CD103~+DC Regulate The Activity Of NK Cells In Melanoma

ObjectiveAs heterogeneous population,dendric cells（DCs）are mainly divided into CD103⁺D-Cs and CD103^-DCs in non-lymphoid tissues.It has been reported that CD103^-DCs contrib-ute to anti-tumor immunity primarily by presenting antigen to CD8⁺T cells.However,whe-ther CD103⁺DCs are involved in anti-tumor immunity remain poorly understand.Accordi-ng to the ability of DCs to cross-talk with NK cells,we hypothesized that CD103⁺DCs mi-ght contribute to anti-tumor immunity by regulating NK cells.In this study,B16 cells were inoculated subcutaneously in C57BL/6 and Batf3^-/-mice to induce melanoma.We researched the role of CD103⁺DCs in melanoma as well as the regulation of infiltrated NK cells within the tumor by CD103⁺DCs.Methods1.We inoculated subcutaneously B16 cells（1?10⁶ per mouse）on the back of C57BL-/6 and Batf3^-/-mice.Then we measured dynamically the diameter of melanoma to make the tumor growth curve.14 days later,mice were sacrificed and tumors were taken photos and weighed.These experiments were designed to explore the role of CD103⁺DCs in anti-tum-or immunity.2.2.43 hybridoma cells were cultured to collect supernatant,which were then intrap-ertoneally injected into C57BL/6 and Batf3^-/-mice every two days（3ml per mouse）to dele-te CD8⁺T cells in vivo.We also cultivated PK136 hybridoma cells to collect supernatant a-nd intraperitoneally injected them into C57BL/6 and Batf3^-/-mice（3ml per mouse）for 3 d-ays to delete NK cells in vivo.Mice were then inoculated subcutaneously by B16 cells and diameters of melanoma were measured at indicated time to make the tumor growth curve.These experiments were designed to explore how CD103⁺DCs regulate CD8⁺T cells and NK cells in anti-tumor immunity.3.We tested the cytotoxicity of splenic NK cells against B16 cells by FACS with e450/7-AAD double staining in vitro.4.To explore whether CD103⁺DCs influences the infiltration of NK cells in the tumor,we tested the number of NK cells within the tumor of C57BL/6 and Batf3^-/-mice by FACS.We also extracted the mRNA of the tumor to detect the expression level of proinflammator-y cytokines and certain chemokines that have the potential to regulate NK cells.5.To explore how CD103⁺DCs influence the function of NK cells,we tested the sub-sets of NK cells within the tumor and examined the FasL and surface receptors expression on NK cells.Results1.Four days after inoculation of B16 cells,we could touch tumor on the back of mice.12 days later,there had a difference on the tumor size between WT mice and Batf3^-/-mice.After 14 days of inoculation,the size and weight of tumor were bigger in Batf3^-/-mice than that in WT mice.The data indicated that tumor grew faster in Batf3^-/-mice compared with WT mice.2.After clearing CD8⁺T cells in vivo,tumor grew faster in WT mice,indicating the anti-tumor immune response mediated by CD8⁺T cells.However,depletion of CD8⁺T cells did not affect tumor growth in Batf3^-/-mice.This data implied that activity of CD8⁺T cells were dependent on CD103⁺DCs and CD103⁺DCs may regulate CD8⁺T cells in the upstream.Importantly,there displayed an accelerated tumor growth in Batf3^-/-mice compared with CD8⁺T cell-depleted mice,which suggested that CD103⁺DCs participated in anti-tumor response not only by regulating CD8⁺T cells.Through analysis of tumor growth after depletion of NK cells in vivo,our data also showed that NK cells contributed to anti-tumor immune response.Similarly,the growth rate of melanoma did not change in Batf3^-/-mice in spite of NK cell depletion.So,the date suggested that NK cell-mediated anti-tumor effect were regulated by CD103⁺DCs in the upstream.3.The cytotoxicity of splenic NK cells in Batf3^-/-mice was significantly impaired compared with that in C57BL/6 mice in vitro,indicating that CD103⁺DC could promote the cytotoxicity of NK cells.4.We next tested the number of NK cells within the tumor of C57BL/6 and Batf3^-/-mice,and found that tumor-infiltrated NK cells significant decreased in Batf3^-/-mice when compared with that in WT mice.We also detected the expression level of chemokines for recruiting NK cells,and found that there existed significant decrease in XCL1 and CXCL10 mRAN expression.Additionally,our data showed that mRNA expression of TNF-αand IFN-γin tumor significantly decreased in Batf3^-/-mice compared with WT mice.These results suggested that deficiency of CD103⁺DCs could affect tumor immune microenvironment,which would regulate NK cells infiltration.5.Through FACS analysis,we found that there had two NK cell subsets in melanoma,including CD49a⁺DX5^-NK subset and CD49a^-DX5⁺NK subset.In Batf3^-/-mice with melonoma,CD49⁺DX5^-NK subset expressed lower Fasl and activating receptor 2B4 and NKG2D;while CD49a^-DX5⁺NK subset expressed lower activiating receptor 2B4,and higher inhibitory receptor KIRG1 and NKG2A.These results indicated that CD103⁺DCs could affect the expression of surface receptor on NK cells within the tumor.This regulation may lead to predominant inhibitory signals for NK cells,which helps to promote tumor genesis.ConclusionCD103⁺DC plays an important role in the anti-tumor immunity by regulating NK cells.In melanoma,there has two NK cell subsets,including CD49a⁺DX5^-NK subset and CD49a^-DX5⁺NK subset.CD103⁺DC could affect not only the number of NK cells within the tumor but the function of NK cells.