Temporal evolution of anti-tumor responses by proximal versus distal LN-primed CD8 T cells. (A) Representative flow plots demonstrating gating of the retransferred OT-I T cells in B16-OVA tumors and pooled tumor-draining LNs. Quantification of the frequency of OT-I T cells 3 days after retransfer into mice bearing both B16.OVA and B16 tumors on contralateral flanks (n = 4–5/group, two experiments). (B) Quantification of IFNγ expression following ex vivo restimulation by the OT-I T cells (n = 5, four experiments). (C) Representative flow plots and quantification of TIM3 and Ly108 expression by OT-I T cells in tumor-draining LNs 1 day after retransfer (n = 4–5, three experiments). (D) Quantification of Ki67 expression by the proximal and distal OT-I T cells 1 and 11 days after retransfer (n = 5, four experiments). (E) Quantification of percent of total transferred OT-I CD8 T cells that are either of proximal or distal origin across different time points after retransfer of 5–10 × 10⁴ cells (n = 5, four experiments). (F) Quantification of total number of T_EX (GranzB⁺TCF1⁻) and T_PEX (Ly108⁺TCF1⁺) cells in the tumors day 5 after retransfer of 5–10 × 10⁴ cells (n = 3–5, two experiments). (G) Representative flow plots and quantification of Ly108 and TCF1 expression by OT-I T cells in tumor-draining LNs 5 days after retransfer (n = 4, two experiments). (H) Quantification of percent of total transferred OT-I CD8 cells that are either of proximal or distal origin across different time points after retransfer of 5–10 × 10³ cells (n = 3–5/group, two experiments). (I) Quantification of the total CD44⁺ endogenous CD8 T cells in the tumor and tumor-draining LN following treatment with anti-PDL1 or isotype control antibody (n = 4–7/group, three experiments). (J) Quantification of the ratio of distal to proximal IC-LN–derived OT-I T cells following treatment (n = 4–7/group, three experiments). (K and L) (K) Quantification of the total OT-I T cell numbers in the tumor-draining LNs and (L) the frequency of cells expressing the indicated markers in the tumor and tumor-draining LNs following treatment (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).