Apc cy7 conjugated anti cd3

A CNS and spleen leukocyte suspension was obtained as described previously (Carrillo‐Salinas et al., 2017 (link)). Isolated cells were incubated with anti‐CD16/CD32 (Affymetrix Inc.) for FcR blockade and labeled with anti‐mouse antibodies: PE‐conjugated anti‐CD44 (2.4 μg/ml), PE‐conjugated anti‐CD274 (2.4 μg/ml), PerCP‐Cy5.5‐conjugated anti‐CD4 (1.2 μg/ml), PECy7‐conjugated anti‐Ly6c (1.2 μg/ml), APC‐Cy7‐conjugated anti‐CD11b (1.2 μg/ml), APC‐conjugated anti‐CD62L (2.4 μg/ml), APC‐conjugated anti‐CD25 (2.5 μg/ml), and APC‐conjugated anti‐CD1d (2.4 μg/ml; all from eBioscience); APC‐conjugated anti‐P2yR12 (2.5 μg/ml) and APC‐Cy7‐conjugated anti‐CD3 (1.2 μg/ml) (both from Biolegend); PE‐conjugated anti‐CD5 (2.2 μg/ml), PerCP‐Cy5.5‐conjugated anti‐B220 (2.4 μg/ml), PerCP‐Cy5.5‐conjugated anti‐CD45 (1.2 μg/ml), PECy7‐conjugated anti‐CD8 (1.2 μg/ml) and PECy7‐conjugated anti‐CD19 (2.4 μg/ml; all from BD Pharmingen). The cells were fixed for 30 min with fixation buffer (Affymetrix Inc.). For FoxP3 detection, the cells were suspended in Fixation/Permeabilization buffer for 30 mins prior to staining with an anti‐FoxP3 antibody (3 μg/ml; BD Pharmingen). At least 50,000 events were registered in each experiment on a FACSAria flow cytometer (BD Biosciences), excluding duplets from the analysis. The data were analyzed using FACSDiva analysis software (BD Biosciences).

Cells were washed in PBS and resuspended in PBS with Zombie Fixable Viability Stain (Biolegend, #423102, #423103) as per the manufacturer’s protocol. Antibodies for surface staining were then diluted into FACS buffer (PBS + 2% FBS + 2 mM EDTA) and added to the cells. The staining panel consisted of APC/Cy7-conjugated anti-CD3 (Biolegend, #344818), PE/Cy7-conjugated anti-CD4 (Biolegend #357410) and APC-conjugated anti-CD8 (Biolegend, #344722). Stained cells were washed in FACS buffer and fixed in 1% paraformaldehyde prior to analysis. Samples were run on an LSRII cytometer (BD Biosciences). Data were analyzed using the FlowJo V10 Software (Treestar). All flow cytometric datasets were pre-gated from a lymphocyte scatter gate (FSC vs. SSC) to identify singlet cells, and then sequentially gated on live cells (Zombie negative/low) and CD3⁺CD8⁻ T cells. Productively infected cells were identified as GFP+ cells. The gating strategy is illustrated in Supplementary Fig. S2.

Thawed PBMCs were rested overnight in media supplemented with 10 % fetal bovine serum. Cells were washed and 1 × 10⁶ cells were labeled with 1 mL of 5 μM CFSE (BioLegend) following an established protocol reported elsewhere [41 (link)]. CFSE-labeled PBMCs were incubated in a 96-well culture plate and stimulated with media, uRBCs, iRBCs, and plate-bound anti-CD3 (BioLegend) at a density of 2.5 × 10⁵ cells per condition. As before, an effector-to-target ratio of 1:3 was used with uRBCs and iRBCs. Anti-CD28 and anti-CD49d were added for costimulation (3 μg/mL BioLegend). At day 7, supernatants were collected and frozen for downstream cytokine analysis and the cells washed and stained with surface antibodies (Brilliant Violet 421-conjugated anti-CD4, APC-conjugated anti-CCR7, APC-Cy7-conjugated anti-CD3 (BioLegend), PE-Cy7-conjugated anti-CD27, and PerCP-Cy5.5-conjugated anti-CD8 (BD Pharmingen)) before acquisition. Once again, LIVE/DEAD aqua amine was included to exclude dead cells from downstream analysis.