Characterization of tumor-infiltrating T cells and TCR clonotypes in Calr KO tumors. (A) UMAP plot of CD8⁺ T cells from single-cell RNA-seq with paired TCR analysis, combined from Calr KO and control LLC tumors. Data are pooled from five mice for each group. (B and C) Single-cell TCR clonotype size projected onto UMAP plot of CD8⁺ cells from control (B) and Calr KO (C) tumor. Clonal size (%) is calculated as the proportion of cells of each clonotype in total cell count. (D) TCR antigen recognition patterns in Calr KO and control tumors analyzed by the GLIPH2 algorithm based on CDR3 sequences. (E) Schematic representation of the Jurkat-tumor cell coculture system used to assess TCR reactivity to tumor cells. The Jurkat cells are engineered to express a chimeric CD8α/β receptor with a murine extracellular domain. (F) Chart displaying the count of candidate TCRs enriched in Calr KO LLC tumors. (G and H) Jurkat cells expressing candidate TCRs, including TCR-1, TCR-2, TCR-5 (G), and TCR-3 (H) are cocultured with H2-Kb H2-Db KO (MHC-I KO), Calr KO, and control tumors, respectively. The level of CD69 in Jurkat cells was determined by FACS. Data are presented as the mean ± SD and representative of at least two independent experiments. ns, not significant; ***P < 0.001; ****P < 0.0001 by an unpaired t test. Representative flow cytometry histograms of Jurkat cells expressing TCR-3 cocultured with Calr KO and control tumors are shown in H. gMFI, geometric mean fluorescence intensity.