SAHA-treated Breg-depleted PBMC from HIV_EC and HIV_ART exhibit upregulated expression of antigen-presenting molecules and proliferation of CD4+ T cells.

<p>After 4 days in culture, (<b>a</b>) the expression of MHC-II and MHC-I/II on B cells and dendritic cells (LIN⁻CD11c⁺HLA-DR⁺) respectively was determined by flow cytometry in (<b>b</b>) SAHA-treated total or Breg-depleted PBMC from HIV_EC, (n = 4, upper panel) and HIV_ART, (n = 4, lower panel). The gating strategy and representative histogram overlays are depicted in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0092934#pone-0092934-g003" target="_blank">Figure 3a</a>. (<b>c</b>) VPD450-proliferation dye labeled total or Breg-depleted PBMC were stimulated with SAHA (500 nM, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0092934#pone-0092934-g001" target="_blank">Figure 1b</a> right panel, n = 5) and after 4 days in culture proliferation of CD4⁺ T cells was determined by flow cytometry. p values for differences in CD4⁺ T cell proliferation as calculated by paired two-tailed t test (Graphpad Prism software) are indicated.</p

HIV_EC and HIV_NEG have comparable Bregs frequency.

<p>PBMC from HIV_EC (n = 15), HIV_ART (n = 20), HIV_NEG, (n = 20) and HIV_VIR, (n = 17) were cultured for 48H and during the final 5H supplemented with PMA (25 ng/ml), Ionomycin (1 ug/ml), Brefeldin A (1∶100) and by flow cytometry, the (<b>a,b</b>) frequency of CD19⁺CD24^hiCD38^hi Bregs and (<b>c</b>) IL-10-positive Bregs (intracellular cytokine staining) determined. p values for differences as calculated by Mann Whitney test two-tailed t test (Graphpad Prism software) are indicated; * = p<0.05, ** = p<0.01, lines indicate mean with SEM.</p

SAHA treated Breg-depleted PBMC from HIV_EC and HIV_ART exhibit higher frequencies of anti-HIV CTL-competent CD8⁺ T cells.

<p>(<b>a</b>) 500 nM SAHA-treated total or Breg-depleted PBMC from HIV_EC (n = 4) and HIV_ART (n = 6) were cultured for 4 days and the frequency CD107a⁺CD8⁺ T cells determined by flow cytometry. (<b>b</b>) After 4 days in culture, by flow-cytometry using an HLA-A*0201 MHC-I HIV Dextramer® (Immudex), the frequency of HIV_gagSL9⁺ CD8⁺ T cells was determined; left panel depicts representative dot-plots demonstrating specific binding and right panel shows the summary of results (A2^pos = HLA-A*2 positive, A2^neg = HLA-A*2 negative). p values for differences as calculated by paired one-tailed t test (Graphpad Prism software) are indicated.</p

Association between elevated frequency of CTL–competent T cells, clearance of infected CD4⁺ T cells and reduced viral DNA.

<p>(<b>a</b>) 500 nm SAHA-treated total or Breg-depleted PBMC from HIV_EC (n = 4) and HIV_ART (n = 5) were cultured for 4 days and the frequency of infected CD4⁺ T cells was determined by binding to KC57-antibody. (<b>b</b>) In HIV_ART subjects (n = 5), relative levels of HIV DNA between SAHA-treated total or Breg-depleted PBMC were determined by qPCR with LTR hybridizing primers after 4 days in culture. p values as calculated by paired two-tailed t test (Graphpad Prism software) are indicated.</p