Cd8 clone rpa t8

T cells were isolated from the non-adherent fraction of the osteoclast differentiation cultures and activated with CD3/CD28 mAbs coated beads (T cell TransAct™, Miltenyi Biotech, Auburn, CA). After 3 days of stimulation, IL-2 (50 U/ml, TECIN™, National Cancer Institute, Frederick, MD) was added to the media. At day 6, the phenotype of the expanded T cells was analyzed by flow cytometry. Prior to surface staining, T cells were stained with LIVE/DEAD staining (Invitrogen, MA), followed by incubation with 1 μg/ml human IgG (Sigma, MO) to block Fc receptors. Cell surface staining were performed using a cocktail of mAbs: CD3 (clone UCHT1) and CD8 (clone RPA-T8) both from BD Biosciences, CA. Cells were acquired using a BD FACS Symphony flow cytometer and analyzed using FlowJo.

T-cell lines were analyzed after stimulation with “WT CD4⁺ pool” or “Omicron CD4⁺ pool” (1 μM/peptide) for 6 h. During the last 5 h, a mixture of Brefeldin A and Monensin (Biolegend, San Diego, CA, USA) was added. Cells were stained for antihuman CD3 (clone HIT3A; BioLegend), CD4 (clone SK3), and CD8 (clone RPAT8; both BD Biosciences, Franklin Lakes, NY, USA). After fixation and permeabilization, using FoxP3/Transcription Factor Staining Buffer Set (eBioscience, San Diego, CA, USA, Thermo Fisher Scientific, Waltham, MA, USA), cells were stained intracellularly for antihuman, CD154 (clone TRAP1; BD Bioscience), and cytokines: IFN-ɣ (clone 4S.B3; BD Bioscience), IL-2 (clone MQ1–17H12; Thermofisher), or TNF-α (clone Mab11; Thermofisher). Cells were acquired on a FACS Symphony A3 analyzer (BD) and analyzed using FlowJo (V10, Tree Star, Ashland, OR, USA). On average, 40,000 events were acquired; however, in the T-cell lines obtained from the convalescent subjects, the number of events was often somewhat lower (i.e., 8000 events).

Cryopreserved PBMCs were thawed, washed, and stained with an antibody cocktail (1:100 dilution) of CD3 (clone SP34-2, BD Biosciences), CD4 (clone OKT4, Biolegend), CD8 (clone RPA-T8, BD Biosciences), CD14 (clone M5E2, BD Biosciences), CD20 (clone 2H7, Biolegend), IgM (MHM-88, Biolegend), IgG (clone G18-145, BD Biosciences) and fluorescently labeled biotinylated SIVmac239 SOSIP.664 Env at room temperature for 20 min in the dark. SIVmac239 Env trimer probes were labeled with two different fluorophores, from which SIVmac239 Env dual positive memory B cells (CD3⁻CD4⁻CD8⁻CD14⁻CD20⁺IgM⁻IgG⁺SIVmac239 SOSIP²⁺) were analyzed with BD FACSMelody or BD FACSFusion, and single-cell sorted, cultured, expanded in 384-well plates as described previously^{28 (link),29 (link)}. Briefly, sorted B cells were cultured with Iscove’s modified Dulbecco’s medium (IMDM) with GlutaMAX (Gibco) supplemented with 10% heat-inactivated fetal bovine serum (FBS), 1× MycoZap Plus-PR (Lonza), 100 U/mL human IL-2 (Roche), 50 ng/mL human IL-21 (Invitrogen), 50 ng/mL human IL-4 (Miltenyi), 0.1 μg/mL anti-rhesus IgG (H + L) (BioRad), and irradiated 3T3msCD40L feeder cells. Flow cytometric data were subsequently analyzed using FlowJo (v10.7.1).