| Myeloid-derived suppressor cells(MDSCs)represent a heterogeneous population of immature myeloid cells consisting of precursors for granulocytes,macrophages or dendritic cells that are accumulated during chronic inflammation and tumor progression.In tumorbearing mice,MDSCs are characterized by a CD11b+Grl+ phenotype,and can be further classified into granulocytic(PMN-MDSCs,CD11b+Ly6G+Ly6Clow)and monocytic(MMDSCs,CD11b+Ly6ChighLy6G-)subpopulations.In cancer patient,MDSCs are characterized by a CD33+HLA-DR-phenotype,and can be further classified into PMN-MDSCs(CD33+HLA-DR-CD66b+CD14-)and M-MDSCs(CD33+HLA-DR-CD14+CD66b-).PMNMDSCs and M-MDSCs inhibit T cell function.PMN-MDSCs has increased NADPH oxidase(Nox)activity,which results in high levels of ROS and low levels of nitric oxide(NO)production,and expressed arginase 1.M-MDSCs express high levels of NO and arginase 1,but show low ROS production.Lymphocyte adaptor protein(Lnk),also known as SH2B3,is a member of the SH2B family and a lymphocyte adaptor protein with no enzyme activity.Lnk is highly expressed in hematopoietic stem cells,endothelial cells,cancer epithelial cells.Studies have shown that Lnk is a key negative modulator of the hematopoietic receptor signaling pathway activated by growth factors,while it was not clear whether Lnk can regulate the differentiation and function of MDSCs.Our research group constructed subcutaneous transplant tumor models of B16F10 melanoma and 3LL lung cancer in WT and Lnk-/-mice,and found that MDSCs expressed Lnk;Lnk-/-mice noted slower tumor growth and reduced tumor weight;The proportion of splenic and GM-CSF-induced Lnk-/-MDSCs decreased;Lnk-/-MDSCs decreased expression of ARG1 and increased secretion of TNF-α.These results suggested that Lnk may inhibit tumor growth by regulating the differentiation and function of MDSCs.This paper further focused on the differentiation and function of MDSCs and discussed the possible mechanism.Previous studies showed that MDSCs expressed Lnk in mice.Then,we explored the expression of Lnk in MDSCs of clinical cancer patients.Compared with healthy donors,the proportions of MDSCs and PMN-MDSCs in PBMCs of lung cancer patients were increased,while the proportion of M-MDSCs was not significant different.After,the Lnk expression of MDSCs in Peripheral blood of healthy donors and lung cancer patients was determined by flow cytometry.The results showed that the expression of Lnk in MDSCs may be closely related to the occurrence of cancer.Next,we examined the percentages of associated immune cells in spleens between tumorfree WT and Lnk-/-mice.We found that the percentages of associated immune cells in spleens were not different between tumor-free WT and Lnk-/-mice by flow cytometry.Subsequently,the proportion and number of associated immune cells in spleens and tumor tissues of WT and Lnk-/-B16F10 and 3LL tumor-bearing mice were measured by flow cytometry and immunofluorescence.Compared with WT mice,the percentages and numbers of MDSCs,mainly PMN-MDSCs,in spleens and tumor tissues of tumor-bearing Lnk-/-mice were decreased.Besides,the percentages of MDSCs were also decreased in the Peripheral Blood(PB),Lymph Node(LN)and Bone Marrow(BM),of B16F 10-bearing Lnk-/-mice,compared with those of WT controls.To explore the mechanism of the decrease of MDSCs,we discussed from three aspects:decreased proliferation,increased apoptosis and decreased differentiation ability.The results showed that Lnk deficiency did not affect the proliferation and apoptosis of MDSCs,while the ability of bone marrow differentiation into MDSCs,mainly PMNMDSCs,was decreased.These results indicated that Lnk deletion reduces the accumulation through differentiation.To