PD-1/PD-L1 pathway: an adaptive immune resistance mechanism to immunogenic chemotherapy in colorectal cancer

Abstract

Chemotherapy is currently evaluated in order to enhance the efficacy of immune checkpoint blockade (ICB) therapy in colorectal cancer. However, the mechanisms by which these drugs could synergize with ICB remains unclear. The impact of chemotherapy on the PD-1/PD-L1 pathway and the resulting anticancer immune responses was assessed in two mouse models of colorectal cancer and validated in tumor samples from metastatic colorectal cancer patients that received neoadjuvant treatment. We demonstrated that 5-Fluorouracil plus Oxaliplatin (Folfox) drove complete tumor cure in mice when combined to anti-PD-1 treatment, while each monotherapy failed. This synergistic effect relies on the ability of Folfox to induce tumor infiltration by activated PD-1⁺ CD8 T cells in a T-bet dependent manner. This effect was concomitantly associated to the expression of PD-L1 on tumor cells driven by IFN-γ secreted by PD-1+ CD8 T cells, indicating that Folfox triggers tumor adaptive immune resistance. Finally, we observed an induction of PD-L1 expression and high CD8 T cell infiltration in the tumor microenvironment of colorectal cancer patients treated by Folfox regimen. Our study delineates a molecular pathway involved in Folfox-induced adaptive immune resistance in colorectal cancer. The results strongly support the use of immune checkpoint blockade therapy in combination with chemotherapies like Folfox.

Keywords: CD8 T cells; PD-1/PD-L1 pathway; chemotherapy; colorectal cancer, adaptive immune resistance; immunotherapy.

Figure 1.

Addition of Folfox to anti-PD-1 therapy promotes complete tumor regressions. CT26 tumor-bearing Balb/c mice (n = 6-8/group) were treated with a single injection of Glucose 5% (control), 5-Fluorouracil (5-FU), Mitomycin C (MMC), Oxaliplatin (OX), 5-FU plus MMC (5-FU/MMC) or 5-FU plus OX (Folfox) combined or not with anti-PD-1 therapy. (A) Tumor growth. Each line represents an individual mouse. (B) Survival (Log-rank test). (C) Results of all experiments performed in the same conditions were analyzed and pooled (three experiments for control, anti-PD-1, Folfox and Folfox/anti-PD1 groups; two for the other groups). Number of survivors 17 day post-chemotherapy and number of tumor-free mice among total are indicated. **p < 0.001; ns, not significant. Data are representative (A,B) or pooled (C) of 2 to 3 independent experiments. See also Supplementary Fig. 1.

Figure 2.

Chemotherapies differently modulate CD8 T cell function in the tumor. CT26 tumor-bearing mice were treated with different chemotherapies. Tumors were harvested 8 days after treatment (n = 3-4/group). (A) Frequency of CD8 TILs measured by flow cytometry (Kruskal-Wallis test). (B) IFNɣ secreted by CD8 TILs ex vivo (Kruskal-Wallis test). (C) IFNɣ-expressing CD8 TILs in response to AH-1/H-2L^d tumor peptide (Mean±s.d., Sidak test). **p < 0.01; ns, not significant. Data are representative of two independent experiments. See also Supplementary Figs. 2 and 3.

Figure 3.

