Lrsii

To determine endogenous intracellular IL-10 production, PBMC were cultured for 48 hours; during the final 5 hours of incubation the cultures were supplemented with Brefeldin A (1∶100, BD), PMA (50 ng/ml, Invivogen) and Ionomycin (1 ug/ml, Invivogen). After incubation the cells were washed, stained for viable cells (LIVE/DEAD Aqua Fixable Dead Cell Stain Kit, Invitrogen), surface stained, fixed/permeabilized (Fix/Perm Kit BD Biosciences) and stained for intracellular IL-10 (IL-10-AF-647, eBioscience). To determine spontaneous expression of IL-10 by Bregs from HIV-infected individuals and healthy controls, PBMCS were incubated overnight, stimulated for the final 5 hours and stained as described for healthy controls. The following antibodies were used for immunophenotyping of PBMC: CD19-ECD (Beckman Coulter), PD-L1-PE-Cy7 (eBioscience), CD24-PE, CD38-FITC, HLA-DR-PE-Cy7, CD4-Pacific Blue, CD8-APC-H7, Lineage-1-FITC, CD11c- AF-700, HLA-ABC-PE-C7 and CD107a-PE-C5 (BD, Bioscience). HIV-specific CD8⁺ T cells were identified by binding to MHC-1-APC Dextramer® (Immudex) and HIV-infected CD4+ T cells were identified by binding to KC-57-PE antibody (Beckman Coulter). All samples were acquired on an LRSII (BD, Bioscience) flow cytometer and the data was analyzed using FlowJo software (Tree Star Inc).

Annexin-APC and PI (Thermo Fisher Scientific) were used for apoptosis analysis. CD11b-PE (Beckman Coulter, Tokyo, Japan) was used for granulocytic differentiation analysis. CD14-Alexa Fluor (Thermo Fisher Scientific) was used for monocytic cell differentiation. Cells were incubated for 15 min with indicated antibodies and then detected using LRSII (BD Biosciences, San Jose, CA). Results were analyzed using the FlowJo software (version 9.3.2).