Il 17 alexa fluor 488

6-day stimulated, CFSE-labeled PBMCs were subjected to three different panels of staining to measure: phenotypic and cytokine profile, HIV infectivity (p24) and intracellular transcription factors (T-bet and EOMES) in CFSE-low, Ag-specific CD4 T cells. Briefly, cells were first stained for viability with LIVE/DEAD Fixable Aqua Blue (Life Technologies), followed by surface makers staining, including CD3-APC-H7 (BD Bioscience), CD4-PE-Cy5 (BD Bioscience), CD8-BV785 (Biolegend), CD25-PE-Cy5 (Biolegend), CCR5-Pacific Blue (BD Bioscience), α4β7-APC (NIH AIDS Reagent Program), CCR6-APC (R&D Systems), CCR9-PerCP-Cy5.5 (Biolegend). After surface staining, cells were fixed, permeabilized (BD Bioscience) and subjected to intracellular staining, including HIV p24-PE (Beckman Coulter), IL-17-Alexa Fluor 488 (eBioscience), IL-22-APC (eBioscience), IL-2-PerCP-Cy5.5 (Biolegend), IFN-γ-Alexa Fluor 700 (eBioscience), MIP-1β-PE-Cy7 (BD Bioscience). Combination of surface and intracellular staining antibodies may vary according to different assays. For intracellular staining for T-bet and EOMES, cells were fixed and permeabilized using eBioscience’s transcription factor staining buffer set (eBioscience) according to manufacturer’s instructions. Cells were then subjected to intracellular staining with anti-T-bet-Pacific Blue (Biolegend) and anti-EOMES PE-eFluor 610 (eBioscience).

Following overnight stimulation as described above, PBMCs (RV21 HIV-infected subjects) were subjected to staining and flow cytometric analysis for measuring de nova frequencies of C. albicans and CMV-specific CD4 T cells. Cells were first stained for viability with LIVE/DEAD Fixable Aqua Blue (Life Technologies), followed by surface staining, including CD3-APC-H7 (BD Bioscience), CD4-PE-Cy5 (BD Bioscience), CD8-BV785 (Biolegend). Cells were then fixed, permeabilized (BD Bioscience) and subjected to intracellular staining, including IL-17-Alexa Fluor 488 (eBioscience), IL-22-APC (eBioscience), IL-2-PerCP-Cy5.5 (Biolegend), IFN-γ-Alexa Fluor 700 (eBioscience), MIP-1β-PE-Cy7 (BD Bioscience). Following staining, cells were acquired using the LSRII Fortessa Analyzer (BD) and data were analyzed using FlowJo (Tree Star). Where appropriate, Boolean gating and Spice analysis were performed using the FlowJo program to analyze the poly-functional characteristics of antigen-specific CD4 T cells.