Folfox favors the infiltration of tumors by functional PD-1⁺ CD8 T cells. (A) CT26 tumor-bearing mice were treated with glucose 5% (control) or Folfox. FACS-sorted CD8 TILs were pooled (n = 10/group) and subjected to RNA-sequencing. Naïve CD8 T cells were used as reference. Heatmap of expression of genes associated with inhibitory receptors is shown (two samples per condition). (B-C) CT26 tumor-bearing Balb/c mice (n = 6/group) were treated with the different chemotherapies. (B) Frequency of PD-1 and Tim-3 was determined by flow cytometry (Kruskal-Wallis test). (C) Representative dot plot of PD-1 and Tim-3 expression on CD8 TILs. (D) Percoll-isolated TILs were harvested from Folfox-treated mice. (Left) CD8 TILs (n = 4) were FACS-sorted according to PD-1 and Tim-3 expression. mRNA IFNγ (ifng), Perforin (prf1) and Granzyme B (gzmB) expression was measured in each subset by RT-PCR. β-Actin was used as reference (Mean ± s.d of experimental replicates, Kruskal-Wallis test). (Right) Frequency of IFNγ, TNF-α, and CD107a produced by CD8 TILs after anti-CD3 stimulation (Mean ± s.d, Kruskal-Wallis test). (E) CD8 TILs were FACS-sorted according to PD-1 and Tim-3 expression. Relative mRNA expression to actin of IFNγ (ifng), Perforin (prf1), Granzyme B (gzmB) and TNF-a (tnfa) by RT-PCR at day 8 and day 17 post-treatment (Mean ± s.d. of experimental replicates, Mann-Whitney test). (F) CT26 tumor-bearing Balb/c mice (n = 3-4/group) were treated with glucose 5% (control) or Folfox combined or not with anti-PD1 therapy. IFNɣ and CD107 a produced by CD8 TIL 12 and 20 days after treatment (Mean ± s.d.,Sidak test). **p < 0.01; ns, not significant. Data are representative of one (A), two (E,F) or at least three (B-D) independent experiments. See also Supplementary Figs. 4 and 5.

Figure 4.

T-bet drives the induction of functional CD8 TILs after Folfox therapy. (A) CT26 tumor-bearing mice were treated with Folfox (n = 4/group) and FACS-sorted CD8 TILs were pooled. T-bet (Tbx21) and Eomes (Eomes) mRNA expression was assessed by RT-PCR. β-Actin was used as reference and data were normalized to control (Mean ± s.d of four experimental replicates, Mann-Whitney test). (B-F) MC38 tumor-bearing C57BL/6 mice deficient in CD8 T cells for Tbx21 (CD8^Tbet−/−), Eomes (CD8^Eomes−/−) and their respective control mice CD8^Tbet+/+ or CD8^Eomes+/+ were treated with Folfox (n = 4/group). (B) CD8⁺ TILs from each group were FACS-sorted then pooled and the relative expressions of PD-1 (Pdcd1) and Tim-3 (Havcr2) mRNA were analyzed by RT-PCR. β-Actin was used as reference (Mean ± s.d. of technical replicates, Mann-Whitney test). (C) Expression of PD-1 (left) and Tim-3 (right) on CD8 TILs by flow cytometry. Each dot represents one individual (Mann-Whitney test). (D-E) Relative mRNA expression of IFNγ (Ifng), Perforin (Prf1) and Granzyme B (Gzmb) in FACS-sorted CD8 TILs from controls and (D) CD8^Tbet−/− and (E) CD8^Eomes−/− mice. β-Actin was used as reference (Mean ± s.d. of technical replicates, Mann-Whitney test). (F) Tumor growth measured 8 days following treatment. Each dot represents one individual (n = 4/group, Mann-Whitney test). *p < 0.05; **p < 0.01; ns, not significant. Data are representative of two independent experiments. See also Supplementary Fig. 6.

Figure 5.

Folfox induces PD-L1 expression on tumor cells in vivo. (A-C) CT26-tumor bearing mice were treated either with glucose 5% (control) or Folfox and tumors were harvested 8 days after treatment. (A) Scheme of the experiment and gating process to isolate viable tumor cells (CD45/7AAD-negative) by FACS (See also Materials and Methods). (B) Relative expression level of PD-L1 (Cd274) mRNA analyzed by RT-PCR. β-Actin was used as reference (n = 3/group, mean ± s.d., Mann-Whitney test). (C) Expression of PD-L1 determined by flow cytometry on viable tumor cells (n = 6/group, each dot represents one individual, Mann-Whitney test). (D) MC38 tumor-bearing C57BL/6 mice were treated with glucose 5% (control) or Folfox (n = 3/group). Expression of PD-L1 on viable tumor cells by flow cytometry 8 days after treatment (Mean ± s.d, Mann-Whitney test). (E) Monitoring of PD-L1 expression on viable tumor cells from Folfox-treated mice over seventeen days post-treatment (n = 4/group, mean ± s.d.). (F) CT26-tumor bearing Balb/c mice were treated as in Fig. 2C (n = 5/group). Expression of PD-L1 on viable tumor cells by flow cytometry 8 days after treatment is shown. Dashed line delineates the FMO control (Mean ± s.d.,Kruskal-Wallis test). Data are representative of 2 experiments (B, D-F) or more than 4 independent experiments (C). *p < 0.05, **p < 0.01. See also Supplementary Fig. 7.