explore the influence of Lnk deficiency on the phenotype and function of MDSCs(subsets),we constructed a B16F10 melanoma tumor model in WT and Lnk-/-mice.After 15 days,splenic MDSCs were taken for RNA sequencing(RNA-Seq).We found that Lnk deficiency up-regulated gene expression of immunostimulatory phenotype,and downregulated gene expression of immunosuppressive phenotype in MDSCs.By flow cytometry,compared with WT MDSCs,the expression levels of MHC Ⅰ and MHC Ⅱ were increased in splenic MDSCs of 3LL tumor bearing Lnk-/-mice.Next,we examined the expression of MDSCs(subsets)cytokine and enzyme activity for further verification.Compared with WT mice,ROS production in splenic MDSCs in 3LL-bearing Lnk-/-mice did not change significantly;the activities of NOS and Arglfrom splenic MDSCs in 3LL-bearing Lnk-/-mice were decreased,mainly decreased by PMN-MDSCs;the expressions of IFN-γ and TNF-αfrom splenic MDSCs in 3LL-bearing Lnk-/-mice were increased,increased by PMN-MDSCs and M-MDSCs.We further examined the effect of MDSCs(subsets)on T cell function.Compared with WT MDSCs,Lnk-/-MDSCs had no significant effect on the proliferation level of CD4+T/CD8+T cells.However,Lnk-/-MDSCs,mainly Lnk-/-PMN-MDSCs,showed loss inhibition of IFN-γ secretion by CD8+T cells.In order to explore the mechanism that Lnkdeficient MDSCs(subsets)failed to inhibit IFN-γ production from CD8+T cells,we selected splenic MDSCs/PMN-MDSCs from 3LL tumor-bearing WT and Lnk-/-mice were co-cultured with anti-CD3/CD28 activated CD8+T cells in a ratio of 1:2,adding iNOS inhibitor SMT or Argl inhibitor nor-NOHA.The ability of CD8+T cells to produce IFN-γ was detected by intracellular staining flow cytometry after 2 days.The results showed that WT MDSCs/PMNMDSCs failed to inhibit IFN-γ production from CD8+T cells after adding SMT or nor-NOHA,while Lnk-/-MDSCs/PMN-MDSCs could not inhibit IFN-γ production from CD8+T cells,with or without SMT or nor-NOHA.Next,we found that Lnk-deficient BM-derived MDSCs showed decreased NOS and Argl activity,increased TNF-α and IFN-γ expression,and decreased the inhibition to IFN-γ production from CD8+T cells.To further confirm whether Lnk-deficient MDSCs possess antitumorigenic properties,splenic MDSCs in 3LL tumorbearing WT and Lnk-/-mice were adopted back into tumor-bearing WT mice through tail vein,then we observed tumor growth.Compared with adoptive transfer of WT MDSCs,adoptive transfer of Lnk-/-MDSCs did not promote tumor growth.These results suggested that Lnk-/MDSCs weakened immunosuppressive function and did not promote tumor progression.Next,we primarily explored the mechanism regulating the function of Lnk-deficient MDSCs.KEGG classification analysis of MDSCs differential genes was performed using RNA-Seq data.KEGG Classification analysis identified that MAPK signaling pathway,JAK/STAT signaling pathway and PI3K-Akt signaling pathway were enriched in Lnk-/MDSCs.We extracted MDSCs total proteins from spleen of 3LL-bearing WT and Lnk-/-mice,and detected MAPK signaling pathway activation by Western blot.The results showed Lnk-/MDSCs increased the expressed of p-p38 and p-ERK,compared with WT MDSCs.After p38 or ERK inhibitor were added respectively for 24 h,we examined the secretion of TNF-α from MDSCs.The result showed that Lnk-/-MDSCs decreased TNF-α secretion,after added p38 or ERK inhibitor.These results suggested that splenic MDSCs in tumor-bearing Lnk-/-mice can promote the secretion of TNF-α by activating p38 and ERK.In conclusion,Lnk deficiency can lose tumorigenic properties by inhibiting the function of MDSCs.Lnk deficiency regulated the function of MDSCs through the ERK and p38 pathways. |
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