Anti cd3 clone okt3

PBMCs were plated in a 96-well flat-bottom plate and cultured in RPMI-1640 supplemented with 10% FBS at a density of 2 × 10⁶ cells per well. Cells were stimulated with coated anti-CD3 (Clone: OKT3; 2 µg/mL, Biolegend, San Diego, CA, USA) or PBS control and measured at 4 h and 24 h time points. For the final one hour of stimulation MitoTracker Green (100 nm, Thermo Fisher Scientific, Waltham, MA, USA) and TMRM (25 nM, Sigma-Aldrich, St. Louis, MI, USA) were added to the wells at 37 °C/5% CO₂. After culture, cells were stained with cell surface markers anti-CD3, -CD4, -CD27, -CD28, -CD45RO and -CD45RA (BioLegend, San Diego, CA, USA). Cell viability was assessed using Live/Dead Aqua (ThermoFisher, Waltham, MA, USA). Cells were immediately analysed using the BD LSRFortessa^TM X-20 cell analyser (BD Biosciences, Franklin Lakes, NJ, USA). Separately stained cells were used to define compensation parameters.

CD3⁺ T cells were isolated from healthy adult donors. All experiments using primary human cells were conducted in accordance with the Declaration of Helsinki and IRB approval to the French Blood Bank (Etablissement Français du Sang). T lymphocytes were purified by negative-selection using Rosette tetramers (StemCell Technologies) and the purity was monitored by flow cytometry. Lymphocytes were cultured in RPMI medium 1640 + GlutaMAX (Gibco-Life technologies) supplemented with 10% FCS and 1% penicillin/streptomycin (Gibco-Life technologies).
T cell activations were performed using plate-bound anti-CD3 (clone OKT3, Biolegend) and anti-CD28 (clone 9.3, kindly provided by Carl June) mAbs at a concentration of 1 μg/ml. rIL-2 (100 U/ml) was added every other day starting at day 2 post-activation. Cells were maintained in a standard tissue culture incubator containing atmospheric (20%) O₂ or an air-controlled incubator where O₂ conditions were maintained at 1–2% by nitrogen injection (Heraeus incubator; Sanyo) and 5% CO₂.

Mononuclear cells were resuspended at 1×10⁶/mL in RPMI supplemented with 10% FBS (R10) and plated 200 μL per well in Immobilon-P 96-well microtiter plates (Millipore) pre-coated with 2 μg/mL anti-IFN-γ (clone DK1, Mabtech). Individual HLA-optimal HIV peptides matched to each subject’s HLA genotype (listed in table S1) were added at 1 μM and incubated at 37°C overnight. Negative control wells did not receive peptide and positive control wells were treated with 1 μg/mL anti-CD3 (clone OKT3, Biolegend) and 1 μg/mL anti-CD28 (clone CD28.8, Biolegend) antibodies. ELISPOT assay was performed using manufacturer’s protocol with anti-IFN-γ (clone 1-DK1, Mabtech) capture, biotinylated anti-IFN-γ (clone B6–1, Mabtech) detection, Streptavidin-ALP (Mabtech) and AP Conjugated Substrate (BioRad) followed by disinfection with 0.05% Tween-20 (Thermo Fisher) and analysis using S6 Macro Analyzer (CTL Analyzers). Responses greater than 10 spots per well or 3-fold above negative controls were scored as positive. The largest responses with available pHLA tetramer reagents were selected for downstream investigation of HIV-specific CD8⁺ T cell responses in each individual, listed in table S2.

PBMCs were isolated using Ficoll-Hypaque gradient and CD4⁺ T cells were then purified using the untouched CD4 isolation kit (EasySep Human CD4⁺ T cell Enrichment Kit; StemCell Technologies, Vancouver, BC, Canada), allowing for more than 94% purification without any cell stimulation and apoptosis. CD4⁺ T cells were then activated 72 hours in RPMI complete in the presence of 1 µg/mL anti-CD28 (BD Biosciences) in 6 well plates pre-coated 24 hours earlier with 0.5 µg/mL anti-CD3 (clone: OKT-3, BioLegend; 2.10⁶ cells/well).