MDSCs-derived GPR84 induces CD8+ T-cell senescence via p53 activation to suppress the antitumor response

Abstract

Backgrounds: G-protein-coupled receptor 84 (GPR84) marks a subset of myeloid-derived suppressor cells (MDSCs) with stronger immunosuppression in the tumor microenvironment. Yet, how GPR84 endowed the stronger inhibition of MDSCs to CD8⁺ T cells function is not well established. In this study, we aimed to identify the underlying mechanism behind the immunosuppression of CD8⁺ T cells by GPR84⁺ MDSCs.

Methods: The role and underlying mechanism that MDSCs or exosomes (Exo) regulates the function of CD8⁺ T cells were investigated using immunofluorescence, fluorescence activating cell sorter (FACS), quantitative real-time PCR, western blot, ELISA, Confocal, RNA-sequencing (RNA-seq), etc. In vivo efficacy and mechanistic studies were conducted with wild type, GPR84 and p53 knockout C57/BL6 mice.

Results: Here, we showed that the transfer of GPR84 from MDSCs to CD8⁺ T cells via the Exo attenuated the antitumor response. This inhibitory effect was also observed in GPR84-overexpressed CD8⁺ T cells, whereas depleting GPR84 elevated CD8⁺ T cells proliferation and function in vitro and in vivo. RNA-seq analysis of CD8⁺ T cells demonstrated the activation of the p53 signaling pathway in CD8⁺ T cells treated with GPR84⁺ MDSCs culture medium. While knockout p53 did not induce senescence in CD8⁺ T cells treated with GPR84⁺ MDSCs. The per cent of GPR84⁺ CD8⁺ T cells work as a negative indicator for patients' prognosis and response to chemotherapy.

Conclusions: These data demonstrated that the transfer of GPR84 from MDSCs to CD8⁺ T cells induces T-cell senescence via the p53 signaling pathway, which could explain the strong immunosuppression of GPR84 endowed to MDSCs.

Keywords: CD8+T cells; GPR84; MDSCs; exosome; p53 signaling pathway.

Figure 1

MDSCs inhibits CD8⁺ T-cell cytotoxicity depends on the GPR84 transfer. (A) The apoptotic rates of B16-OVA cells co-cultured with OT-I CD8⁺ T cells and GPR84^+/– MDSCs for 6 hours (CD8⁺ T cells: MDSCs: tumor cells=1:4:1). CD8⁺ T cells and GPR84^+/− MDSCs were co-cultured for 24 hours. Flow cytometry was used to analyze the proliferation (B) and function (C) of CD8⁺ T cells (CD8⁺ T cells: MDSCs=1:4). (D) The percentage of GPR84⁺ CD8⁺ T cells before and after being co-cultured with MDSCs (CD8⁺ T cells: MDSCs=1:4) was examined using flow cytometry. (E) The ratio of GPR84⁺ CD8⁺ T cells in blood and tumor tissues was detected in mice models bearing LLC cells. (F) CD8⁺ T cells were purified from C57 mice spleens after activation with an anti-CD3/CD28 antibody; the GPR84 plasmid was transfected, and GPR84-overexpressed CD8⁺ T cells were constructed. The transfection efficiency was examined using qRT-PCR and western blotting. Flow cytometry was used to analyze the proliferation (G) and function (H) of mock or GPR84-overexpressed CD8⁺ T cells. (I–J) The expression levels of functional and exhaustion-related genes on GPR84 overexpressed or mock CD8⁺ T cells were examined using qRT-PCR. Data were represented in at least three independent experiments. Data were presented as the mean±SEM. ns, not significant, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. GPR84, G-protein-coupled receptor 84; MDSC, myeloid-derived suppressor cell; qRT-PCR, quantitative real-time PCR.

Figure 2

Loss of GPR84 enhances antitumor immunity of CD8⁺ T cells. GPR84, G-protein-coupled receptor 84; MDSC, myeloid-derived suppressor cell.

Figure 3

GPR84 suppresses the infiltration and reduces the antitumor ability of CD8⁺T cells. GPR84, G-protein-coupled receptor 84; IFN, interferon; IL, interleukin; MACS, magnet-activated cell sorting; s.c., subcutaneous under the skin; WT, wild type.

