Cd4 bv786 clone sk3

PBMCs from healthy human donors were obtained from Miltenyi Biotec, and naive CD4⁺ cells were negatively selected using the naive CD4⁺ T cell isolation kit II by MACS according to the manufacturer’s instructions. CD4⁺ cells were activated using anti-CD3 (clone OKT3, 5 μg/mL) and anti-CD28 (clone CD28.2, 10 μg/mL) mAb–precoated 96-well plates. Next, 2 × 10⁵ T cells and 4 × 10³ DLBCL cells (SU-DHL-4 or Karpas-422) were cocultured at a 50:1 ratio in AIM-V medium (Invitrogen) supplemented with HLA-DR/DP/DQ mAb (clone WR18, 10 μg/mL) for MHC II blockade for 3 days. T cell proliferation was assessed using CellTrace violet dye (Invitrogen). Prior to coculturing, CD4⁺ cells (1 × 10⁷ cells/mL) were stained with 5 μM CellTrace violet dye and incubated at 37°C for 15 minutes with gentle vortexing every 5 minutes. Flow cytometry was performed on the FACSSymphony flow cytometer (BD Biosciences) using the following Abs/stains: LIVE/DEAD Yellow Fixable dye 1:1000 (Invitrogen), CD4 BV786 clone SK3 1:200 (BD Biosciences), CD8 APC-H7 clone SK1 1:200 (BD Biosciences), and CD69 FITC clone FN50 1:50 (BioLegend).

The following antibodies were used: CD161-BV605 (HP-3G10, BioLegend), CD11c-BV650 (clone B-ly6, BD Biosciences), CD107a-BV711 (clone H4A3, BioLegend), CD4-BV786 (clone SK3, BD Biosciences), Vα7.2-AF-647 (clone 3C10, BioLegend), HLA-DR-AF-700 (clone L243, BioLegend), CD8-APC-H7 (clone SK1, BD Biosciences), IFNγ-V450 (clone B27, BD Biosciences), CD40-PerCp-Cy5.5 (clone 53C, BioLegend), CD3-ECD (clone UCHT1, Beckman Coulter), and PD1-BV711 (Clone EH12.2H7, BioLegend). Aqua fluorescent dye (Invitrogen) was used as a viability dye.
Following stimulation, cells were stained for viability with the Aqua fluorescent dye and incubated for 10 min at room temperature. After viability staining, surface staining was performed with CD161, Vα7.2, CD4, and CD40. Cells were then fixed and permeabilized with Cytofix/Cytoperm buffer (BD Biosciences) followed by intracellular staining with CD3, CD8, CD14, CD11c, HLA-DR, and IFNγ. The stained samples were acquired on a BD LSR Fortessa FACSDiva Software. A total of 500,000 events were collected and results analyzed using FlowJo (version 9.9.6, FlowJo LLC). We defined MAIT cells as CD3⁺CD161⁺Vα7.2⁺ T cells, after excluding doublets and dead cells.

Fixable Viability Stain 510 was obtained from ThermoFisher Scientific (#L34957, 1/1000). The following antibodies were used in staining experiments: CD3 AF700 Clone UCHT1 (BD Biosciences, #557943, 1/50), CD4 PE-Cy7 Clone L200 (BD Bioscience, #560644, 1/200) and CD4 BV786 Clone SK3 (BD Biosciences, #563881, 1/200), CD8 BV510 Clone RPA-T8 (BioLegend, #301047, 1/200), CD45RO BV421 Clone UCHL1 (BD Biosciences, #562649, 1/100) or CD45RO PE Clone HI100 (BD Biosciences, #5555493, 1/20), CD27 BV605 Clone L128 (BD Biosciences, #562656, 1/100), PD1 BB700 Clone EH12.1 (BD Biosciences, #566461, 1/100), CD69 PerCP-Cy5.5 Clone FN50 (BD Biosciences, #560738) and CD69 APC (BioLegend, #310909, 1/50), CD25 BV421 Clone M-A251 (BD Biosciences, #562443, 1/50), HLA-DR FITC Clone REA805 (Miltenyi Biotech, #130-111-788, 1/50), GZMA PE-Cy7 Clone CB9 (BioLegend, #507221, 1/25), GZMB PB Clone GB11 (BioLegend, #515407, 1/50), CCL5 PerCP-Cy5.5 Clone VL1 (BioLegend, #515507, 1/50), CD127 PE-CF594 Clone HIL-7R-M21 (BD Biosciences, #562397, 1/50). For p24 staining, we used a combination of two antibodies: p24 KC57-FITC (Beckman Coulter, #6604665, 1/500) and p24 28B7-APC (MediMabs, #MM-0289-APC, 1/400).