Reprogramming the Intrahepatic Cholangiocarcinoma Immune Microenvironment by Chemotherapy and CTLA-4 Blockade Enhances Anti-PD-1 Therapy

Abstract

Intrahepatic cholangiocarcinoma (ICC) has limited therapeutic options and a dismal prognosis. Adding blockade of the anti-programmed cell death protein (PD)-1 pathway to gemcitabine/cisplatin chemotherapy has recently shown efficacy in biliary tract cancers but with low response rates. Here, we studied the effects of anti-cytotoxic T lymphocyte antigen (CTLA)-4 when combined with anti-PD-1 and gemcitabine/cisplatin in orthotopic murine models of ICC. This combination therapy led to substantial survival benefits and reduction of morbidity in two aggressive ICC models that were resistant to immunotherapy alone. Gemcitabine/cisplatin treatment increased tumor-infiltrating lymphocytes and normalized the ICC vessels and, when combined with dual CTLA-4/PD-1 blockade, increased the number of activated CD8+Cxcr3+IFNγ+ T cells. CD8+ T cells were necessary for the therapeutic benefit because the efficacy was compromised when CD8+ T cells were depleted. Expression of Cxcr3 on CD8+ T cells is necessary and sufficient because CD8+ T cells from Cxcr3+/+ but not Cxcr3-/- mice rescued efficacy in T cell‒deficient mice. Finally, rational scheduling of anti-CTLA-4 "priming" with chemotherapy followed by anti-PD-1 therapy achieved equivalent efficacy with reduced overall drug exposure. These data suggest that this combination approach should be clinically tested to overcome resistance to current therapies in ICC patients.

Figure 1.

Standard chemotherapy converts ICB-resistant ICCs to ICB-responsive tumors, increases the infiltration of CD8⁺ T cells, and normalizes tumor vasculature. A and D, Orthotopic ICC models in mice: p53^KOKras^G12D murine 425-ICC in C57Bl/6/FVB F1 mixed background mice (A) and Idh2^R172K/Kras^G12D murine SS49-ICC in C57Bl/6 mice (D). Upper, high-frequency ultrasound images. B-mode image of a tumor 6 days after orthotopic implantation in a C57Bl/6/FVB F1 mouse and C57Bl/6 mouse. Scale bar, 2 mm. Lower, macroscopic appearance. B and E, Tumor growth curves after treatment: GC/dual ICB therapy induced a tumor growth delay significantly superior to GC alone, whereas ICB alone was ineffective in murine 425-ICC (B) and SS49-ICC (E) models. C and F, Overall survival of ICC-bearing mice after treatment: GC/dual ICB therapy induced a survival advantage that was significantly superior to GC alone, whereas ICB alone showed no efficacy in the murine 425-ICC (C) and SS49-ICC (F) models. G and H, Representative IF for the T-cell marker CD8 (scale bar, 25 μm; G) and the endothelial and perivascular cell markers CD31 and α-SMA (scale bars, 100 μm in low-magnification and 50 μm in high-magnification images; H) in the 425-ICC model at day 8, respectively. I, In the 425-ICC model, ICC tissue infiltration by CD8⁺ T cells was increased mice treated with GC alone or with GC/dual ICB at day 8. J and K, GC can increase CD31⁺ in the tumor and induce normalization of ICC vessels; both were quantified by IF in 425-ICC tissue at day 8. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001 from Dunnett multiple comparisons tests (B, E), log-rank (Mantel–Cox) test (C, F). In vivo studies were performed in duplicate; n = 14 mice (murine 425-ICC) and n = 10 (murine SS49-ICC). The box plots depict mean values and standard error of the mean (SEM).

Figure 2.

GC/dual ICB therapy reprograms the immune microenvironment of 425-ICC. A, Volcano plot showing the differential gene expression between GC/dual ICB and GC groups by scRNA sequencing at day 10. B, Expression levels of angiogenesis-related genes. The P-value was calculated by a two-sided Wilcoxon rank-sum test. C, Top 10 enriched pathways of differential gene expression between GC/dual ICB and GC groups. D and E, Representative multicolored images for every sample in the tissue microarray for DNA, CD31, CD3e, PD-L1, and PD-1 (D), Pan-Keratin, CD3e, CD8a, TCF1, and granzyme B (E) at day 20, N = 6 mice/group. Scale bar, 100 μm. F and G, GC combined with ICB can increase CD31⁺ (F) and CD8⁺ (G) cells in ICC tissues, as quantified by IMC at day 20. H and I, The abundance of CD8⁺CD3⁺TCF1⁺ cells and CD8⁺CD3⁺GZMB⁺ cells assessed by IMC at day 20 showed a significant increase in the GC/ICB group. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001, Kruskal–Wallis test and 3 high-power fields for each sample. The box plots depict mean values and SEM.

