Figure 5.

Low neoantigen expression limits T cell cross priming. (a) Efficiency of tumor formation 6 weeks post-transplant of hi^SIIN organoids into WT, Batf3^−/−, and WT mice with continuous anti-CD4 treatment. N = independent animals. (b-e) Cross priming of 10,000 naïve OT-1s co-cultured with 50,000 activated BM-DCs loaded with lysed no^SIIN (grey), lo^SIIN (blue), and hi^SIIN (red) cells. Flow cytometric quantification of CD44⁺, Ki67⁺, GZMB⁺, and TNFα⁺/IFNγ⁺ OT-1s. N = 4 co-cultures per line and condition. (f) Images of day 4 organoid and activated OT-1 co-cultures, representative of (g). (g) Quantification of organoid confluence. E:T = effector-to-target ratio. N = 3 co-cultures per line and condition. Significance assessed by two-tailed t-test. (h) Efficiency of tumor formation 6 weeks post-transplant of lo^SIIN, lo^SIIN 4 weeks post-transplant of hi^SIIN (Re-challenge), lo^SIIN and hi^SIIN at separate sites or mixed, and lo^SIIN concurrent with retro-orbital injection of activated OT-1s. N = independent animals. (i) Schematic of co-transplant of lo^SIIN (green) and hi^SIIN (red) organoids (top), and stereoscopic images 8 days post-transplant (bottom) representative of N = 12 animals. (j-k) Flow cytometric analysis of antigen-specific T cells from DLNs and lesions 8 days post-co-transplant of hi^SIIN (red) and lo^SIIN (blue) organoids at separate sites. N = 12 independent animals. (j) Total antigen-specific T cells. Significance assessed by two-tailed Wilcoxon Rank Sum. (k) Expression of GZMB and TCF1, representative of animals in (j). (l) Efficiency of tumor formation 6 weeks post co-transplant of lo^SIIN with indicated organoids mixed or at separate sites. N = independent animals. (m) Expression of H-2K^b/H-2D^b on hi^SIIN organoids after electroporation with Cas9 complexes targeting H2-k1 (pre-sorting). Purple: targeted; grey: untargeted. Organoids pre-treated with IFNγ. N = 1 experiment. (n) Efficiency of tumor formation 6 weeks post-transplant of K^b-KO^SIIN, hi^SIIN or co-transplant of lo^SIIN and K^b-KO^SIIN in WT and Batf3^−/− mice. N = independent animals. (o-p) Stereoscopic images of tumors 6 weeks post co-transplant of K^b-KO^SIIN and lo^SIIN organoids in WT (o) and Batf3^−/− mice (p), representative of animals transplanted in (n). Significance in (a), (h), (l) and (n) assessed by 2×2 Fisher’s exact test, with Holm’s correction applied in (h).