Characterization of γδ T Cell Subsets in HIV-1 and Syphilis

For surface staining, PBMCs were isolated from HC and HIV-1-infected patients with or without syphilis infection. Cells were washed with 1% bovine serum albumin in PBS and were labeled with LIVE/DEAD fixable viability stain 510 (BD Biosciences, San Jose, CA, USA), and dead cells were excluded. Then, cells labeled with specific surface antibodies: gating strategy on CD3⁺γδTCR⁺ cells, Vδ₁ and Vδ₂ T cells, or γδT_Naive (CD27⁺CD45RA⁺), γδT_CM (CD27⁺CD45RA⁻), γδT_EM (CD27⁻CD45RA⁻), and γδT_EMRA (CD27⁻CD45RA⁺) were displayed (Figure 2). Cytometer setup and tracking calibration particles were used to ensure that fluorescence intensity measurement was consistent in all experiments. Flow cytometry Comp-Beads kits (BD Biosciences, San Jose, CA, USA) were used for compensation. Forward angle and side scatter light gating were gated on lymphocytes and were used to exclude cell debris from the analysis. Forward height and forward area were used to exclude doublet cells. Cells were performed with a FACScan flow cytometer, as previously described (33 (link), 34 (link)). The final analysis was performed with FlowJo software (version 7.6.2; Tree Star Inc., Ashland, OR, USA), which generated a graphical output. The strategies for the analysis of flow cytometry data are detailed in Figure 2.