Extended Data Fig. 7: Characterization of TPEX cells in the tumor-draining lymph nodes.

(a) CD8⁺ T cells from AT3-OVA tumor-bearing mice were analyzed. Contour plots of CD44 versus PD-1 expression pre-gated on CD8⁺ (left) or OVA-specific (Tet⁺) (left) from tdLNs or tumor, with quantification of PD-1⁺ and Tet⁺ among CD44⁺ on the right (n = 7). (b,c) C57BL/6 mice were subcutaneously inoculated with AT3-OVA tumors and tumors were analyzed at tumor endpoint. Contour plots (left) show TCF1 vs TIM-3 expression; quantification (right) (n = 5). (c) Histograms (left) and quantification (right) of expression of ID3 (n = 6) and Ly108 (n = 7) as in CD8⁺ T cells within the tdLN and tumor. (d) Irf4^Tomato mice were subcutaneously inoculated with AT3-OVA tumors and tumors were analyzed at tumor endpoint. Histograms (left) and quantification (right) of expression of IFR4-Tomato (n = 7) and TOX (n = 8) in specified CD8⁺ T cell subsets in tdLN are shown. (e) UMAP representation of TPEX and TEX¹⁷ and TRM⁴⁴ gene signatures projected onto shared and expanded CD8⁺ T cell clones within tdLN and tumors of ICB-treated AT3-OVA tumor bearing mice. (f,g) AT3-OVA tumor-bearing mice were treated with ICB (n = 5) on days 11, 13, 15 or left untreated (n = 7) and CD8⁺ T cells in tdLN were analyzed on day 17. (f) Flow cytometry plots (upper), frequencies and numbers per LN (lower) of OVA-specific (Tet⁺) cells among PD-1⁺ cells. (g) Flow cytometry plots (upper), frequencies, and numbers per LN (lower) of CX3CR1⁺ cells among Tet⁺ cells. (h-j) B16-GP33 tumor-bearing mice were treated with ICB (n = 11) when tumors became palpable, receiving three doses every three days, or left untreated (n = 12). CD8⁺ T cells in tdLN were analyzed two days after the last treatment. Frequencies (left) and numbers per LN (right) of CD44⁺ PD-1⁺ among CD8⁺ T cells (h), of CD62L⁺ and CD62L⁻ TPEX cells among PD-1⁺ CD8⁺ T cells (i), and of CX3CR1⁺ among PD-1⁺ CD8⁺T cells (j). (k) Numbers of specified CD8⁺ T cell subsets per mg of AT3-OVA tumor in untreated or ICB ± FTY720 treated mice (untreated n = 11, ICB n = 7, ICB + FTY n = 8). GMFI, geometric mean fluorescence intensity. Dots in graphs represent individual mice; horizontal lines and error bars of bar graphs indicate means and ±SEM. Histograms and flow cytometry plots are representative. Data are representative of at least two or pooled from two independent experiments. P values are from two-tailed unpaired t-tests (a-j) or one-way ANOVA with Tukey’s comparisons test (k).