(A) Tumor-free survival after rechallenge with B16.gp33 (middle) or MC38.gp33 (right) in mice with complete regression of MC38.gp33 and B16.gp33 (+αPD-1) tumors, respectively (see Figure 1B and Figure 5A), n = 8 WT, n = 9 VHL-KO, n = 6 control (middle); n = 9 experimental groups, n = 5 control (right). (B) Recovered memory WT or VHL-KO P14 cell counts in animals in A were adjusted to the respective mean counts recovered in primary tumor models (see Figure 1E). (C) Weight-adjusted donor TIL and splenocyte numbers between animals receiving WT (black) and VHL-KO (red) memory cells, n = 4 VHL-KO, n = 5 WT. (D) CD69 and CD103 expression (left) and quantification (right) on recovered memory cells in C. (E) Frequency of Tcm (CD62L^hi), Tem (CD62L^loCD103^lo), and Trm (CD103^hiCD69^hiCD62L^lo) phenotypes within the donor memory populations. (F) Recall function of memory cells recovered from mice bearing B16.gp33 rechallenge tumors. Representative flow cytometry (left) of IFN-γ and TNF-α coproduction by indicated memory TILs (top row) and memory splenocytes (bottom row), and quantification by frequency (top right) and weight-adjusted cell numbers (bottom right), n = 2 VHL-KO, n = 3 WT. (G) Tumor-free survival after FTY720 or vehicle and rechallenge with B16.gp33 tumor cells in mice that resisted the rechallenge tumor growth in A. RAG-KO mice were controls, n = 14 VHL-KO, n = 5 WT, n = 6 naive control, n = 5 RAG-KO. Data are representative of 2 independent (A–F) or pooled (G) experiments. Circle and square symbols in B–F indicate rechallenge with B16.gp33 and MC38.gp33, respectively, error bars represent mean ± SEM. NS, not significant, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001; (A and G) Bonferroni-corrected log-rank test, (B–D, and F) 1-way ANOVA with Bonferroni correction for multiple comparisons, (E) Student’s t test. (*) failed to reach Bonferroni-corrected threshold although showing P < 0.05.