Study On The Immunosuppressive Function Of CD73~+ NK Cells

Objective:The innate immunity constitutes the first line of defense against invading pathogens and provides the fundamentals of the immune system.NK cells are a group of innate lymphoid cells that can directly eliminate malignant cells without prior stimulation.They can also trigger anti-tumor immune responses by secreting IFN-γ.CD73⁺NK cells are a recently discovered subset of NK cells with immunosuppressive function,and their role remains to be studied.This study explores the way CD73⁺NK cells exert immunosuppressive effects,to provide a theoretical basis for the clinical application of CD73⁺NK cells under different conditions.Method:（1）The expression of key adenosine signaling pathway genes in HNSCC was analyzed using the TCGA database.The relationship between CD73,a key enzyme for adenosine production,and HNSCC patient survival was investigated.These bioinformatics findings were validated by flow cytometric analysis of CD73 expression in primary HNSCC tissues.（2）HNSCC cell lines were co-cultured in vitro with NK cells isolated from healthy human peripheral blood.Differences in the biological behaviors of CD73⁺and CD73^-NK cells were compared by assessing the effects of HNSCC cell lines on CD73 surface expression on NK cells.（3）The expression of HIF-1α,a putative upstream regulator of CD73,in HNSCC was analyzed using the TCGA database.Relevant mechanisms by which CD73⁺NK cells contribute to HNSCC pathogenesis were explored by simulating hypoxic tumor microenvironment conditions in vitro.（4）To further investigate the safety of allogeneic mesenchymal stem cell therapy,NK cells were co-cultured with bone marrow-derived and dental pulp-derived mesenchymal stem cells.The effects on the biological functions of allogeneic NK cells were studied.（5）By examining the effects of the CD73 metabolite adenosine on NK cell cytotoxicity and expression of activating and inhibitory surface receptors,we aimed to delineate the potential mechanisms by which CD73⁺NK cells contribute to the efficacy of allogeneic mesenchymal stem cell therapy for autoimmune diseases.Results:（1）Expression analysis of primary HNSCC tissues revealed upregulation of NT5E（encoding CD73）,ENTPD1（encoding CD39）,and the adenosine receptors ADORA2A and ADORA2B in the adenosine pathway.In contrast,expression of the adenosine receptor ADORA1 was downregulated.Elevated NT5E expression in HNSCC patients was significantly associated with reduced overall survival,disease-specific survival,and progression-free interval.Immune profiling indicated increased infiltration of NK cells and dendritic cells in HNSCC tumors with high NT5E expression.A significant positive correlation was observed between intratumoral NK cell abundance and NT5E expression.Flow cytometric analysis of OSCC tissues demonstrated CD73 surface expression on tumor-infiltrating NK cells.（2）Compared to CD73^-NK cells,CD73⁺NK cells exhibited increased expression of the inhibitory receptors NKG2A and KLRB1 and decreased expression of the activating receptors NKP30 and NKp44.CD73⁺NK cells also displayed reduced cytotoxicity,Fas L expression,and IFN-γsecretion relative to CD73^-NK cells.Conversely,CD73⁺NK cells showed greater production of the immunosuppressive cytokine IL-10 versus CD73^-NK cells.Collectively,these data demonstrate that CD73 expression on NK cells is associated with diminished anti-tumor functionality.（3）Bioinformatics analysis revealed elevated expression of the hypoxia-inducible factor HIF-1A in HNSCC tissues.A positive correlation between HIF-1A and NT5E expression was observed in HNSCC.Simulating hypoxic conditions in vitro with CoCl₂ treatment induced increased CD73 expression on NK cells,indicating hypoxia as a driver of CD73 upregulation.（4）Co-culture experiments demonstrated bidirectional interactions between mesenchymal stem cells（MSCs）and NK cells.Both dental pulp stem cells（DPSCs）and bone marrow-derived MSCs（BMMSCs）inhibited NK cell proliferation and promoted NK cell apoptosis.Reciprocally,activated NK cells exhibited cytotoxicity against DPSCs.MSC co-culture led to decreased expression of the NK cell activation markers CD69,DNAM-1,NKp30,NKp44,NKp46,and NKG2D,increased expression of the inhibitory receptor CD158b,and acquisition of CD73 expression by NK cells.（5）CD73⁺NK cells can catalyze the conversion of extracellular ATP to adenosine via surface CD39 and CD73.Co-culture with the adenosine analog 2-chloroadenosine significantly impaired NK cell cytotoxicity,supporting a role for CD73-generated adenosine in inhibiting NK cell function.Conclusions:（1）Our findings suggest the adenosine signaling pathway may play an important role in HNSCC pathogenesis and prognosis,as high CD73expression significantly correlated with poor clinical outcomes.CD73⁺NK cells represent a subset of tumor-infiltrating regulatory NK cells that appear intricately linked with HNSCC development.The HIF-1α-CD73signaling axis appears to be a key driver of increased intratumoral CD73⁺NK cells in HNSCC.（2）Co-culture experiments demonstrated that CD73 expression can be acquired by NK cells following interactions with mesenchymal stem cells.These CD73⁺NK cells are capable of converting pro-inflammatory ATP to immunosuppressive adenosine via surface CD39/CD73,thereby inhibiting their effector functions in an autocrine manner.This represents a potential mechanism by which mesenchymal stem cell therapy may modulate allogeneic NK cell responses.Overall,these findings elucidate CD73 expression on NK cells as a critical determinant of anti-tumor and alloreactive responses,with important implications for prognostication and therapeutic manipulation in cancer and autoimmunity.Further investigation of the factors governing CD73 induction and activity in NK cells is warranted.