Figure 4

MDSCs transfer GPR84 to CD8⁺ T cells in an exosome-dependent way to repress CD8⁺ T-cell function. (A) After co-culturing with WT or GPR84^-/-MDSCs for 48 hours, the expression of GPR84 on CD8⁺ T cells was determined using quantitative real-time PCR (qRT-PCR). (B–C) CD8⁺ T cells were purified from GPR84-KO mice and co-cultured with GPR84⁺ MDSCs. Then, flow cytometry (B) and imaging flow cytometry (C) were used to analyze the expression of GPR84 on CD8⁺ T cells. (D–E) Co-cultured CD8⁺ T cells with WT or GPR84^–/– MDSCs directly (D) or indirectly (E). Flow cytometry was used to analyze the percentage of GPR84⁺CD8⁺ T cells at 48 hours and 72 hours. (F) CD8⁺ T cells were cultured in the cultured supernatant of WT or GPR84^–/– MDSCs. Flow cytometry and imaging flow cytometry were used to analyze the expression of GPR84 on CD8⁺ T cells. (G) Flow cytometry was used to analyze the percentage of GPR84⁺ MDSCs after co-culturing with or without CD8⁺ T cells. (H) GPR84⁺ MDSCs were pretreated with GW4869 for 2 hours and then co-cultured with CD8⁺ T cells. After 48 hours, the percentage of GPR84⁺CD8⁺ T cells was analyzed using flow cytometry. (I) The morphology of exosomes obtained from WT or GPR84^–/– MDSCs was detected using electron microscopy. The size distribution of the exosomes obtained from WT or GPR84^–/– MDSCs was measured by using NTA assay. (J) The expression levels of Tsg101, CD9, GPR84, and GAPDH in MDSCs and MDSC-derived exosomes were examined using western blotting. (K) Representative images of PKH-67 (green) on CD8⁺ T cells co-cultured for 24 hours with PKH-67-tagged MDSC-derived exosomes. Nucleus was counter-stained with Hoechst (blue). (Bar, 10 µm). (L) CD8⁺ T cells were co-cultured with WT or GPR84^–/– MDSCs-derived exosome for 24 hours. Flow cytometry was used to analyze the expression of GPR84 on CD8⁺ T cells. (M) CD8⁺ T cells were co-cultured with GPR84 antibody-tagged MDSCs for 24 hours. Then, flow cytometry was used to analyze the expression of GPR84 on CD8⁺ T cells. (N) GPR84⁺ MDSCs were pretreated with GW4869 for 2 hours and then co-cultured with CD8⁺ T cells. The killing ability (N) proliferation (O) and function-related cytokines (P) of CD8⁺ T cells were analyzed using flow cytometry. Data were represented in at least three independent experiments. Data were presented as the mean±SEM. ns, not significant, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. GPR84, G-protein-coupled receptor 84; IFN, interferon; IL, interleukin; MDSC, myeloid-derived suppressor cell; NTA, nanoparticle tracking analysis; WT, wild type.

Figure 5

GPR84 induces the senescence of CD8⁺ T cells via the p53 pathway. CM, cultured medium; DAPI, 4,6-diamino-2-phenyl indole; GPR84, G-protein-coupled receptor 84; KEGG, Kyoto Encyclopedia of Genes and Genomes; MDSC, myeloid-derived suppressor cell; ROS, reactive oxygen species.

Figure 6

Clinical significance of GPR84 levels in CD8⁺ T cells. (A–C) Overall survival of skin cutaneous melanoma (SKCM), lung adenocarcinoma (LUAD), and lung squamous cell cancer (LUSC) was analyzed in GPR84^high CD8^high and GPR84^low CD8^high groups based on the data from The Cancer Genome Atlas. (D) The percentage of CD8⁺ T and GPR84⁺ CD8⁺ T cells in peripheral blood mononuclear cells from healthy donors (n=50) and patients with cancer (n=50) was analyzed using flow cytometry. (E) The correlation between CD8⁺ T cells and GPR84⁺ MDSCs was analyzed based on the flow cytometry data from healthy donors (n=63). (F) The correlation between GPR84⁺ CD8⁺ T cells and GPR84⁺ MDSCs was analyzed based on the flow cytometry data from healthy donors (n=63). (G) The expression of function-related markers in GPR84^high and GPR84^low CD8⁺ T cells was analyzed using flow cytometry from healthy donors (n=63). (H) The percentages of MDSCs, GPR84⁺ MDSCs, CD8⁺ T cells, and GPR84⁺ CD8⁺ T cells were analyzed in chemotherapy-resistant (n=11) or sensitive (n=11) patients with cancer using flow cytometry. Data were represented in at least three independent experiments. Data were presented as the mean±SEM. ns, not significant, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. GPR84, G-protein-coupled receptor 84; HD, healthy donor; IFN, interferon; IL, interleukin; MDSC, myeloid-derived suppressor cell; TNF, tumor necrosis factor.