Regulation Of Stress-induced T Cell Exhaustion In Cancer Immunosurveillance By Kisspeptin/GPR54 Signaling

The fast pace of life and increasing work pressure causes substantial mental depression and endocrine disorders.Epidemiological studies shows that chronic psychological stress is associated with the poor prognosis of cancer patients.Although the role of the neuroendocrine system on tumorigenesis and development has been partially clarified,the underlying mechanism of stress-induced tumorigenesis and tumor suppressive immune microenvironment need further exploration.To investigate the influence of mental disorders on the immune system,we adopted acute restraint（AR）to construct chronic mental stress mice model and analyzed the change of immune cells in the spleen.The results showed that the ratio of DCs and macrophage were comparable after AR treatment,but the ratio of splenic central memory T cells(T_cm)and effector memory T cells(T_em)were significantly reduced in AR-mice.To investigate the influence of T cells in chronic mental stress,splenic T cells were analyzed by RNA-seq.AR treatment significantly upregulated Gpr54 expression in T cells,whereas T cell activation-related genes were downregulated significantly.In addition,AR treatment increased the level of plasma neuropeptide hormones kisspeptin and the expression of its receptor Gpr54 in the hypothalamus and splenic,suggesting that kisspeptin/GPR54 signaling plays an important role in AR-induced imbalances of neuroendocrine and immune homeostasis.To further investigate the potential impact of neuroendocrine on cancer development and the tumor microenvironment,AR-treated mice were implanted with tumors.AR treatment promoted tumor growth,increased serum levels of kisspeptin,and enhanced Gpr54 expression in tumor-infiltrating lymphocytes.Meanwhile,AR treatment decreased the ratio of CD4⁺and CD8⁺T cells in the tumor immune microenvironment.Additionally,CD8⁺T cells in the tumor of the AR group showed increased expression of exhausted genes.These data imply that kisspeptin/GPR54 signaling plays a dominant role in neuroendocrine imbalance facilitated tumor growth.To clarify the role of kisspeptin/GPR54 on tumorigenesis and development,we analyzed the correlation between GPR54 expression and tumor prognosis in clinical samples.The bioinformatic analysis showed that GPR54 expression is negatively associated with CD8⁺T cell infiltration in lung adenocarcinoma（LUAD）and lung squamous cell carcinoma（LUSC）.Meanwhile,higher GPR54 expression from lung adenocarcinoma patients significantly correlated with poor prognosis.Accordingly,administration of kisspeptin-10（KP-10）significantly impaired T cell function and promoted T cell exhaustion.These data demonstrate that kisspeptin/GPR54 signaling may be an important indicator of immune escape in lung cancer.To elucidate the influence of Gpr54 deprivation on T cell function,we constructed Gpr54^{fl/fl CD4Cre} mice using the CRISPR/Cas9 system.Gpr54^{fl/fl CD4Cre} mice were subjected to chronic stress and subcutaneous LLC tumors.We found that T cell conditional deletion of Gpr54 inhibited T cell dysfunction and exhaustion,impaired lung cancer growth,in both chronic mental stress and normal mice.To further explore whether GPR54 is involved in CD8⁺T cell-mediated cytotoxicity,Gpr54 knockout OT-1 mice were used for verification.Chronic stress impaired OT-1 CD8⁺T cell-mediated cytotoxicity to OVA positive tumor cells,and CD8⁺T cells showed a better killing effect at low effector-to-target ratios after Gpr54 deletion.These results suggest that GPR54has a superior inhibitory effect on CD8⁺T cell-mediated anti-tumor immune function.Mechanistically,Gpr54 conditional deletion did not affect the phosphorylation of LCK,Src,and Zap70,but significantly impaired exhaustion-related gene NR4A1expression.In addition,kisspeptin promoted ionomycin-induced ERK5phosphorylation,and this activation could be restricted by chelating intracellular Ca²⁺through 2-APB.Furthermore,Gpr54 conditional deletion in T cells also decreased ionomycin-induced phosphorylation of ERK5.Meanwhile,pharmacologic inhibition of ERK5 signaling by XMD8-92 significantly reduced tumor growth by enhancing CD8⁺T cell antitumor function.These results imply that ERK5 mediated NR4A1activation was found essential for kisspeptin/GPR54 facilitated T cell dysfunction.To further examine the potential value of GPR54 in cancer immunotherapy,we generated an adoptive transfer model using Gpr54 knockout and wildtype OT-1 cells.Phenotypically,adoptively transferred Gpr54 knockout T cells impaired LLC-OVA tumor growth and decreased the expression of inhibitory receptors in CD8⁺T cells.To further explore the clinical application of GPR54 and ERK5,we used T cells from healthy donors to construct CD19-CAR-T and PSMA-CAR-T cells,and knock out GPR54 and ERK5 genes respectively through the CRISPR/Cas9 system.Knockout of GPR54 enhanced PSMA-CAR-T cell proliferation and cytokine release,as well as increased the cytotoxicity to tumor cells.Accordingly,PC3-PSMA cells induced CAR-T cell exhaustion was also decreased in GPR54 deleted PSMA-CAR-T cells.In addition,ERK5 depletion in PSMA-CAR-T or CD19-CAR-T cells also enhanced cytotoxicity to tumor cells and increased cytokine release.Moreover,ERK5^-/-CAR-T cells presented a lower exhausted phenotype.These results suggest that GPR54 or ERK5 depletion increases T cell-mediated cytotoxicity in TCR-T or CAR-T cell therapy which has great clinical potential in adoptive cell therapy.In summary,we demonstrate the crucial role of kisspeptin/GPR54 signaling in chronic stress-induced immune suppression in the tumor microenvironment.And depletion of GPR54 or downstream ERK5 signaling not only rescues the chronic stress facilitated tumor growth but also restricts T cell exhaustion,and strengthens T cell-mediated immune therapy.Thus,our data provide an important theoretical basis for a better understanding of the regulation of neuroendocrine-immune homeostasis and tumor immune escape.Furthermore,our data also provide new targets and ideas for T cell-based tumor immune cell therapy.