Pe conjugated anti cd3

To evaluate CD5 expression on peptide-specific CD8⁺ T cells, 2×10⁶ PBMC were labeled with 1 μM carboxyfluorescein succinimidyl ester (CFSE, Invitrogen) as per the manufacturer’s instructions, washed, and incubated in a small volume with either lymphocyte medium alone or 100 μM peptide of interest for 1 hour, then resuspended and cultured in 1 mL lymphocyte medium (with human serum substituted for FCS) for 7 days. Peptide-specific T cells were identified by CFSE fluorescence intensity dilution using a Becton Dickinson FacsScan flow cytometer. CD8⁺CD3⁺ cells were identified using phycoerythrin (PE)-conjugated anti-CD3 and peridinyl chlorophyll protein (PerCP)-conjugated anti-CD8 (clones UCHT1 and HIT8a respectively, BioLegend). The fraction of proliferating cells expressing CD5 was estimated by co-staining the cells with allophycocyanin-conjugated anti-CD5 (clone L17F12, eBioscience), gating on CD8⁺CD3⁺ cells and plotting CD5 versus CFSE intensity. Positive peptide-specific proliferation was defined as at least twice the percentage of proliferating cells compared to the control culture. If background proliferation in the absence of stimulation was > 2.5%, results were excluded. The baseline fraction of CD8⁺ T cells expressing CD5 was measured by flow cytometry with fresh PBMC using the same antibodies.

To detect IFN-γ production by NK cells, cells were stimulated for 4 h with 5 μg/ml Brefeldin A (Yeasen, 50502ES03), then stained with FITC-conjugated anti-CD45 (Biolegend, 147710, dilution 1:200), PE-conjugated anti-CD3 (Biolegend, 100205, dilution 1:200), and BV421-conjugated anti-NK1.1 (Biolegend, 108741, dilution 1:200) antibodies. The cells were then fixed with 4% paraformaldehyde and permeabilized using permeabilization buffer (Invitrogen, 88-8824-00). Finally, the cells were stained with APC-conjugated anti-IFN-γ antibody (Biolegend, 505810, dilution 1:100). Data were acquired on a cytometer (FACSCelesta, BD Biosciences) and analyzed using FlowJo X software (Tree Star, Ashland, OR).