Recombinant HLA-A∗02 monomer with UV exchangeable peptide were either synthesized as previously described (Altman and Davis, 2003 ) or purchased commercially (Biolegend). UV peptide exchange was performed over 20 min with 1 mM of peptide in PBS using Strategene UV Stratalinker 2400. Streptavidin conjugated fluorophore was added incrementally the following day for a final 4:1 molar ratio of MHC:streptavidin. Tetramer staining was performed in PBS plus 2% FBS in Fc Blocking solution (Biolegend) at room temperature for 1 h. For peripheral blood samples, cells were subsequently stained with anti-TCRγδ (B1, Biolegend), anti-CD19 (H1B19, Biolegend), anti-CD14 (M5E2, Biolegend), anti-CD3 (OKT3, Biolegend), anti-CD4 (RPA-T4, Biolegend), anti-CD8 (HIT8a, Biolegend), and live/dead near-IR dye (Invitrogen). For tumor samples, cells were stained with anti-CD4 (OKT4, Biolegend), anti-CD8 (HIT8a, Biolegend), anti-CD3 (UCHT1, Biolegend), anti-CD45 (H130, Biolegend).
Chiou S.H., Tseng D., Reuben A., Mallajosyula V., Molina I.S., Conley S., Wilhelmy J., McSween A.M., Yang X., Nishimiya D., Sinha R., Nabet B.Y., Wang C., Shrager J.B., Berry M.F., Backhus L., Lui N.S., Wakelee H.A., Neal J.W., Padda S.K., Berry G.J., Delaidelli A., Sorensen P.H., Sotillo E., Tran P., Benson J.A., Richards R., Labanieh L., Klysz D.D., Louis D.M., Feldman S.A., Diehn M., Weissman I.L., Zhang J., Wistuba I.I., Futreal P.A., Heymach J.V., Garcia K.C., Mackall C.L, & Davis M.M. (2021). Global analysis of shared T cell specificities in human non-small cell lung cancer enables HLA inference and antigen discovery. Immunity, 54(3), 586-602.e8.
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