Cd8a efluor 450

Blood cells from 300 μl blood were incubated in RBC-Lysis buffer (Biolegend) to lyse the red blood cells. Remaining cells were washed and incubated with a cocktail of fluorochrome-conjugated antibodies (Cd4-PE-Cy7 (#552775) and Cd62L-FITC (#561917) from BD Pharmingen; Cd3e-PE (#12–0031), Cd8a-eFluor 450 (#48–0081) and Cd44-APC (#17–0441) from eBioscience.), incubated with propidium iodide for the detection of dead cells and analysed using the FACSCanto II analyser (BD Biosciences). The following T cell subsets were quantified: Cd3⁺, Cd8⁺, Cd44^high cytotoxic memory T cells; Cd3⁺, Cd8⁺, Cd44^low, Cd62L^high cytotoxic naïve T cells, Cd3⁺, Cd4⁺, Cd44^high helper memory T cells and Cd3⁺, Cd4⁺, Cd44^low, Cd62L^high helper naïve T cells.

Splenocytes or PBMCs isolated from whole blood were stimulated with peptide at 2 μg/ml, ionomycin and phorbol myristate acetate (PMA) at 2.0 μg/ml and 0.5 μg/ml, respectively, or tissue culture media with 1% DMSO as a negative control. The cultures were supplemented with anti-CD107a PE-conjugated mAb (eBioscience). The cells were incubated at 37 ^oC, 5% CO₂ for 2 hours prior to the addition of Brefeldin A and monensin (BD Biosciences) and then left in culture overnight. The cells were centrifuged briefly, washed in PBS plus 5% BSA (Sigma-Aldrich) and the pellet re-suspended in 40 μl of CD16/32 with LIVE/DEAD fixable aqua stain (Molecular Probes, Invitrogen). Cells were washed, a mastermix of anti-membrane marker mAbs was prepared containing CD4 APC/Cy7 (Biolegend), CD3 PerCP-eFluor710 and CD8a eFluor 450 (both from eBioscience) and 40 μl added to each tube. The cells were incubated at 4 ^oC for 30 min and then permeabilized using Fix/Perm solution (Becton-Dickinson) for 20 min at 4 ^oC. The cells were washed with Perm Wash buffer (Becton Dickinson) and a mastermix of anti-intracellular molecule mAbs was prepared containing IFN-γ PE-Cy7, IL-2 APC and TNF-α FITC (all from eBioscience). The cells were incubated at 4 ^oC for 30 min, washed and resuspended in Perm Wash buffer prior to running on an LSRII flow cytometer (Becton-Dickinson).

Refolded MHC-peptide monomers (immunAware, Denmark) were tetramerized by adding streptavidin-conjugate-APC (Life Technologies) at 4°C. Splenocytes were stained with 30 μL of the optimal tetramer concentration for 20 min at room temperature and washed followed by the addition of 40 μL of a mastermix of anti-membrane marker mAbs containing LIVE/DEAD fixable aqua stain (Molecular Probes, Invitrogen), CD4 APC/Cy7 (BioLegend), CD3 PerCP-eFluor710, and CD8a eFluor 450 (both from eBioscience). The cells were incubated at 4°C for 30 min, washed, and fixed with 1% paraformaldehyde in PBS prior to running on an LSRII flow cytometer (Becton Dickinson).

Splenocytes and PBMCs isolated from whole blood were stimulated with specific tHIVconsvX-derived BALB/c peptide pool and stained with 100 μL of a mastermix of anti-membrane marker mAbs containing LIVE/DEAD fixable aqua stain (Molecular Probes, Invitrogen), CD3-APC, CD4-FITC, CD8a-eFluor 450, CD44-PE, and CD62-L-PE-Cy7 605 (all from eBioscience). The cells were incubated at 4°C for 30 min, washed, and fixed with 1% paraformaldehyde in PBS prior to running on an LSRII flow cytometer (Becton Dickinson). The frequencies of the subtypes in CD8⁺ and CD4⁺ T cells represent the differences in stimulated and unstimulated immune cells.