Figure 3.

The significant role of CTLA-4 blockade in GC/dual ICB therapy efficacy and increase in CD8⁺ T-cell frequency in the murine 425-ICC model. A and B, Tumor growth kinetics and survival distributions after treatment in the orthotopic murine 425-ICC model: GC/anti–CTLA-4 combination is significantly superior to GC alone and in delaying tumor growth (A) and increasing survival (B); addition of anti–PD-1 to GC/anti–CTLA-4 (GC+ICB) but not to GC alone induces a more significant delay in tumor growth (A) and increase in survival (B). C–H, Immunophenotyping of treated ICC tissues at days 10 (control and ICB groups) and 20 (GC-containing groups): TCR⁺ TIL frequencies were higher in all GC-treated groups and significantly increased in the groups receiving GC and anti–CTLA-4 (C). CD8⁺ T cells represented approximately 60% of TILs in all groups (D). CD8⁺IFNγ⁺ T-cell frequencies were higher in all GC-treated groups and significantly increased in the GC/dual ICB group (P = 0.0044 vs. GC alone; E). Ki67⁺CD8⁺ T-cell frequency (F). CD8⁺Cxcr3⁺ T-cell frequencies were significantly increased in the groups receiving GC/dual ICB treatment (G). CD8⁺Cxcr3⁺IFNγ⁺ T-cell frequencies were significantly increased in the GC/dual ICB group (H). I–K, Anti–PD-1 therapy (alone or in combination with GC) significantly increased the frequency of CTLA-4⁺PD-1^– cells among conventional CD4⁺ T cells (I). GC reduced the proportion of CTLA-4⁺PD-1⁺ conventional CD4⁺ T cells and Tregs compared with control, and the addition of ICB (but not anti–PD-1) further decreased these populations compared with GC (J, K). L and M, Depletion of CD8⁺ (M) but not CD4⁺ (L) T cells compromised the survival benefit of both GC/anti–CTLA-4 and GC/dual ICB combination treatments. *, P < 0.05; **, P < 0.01; ****, P < 0.0001 from Dunnett multiple comparisons tests (A) and log-rank (Mantel–Cox) test (B, J, K) and Tukey multiple comparisons tests (C–K). In vivo studies were performed in duplicate; n = 14 mice per group. The box plots depict mean values and SEM.

Figure 4.

CD8⁺Cxcr3⁺ T cells and anti–CTLA-4 therapy priming mediate the benefit of GC/dual ICB combination therapy in the murine 425-ICC model. A and B, The tumor growth delay (A) and overall survival (B) benefits of GC/dual ICB over GC alone therapy are significantly reduced in Rag1^–/–/C57Bl/6 mice that received CD8⁺ T-cell transfer from Cxcr3^–/–/C57Bl/6 mice prior to ICC implantation (n = 8 mice). C and D, Tumor growth delay (C) and overall survival (D) after treatment with GC/anti–CTLA-4 priming for a week followed by GC/anti–PD-1 maintenance were comparable to full dose GC/dual ICB and significantly superior to GC/anti–PD-1 priming for a week followed by GC + anti–CTLA-4 maintenance and to GC alone (n = 10). E, Proposed mechanism of benefit: standard GC therapy reprograms the immune microenvironment and normalizes vessels in ICB-resistant ICC, and combination with anti–PD-1/CTLA-4 dual ICB therapy increased efficacy mediated by anti–CTLA-4 therapy priming and tumor infiltration by activated CD8⁺Cxcr3⁺ T cells. *, P < 0.05; **, P < 0.01; ****, P < 0.0001 from Dunnett multiple comparisons tests (A, C) and log-rank (Mantel–Cox) test (B, D). The tumor growth curves depict mean values and SEM at each time point.