Figure 1.
HIVEC and HIVNEG have comparable Bregs frequency.
PBMC from HIVEC (n = 15), HIVART (n = 20), HIVNEG, (n = 20) and HIVVIR, (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 (a,b) frequency of CD19+CD24hiCD38hi Bregs and (c) 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.
Figure 2.
SAHA treated Breg-depleted PBMC from HIVEC and HIVART exhibit higher frequencies of anti-HIV CTL-competent CD8+ T cells.
(a) 500 nM SAHA-treated total or Breg-depleted PBMC from HIVEC (n = 4) and HIVART (n = 6) were cultured for 4 days and the frequency CD107a+CD8+ T cells determined by flow cytometry. (b) After 4 days in culture, by flow-cytometry using an HLA-A*0201 MHC-I HIV Dextramer® (Immudex), the frequency of HIVgagSL9+ CD8+ T cells was determined; left panel depicts representative dot-plots demonstrating specific binding and right panel shows the summary of results (A2pos = HLA-A*2 positive, A2neg = HLA-A*2 negative). p values for differences as calculated by paired one-tailed t test (Graphpad Prism software) are indicated.
Figure 3.
Association between elevated frequency of CTL–competent T cells, clearance of infected CD4+ T cells and reduced viral DNA.
(a) 500 nm SAHA-treated total or Breg-depleted PBMC from HIVEC (n = 4) and HIVART (n = 5) were cultured for 4 days and the frequency of infected CD4+ T cells was determined by binding to KC57-antibody. (b) In HIVART 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.
Figure 4.
SAHA-treated Breg-depleted PBMC from HIVEC and HIVART exhibit upregulated expression of antigen-presenting molecules and proliferation of CD4+ T cells.
After 4 days in culture, (a) 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) SAHA-treated total or Breg-depleted PBMC from HIVEC, (n = 4, upper panel) and HIVART, (n = 4, lower panel). The gating strategy and representative histogram overlays are depicted in Figure 3a. (c) VPD450-proliferation dye labeled total or Breg-depleted PBMC were stimulated with SAHA (500 nM, Figure 1b 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.
Figure 5.
Bregs from HIVEC and HIVART subjects exhibit comparable endogenous levels of PD-L1.
In PBMC (n = 15) of HIVEC, HIVART, HIVNEG, and HIVVIR, endogenous levels of PD-L1 expression on (a) Bregs and (b) Bregs compared to the other B-cell subsets were determined by flow cytometry. Boxes represent 25th to 75th percentiles, whiskers indicate minimum and maximum values and the lines indicate the median. P values (Graphpad Prism software) are indicated; * = p<0.05, ** = p<0.005, *** = p<0.0005.
Figure 6.
In HIVART subjects Breg-mediated inhibition of CD4+ T cell proliferation is dependent on a synergy between PD-L1 and IL-10.
Bregs or non-Bregs B cells were co-cultured for 72 hours with VPD450-labeled FACS-purified CD4+ T cells, activated using Anti-Biotin MACSiBead (Miltenyi, T Cell Activation/Expansion Kit) and IL-2 (2 U/ml) and proliferation of CD4+ T cells determined by flow cytometry. (a) Right panel, shows a representative overlay and right panel depicts compilation of results, whereby the reduction in proliferation is normalized to CD4+ T cell proliferation alone (n = 4). (b) In antibody blocking experiments, labeled and activated CD4+ T cells were co-cultured with Bregs and under conditions shown above the representative histograms (n = 4) (α-IL-10 = IL-10R blocking antibody (20 ug/ml, Biolegend), α-PD-1 = PD-1 blocking antibody (10 ug/ml, Biolegend).