| Summary: | 碩士 === 國立成功大學 === 微生物及免疫學研究所 === 102 === CD4+ T lymphocytes are important for immunity to monitor pathogens, tumors, and allografts. Cytokine production is the major regulators released from CD4+ T lymphocytes. While glycogen synthase kinase (GSK)-3, a serine/threonine kinase, has been speculated for facilitating cytokine production probably by controlling several transcription factors, this study aimed to investigate the involvement of GSK-3 for cytokine production in CD4+ T lymphocytes. In an experimental model of CD4+ T lymphocyte activation, a combination of 12-O-tetradecanoylphorbol-13-acetate (TPA), used as an activator of protein kinase C (PKC), and ionomycin, used for calcium influx, so called T/I model, was utilized for this work. Here we showed that T/I treatment induced the production of cytokines IFN-γ, TNF-α, and IL-2 only in human Jurkat T cells but not in another human MOLT-4 cells. Immunostaining followed by flow cytometry analysis showed that MOLT-4 cells were mostly CD4+CD8+ double positive, whereas Jurkat T cells were CD4lowCD8-. However, T/I treatment was able to activate all mouse CD4-bearing thymocytes to produce IFN-γ ex vivo. For this study, purified human T lymphocytes from peripheral blood were specifically utilized. T/I treatment significantly increased the production of IFN-γ, TNF-α, and IL-2 in human CD4+ T lymphocytes. Treating cells with PKC inhibitor bisindolymaleimide or calcium chelator BAPTA blocked T/I-induced cytokine production. It is notable that treatment of GSK-3 inhibitor BIO and short hairpin RNA against GSK-3β decreased T/I-induced IFN-γ, TNF-α, and IL-2. These results demonstrate the common upstream role of GSK-3β for IFN-γ, TNF-α, and IL-2 production in T/I-activated human CD4+ T lymphocytes. Moreover, proline-rich tyrosine kinase 2 (Pyk2) inhibitor Tyrphostin A9 and calcineurin (PP2B) inhibitor cyclosporine A treatment also blocked cytokine production as well as GSK-3 activation in T/I-activated human CD4+ T lymphocytes. It is speculated that Pyk2 and PP2B positively regulate GSK-3β activation in T/I-stimulated human CD4+ T lymphocytes. Activated GSK-3β regulated transcription factor T-bet, which was specifically and individually regulated for these cytokines under T/I stimulation. In a mice model, BIO treatment significantly inhibited T/I-induced mortality and the serum levels of cytokines. According to these results obtained from this work, targeting GSK-3 confers the therapeutic efficacy against CD4+ T cell activation and cytokine production.
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