Temporal evolution of anti-tumor responses by proximal versus distal IC-LN–primed CD8 T cells. (A–G) Congenically disparate (CD45.1⁺/CD45.1⁺.2⁺) OT-I T cells were primed in B6 recipients, isolated from proximal and distal IC-LNs, and quantified. 5–10 × 10⁴ proximal and distal OT-I CD8 T cells each were retransferred at a 1:1 ratio into new B16.OVA (or also B16 on contralateral side) tumor-bearing mice. Tumors and tumor-draining LNs were harvested at the indicated time points after transfer. (A) Schematic of experimental design (n = 4–5/group, four experiments). (B) Proximal and distal OT-I T cell numbers were quantified 1 day following retransfer. (C) Representative plots and quantification of granzyme B and IFNγ expression following ex vivo restimulation by the OT-I T cells relative to endogenous CD44⁻ CD8 T cells. (D) Representative flow plots and quantification of TIM3 and Ly108 expression by tumor-infiltrating OT-I T cells 1 day after retransfer. (E) Quantification of the ratio of distal to proximal IC-LN–derived OT-I T cells in B16.OVA tumors and tumor-draining LNs across different time points. (F) Quantification of the fold expansion of proximal and distal OT-I T cells from day 1 to day 11 following retransfer. (G) Expression of Ly108, granzyme B, and TCF1 by OT-I T cells in the tumor 5 days after retransfer. (H) Quantification of the ratio of distal to proximal IC-LN–derived OT-I T cells in B16.OVA tumors and tumor draining LNs at indicated time points after retransfer of 5–10 × 10³ OT-I T cells from each source (n = 3–5/group, two experiments). (I) Expression of TIM3 by proximal and distal OT-I T cells 7 and 11 days after retransfer. (J) Representative plots and quantification of PD1 and TCF1 expression by OT-I T cells at the indicated time point. (K) Schematic of experimental design. Following retransfer of 5–10 × 10³ congenically disparate proximal and distal IC-LN–derived OT-I T cells into B16.OVA-bearing mice, recipients were treated with anti-PDL1 or IgG isotype control antibody at day 10 and day 12. Tumors and LNs were harvested on day 14 for analysis by flow cytometry (n = 4–7/group, three experiments). (L) Quantification of the total proximal- or distal-derived OT-I T cell numbers in the tumor following treatment. (M and N) Frequency and total cell numbers of responding proximal- or distal-derived OT-I T cells in the tumor that are of a (M) T_PEX (Ly108⁺TIM3⁻) or (N) T_EX (TIM3⁺Ly108⁻) phenotype (n = 4–7/group, three experiments). Data from multiple pooled experiments are denoted by different symbols within the same group. Graphs show mean ± SD and were analyzed using paired Student’s t test for comparison within the same mouse or unpaired between separate mice. For anti-PDL1–treated versus isotype control samples, nonparametric unpaired t test was used. ****P < 0.0001; ***P < 0.001; **P < 0.01; *P < 0.05; P > 0.05 not significant (ns). Wilcoxon signed-ranked test was used to compare new ratios of distal to proximal cells to a hypothetical value of 1 (ratio at time of transfer).