CCR5 blockade inflames antitumor immunity in BAP1-mutant clear cell renal cell carcinoma
https://jitc.bmj.com/content/8/1/e000228
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.
C-C chemokine receptor 5 (CCR5) is found to associate with renal cell carcinoma (RCC) development. It was reported that CCR5 and its ligands was significantly upregulated in high-stage or high-grade tumors.9 Thus, CCR5 might be a potential therapeutic target.
Moreover, we found that the use of anti-CCR5 antibody caused extensive necrosis of tumor cells (figure 2D) and a similar result was observed by flow cytometry analysis.
CCR5 blockade activates the antitumor immunity in BAP1-mutant ccRCC
We found that the proportions of tumor-infiltrating CD8+ T cells and dendritic cells (DCs) were increased in mice treated with anti-CCR5 antibody and the corresponding proportions of interferon (IFN)-γ+, GZMB+, PRF1+, CD80+ and CD86+ cells were also increased, indicating enhanced cell cytotoxicity and antigen presentation (figure 3A).
Meanwhile, the proportions of programmed cell death ligand 1 (PD-L1)+ and CTLA-4+ cells were significantly decreased (figure 3A).
the proportion of CD4+ T cells secreting the functional effector molecule IFN-γ and CD8+ T cells secreting IFN-γ and GZMB or expressing CD107a were significantly increased. The number of DCs increased together with elevated expression of CD80 and CD86;
The proliferation ability of CD8+ T cells and DCs were enhanced as well (figure 3B). In addition, interleukin-10 (IL-10), transforming growth factor-β (TGF-β), arginase and inducible nitric oxide synthase expression levels in tumor supernatants were measured. We found that the expression levels of all these inhibitory molecules decreased following maraviroc treatment (figure 3C).
CCR5 blockade suppresses the recruitment of regulatory T cells
A previous study indicated that CCR5 was partially expressed on the surface of Tregs and blockade of CCR5 affected the recruitment of Tregs.19 Likewise, we found that the total number of Tregs, especially CCR5+ Tregs, was decreased after treatment with the CCR5-blocking antibody compared with the control group, but no obvious change was found in CCR5− Tregs (figure 4B,C).
CCR5 blockade represses PD-L1 expression by tumor cells in BAP1-mutant ccRCC
Given that research has shown positive PD-L1 expression in tumor cells20 and that our study revealed a decreased number of tumor-infiltrating PD-L1+ cells after treatment with the CCR5-blocking antibody (figure 3A), we analyzed the distribution of PD-L1 in BAP1-mutant tumor tissues and found that tumor cells had a significantly higher proportion of PD-L1+ cells than non-tumor cells (figure 5A). The proportion of PD-L1+ tumor cells decreased, while the proportion of PD-L1+ non-tumor cells did not change substantially after maraviroc treatment (figure 5B).
Interestingly, tumor cells also included a relatively high proportion of CCR5+ cells (figure 5C). We stimulated the isolated BAP1-mutant renal tumor cells with CCR5 ligands in vitro with addition of maraviroc. Subsequent analysis revealed CCL5 could significantly enhanced PD-L1 expression by tumor cells, followed by CCL3 and CCL8. However, this effect was reversed after blocking CCR5 (figure 5D). Moreover, blockade of CCR5 caused PD-L1 expression to decrease without addition of exogenous chemokines.
Previous studies have reported the high expression of CCR5 and its ligand CCL5 in basal and HER-2 breast cancer, revealing that CCR5 plays a crucial role in cancer progression.11 23 Taken together, these results suggested that targeting CCR5 might serve as a novel strategy in cancer treatment.
In the present study, we noted that immunosuppressive microenvironment was interrupted after CCR5 blockade. Specifically, the number of cells related to cytotoxicity and antigen presentation was increased, while the number of cells mediating immunosuppression was decreased. In vitro studies revealed an elevated expression of functional effector molecules by lymphocytes and antigen-presenting molecules by DCs after maraviroc treatment.
Taken together, these results indicated that CCR5 blockade could enhance the ability of the host immune system to eliminate tumor cells.
Similarly, Zhang et al found that colon cancer cells recruited CCR5+ Tregs to local tumors by secreting CCL5, which in turn suppressed the cytotoxicity of CD8+ T cells and mediated immune evasion.19 28
https://jitc.bmj.com/content/8/1/e000228
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.
