^*, Katsuya Suzuki^*, Tohru Abe^* and Tsutomu Takeuchi^*

^* Division of Rheumatology, Department of Internal Medicine, Saitama Medical Center, Saitama Medical School, Saitama, Japan; and Project Research Division, Research Center for Genomic Medicine, Saitama Medical School, Saitama, Japan

We have reported that the TCR mRNA with alternatively spliced 3' UTR ( mRNA/as-3'-untranslated region (UTR)) and mRNA lacking exon 7 ( mRNA/exon 7–) observed in systemic lupus erythematosus patient T cells can lead to down-regulation of both and TCR/CD3 complexes. To determine whether these T cells expressing decreased exhibit differential transcription patterns, we transfected retrovirus vectors containing wild-type cDNA, cDNA/as-3' UTR, and cDNA/exon 7– into murine T cell hybridoma MA5.8 cells which lack expression to construct the MA5.8 mutants WT, AS3' UTR, and EX7–, respectively. FACS analyses demonstrated reduced cell surface expression of and TCR/CD3 complexes on the AS3' UTR mutant and the EX7– mutant in comparison to that on the WT mutant. Total RNA was collected after stimulating the MA5.8 mutants with anti-CD3 Ab. Reverse-transcribed cDNA was applied to the mouse cDNA microarray containing 8691 genes, and the results were confirmed by real-time PCR. The results showed that 36 genes encoding cytokines and chemokines, including IL-2, IL-15, IL-18, and TGF-2, were down-regulated in both the AS3' UTR mutant and the EX7– mutant. Another 16 genes were up-regulated in both, and included genes associated with membranous proteins and cell damage granules, including the genes encoding poliovirus receptor-related 2, syndecan-1, and granzyme A. Increased protein expression of these genes was confirmed by Western blot and FACS analyses. Identification of these responsive genes in T cells in which the and TCR/CD3 complexes were down-regulated may help to better understand the pathogenesis of systemic lupus erythematosus.