Phenotypically naive CD4+ T-cells from CI on ART subjects are impaired in their Th17-polarization potential in vitro. Total CD4+ T-cells were isolated by negative selection using magnetic beads (Miltenyi) and stained with a cocktail of CD8, CD19, CD56, CD45RA, and CCR7 Abs and the viability dye Vivid. (A) Shown is the schematic experimental design. Briefly, naive-like CD4+ T-cells (CD45RA+CCR7+ phenotype) lacking CD8, CD19, and CD56 expression, were sorted by flow cytometry (as in Additional file 1: Figure S1) and stimulated via CD3/CD28 under Th17 polarizing conditions for 12 days. Media containing polarizing cytokines and Abs together with IL-2 was refreshed at day 4 and 8 post-culture. At day 12, cells were stimulated with PMA and Ionomycin in the presence of Brefeldin A for 17 hours. Cells were analyzed by flow cytometry for cytokine expression upon intracellular staining with specific Abs. Vivid-positive cells were excluded from the analysis. (B) Shown is the frequency of cells expressing intracellular IL-17A, IFN-γ, and/or TNF-α in representative HIV- control and CI on ART subject. (C-D) Shown are statistical analysis of single cytokine expression (C) and cytokine co-expression (D) in Th17-polarized cells from HIV- controls (n = 8) and CI on ART (n = 10) subjects. (E-F) At day 8 of polarization cell pellets and culture supernatants were harvested for the quantification of RORC mRNA (n = 3 HIV- and n = 5 CI on ART) and IL-17A production (n = 6 HIV- and n = 6 CI on ART), respectively. The Mann Whitney p-values are indicated on the graphs. (G) Shown is statistical analysis of cell viability. Each symbol represents a different subject. The Mann–Whitney p-values are indicated on the graphs. Clinical parameters of subjects included in these studies are included in Table 1 (HIV- #03, 06, 07, 09, 14, 15, 19, 22) and Table 3 (CI #03, 04, 06–10, 16–18).