Pe conjugated anti human hla a2 antibody

This study was approved by the Institutional Review Board of the School of Medicine of Jinan University (JNUKY-2021-009 and JNUKY-2022-101). Peripheral blood samples (approximately 10 mL per sample) were extracted from NSCLC patients diagnosed at the First Affiliated Hospital of Jinan University (Table S1). The peripheral blood from the healthy donors were collected at Guangzhou Blood Center (200-400 mL per sample). Peripheral blood mononuclear cells (PBMCs) were isolated by density gradient centrifugation using lymphocyte separation medium (GE, US) and stained with PE conjugated anti-human HLA-A2 antibody (BioLegend, Cat#343305, US) to identify the HLA-A2+ donors.

PBMCs were isolated from peripheral venous blood of healthy donors and SARS-CoV-2 vaccinees. The HLA-A2⁺ donors were identified by using flow cytometry without the subtype identification. In brief, 10⁶ PBMCs were stained with PE-conjugated anti-human HLA-A2 antibody (BioLegend, 343305) at 4 °C in the dark for 30 minuntes and acquired using a flow cytometer. HLA-A2⁺ PBMC samples were stimulated with T2 cells presenting SARS-CoV-2 epitopes for 16 hours and then stained with PE-labeled tetramer (produced in-house) plus APC-labeled human CD8 antibody (BioLegend, 344721). CD8 T cells isolated from vaccinated donors 50 days after second doses were co-cultivated with T2 cells loaded with SARS-CoV-2 epitopes (ORF1a 1707–1716, ORF1a 2225–2234, ORF1a 2230–2238, S 2–11, M 82–90, ORF1a 2340–2349 and ORF1a 3683–3692) at a 1:1 ratio, and PE-labeled tetramer with FITC-conjugated anti-human GZMB (BioLegend, 515403) were added after 7 days and acquired with a FACSCanto flow cytometer (BD Biosciences).