Phorbol myristate acetate (pma)

In this study, we obtained whole blood from 15 patients with SLE and 15 HCs, and PBMCs were extracted via Ficoll separation (Dakewei, Shenzhen, China). PBMCs (5 × 10⁵) were then washed twice with PBS and incubated in 96-well plates. The culture medium comprised of RPMI 1640 medium with 10% fetal bovine serum, with the addition of ionomycin (50 ng/ml, InvivoGen, San Diego, CA, USA), phorbol myristate acetate (PMA, 1 μg/ml, InvivoGen), and brefeldin A (BFA, 1:1000, Biolegend, San Diego, CA, USA). ionomycin and PMA were used to stimulate TFH cells to secrete IL-21 and TFR cells to secrete IL-10, while BFA was used to inhibit the expression of IL-21 and IL-10 to extracellular. Cells were then cultured for 5 h at 37 °C in an atmosphere of 5% carbon dioxide and harvested for flow cytometry analysis.

THP-1 X-Blue™ monocytes were seeded in 100 mm cell culture dishes (Corning, Borre, France), 5 × 10⁶ cells per dish in 10 mL of medium, and differentiated to TDM by exposure to 100 ng/mL of phorbol 12-myristate 13-acetate (PMA, InvivoGen, San Diego, CA, USA) for 3 days, as previously described [57 (link),58 (link)]. Monocyte-to-macrophage differentiation was assessed by optical microscopy inspection. Macrophage-like cells adhered to the support and displayed a flattened and elongated morphology compared to floating round-shaped monocytes. Following differentiation, the PMA containing medium was removed and replaced by PMA-free fresh medium immediately prior to treatment.

Calcium nitrate dihydrate (Ca(NO₃)₂.2H₂O), diammonium phosphate ((NH₄)₂HPO₄), boric acid (H₃BO₃),
ammonia (NH₄OH), acetic acid (glacial), l-ascorbic
acid, β-glycerophosphate,
and dexamethasone were purchased from Sigma, USA. DMEM:F12, fetal
bovine serum, penicillin–streptomycin cocktail, RPMI 1640,
EndoGo XF, and EndoGoXF supplements were purchased from Biological
Industries, Israel. PMA and Normocin were obtained from Invivogen,
USA. Other chemicals and solutions used in the study are reagent-grade.

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).