performed statistical analysis
performed statistical analysis. endogenous HIV-1-specific CD8+ T cell response and/or redirect the response toward conserved epitopes. However, since both HDACis and PKCms have been reported to have immunomodulatory effects13,14,15,16, it is important to consider whether these brokers may impact the ability of CD8+ T cells to respond to viral antigen. Recently, Jones reported that HDACis impaired HIV-1-specific CD8+ T cell responses from participants receiving oral Vorinostat. We report that changes in T cell phenotype and function were significantly greater and more sustained in PBMC treated with PKCms compared to HDACis, but that even within the same class, compounds differed in their effects. Interestingly, some effects were only evident 48?hours or more after a short 3?hour exposure to drug. We conclude that this timing of antigen presentation by reactivated cells will be critical in determining whether their clearance by CD8+ T cells is usually impaired following treatment with a LRA. Results HDACis minimally activate T cells The plasma half-lives of Vorinostat, Romidepsin, and Panobinostat are reported as approximately 2?hours, 3.5?hours, and 30?hours, respectively21,22,23. To mimic exposure of cells to drug we tested the effect of different periods of exposure, 3, 6, 12, or 24?hours, to Vorinostat, Romidepsin, and Panobinostat on T cell activation. The concentration used for each drug was determined by previously reported plasma Cmax levels that also elicited HIV-1 reactivation antigen-specific CD8+ T cell function. Following stimulation, CD8+ T cells rapidly release pre-formed perforin-containing granules, resulting in a reduction in intracellular perforin and the accumulation of CD107a contained in the granule membrane at the cell surface30. To assess cytotoxic T cell function and maximize assay stringency, we gated on cells that produced IFN- in response to peptide stimulation and were CD107a+ and perforinlow?31 (Supplementary Fig. S10). We observed that relative to vehicle, pre-exposure to Panobinostat modestly but consistently reduced the frequency of antigen-specific CD8+ T cells exhibiting cytotoxic potential in both seropositive and seronegative individuals (mean 1.4-fold (range 1.1C1.7) decrease in seropositive; 2.0-fold (range 1.4C2.7) decrease in seronegative; p?=?0.002 by exact Wilcoxon Signed Rank test stratified by HIV-1 serostatus (S)-10-Hydroxycamptothecin (Fig. 5c)). Ingenol-db, on the other hand, increased the frequency of antigen-specific perforinlow CD107a+ IFN-+ CD8+ T cells (mean 1.6-fold (range 1.2C2.4) increase in seropositive; 1.9-fold (range 0.8C2.9) in seronegative; p?=?0.006). None of the other drugs tested significantly altered the magnitude of this T cell response. When antigen-specific CD8+ T cell responses involving other combinations of cytokines were assessed, Panobinostat exposure consistently decreased antigen-specific CD8+ T cell responses. While Vorinostat had no effect on production of lytic markers, exposure modestly reduced the frequency of antigen-specific CD8+ T cell responses involving the production of TNF (p? ?0.05; Table 2). (S)-10-Hydroxycamptothecin Romidepsin did not significantly affect any of the functional parameters studied. Among the PKCms, Prostratin did not affect antigen-specific CD8+ T cell responses. Ingenol-db and Bryostatin-1, while both strongly inducing non-specific cytokine production (Fig. 5a,b), also increased the frequency of CD8+ T cells producing IFN- and/or TNF in response to antigen after subtraction of the non-specific response (Table 2). In summary, Panobinostat was the only HDACi, when administered at a physiologically-achievable dose that significantly impaired antigen-specific lytic responses in primary CD8+ T cells. Among the PKCms, Ingenol-db and Bryostatin-1 enhanced some antigen-specific T cell (S)-10-Hydroxycamptothecin responses. Table 2 Effects of pre-exposurea to LRAs on antigen-specific CD8+ T cell functionb. effects of Vorinostat on T cell phenotype and function are minimal We also examined (S)-10-Hydroxycamptothecin the phenotype and function of T cells from three durably-suppressed HIV-1-seropositive donors who received a single Rabbit Polyclonal to TLK1 400?mg oral dose of Vorinostat. Plasma Vorinostat levels were monitored for 10?hours post-dose (Fig. 7a). PCR analysis using cells obtained by leukapheresis 4?hours after the dose indicated that Vorinostat had induced viral reactivation (defined as a significant increase in cell-associated viral RNA from baseline) in participant A but not participant B (no data available for participant C) (Fig. 7b). T cell activation was examined on cells freshly isolated from peripheral blood obtained immediately prior to the dose (time 0) and at 4, 7, 10, and 24?hours post-dose. Most markers were unchanged from baseline (Fig. 7c, Supplementary Fig. S11). However, we observed a consistent increase in the frequency of CD25-expressing CD4+ T cells (2.9-fold for participant A, 1.7-fold for B, and 6.0-fold.