Cyclosporin A inhibits the early phase of NF-κB/RelA activation induced by CD28 costimulatory signaling to reduce the IL-2 expression in human peripheral T cells

Cyclosporin A (CsA) inhibits interleukin (IL)-2 production, activation and proliferation of human peripheral T cells (HPTC) costimulated with simultaneous engagement of T cell receptor (TCR)/CD3 and CD28. We demonstrated that 10 ng/ml CsA, which reduced the proliferation of HPTC costimulated with anti-CD3 and anti-CD28 by half, prevented NF-AT and NF-kappaB from migrating into the nucleus. Whereas CsA added even 30 min after the costimulation caused NF-AT to remain in the cytoplasm, the delayed addition of CsA could not prevent NF-kappaB from translocating into the nucleus. CsA, which was added to HPTC simultaneously with the engagement of both CD3 and CD28 or the 1-h-delayed engagement of CD28 after prior TCR/CD3-triggering, inhibited NF-kappaB p65/RelA from binding to the target DNA fragment, followed by reduction of HPTC proliferation in response to the costimulation. When CsA was added 30 min after the delayed engagement of CD28 following the prior engagement of TCR/CD3, these inhibitory effects were diminished. Antisense NF-kappaB p65/RelA oligonucleotides inhibited p65/RelA mRNA expression, diminished IL-2 mRNA expression in the costimulated HPTC and reduced HPTC proliferation to the same extent as CsA added simultaneously with the costimulation. The CsA- or antisense p65/RelA oligonucleotide-induced reduction in the proliferation of costimulated HPTC was overcome by the addition of exogenous IL-2. These findings indicate that the major effects of CsA on the early phase of CD28-mediated costimulation in the presence of TCR/CD3 signaling are to inhibit NF-kappaB/RelA from translocating into the nucleus and binding to the target DNA sequence in the IL-2 gene promoter region, which induces IL-2 expression leading to HPTC proliferation.