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. 2021 Jun 23;12:3862. doi: 10.1038/s41467-021-24132-6

Fig. 1. Quantification of SPAS-1 neoantigen-specific CD8+ T cells following TRAMP-C2 tumor challenge.

Fig. 1

a TRAMP-C2 cells were injected s.c. in the flank of C57BL/6J mice and tumor growth measured. Average of four mice from one representative experiment of four performed. b Representative flow cytometry staining of blood collected from mice before (naive) and at the indicated times after TRAMP-C2 tumor challenge. SPAS-1+ T cells within the CD8+ T cell gate were identified by dual staining with phycoerythrin (PE)- and allophycocanin (APC)- labeled peptide-MHC-I tetramers loaded with STHVNHLHC peptide specific for the H8 neoantigen of SPAS-1 in TRAMP-C2 cells. c Quantification of staining shown in (b), compiled in multiple mice. Kruskal–Wallace test was performed, p = 0.0005, with exact p values by Dunn’s multiple comparision test; ***p < 0.001. Results in (a–c) are representative of four independent experiments performed. (d, e) Single-cell suspensions of pooled spleen and peripheral lymph nodes from naive tumor-bearing mice were incubated with PE- and APC- fluorochrome tetramerized H2-Db SPAS-1. Tetramer-binding cells were captured by enrichment over anti-flurochrome magnetic beads and then stained for flow cytometry. d Representative flow cytometry staining of gated CD8+ T cells after tetramer enrichment showing expression of PE-labeled tetramer and CD44. e Quantified and pooled data showing values for individual naive (n = 21) or TRAMP-C2 challenged mice at days 7, 14, and 28 (n = 11, 11, and 14, respectively). Data are derived from 5, 3, and 4 independent experiments, respectively, for naive, day 7 and 14, and day 28 following tumor challenge. For SPAS-1 tetramer enrichment from naive mice injected i.v. with the SPAS-1 peptide in the presence of Poly I:C and anti-CD40 (TriVax), one representative experiment with three biological replicates is shown, of two similar experiments performed. Kruskal–Wallace test was performed, p < 0.0001, with exact p values by Dunn’s multiple comparison test; *p < 0.05, **p < 0.001, ****p < 0.0001. Bars represent mean ± S.E.M. Source data are provided as a Source Data File.