Anti human cd4 clone a161 a1

A total of 20 × 10⁶ PBMCs from 2 PWH and 4 previously cryopreserved leuko-paks were thawed, washed with warm R-10 medium (RPMI 1640 [Thermo Fisher Scientific] supplemented with 10% FBS, l-glutamine, 10 mM HEPES, and penicillin/streptomycin) twice, and placed at a concentration of 4 × 10⁶ cells/mL at 37°C and 5% CO₂ in 2 mL per condition with 12 conditions in a 12-well plate. PBMCs from each participant were treated for 4 days with increasing amounts of DMSO, HODHBt (50 μM, or 100 μM, or 150 μM), IL-15 (1 ng/mL, or 10 ng/mL, or 25 ng/mL), or the combination of HODHBt with IL-15 (50 μM, or 100 μM, or 150 μM + 1 ng/mL, 10 ng/mL, or 25 ng/mL, respectively). Flow cytometry was performed at day 1 and day 4 after adding stimuli, using the following: fixable viability dye (aqua; Thermo Fisher Scientific, catalog L34966); anti-human CD3 (clone SK7; BioLegend, catalog 344842); anti-human CD4 (clone A161 A1; BioLegend, catalog 357418); anti-human CD8 (clone RPA-T8; BioLegend, catalog 301040); anti-human CD56 (clone HCD56, catalog 318336) anti–human HLA-A,B,C (clone W6/32, catalog 311426); anti-human perforin (clone dG9; BioLegend, catalog 308126); and anti-human granzyme B (clone GB11; BioLegend, catalog 515403). Cytofix/Cytoperm (BD Biosciences; 554722) was used to fix and permeabilize cells, and 1× Perm Wash buffer was used to dilute intracellular antibodies.

At each time point sampled, cells were stained with 1:100 dilutions of the antibodies in PBS with 2% FBS, 2 mM EDTA, fixable viability dye (aqua; Thermo Fisher Scientific, catalog L34966), anti-human CD3 (clone SK7; BioLegend, catalog 344842), anti-human CD4 (clone A161 A1; BioLegend, catalog 357418), anti-human CD8 (clone RPA-T8; BioLegend, catalog 301040), anti-human CD69 (clone FN50; Invitrogen, catalog 47-0699-42), anti-human GZMA (clone CB9; BioLegend, catalog 507215), anti-human GZMB (clone GB11; BioLegend, catalog 515403), anti-human HLA-DR (clone LN3, BioLegend, catalog 327002), anti-human perforin (clone dG9; BioLegend, catalog 308126), and anti-Bcl2 (clone 100; BioLegend, catalog 658709). Cytofix/Cytoperm (BD Biosciences, catalog 554722) was used to fix and permeabilize cells, and 1× Perm Wash buffer was used to dilute intracellular antibodies.