Anti human cd3 pe cy7

The CD4⁺ TIL clone was generated by limiting dilution and confirmed using the IO Test^® Beta Mark TCR V beta Repertoire Kit (Beckman Coulter, Brea, CA) by flow cytometry. A total of 2 × 10⁵ T cells were co-cultured with 4 × 10⁴ autologous tumour cells for 5 h at 37 °C (and 5% CO₂) in a 96-well tissue culture plate containing 200 μl assay medium/well (RPMI 1640 with 10% FBS and penicillin/streptomycin; both from Thermo Fisher Scientific, Waltham, MA). During the incubation period, 1.3 μg/ml of monensin (Merck KGaA, Darmstadt, Germany), and 4 μl of the anti-human CD107a-Alexa Fluor 700 antibody (Clone H4A3; BD Biosciences, Franklin Lakes, NJ) were added. PMA = phorbol 12-myristate 13-acetate (PMA) was used as the positive control and assay medium alone without tumour cells was used as negative control. After 5 h of incubation, the cells were stained with anti-human CD3-PE/Cy7 (Clone HIT3A; BioLegend, San Diego, CA), anti-human CD4-V450 (Clone RPA-T4) and anti-human CD8-APC/Cy7 (Clone SK1) (both from BD Biosciences, Franklin Lakes, NJ), and analysed by flow cytometry.

PBMCs (1 × 10⁶ cells/ml) resuspended in 50 µl of PBS were stained with anti-human CD3 PE cy7, anti-human CD4 Violet and anti-human CD25 APC (clone: PC61; Biolegend, UK) for 30 min at 4 °C. Post incubation, the cells were washed in PBS twice and fixed with Foxp3 Fix Perm solution (Thermo Fischer) for 30 min at room temperature, followed by a wash and staining with anti-human Foxp3 PE (clone: PCH101; Thermo Fischer) in diluted permeabilization buffer (Thermo Fischer) for 30 min at 4 °C. Post incubation, the cells were washed and analysed using a Cyan ™ ADP flow cytometer (Dako). Regulatory T cells were defined as CD3⁺ CD4⁺ CD25⁺ Foxp3⁺ cells [ gating strategy Supplementary Fig. 1] [32 (link)].

The activation markers expression on resting memory CD4⁺ T cells were detected after IP-10 or CD3/CD28 stimulated and measured using the following fluorophore-conjugated antibodies: phycoerythrin (PE)-Cy7 anti-human CD3, allophycocyanin (APC)-Cy7 anti-human CD4, peridinin-chlorophyll-protein anti-human CD197/CCR7, fluorescein isothiocyanate (FITC) anti-human CD45RA, APC anti-human CD69, APC anti-human CD25, APC anti-human HLA-DR, APC mouse IgG1 κ isotype control, and PE mouse IgG1 κ isotype control (all from Biolegend). Samples were analyzed using an LSR II flow cytometer (BD Biosciences), which was adjusted with 10-peak color rainbow beads (Spherotech, Lake Forest, IL, USA). Gates were defined using appropriate isotype controls. Expression levels in each sample were analyzed with FlowJo v10 software (Ashland, OR, USA).

PBMC was purified by ficoll gradient from HIV infected patient, incubated with 1 μg/mlbiotinylated SF162gp140 trimer at 4°C for 30 min. The PBMC was then washed by PBS and stained with second antibodies:Pacific Blue™ anti-human CD19 (Biolegend, 302224), PE/Cy7 anti-human CD3(Biolegend, 300316), APC/Cy7 anti-human CD14 (Biolegend, 325620), APC anti-human IgG(Biolegend, 409306), 7-AAD(7-Aminoactinomycin D) (Invitrogen, 302232), FITC anti-human CD27(Biolegend, 302806), PerCP/Cy5.5 anti-human IgM (Biolegend, 314512), and R-Phycoerythrin Streptavidin (Jackson immunolab, 016-110-084). SF162gp140-trimerbinding memory B cells (CD19+, IgG+, SF162gp140+7AAD-IgM-CD14-CD3-) were sorted in 96 well PCR plate by FACS AriaIII(BD). The sorted cells were then lysedwith RNA directly reverse transcribed into cDNA according SuperScript™ III CellsDirectcDNA Synthesis System (Invitrogen, 18080-300) manual. Single cell derived cDNA was used for antibody variable gene amplification.