Extended Data Fig. 3: Low neoantigen expression drives reduced T cell function and diversity.

(a-m) Flow cytometry of CD44⁺/CD8⁺ antigen-specific T cells from lesions and DLNs post-transplant of hi^SIIN (red) and lo^SIIN (blue) organoids. (a) Total antigen-specific T cells and (b) percent Ki67 positive in DLNs at 8 days. N = 10 hi^SIIN and 9 lo^SIIN-transplanted animals. (c-d) TCF1 and GZMB expression in antigen-specific T cells in lesions at 8 (c) and 14 (d) days. Representative of N = 59 animals per line and timepoint. (e) Percent of antigen-specific T cells double-positive for TNFα and IFNγ, and (f) and representative expression of TCF1 and GZMB in this subset within DLNs at 8 days. N = 10 hi^SIIN and 9 lo^SIIN-transplanted animals. (g) Inhibitory receptor expression on TCF1^-/GZMB^- antigen-specific T cells from tumors at 14 days. Representative of N = 6–7 animals per line. (h) Median percent of TCF1^-/GZMB^- antigen-specific T cells from tumors expressing 0 through 4 inhibitory receptors (PD-1, TIM3, LAG3, and 2B4) at 8 days. N = 9 hi^SIIN and 9 lo^SIIN-transplanted animals. Bars = standard deviation. (i-j) Percent of SIINFEKL-loaded “target” splenocytes killed in DLNs and spleens from killing assay at 8 (i) and 14 (j) days post-transplant of hi^SIIN and lo^SIIN organoids. N = 6–7 animals per line and timepoint. (k-l) Frequency of most common clonotypes (k) and Simpson diversity score (l) from TCRβ chain sequencing of antigen-specific T cells from hi^SIIN (down-sampled) and lo^SIIN lesions at 8 days. N = 4 independent animals per line. (m-n) Total antigen-specific T cells isolated from lesions at 14 days across all lines (m) and versus mScarlet^SIIN MFI (n). (o-p) Percent of antigen-specific T cells from lesions at 8 days double-negative for TCF1 and GZMB across lines (o) and versus mScarlet^SIIN MFI (p). Dashed lines connect medians. Significance assessed by Spearman’s rank correlation. N = 5–9 independent animals per line in (m-p). Significance in (a-b), (e), (i-j), (l-m) and (o) assessed by two-tailed Wilcoxon Rank Sum. Holm’s correction applied in (m) and (o). Source data for panels (a, h-j, m-p) can be found in Source Data Figure 3a, i, n–r.