Cd8 apc

Cryopreserved lymphocytes isolated from the blood samples obtained from HIV-infected or normal patients were quickly thawed in a 37°C water bath and washed with PBS. Cells were then stained for cell surface markers using specific antibodies. The immune activation panel consisted of antibodies CD3-Cy7, CD4-Tx red, CD8-APC along with immune activation markers CD38 PE and HLA-DR FITC (BD Pharmingen) (Figure S1A). The apoptosis panel comprised of the following antibodies CD3-Cy7, CD4-Tx red, CD8-APC (Beckman Coulter) along with CaspACE FITC-VAD-FMK (Promega) (Figure S1B). Stained cells were washed and fixed using Cytofix reagent (Beckman coulter) and acquired on a 10 color Beckman Coulter Gallios flow cytometer. At least 20,000 events for each sample were acquired. Data was analyzed using FlowJo software (Tree Star). Cells were first gated on CD3+ population and immune activation/apoptosis on CD4+ and CD8+ T cell subsets determined (Figure S1A & B). For in vitro T cell activation, lymphocytes were cultured in RPMI-1640 medium supplemented with 20% FBS and Phytoheamagglutinin (Sigma) at 2.5 μg/ml and IL-2 (Roche) at 10U/ml for 48h, stained and analyzed as above for immune activation markers and CD4:CD8 ratios (Figure S2).

Cryopreserved PBMCs from each patient obtained at day 0 and day 56 were assayed for phenotypic markers consistent with NK cells, T lymphocytes, myeloid derived suppressor cells (MDSC) and T regulatory cells as previously described (26 (link), 32 (link)). Briefly, PBMC from each patient were suspended at a concentration of 1x10⁷/mL in flow staining buffer (PBS containing 1% FBS). Cells were incubated with fluorochrome-labeled antibodies for one hour at 4°C. Specific antibodies include CD4-APC (Beckman Coulter), CD8-APC (Beckman Coulter), NKRD1 (Beckman Coulter), CD33-PE (BD Biosciences), HLA-DR PERCP-Cy5.5 (eBioscience), and CD14-Pacific Blue (BD Biosciences). PBMC were also labeled with the appropriate isotype control antibodies for each fluorochrome to use as negative controls. Cells were then washed with flow buffer, fixed with 1% formalin, and stored at 4°C until analysis. All samples were run on a BD LSR II flow cytometer, and were subsequently analyzed with FlowJo software (Tree Star, Inc.). Monocytic MDSC were defined as cells with a CD33⁺HLA-DR^negCD14⁺ phenotype. T regulatory cells were defined as CD4⁺CD25⁺FoxP3⁺ and assessed using the commercially available Human T regulatory cell staining kit per manufacturer’s recommendations (eBioscience).