For selected neopeptides, pMHC tetramers were generated for staining of neoepitope-specific T cells. Neopeptides were selected based on the observed NART responses from the DNA barcode-labelled multimer screening. Following the observed increase in NART responses at 3 weeks post-treatment, NART responses in 3-week post-treatment PBMC samples were interrogated wherever sufficient patient material remained, otherwise 9-week post-treatment samples were analyzed. Single-fluorochrome pMHC specificity tetramers using were generated as described in detail previously
81 (link),82 (link), using a library of streptavidin (SA)-conjugated flourochromes consisting of PE-SA (BioLegend, cat. #405204),
APC-SA (BioLegend, cat. #405207),
BV421-SA (BD, cat. #563259),
PE-Cy7-SA (BD, cat. #557598), BV605-SA (BD, cat. #563260), PE-CF594-SA (BD, cat. #562284), BV650-SA (BD, cat. #563855), BUV395-SA (BD, cat. #564176). Up to eight patient-specific pMHC tetramers per sample were investigated. PBMC samples were stained with respective library of pMHC tetramers and with an antibody mix consisting of CD8-BV480, dump channel antibodies and a dead cell marker, as above. Tetramer-specific T cells analyzed as lymphocytes, single, live, CD8
+, FITC
‒ and tetramer
+ cells. Due to staining strategy, tetramer
+ cells were gated by being CD8
+.
Holm J.S., Funt S.A., Borch A., Munk K.K., Bjerregaard A.M., Reading J.L., Maher C., Regazzi A., Wong P., Al-Ahmadie H., Iyer G., Tamhane T., Bentzen A.K., Herschend N.O., De Wolf S., Snyder A., Merghoub T., Wolchok J.D., Nielsen M., Rosenberg J.E., Bajorin D.F, & Hadrup S.R. (2022). Neoantigen-specific CD8 T cell responses in the peripheral blood following PD-L1 blockade might predict therapy outcome in metastatic urothelial carcinoma. Nature Communications, 13, 1935.
