Fig 2: FBCs from HIV-infected MSM induce high levels of T cell activation in PBMC. Healthy PBMC was stimulated with FBC from 6 SN MSW, 13 SN MSM, 8 HIV+ART+MSM or 11 HIV+ART−MSM subjects for 4 days. (A) Representative dot plots and accumulative data showing HLA-DR+ CD38+ CD4+ activated T cells. (B) Levels of cytokines in the supernatant. Each dot represents the median of 6 different PBMC stimulated with the same FBC. (C) Whole PBMC or isolated CD4+ T cells were stimulated with FBC isolated from 10 HIV+ART−MSM for 4 days. Red: FBC from HIV-infected subjects, Black: FBC from HIV negative. ∗ = p < 0.05, ∗∗ = p < 0.005, ∗∗∗ = p < 0.0005.

Fig 3: Application of the Pre-GC tube, showing the major B-cell subsets in blood of healthy controls, and PID patients using classical 2-dimensional plots vs. n-dimensional PCA. Analysis of circulating B-cell subsets (FS/SSloCD19+) identified within 2 × 106 blood leukocytes using the markers from the Pre-GC tube: Immature (CD5+ CD27− CD38++ smIgM++ smIgD+), CD5+ and CD5− naïve B-cells (CD27− CD38+d smIgM+ smIgD++), Ig non-switched and Ig-switched memory B-cells (CD5− CD27+ CD38− smIgM+ smIgD++ and CD5− CD27+/− CD38− smIgM− smIgD−, respectively), and plasmablasts (CD5− CD27++ CD38+++). (A) The identification of biologically relevant subsets of B-cells using the minimum number of bivariate plots required for a 5 marker-combination (three bivariate plots/cell population). (B) The same data via PC1 vs. PC2 representation of a 5-dimensional space in blood of six healthy controls (left panel), in blood of a patient with common variable immunodeficiency (CVID; middle) and in blood of a patient with agammaglobulinemia (right), all stained with the Pre-GC tube under comparable conditions. The CVID patient lacks plasmablasts in blood (<0.01 cell/μL blood) and has reduced IgH-class switched MBC. The agammaglobulinemia patient has no plasmablasts, no MBC, and also the naïve B-cell compartment is strongly reduced.

Fig 4: Levels of sCD14, LPS Binding Protein (LBP) and CD8+ T cell activation in plasma and peripheral blood. (A) Plasma levels of soluble CD14. (B) Plasma levels of LBP. (C) PBMC was stained directly ex vivo for CD8 + HLA-DR + CD38+ activated T cells. Red: FBC from HIV-infected subjects, Black: FBC from HIV negative. ∗ = p < 0.05, ∗∗ = p < 0.005, ∗∗∗ = p < 0.0005.

Fig 5: FBCs isolated from HIV-infected females induce monocyte and T cell activation. Healthy PBMC was stimulated with FBCs isolated from 5 HIV-negative, and 7 HIV-infected treated females. (A) Percent change of CD14+ and CD80+ CD33+ cells after 24 h stimulation. (B) Frequency of CD4 + HLA-DR + CD38+ T cells after 4 days of culture. (C) Levels of IFN-γ and IL-17 in PBMC after 4 days of culture in the supernatant. Red: FBC from HIV-infected subjects, Black: FBC from HIV negative. ∗ = p < 0.05, ∗∗ = p < 0.005, ∗∗∗ = p < 0.0005.