H10/0.05% DMSO was used as a negative control and PHA was used as a positive control. The following day, the cells were discarded and the plate was incubated with
biotinylated anti-IFN-γ antibody (Mabtech) for 3 h at 37°C, followed by streptavidin-conjugated alkaline phosphatase (Mabtech) for 1 h at 37°C. The plate was developed with alkaline phosphatase conjugate substrate (Bio-Rad). Spots were counted using an automated ELISpot plate reader Enzalutamide research buy (AID Systems, Germany) and the frequencies of IFN-γ-producing cells were expressed as IFN-γ SFU per 106 PBMCs. The Kruskal–Wallis test followed by Dunn’s multiple see more comparisons post-test (multiple group comparisons), Wilcoxon matched-pairs
test, Spearman’s rank test and paired t-test were performed using GraphPad Prism version 5. p values of <0.05 were considered statistically significant. This work was supported by the Oxford NIHR Biomedical Research Centre, UK. A.M., P.B. and L.D. are Jenner Investigators. The authors declare no financial or commercial conflict of interest. As a service to our authors and readers, this journal provides supporting information supplied by the authors. Such materials are peer reviewed and may be re-organized for online delivery, but are not copy-edited or typeset. Technical support issues arising from supporting information (other than missing files) should be addressed to the authors. Supplementary Figure 1 Progressive gating strategy used to identify CD4+ T cells, CD8+ T cells and CD19+ B cells
within the lymphocyte population and CD3- CD14+ cells within the monocyte population. Supplementary Figure 2 Frequencies of CD4+ T cells, CD8+ T cells, CD19+ B cells and CD14+ monocytes that constitutively express IL-10 among ART-naive patients (n=25), ART-treated patients (n=20) and uninfected controls (n=5). Supplementary Figure 3 Effect of depletion of HIV-1 gag-specific IL-10+ CD8+ T cells on HIV-1 gag-induced expression of HLA-DR and CD38 on CD4+ T cells. Supplementary Figure Methane monooxygenase 4 (A) CD38 expression on CD14+ monocytes from infected (p24 Ag+) and mock-infected PBMC (n = 4) after 3 and 5 days’ culture. CD8+ T cell-depleted PBMC from four HIV-negative subjects were activated for 3 days with phytohaemagglutinin and then infected with HIV-1BaL in the presence of IL-2, using a MOI to achieve infection of 5-10% CD4+ T cells, as indicated by expression of p24 antigen (p24 Ag). (B) Representative plots showing p24 Ag expression in monocytes from mock-infected PBMC cultures (left) and HIV-1BaL-infected PBMC cultures (right) after 3 days.