Figure 6.

PD-L1 tumor expression upon Folfox chemotherapy is driven by IFNγ produced by CD8 T cells. (A) CT26 tumor-bearing immunocompetent Balb/c or immunodeficient Nude Balb/c mice were treated with glucose 5% (control) or Folfox (n = 4/group). Expression of PD-L1 determined by flow cytometry on viable tumor cells 8 days after treatment (Kruskal-Wallis test). (B-C) CT26 tumor-bearing mice (n = 5-6/group) were treated with Folfox with or without (B) anti-CD4 or anti-CD8 depleting antibodies or (C) anti-IFNγ antibody. PD-L1 expression on tumor cells 8 days after treatment (Kruskal-Wallis test). (D-F) CT26 tumor cells were cultured with or without recombinant mouse IFNγ (10 ng/mL). (D) Kinetic analysis of indicated proteins by Western Blot. β-actin was used as control. (E) ChIP analysis of the binding of IRF1 to the putative binding site −344 of the Cd274 (PD-L1) promoter. (F) CT26 tumor cells were transfected with siRNA IRF1 or siRNA control then cultured with or without IFNγ for 24 H. Relative expression level of Irf1 and Cd274 to untreated siRNA control-transfected cells is shown. β-Actin was used as the internal control and data were normalized to untreated siRNA control (Mean ± s.d. of technical replicates, one-way anova test).**p < 0.01; ns, not significant. Data are representative of two independent experiments.

Figure 7.

The induction of immunogenic tumor cell death drives PD-L1 expression on tumor cells in vivo. (A-B) CT26 colorectal tumor cells were treated in vitro with or without 5-Fluorouracil (5-FU, 10 µM), Mitomycin (MMC, 20 µM), Oxaliplatin (OX, 50 µM) or Folfox (5-FU, 10 µM + OX, 50 µM). (A) HMGB1 release by ELISA 24 H after treatment (Mean ± s.d of experimental replicates, Kruskal-Wallis test). (B) Immunofluorescence analysis of calreticulin (CRT) exposure at the membrane 6 h after treatment. (C) Correlation between calreticulin (CRT) exposure on CT26 tumor cells treated in vitro with the different chemotherapies and PD-L1 expression on tumor cells in vivo (Pearson correlation) (n = 3/group). (D) MC38 tumor-bearing C57BL/6 and TLR4-deficient mice were treated with glucose 5% (-) or Folfox (+). PD-L1 expression on viable tumor cells 8 days after treatment is depicted (n = 4-5/group, Kruskal-Wallis test). Each dot represents an individual measurement in one individual. *p < 0.05; **p < 0.01; ns, not significant. Data are representative of two independent experiments. See also Supplementary Fig. 8.

Figure 8.

Impact of Folfox neoadjuvant chemotherapy in metastatic colorectal cancer patients. Tumor samples from 9 metastatic colorectal cancer patients were harvested before and after Folfox neoadjuvant chemotherapy. (A) Representative images of patients' tumor biopsies showing PD-L1, LC3B and CD8 labeling by immunohistochemistry. L: liver, I: infiltrate, T: tumor. (B) Individual representation of patients for each parameter. Scores of 2+ and 3+ are classified as high expression. (C) Heatmap of patients before and after Folfox chemotherapy.