Apc conjugated anti cd3 145 2c11

Intracellular cytokine staining was performed as previously reported [1 (link),5 (link)]. In brief, immune cells isolated from the lung and spleen were stimulated with a 9-mer Ag85B CD8 epitope peptide (YYQSGLSIV) (Pep8) [1 (link)] and a 15-mer Ag85B CD4 epitope peptide (peptide-25: FQDAYNAAGGHNAVF) [6 (link)] for 6 h. The cells were then incubated with LIVE/DEAD Fixable Dead Cell Stains (Thermo Fisher scientific, Waltham, MA, USA) to identify dead cells, followed by surface staining with the antibodies APC-conjugated anti-CD3 (145-2C11, BioLegend, San Diego, CA, USA), PerCP-Cy5.5-conjugated anti-CD8 ((53-6.7, BioLegend), and PE-Cy7-conjugated anti-CD4 (GK1.5, BioLegend). The cells were then fixed and permeabilized using BD Cytofix/Cytoperm (BD Biosciences, Franklin Lakes, NJ, USA) and stained for IFN-γ (PE) (XMG1.2, BD Biosciences), IL-2 (APC-Cy7) (JES6-5H4, BD Biosciences), and TNF (Alexa Fluor 488) (MP6-XT22, BioLegend). The gating strategy used to identify cytokine-producing CD4⁺ and CD8⁺ T cells is shown in Figure 3C. Polyfunctional cells were defined as those producing two or more cytokines using Boolean combinations Figure 3D. FACS analysis was performed using a FACSVerse flow cytometer (BD Biosciences) with FlowJo software (version 10) (BD Biosciences).

The following antibodies were used for the flow cytometry analysis: phycoerythrin (PE)- and Cy7-conjugated anti-CD8 (clone 53-6.7; eBioscience), allophycocyanin (APC)-conjugated anti-CD3 145-2C11; BioLegend), fluorescein isothiocyanate (FITC)-conjugated-CD25 (clone PC 61; eBioscience), APC-anti-CD44 (clone IM7; eBioscience), PE-anti-CD62L (clone M1L-14; Tonbo), PE-anti-CD122 (clone TM-b1; eBioscience), PE-anti-CD127 (clone A7R34; BioLegend), PerCP- and Cy5.5-conjugated-ScaI (clone D7; eBioscience), APC-anti-KLRG1 (clone 2F1; BioLegend), PE-anti-NK1.1 (clone PK136, eBioscience), FITC-anti-PD-1 (clone J43; eBioscience), PE-anti-B7H1 (clone 10F.9G2), PerCP-Cy5.5-anti-CD45 (clone 30-F11; BioLegend), FITC-anti-CD90.1 (clone HIS51; eBioscience), APC-anti-Flag (clone L5; BioLegend), PE-Cy7-anti-Bcl-2 (clone 10C4; eBioscience), PE-anti-TCF1 (clone 14456S; Cell Signaling Technology), FITC-anti-CD95 (clone SA367H8, BioLegend), PE-anti-IFN-γ (clone XMG1.2; eBioscience), and APC-anti-IL-2 (clone JES6-5H4; eBioscience). For the CFSE dilution assay, T cells were labeled with 5 μM CFSE before being cultured. Dead cells were discriminated with the Fixable Viability Dye eFluor™ 780 (Thermo Fisher Scientific). The stained cells were analyzed with a cytoflex instrument (Beckman coulter). The data analysis was performed using FlowJo X software (Tree Star).