Joshua L. D. Parris

S47-derived T cells display a steady-state deficit in effector T-cell function after stimulation. RBC-depleted splenocytes from either S47 or P47 mice were stimulated with PMA/Ionomycin, Concanavalin A, or anti-CD3/CD28 Dynabeads, then assayed for cytolytic markers and effector cytokine production via multiparametric flow cytometry. Values were normalized to negative control wells receiving no stimulation. Data are derived from four independent experiments. A–C, Differences in CD4+ and CD8+ T-cell degranulation (CD107a), IFNγ production, and TNFα production between S47 and P47 mice when stimulated with Concanavalin A, anti-CD3/CD28 beads, or PMA/Ionomycin, respectively. Polyfunctionality profiles for CD4+ and CD8+ T cells double positive (CD107a+/TNFα+) when stimulated with ConA or PMA/Ionomycin (D and F, respectively) or triple positive (CD107a+/Perforin+/TNFα+) when stimulated with anti-CD3/CD28 beads (E) for effector molecules between S47 and P47 mice. Statistics derived from one-sided Wilcoxon rank-sum test. *, P < 0.05; **, P < 0.01; ***, P < 0.001.

Differences in memory T-cell formation and chemokine receptor usage in S47 mice. Splenocytes derived from non–tumor-bearing, male S47 and P47 mice between the ages of 6 and 10 weeks were analyzed by flow cytometry for differences in baseline T-cell phenotypes. Data are derived from five independent cohorts of mice (n = 27 S47, n = 26 P47), and all panels incorporate multiple cohorts. A and B, Frequencies of total memory (CD44+) and central memory (CD44+/CD62L+) CD4+ T cells between P47 and S47 mice. C and D, Frequencies of CX3CR1, or CXCR5 chemokine receptor expression in memory (CD44+) CD4+ T cells between P47 and S47 mice. E and F, Frequencies of total memory (CD44+) and central memory (CD44+/CD62L+) CD8+ T cells between P47 and S47 mice. G, Frequency of CXCR3 chemokine receptor expression in memory (CD44+) CD8+ T cells between P47 and S47 mice. Statistics derived from two-tailed Student t tests. *, P < 0.05; **, P < 0.01; ***, P < 0.001.

TILs in S47 mice exhibit elevated coexpression of multiple IRs. Tumors and spleens were harvested 21 days after injection of MC38 cells and T cells were analyzed for surface expression of multiple IRs by flow cytometry. Comparison of frequency of CD4+ TILs (A) and CD8+ TILs (B) expressing the LAG3, PD1, TIGIT, or Tim3 IRs between S47 and P47 mice. Comparison of the frequency of IR coexpression profiles in CD4+ TILs (C) and CD8+ TILs (D) between S47 and P47 mice. Bar graphs represent frequencies of specific IR coexpression profiles in the total CD4+ or CD8+ T cell populations derived from tumors, whereas pie charts represent relative frequencies of specific IR profiles in the total CD4+ or CD8+ TIL populations expressing any IR. Pie chart slices represent the average relative frequencies of 5 mice.

Differences in memory T-cell formation and chemokine receptor usage in S47 mice. Splenocytes derived from non–tumor-bearing, male S47 and P47 mice between the ages of 6 and 10 weeks were analyzed by flow cytometry for differences in baseline T-cell phenotypes. Data are derived from five independent cohorts of mice (n = 27 S47, n = 26 P47), and all panels incorporate multiple cohorts. A and B, Frequencies of total memory (CD44+) and central memory (CD44+/CD62L+) CD4+ T cells between P47 and S47 mice. C and D, Frequencies of CX3CR1, or CXCR5 chemokine receptor expression in memory (CD44+) CD4+ T cells between P47 and S47 mice. E and F, Frequencies of total memory (CD44+) and central memory (CD44+/CD62L+) CD8+ T cells between P47 and S47 mice. G, Frequency of CXCR3 chemokine receptor expression in memory (CD44+) CD8+ T cells between P47 and S47 mice. Statistics derived from two-tailed Student t tests. *, P < 0.05; **, P < 0.01; ***, P < 0.001.

