Multiparametric Phenotypic Analysis of T and NK Cells

For phenotypic analysis, cells were labeled simultaneously with a panel of anti-CD3-Cy-7APC (clone SP34-2; 1:300 dilution), CD4-APC (clone RPA-T4; 1:300 dilution), CD8-Alexa700 (clone RPA-T8; 1:300 dilution), CD95-FITC (clone DX2; 1:300 dilution) and CD28-Cy-5PE (clone CD28.2; 1:300 dilution) antibodies (BD Biosciences). Memory CD4 T cells were discriminated based on the expression of CD28 and CD95 as described previously^{96 (link),97 (link)}. To identify NK cells, PBMC were surface labeled with a panel of anti-CD3 (1:300 dilution), CD20 (clone 2H7; 1:300 dilution), CD14 (clone M5E2; 1:300 dilution), NKG2A (clone Z199; 1:300 dilution), and KIR2D (clone NKVFS1; 1:300 dilution) antibodies. After the cells were fixed and permeabilized, they were labeled with anti-perforin (clone deltaG9; 1:100 dilution) and Ki-67 (clone B56; 1:300 dilution) antibodies. Labeled cells were fixed with 0.5% paraformaldehyde and analyzed using an LSR II flow cytometer (BD Biosciences). The gating strategy is shown in Supplementary Fig. 5. All the antibodies were titrated using rhesus macaque PBMC.
To determine SIV-env and gag specific CD4 and CD8 T cell responses, cells were stimulated with overlapping peptides as described previously^{41 (link),81 (link),98 (link)}. Control cultures were set up for each sample without SIV peptides. After stimulation, cells were labeled with cell surface markers (anti-CD3, CD4, CD8, CD28 and CD95 at dilutions as above) and Vivid to discriminate live and dead cells⁹⁹ . The cells were fixed (Fix/Perm kit; BD Biosciences), and after permeabilization were labeled with anti-IL-2-PE (clone MQ1-17H12; 1:300 dilution), IFN-γ-FITC (clone B27; 1:300 dilution), and TNF-α-Cy7PE (clone Mab11; 1:50 dilution). Labeled cells were fixed with 0.5% paraformaldehyde and analyzed using an LSR II flow cytometer (BD Biosciences).