Fitc anti human cd38

T cell immunophenotyping and CD4⁺ and CD8⁺ T-cells relative count was performed on fresh blood samples anticoagulated with EDTA, using the antibodies PerCP anti-human CD3 (Clone: HIT3a), PE anti-human CD4 (Clone: RPA-T4), APC anti-human CD8 (Clone: RPA-T8); the samples were stained with Alexa 700 anti-human HLA-DR (Clone: L243) and FITC anti-human CD38 (Clone: HB-7) (All antibodies from BioLegend, San Diego CA, USA) to determine the immune activation. Attune NxT Flow Cytometer (Thermo Fisher Walthman, MA, USA) was used to acquire 10,000 events in the lymphocyte gate. The data was analyzed with Attune NxT Flow Cytometer Software version 2.6 (Thermo Fisher, Walthman, MA, USA). The normalization was determined by the proportion of the HLA-DR⁺, CD38⁺ as well as HLA-DR⁺ and CD38⁺ expression or MFI with the CD4⁺ and CD8⁺ T-cells relative count, respectively. The fold change analysis was calculated by the log₂ fold difference of HLA-DR⁺, CD38⁺ and, HLA-DR⁺ and CD38⁺ expression and MFI in CD4⁺ and CD8⁺ T-cells of HIV+ groups divided by the expression or MFI of the same cell subpopulations in HIV- controls [18 (link), 19 (link)]. The CD4⁺/CD8⁺ ratio was calculated using the previously determined absolute CD4⁺ and CD8⁺ T-cells count. Serum levels of sCD14 and sCD163 were quantified by ELISA (both CUSABIO, Houston, TX, USA) according to the manufacturer instructions.

For analysis of human intracellular cytokine production, PBMCs were stimulated with PMA (50 ng/ml), ionomycin (1 μg/ml), and brefeldin A solution (5 μg/ml) for 5 h. T cells and B cells were stimulated with 0.5 mg/ml purified plate-bound CD3 mAb for 3 days. For intracellular staining, cells were stained with combinations of FITC antihuman CD4 (5 μl/test), PerCP/Cy5.5 antihuman CD19 mAb (5 μl/test), PE antihuman CD24, and FITC antihuman CD38 (Biolegend, San Diego, USA). Cells were washed, fixed, permeabilized (Cytofix/Cytoperm, BD, USA), and stained for the detection of intracellular cytokines with PerCP/Cy5.5 antihuman IFN-γ (5 μl/test), PE antihuman TNF-α (5 μl/test), and APC antihuman IL-10 mAb (5 μl/test) (Biolegend, San Diego, USA). Appropriate isotype controls were used for gate setting for cytokine expression.

Cells (10⁵ cells/staining) were harvested and fixed in 1% paraformaldehyde for 15 min. Cells were then washed in PBS/1% BSA buffer and incubated for 30 min on ice with the following antibodies from Biolegend (San Diego, USA): FITC‐anti‐human CD38 (#303503), PE‐anti‐human CD206 (#321105), PE‐anti‐human CD209 (#330106), and APC‐anti‐human CD86 (#305411). After washes in PBS/1% BSA buffer, cells were acquired on a Novocyte flow cytometer (Agilent Technologies). For blood‐derived monocytes phenotyping, FITC‐anti‐human CD14 (Miltenyi, #130‐110‐576) antibody from Biolegend was also used.