Figure 4.
Improved functions of exhausted CD8+ T cells after in vitro VPA treatment. Splenocytes from Cl-13-infected mice (day 40 p.i.) were cultured in vitro with anti-CD3 and anti-CD28 mAbs in the absence or presence of VPA (1.2 mmol/l) for 48 hours, then washed and rested in medium overnight. The diacetylated histone H3 (diAcH3) level and cytokine expression were measured: (a) the diAcH3 level in CD8+ T cells by FACS; (b) specific enrichment of diAcH3 at the Ifng promoter, enhancer and Il2 promoters of CD8+ T cells by ChIP; (c) mRNA level of Ifng and Tnfa by RT-PCR analysis of CD8+ T cells after stimulation with PMA/I; (d) IFN-γ production by FACS (gated on CD8+) after stimulation with GP33-41 peptide or PMA/I. The data shown are representative of more than three separate experiments (*P < 0.05, **P < 0.01, ***P < 0.001). CFU, colony-forming units; PMA/I, phorbol 12-myristate 13-acetate and ionomycin; VPA, valproic acid.