Extended Data Fig. 2: Single-cell RNA sequencing analysis of CD8 T cells isolated from AT3-OVA tumor bearing mice, and generation and validation of the Hobit^TomatoCre reporter mouse model.

(a,b) CD44⁺ CD8⁺ T cells were sorted from tumors and PD-1⁺ CD8⁺ T cells from tdLN of AT3-OVA-tumor-bearing mice and subjected to scRNA sequencing. (a) UMAP analyses showing expression of exhaustion (Tox, Nr4a2), TEX/effector cell (Gzmb, Prf1), T_PEX cell (Slamf6) and TRM cell down (Klf2, S1pr1) signature genes. (b) Violin plots showing normalized expression of TRM cell signature⁴⁴, TGF-β-induced signaling signature⁴⁵ and Itgae expression within TEX (top) and T_PEX (bottom) tumor-specific clusters. (c) Schematic illustrating the generation of the Hobit^TomatoCre (Hobit^TomCre) allele. A tdTomato reporter cassette and an IRES followed by a Cre recombinase sequence were inserted downstream of exon5 of the Hobit (Znf683) gene locus (mouse chromosome 4). After translation tdTomato is cleaved from the Hobit protein. (d,e) Hobit^TomCre P14 T cells were transferred into congenically marked naive recipient mice, which were infected with LCMV Armstrong (n = 3). CD44⁺CD8⁺ P14 TRM cells at day 30 post-infection were analyzed. (d) Contour plots (left) and quantification (right) showing Hobit versus CD69 expression of CD44⁺ P14 T cells in indicated organs. (e) Contour plots (left) and quantification (right) showing Hobit vs CD103 expression of CD44⁺ P14 T cells in indicated organs. (f) Hobit^TomCre reporter mice were epicutaneously inoculated with B16-gB tumors for analysis of CD8⁺ T cells at tumor endpoint. Contour plots of CD103⁺CD69⁺ cells in CD44⁺ CD8⁺ T cells in indicated organs (upper). Contour plots and quantification of Hobit versus CD103 (middle) and Hobit versus CD69 within the tumor (n = 12), peritumoral skin (n = 14) and tdLN (n = 13). (g,h) Hobit^TomCre reporter mice were orthotopically inoculated with AT3-OVA into the fourth MFP and were treated with ICB on days 11, 13, 16, and 19. (g) Schematic experimental setup. (h) CD44⁺PD-1⁻ CD8⁺ cells at day 50 post-inoculation were analyzed to study TRMs. Contour plots of CD103⁺CD69⁺ cells in CD44⁺ CD8⁺ T cells in indicated organs (top). Contour plots (left) and quantification (right) of Hobit versus CD103 (top), Hobit versus CD69 (middle) and Hobit versus CD49a (bottom) within MFP (n = 6) and spleen (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 in (d-h) are representative of at least two independent experiments. P values are from one-way ANOVA with Tukey’s multiple comparisons test (d-f) and from two-tailed unpaired t-tests (h).