CCR5 blockade inflames antitumor immunity in BAP1-mutant clear cell renal cell carcinoma

Quan Zhou, Yangyang Qi, Zewei Wang, Han Zeng, Hongyu Zhang, Zhaopei Liu, Qiuren Huang, Ying Xiong, Jiajun Wang, Yuan Chang, Qi Bai, Yu Xia, Yiwei Wang, Li Liu, Le Xu, Bo Dai, Jianming Guo, Yu Zhu, Weijuan Zhang, Jiejie Xu, Quan Zhou, Yangyang Qi, Zewei Wang, Han Zeng, Hongyu Zhang, Zhaopei Liu, Qiuren Huang, Ying Xiong, Jiajun Wang, Yuan Chang, Qi Bai, Yu Xia, Yiwei Wang, Li Liu, Le Xu, Bo Dai, Jianming Guo, Yu Zhu, Weijuan Zhang, Jiejie Xu

Abstract

Background: Patients with BRCA1-associated protein 1 (BAP1)-mutant clear cell renal cell carcinoma (ccRCC) have worse prognosis. C-C chemokine receptor 5 (CCR5) plays an important role in ccRCC development and its expression is elevated in BAP1-mutant tumors.

Methods: 533 patients with ccRCC from The Cancer Genome Atlas cohort and 797 patients with ccRCC from the Shanghai cohort were enrolled. In vitro and in vivo studies were conducted with human ccRCC tumors and murine tumor models. The association between BAP1 and CCR5 or its ligands was assessed by immunohistochemistry, flow cytometry, real-time PCR and ELISA. Survival was compared between different subpopulations of patients using Kaplan-Meier curve. Therapeutic effect of CCR5 blockade was validated using human ccRCC tumors and murine models.

Results: Expression of CCR5 and its ligands were elevated in BAP1-mutant patients with ccRCC. High CCR5 expression was indicative of poor prognosis in BAP1-low group of patients. CCR5 blockade prolonged the survival of tumor-bearing mice, resulting in enhanced cytotoxicity of T cells and antigen presentation of dendritic cells but repressed immune checkpoint expression. CCR5 ligands could recruit CCR5+ regulatory T cells to the tumor microenvironment. Additionally, BAP1-mutant ccRCC tumor cells secreted CCR5 ligands, which increased programmed cell death ligand 1 expression. However, both processes could be inhibited by CCR5 blockade. Study limitations include the unclear impact of CCR5 expressed by other cell populations.

Conclusions: CCR5 in BAP1-mutant ccRCC results in an immune-suppressive microenvironment. Targeting CCR5 could provide a potential therapeutic benefit for patients.

Trial registration number: NCT01358721, CA209-009.

Keywords: immunology; oncology; urology.

Conflict of interest statement

Competing interests: None declared.

© Author(s) (or their employer(s)) 2020. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.

