Identification of Pathogen-Specific T Cells

HLA-A2 PE-labeled tetramers loaded with TcTLE peptide (TLEEFSAKL), derived from T. cruzi KMP-11 protein [22 (link)], and also HLA-A2 PE-labeled tetramers with a modified Flu-MP peptide (Flu-MP*; GILGFVTTL) derived from the influenza virus matrix protein [22 (link),26 (link)], and a modified CMV pp65 peptide (CMV*; DLSPMVATV), employed as controls, were produced by the National Institute of Health (NIH) Tetramer Facility (Atlanta, GA, USA). In total, 1 × 10⁶ PBMCs per tube were stained with 0.5 μg/mL tetramers and anti-CD3-PerCP (clone SK7) and anti-CD8-FITC (clone SK1) conjugates (BD Biosciences, San José, CA, USA) for 20 minutes in the dark at room temperature. After the cells were washed with staining buffer (1% fetal bovine serum in 1X PBS), they were resuspended in 500 μL of 1X PBS. At least 50,000 events gated for CD3⁺ CD8⁺ T cells were acquired and analyzed using a FACSAria II flow cytometer (BD Immunocytometry Systems) and FlowJo 9.3.2 software (Tree Star, Inc.). The gating strategy is shown in Fig 1A and 1B. The cut-off point for HLA-A2/TcTLE tetramer CD8⁺ T cells was established at 0.063% after determining the average background value of K1-specific CD8⁺ T cells from HD (0.027%) plus three standard deviations (0.012%). A cut-off point for HLA-A2/Flu-MP* and HLA-A2/CMV* tetramer CD8⁺ T cells was fixed at 0.1% [26 (link)].