C-C chemokine receptor 5 (CCR5) is found to associate with renal cell carcinoma (RCC) development. It was reported that CCR5 and its ligands was significantly upregulated in high-stage or high-grade tumors.9 Thus, CCR5 might be a potential therapeutic target.
Moreover, we found that the use of anti-CCR5 antibody caused extensive necrosis of tumor cells (figure 2D) and a similar result was observed by flow cytometry analysis.
CCR5 blockade activates the antitumor immunity in BAP1-mutant ccRCC
We found that the proportions of tumor-infiltrating CD8+ T cells and dendritic cells (DCs) were increased in mice treated with anti-CCR5 antibody and the corresponding proportions of interferon (IFN)-γ+, GZMB+, PRF1+, CD80+ and CD86+ cells were also increased, indicating enhanced cell cytotoxicity and antigen presentation (figure 3A).
Meanwhile, the proportions of programmed cell death ligand 1 (PD-L1)+ and CTLA-4+ cells were significantly decreased (figure 3A).
the proportion of CD4+ T cells secreting the functional effector molecule IFN-γ and CD8+ T cells secreting IFN-γ and GZMB or expressing CD107a were significantly increased. The number of DCs increased together with elevated expression of CD80 and CD86;
The proliferation ability of CD8+ T cells and DCs were enhanced as well (figure 3B). In addition, interleukin-10 (IL-10), transforming growth factor-β (TGF-β), arginase and inducible nitric oxide synthase expression levels in tumor supernatants were measured. We found that the expression levels of all these inhibitory molecules decreased following maraviroc treatment (figure 3C).
CCR5 blockade suppresses the recruitment of regulatory T cells
A previous study indicated that CCR5 was partially expressed on the surface of Tregs and blockade of CCR5 affected the recruitment of Tregs.19 Likewise, we found that the total number of Tregs, especially CCR5+ Tregs, was decreased after treatment with the CCR5-blocking antibody compared with the control group, but no obvious change was found in CCR5− Tregs (figure 4B,C).
CCR5 blockade represses PD-L1 expression by tumor cells in BAP1-mutant ccRCC
Given that research has shown positive PD-L1 expression in tumor cells20 and that our study revealed a decreased number of tumor-infiltrating PD-L1+ cells after treatment with the CCR5-blocking antibody (figure 3A), we analyzed the distribution of PD-L1 in BAP1-mutant tumor tissues and found that tumor cells had a significantly higher proportion of PD-L1+ cells than non-tumor cells (figure 5A). The proportion of PD-L1+ tumor cells decreased, while the proportion of PD-L1+ non-tumor cells did not change substantially after maraviroc treatment (figure 5B).
Interestingly, tumor cells also included a relatively high proportion of CCR5+ cells (figure 5C). We stimulated the isolated BAP1-mutant renal tumor cells with CCR5 ligands in vitro with addition of maraviroc. Subsequent analysis revealed CCL5 could significantly enhanced PD-L1 expression by tumor cells, followed by CCL3 and CCL8. However, this effect was reversed after blocking CCR5 (figure 5D). Moreover, blockade of CCR5 caused PD-L1 expression to decrease without addition of exogenous chemokines.
Previous studies have reported the high expression of CCR5 and its ligand CCL5 in basal and HER-2 breast cancer, revealing that CCR5 plays a crucial role in cancer progression.11 23 Taken together, these results suggested that targeting CCR5 might serve as a novel strategy in cancer treatment.
In the present study, we noted that immunosuppressive microenvironment was interrupted after CCR5 blockade. Specifically, the number of cells related to cytotoxicity and antigen presentation was increased, while the number of cells mediating immunosuppression was decreased. In vitro studies revealed an elevated expression of functional effector molecules by lymphocytes and antigen-presenting molecules by DCs after maraviroc treatment.
Taken together, these results indicated that CCR5 blockade could enhance the ability of the host immune system to eliminate tumor cells.
Similarly, Zhang et al found that colon cancer cells recruited CCR5+ Tregs to local tumors by secreting CCL5, which in turn suppressed the cytotoxicity of CD8+ T cells and mediated immune evasion.19 28
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