Figures

Figure 1
Figure 1
The expression of C-C chemokine receptor 5 (CCR5) and its ligands increases in BRCA1-associated protein 1 (BAP1)-mutant clear cell renal cell carcinoma (ccRCC). (A) Association between BAP1 RNA expression vs various chemokines and their receptors RNA expression in patients with ccRCC from The Cancer Genome Atlas (TCGA) cohort. ‘Coefficient with BAP1’ means the Spearman's rank correlation coefficients between chemokines or their receptors and BAP1, or ‘Spearman's Rho’. Death risk is the HRs of the overall survival (OS) calculated with Cox model by inputting continuous variables. (B) Association between chemokines and their receptors RNA expression vs risk of death in patients with ccRCC from the TCGA cohort. (C) CCL2-5, CCL8 and CCR5 mRNA expression levels in fresh BAP1-mutant and BAP1-wildtype ccRCC tumor specimens measured by real-time PCR (n=8 per group). Box-and-whisker diagrams were used (median, lower and upper quartiles; horizontal lines define min and max). (D) Representative images showing CCL2-5, CCL8 and CCR5 expression in BAP1-wildtype and BAP1-mutant ccRCC tumor specimens via immunohistochemistry. (E) Correlation between chemokines RNA expression and BAP1 RNA expression from the Shanghai cohort. (F) Left: CCL2-5, CCL8 expression levels in BAP1 knockdown tumor-bearing mice measured by ELISA; right: proportion of CCR5+ cells determined by flow cytometry (n=10 per group).
Figure 2
Figure 2
C-C chemokine receptor 5 (CCR5) antagonist prolongs survival and causes tumor cell death in BRCA1-associated protein 1 (BAP1)-mutant clear cell renal cell carcinoma (ccRCC) mice. (A) Overall survival (OS) and recurrence-free survival (RFS) curves for BAP1-low patients with ccRCC according to immunohistochemical CCR5 staining from a shanghai cohort. (B) Tumor volume from BALB/c mice subcutaneously injected with BAP1-knockdown RENCA cells and treated with anti-CCR5 or isotype (n=10 per group). (C) OS curve for BALB/c mice orthotopically injected with BAP1-knockdown RENCA cells and treated with anti-CCR5 or isotype antibodies (n=10 per group). (D) Typical images of mouse tumorous tissue sections with H&E staining after treatment with anti-CCR5 or isotype antibodies. (E) Left: dead tumor cell fraction in cultured human tumors treated with maraviroc or isotype antibodies (n=8 per group). Dead cells were identified as populations that stained positive for the viability dye (Zombie Violet for 405 nm excitation); right: typical images gated by Epcam+CD45− cells.
Figure 3
Figure 3
Blockade of C-C chemokine receptor 5 (CCR5) reactivates the antitumor immune response. (A) Number of intratumoral CD4+T, CD8+T, dendritic cell (DC), interferon (IFN)-γ+, GZMB+, PRF1+, CD80+, CD86+, MHC-II+, programmed cell death ligand 1 (PD-L1)+, programmed cell death 1 (PD-1)+, CTLA-4+ cells from BALB/c mice orthotopically injected with BRCA1-associated protein 1 (BAP1)-knockdown RENCA cells and treated with anti-CCR5 or isotype antibodies (n=10 per group). (B) Expression of cytotoxic cytokines, Ki67 and CD69 in CD4+ or CD8+ T cells and molecules related to antigen presentation in DC from cultured human tumors treated with maraviroc or isotype antibodies (n=8 per group). Measured by flow cytometry. (C) Interleukin-10 (IL-10), transforming growth factor-β (TGF-β), arginase 1 and inducible nitric oxide synthase (iNOS) levels measured by ELISA and enzyme activity assays in supernatants of tumor tissue cultures treated with maraviroc and the control. ns. not significant. *P<0.05; **p<0.01; ***p<0.001.
Figure 4
Figure 4
Blockade of CCR5 prevents the recruitment of CCR5+ Tregs. (A) The CIBERSORT method was performed to analyze the proportion of 22 immune cells among BAP1-mutant or BAP1-wildtype patients with ccRCC from The Cancer Genome Atlas cohort. (B) Number of Tregs, CCR5+ Tregs and CCR5− Tregs in BALB/c mice orthotopically injected with BAP1-knockdown RENCA cells and treated with anti-CCR5 or isotype antibodies (n=10 per group). (C) Representative images of CCR5+ Tregs and CCR5− Tregs, gated by CD3+CD4+CD25+ cells. (D) Migration of PBMC-derived CCR5+ Tregs in response to CCR5 ligands in the presence of maraviroc or isotype control was analyzed with a migration assay (n=5 per group). (E) PBMC-derived CCR5+ Tregs and CCR5− Tregs were isolated by FACS from BAP1-mutant patients with ccRCC and cultured for 3 days before measurement of interleukin-10 (IL-10) and transforming growth factor-β (TGF-β) concentrations in supernatants via ELISA (n=5 per group). ns. not significant. *P<0.05; **p<0.01; ***p<0.001.
Figure 5
Figure 5
Blockade of C-C chemokine receptor 5 (CCR5) represses the expression of programmed cell death ligand 1 (PD-L1) by tumor cells. (A) Proportion of PD-L1+ tumor and non-tumor cells in fresh BRCA1-associated protein 1 (BAP1)-mutant tumor specimens (n=5). (B) Left: number of PD-L1+ tumor and non-tumor cells from cultured human tumors treated with maraviroc or isotype antibodies (n=8 per group); right: typical image of flow cytometry. (C) Proportion of CCR5+ tumor and non-tumor cells in fresh BAP1-mutant tumor specimens (n=5). (D) Renal tumor cells were isolated from BAP1-mutant patients with clear cell renal cell carcinoma (ccRCC). One group was treated with maraviroc and the other group with isotype antibody. The PD-L1 expression level on the tumor cell surface was measured after 4 days of stimulation with CCR5 ligands in vitro. (A–D) All measured by flow cytometry. (E) Left: renal tumor cells were isolated from BAP1-wildtype and BAP1-mutant patients with ccRCC and cultured in vitro for 3 days and CCL2-5 and CCL8 secretion in supernatants was measured via ELISA; right: CCR5 expression level on the surface of renal tumor cells was measured via flow cytometry. (F) CCL2-5 and CCL8 secretion in culture supernatants of BAP1-knockdown RENCA cell line measured via ELISA. ns. not significant. *P<0.05; **p<0.01; ***p<0.001.

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