Extended Data Fig. 3: Characterization of Hobit⁺ cells in established AT3-OVA tumors.

(a-d) Subcutaneously inoculated, AT3-OVA tumor-bearing Hobit^TomCre mice were analyzed at tumor endpoint. (a) Quantification of Hobit expression within indicated CD45⁺ immune cell populations from indicated organs (n = 7). (b) Proportion (left) and numbers per mg (right) of indicated immune cell subsets within CD45⁺ Hobit⁺ cells within tumors (n = 7). (c) Histograms (right) and quantification of CD103, CD69 and CD49a in Hobit⁺ and Hobit⁻ (n = 8) pre-gated on CD44⁺ (middle) and OVA tetramer⁺ (right) CD8⁺ T cells. (d) Histograms (right) and quantification of TOX, TCF1 and TIM-3 in Hobit⁺ and Hobit⁻ (n = 9) pre-gated on CD44⁺ (middle) and OVA tetramer⁺ (right) CD8⁺ T cells. (e,f) Hobit^TomCreId3^GFP reporter mice were inoculated with AT3-OVA tumors for analysis of CD8⁺ T cells in tumors at endpoint. (e) Contour plot of Hobit-Tomato vs ID3-GFP among PD-1⁺ CD8⁺ T cells (left) and frequencies (right, n = 16) of specified subsets. (f) Frequencies of TCF1⁺(n = 5), TIM-3⁺(n = 8), PD-1⁺(n = 8), CD103⁺ (n = 7) and CD49a⁺ (n = 7) among OVA tetramer⁺ (Tet⁺)-specific CD8⁺ T cells from AT3-OVA tumors. (g,h) ID3 SP, DP, Hobit SP and DN CD8⁺ T cells were sorted from AT3 tumors-bearing Hobit^TomCreId3^GFP reporter mice and subjected to bulk RNA-seq. Heatmaps show expression of T_PEX and TEX cell signature genes¹⁷ (g). Barcode plots show enrichment of the TPEX signature in ID3/Hobit DP vs ID3 SP (left) and the TRM signature in ID3/Hobit DP vs Hobit⁺ ID3⁻ cells (right) derived from tumors (h). Upregulated genes in red and downregulated genes in blue. (i,j) Hobit^TomCreId3^GFP reporter mice were orthotopically inoculated with AT3-OVA into the fourth MFP. (i) Contour plots and frequencies show the expression of ID3-GFP and Hobit-Tomato among CD44⁺ CD8⁺ T cells from tumor (n = 7). (j) Frequencies of indicated markers within ID3⁺ Hobit⁻ (ID3 SP), Hobit⁺ ID3⁺ (DP), Hobit⁺ ID3⁻ (Hobit SP), Hobit⁻ ID3⁻(DN) cells are also shown (n = 6). Flow cytometry plots are representative. Dots in graphs represent individual mice; horizontal lines and error bars of bar graphs indicate means and ± SEM, respectively. Data are representative of or are pooled from 2–3 independent experiments. P values are from one-way ANOVA with Tukey’s comparisons test (b,e-f,i-j), from two-tailed unpaired t-tests (c,d). P > 0.05, not significant (n.s.).