The tumor growth advantage in S47 mice is abolished following CD8+ T-cell depletion. A, Illustration of the experimental approach. Replicate P47 and S47 mice were injected subcutaneously with 1 × 106 MC38 tumor cells. At day 19, spleens and tumors from mice were harvested and analyzed by flow cytometry. B, Tumor growth curves for P47 and S47 mice injected with MC38 cells. n = 5 mice per group. Linear mixed model used for statistical analysis. **, P < 0.01. C, Tumor weights of MC38 tumors harvested from P47 and S47 mice. n = 5 mice per group. Wilcoxon rank-sum test used for statistical analysis. *, P < 0.05. D and E, Tumor growth curves for P47 and S47 mice injected with MC38 cells, ± treatment with anti-CD8b antibody. n = 5 mice per group. Linear mixed model used for statistical analysis. *, P < 0.05. F, The percentage of CD8+ T cells of total T cells (CD45+/CD3+) in P47 and S47 mice before and after CD8+ T-cell depletion is shown on the left. Shown on the right are the total counts of splenic CD8+ T cells in P47 and S47 mice before and after CD8+ T-cell depletion.

The tumor suppressor TP53 is the most frequently mutated gene in cancer and is mutationally inactivated in 50% of sporadic tumors. Inactivating mutations in TP53 also occur in Li Fraumeni syndrome (LFS). In addition to germline mutations in TP53 in LFS that completely inactivate this protein, there are many more germline mutant forms of TP53 in human populations that partially inactivate this protein: we call these partially inactivating mutations “hypomorphs.” One of these hypomorphs is a SNP that exists in 6%–10% of Africans and 1%–2% of African Americans, which changes proline at amino acid 47 to serine (Pro47Ser; P47S). We previously showed that the P47S variant of p53 is intrinsically impaired for tumor suppressor function, and that this SNP is associated with increased cancer risk in mice and humans. Here we show that this SNP also influences the tumor microenvironment, and the immune microenvironment profile in P47S mice is more protumorigenic. At basal levels, P47S mice show impaired memory T-cell formation and function, along with increased anti-inflammatory (so-called “M2”) macrophages. We show that in tumor-bearing P47S mice, there is an increase in immunosuppressive myeloid-derived suppressor cells and decreased numbers of activated dendritic cells, macrophages, and B cells, along with evidence for increased T-cell exhaustion in the tumor microenvironment. Finally, we show that P47S mice demonstrate an incomplete response to anti-PD-L1 therapy. Our combined data suggest that the African-centric P47S variant leads to both intrinsic and extrinsic defects in tumor suppression.Significance:Findings presented here show that the P47S variant of TP53 influences the immune microenvironment, and the immune response to cancer. This is the first time that a naturally occurring genetic variant of TP53 has been shown to negatively impact the immune microenvironment and the response to immunotherapy.

S47-derived T cells display a steady-state deficit in effector T-cell function after stimulation. RBC-depleted splenocytes from either S47 or P47 mice were stimulated with PMA/Ionomycin, Concanavalin A, or anti-CD3/CD28 Dynabeads, then assayed for cytolytic markers and effector cytokine production via multiparametric flow cytometry. Values were normalized to negative control wells receiving no stimulation. Data are derived from four independent experiments. A–C, Differences in CD4+ and CD8+ T-cell degranulation (CD107a), IFNγ production, and TNFα production between S47 and P47 mice when stimulated with Concanavalin A, anti-CD3/CD28 beads, or PMA/Ionomycin, respectively. Polyfunctionality profiles for CD4+ and CD8+ T cells double positive (CD107a+/TNFα+) when stimulated with ConA or PMA/Ionomycin (D and F, respectively) or triple positive (CD107a+/Perforin+/TNFα+) when stimulated with anti-CD3/CD28 beads (E) for effector molecules between S47 and P47 mice. Statistics derived from one-sided Wilcoxon rank-sum test. *, P < 0.05; **, P < 0.01; ***, P < 0.001.

S47 mice are less responsive to PD-L1 blockade therapy. A, Tumor growth curves for P47 and S47 mice treated with rat IgG2b isotype control antibody or anti-PD-L1 antibody following MC38 cell injections. n = 7 P47 IgG2b isotype control; n = 5 S47 IgG2b isotype control; n = 9 P47 anti-PD-L1; n = 10 S47 anti-PD-L1. B, Graph showing the frequency of palpable tumors in S47 or P47 mice receiving anti-PD-L1 antibody. C and D, Individual tumor growth curves for each P47 and S47 mouse treated with rat IgG2b isotype control antibody or anti-PD-L1 antibody following MC38 cell injections. Statistics were derived from Wilcoxon rank-sum test. *, P < 0.05.

Figure S1 shows BMDMs from S47 mice are more anti-inflammatory and polarize towards a M2 phenotype following IL-4 stimulation.