UBC Theses and Dissertations
Selective inhibition of signal transduction pathways in human CD4⁺ tlymphocytes Li, Xiaomei
We utilized an in vitro T cell activation model to investigate the effect of blocking key signaling pathways downstream of TCR and CD28 molecules by using specific kinase inhibitors. Human peripheral CD4⁺ T cells were stimulated with anti-CD3 mAb (OKT3) and anti-CD28 mAb, inducing T cell proliferation with maximum IL-2 secretion at day 1 and maximum IL-2 receptor a chain (CD25) expression at day 3 of cell activation. MAP or ERK kinase (MEK) inhibitor U0126, p38 MAP kinase inhibitor SB203580, protein kinase C (PKC) inhibitor Bisindolylmaleimide I and phosphatidylinositol 3 kinase (PI3- kinase) inhibitor LY294002 were added to cell cultures. These inhibitors did not induce cell apoptosis and they markedly reduced T cell proliferation as well as IL-2 production and CD25 expression. This inhibition was time and dose dependent and more profound than the effects of cyclosporine. The suppression of CD25 was largely independent of IL- 2. All four inhibitors markedly reduced the binding activity of NF-AT and AP-1 for IL-2 gene transcription. Yet their influence on NF-κB and STAT5 binding activity for CD25 gene transcription was variable. LY294002 markedly reduced NF-κB binding activity, while U0126, SB203580 and LY294002 partially reduced STAT5 binding activity. The combination of the CD28 pathway inhibitor LY294002 with one of the TCR pathway inhibitors further reduced T cell proliferation. The dose relationship study showed that LY294002 worked as an antagonist to the TCR pathway inhibitor at the low end of the scale of inhibition and became more synergistic as T cell inhibition increased. Conclusions: Human CD4⁺ T cells activated through the TCR and CD28 molecules induced cell proliferation with an increase of IL-2 secretion and IL-2 receptor a chain (CD25) expression. Specific inhibitors of the PKC, MEK/ERK, p38 MAP kinase and PI3- kinase pathways caused marked inhibition of T cell proliferation as well as IL-2 and CD25 expression, and indicated that these pathways were important for T cell activation but not essential for IL-2 signaling under TCR/CD28 stimulation. This inhibition occurred at the transcription factor level. These data demonstrated the potential value of these important pathways as future targets for immunosuppressant.
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