Specific Decrease In B-cell-derived Extracellular Vesicles Enhances Post-Chemotherapeutic CD8~+ T Cell Responses And The Underlying Mechanisms

Chemotherapy remains one of the most classical treatments for advanced cancers.Chemotherapeutic agents have been reported to enhance antitumor immunity through eliminating immunosuppressive cells or triggering ’immunogenic cell death’ of tumor cells.However,systemic immunosuppression greatly affects the chemotherapeutic antitumor effect.Therefore,amelioration of systemic immunosuppression following chemotherapy is necessary to improve post-chemotherapeutic antitumor immunity.It is well established that extracellular adenosine inhibits T lymphocyte activation and effector function by signaling primarily through A2A adenosine receptors(A2AR)on the surface of T cells.CD39(ectonucleoside triphosphate diphosphohydrolase 1-Entpd1)hydrolyzes ATP and ADP to AMP.AMP can be futher hydrolyzed to adenosine by CD73(ecto-5’-nucleotidase-Nt5e).Chemotherapy induces massive release of ATP from tumor cells and ATP can be hydrolyzed to adenosine by CD39 and CD73 which probably results in inhibition of anti-tumor T cell responses.Extracellular vesicles(EVs)are closely related to regulation of immune functions.Whether CD39+CD73+EVs can hydrolyze ATP from chemotherapy-treated tumor cells into adenosine and affect antitumor immunity remains unclear.Through analyzing the extracellular vesicles obtained from B cells,we find that CD19+ EVs contain high levels of CD39 and CD73.It is confirmed that CD19+EVs can hydrolyze ATP to adenosine and inhibit the proliferation of CD8+ T cells in the presence of ATP.And tumor-bearing mice show bad chemotherapy effect with CD19+EVs treatment.Through building tumor model in thymus-immune-deficient nude mice and using anti-CD4 or CD8 in C57/BL6 mice,it is confirmed that CD19+EVs impair chemotherapy effect depending on CD8+ T cells.Through analyzing tumor infiltrating lymphocytes(TIL),it was further confirmed that CD19+EVs attenuated anti-tumor effect post-chemotherapy mainly by inhibiting the activation and proliferation of CD8+T cells.By comparing serum and splenic CD19+ EVs levels between normal and tumor mice,we find that tumor mice have higher levels of CD19+EVs,due to upregulation of EV release associated protein Rab27a.Through RNA-seq analysis of B cells from normal and tumor mice,we screen hypoxia-inducible factor HIF-la which increases in tumor mice.Through chromatin immunoprecipitation and luciferase reporter experiments,it is confirmed that HIF-1α could bind to Rab27a promoter and further promote Rab27a transcription,and thus promote B cells to release more CD19+EVs and attenuate the anti-tumor effect of chemotherapy.In addition,serum CD19+EVs levels of tumor patients are measured.Compared to healthy people,tumor patients have higher levels of CD19+EVs.And serum CD19+EVs levels of tumor patients are correlated with patients’ chemotherapy effect.Analysis shows that tumor patients with relatively higher levels of CD19+ EVs have shorter progression-free survival.And most patients show a decline in serum CD19+ EVs after tumor tissue removing surgery.Finally,through observation of tumor size and survival rate,anti-tumor effect of chemotherapy is significantly improved by inactivated Epstein-Barr virus(iEBV)carrying Rab27a siRNA in humanized NSG mice.Altogether,our findings provide new insights into the regulation of B cells in post-chemotherapeutic T-cell responses and still reveal a novel mechanism by which tumor cells escape from immune surveillance following chemotherapy and may aid in the design of new antitumor therapies.