Flex t hla a 02 01 monomer uvx

Human donor leukopaks were obtained (Gulf Coast Regional Blood Center) and genotyped for HLA∗A:02 via flow cytometry with purified antihuman HLA-A2 antibody clone BB7.2 (BioLegend). HLA-A∗02–positive samples were selected and dendritic cells (DCs) were generated via plate adherence and pulsed with mHAs not endogenous to the sample (Peptide 2.0 Inc). Naïve CD8 T cells were isolated and cocultures were initiated with mHA-pulsed DCs and naïve CD8 T cells at a 1:4 ratio and maintained for 2 weeks in culture in RPMI, 10% human serum, 1% penicillin-streptomycin, and 1% L-glutamine. The presence of mHA-specific T cells was assessed via flow cytometry with mHA tetramer staining. Tetramers were generated with Flex-T HLA-A∗02:01 Monomer UVX (280004, BioLegend) and fluorophore-conjugated streptavidin. Cells were also stained with the following: FVS700 live/dead (BD Biosciences) and CD8-BV421 (Clone: SK1, BioLegend). Cells cultured with immunodominant influenza A virus M1_58-66 HLA-A∗02:01–binding influenza peptide and stained with Flu-M1_58-66 tetramer (designated as Flu) were used as a positive control, and cells stained with tetramer exposed to UV light with no peptide (UV only) were used as negative control.⁵² (link) Gating strategy is shown in supplemental Figure 4.

HLA-A2-binding epitope of CLDN6 between CLDN6_131–140 was predicted using SYFPEITHI database.^{27 (link)} TLIPVCWTA (CLDN6_132–140) peptide was synthesized by EZBiolab with 82% purity. An HLA-A2/peptide monomer was generated with the Flex-T HLA-A*02:01 monomer UVX reagent followed by tetramerization using PE-streptavidin according to the manufacturer’s instruction (BioLegend). Optimum staining concentration of the A2/CLDN6_132–140 tetramer was determined by titration. CD8-TCR- or mock-transduced T cells were first treated with 50 nM dasatinib (Sigma-Aldrich) for 30 min at 37°C and then stained with the tetramer reagent for 30 min on ice. Tetramer-stained cells was further stained with purified anti-PE (clone PE001, BioLegend) and PE goat anti-mouse Ig (multiple adsorption, BD Biosciences) antibodies.^{28 (link)}