Lkb1 In Dendritic Cells Restricts CD8~+Foxp3~+Treg Expansion In Vivo And Twist1 Maintains Dendritic Cells Antitumor Immune Response

Objective: Objective: To explore the role and mechanism of Lkb1 in dendritic cells(DCs)regulating the expansion of CD8+ Foxp3+ regulatory T cells(Treg).Methods:(1)Cre-loxp knockout gene system was used to generate mice with Lkb1 conditional deletion in DCs(CD11c^Cre Lkb1f/f)through crossing CD11 c Cre mice with Lkb1f/f mice which were purchased from Jackson's laboratory.(2)Then we determined the ratio and number of CD8⁺Foxp3⁺Treg in lymphoid of Lkb1f/f mice and CD11 c Cre Lkb1f/f mice using flow cytometry.The ratios of CD8⁺Foxp3⁺Treg in other organs were aslo determined;(3)Moreover,we determined the expression of CD8⁺Foxp3⁺Treg surface activation markers including CD44 and ICOS in Lkb1f/f mice and CD11 c Cre Lkb1f/f mice;(4)The proliferation and apoptosis of CD8⁺Foxp3⁺Treg were also determined by flow cytometry;(5)Our Previous research showed that Lkb1 in DCs can regulate the differentiation and proliferation of CD4+Foxp3+Treg via inhibiting the OX40/OX40 L signaling pathway.In this study,we performed flow cytometry to detect the expression of OX40 in CD8 +T cell subgroups.The expressions of OX40 L in Lkb1f/f mice and CD11 c Cre Lkb1f/f mice were detected to further verify that Lkb1 in DCs regulates CD8⁺Foxp3⁺ Treg through the OX40/OX40 L signaling pathway;(6)In order to further study the effect of Lkb1-deficit DCs on CD8⁺Foxp3⁺ Treg,we performed LPS subcutaneously on Lkb1f/f mice and CD11 c Cre Lkb1f/f mice,respectively.LPS can specifically inhibit the expression of Lkb1 protein in DCs.CD8⁺Foxp3⁺ Treg in their lymph nodes were determined after 5 days later to verify the role of Lkb1 in DCs which help us to expand CD8⁺Foxp3⁺Treg in vivo.Results:(1)We successfully established Lkb1 knockout mice(CD11c^Cre Lkb1f/f mice),which can specifically knocked out Lkb1 in DCs,and verified that at the gene level;(2)We found that the proportion and number of CD8⁺Foxp3⁺Treg in lymph nodes of CD11 c Cre Lkb1f/f mice were significantly increased compared with that in Lkb1f/f mice.Similar to lymph nodes,CD8⁺Foxp3⁺Treg in other organs such as bone marrow, peripheral blood and lungs of CD11 c Cre Lkb1f/f mice were aslo significantly increased;(3)CD8⁺Foxp3⁺Treg in CD11 c Cre Lkb1f/f mice displayed higher expression of Nrp1 and Helios compared with that in Lkb1f/f mice,indicating that the increasing CD8⁺Foxp3⁺Treg from CD11 c Cre Lkb1f/f mice came from the thymus rather than the peripheral lymphoid organs.ICOS and CD44 are the surface activation markers of CD8⁺Foxp3⁺Treg and the expression of them in CD8⁺Foxp3⁺Treg from CD11 c Cre Lkb1f/f mice were higher than that in Lkb1f/f mice,indicating that more CD8⁺Foxp3⁺Treg from CD11 c Cre Lkb1f/f mice displayed a activated phenotype.The expression of CD103 is related to immunosuppressive function of CD8⁺Foxp3⁺Treg.CD8⁺Foxp3⁺Treg from CD11 c Cre Lkb1f/f mice exhibit higher expression of CD103 indicating that their inhibitory function is stronger;(4)Ki-67 is only expressed in proliferating cells.Both the number of Ki-67+cells and the mean fluorescence intensity(MFI)of Ki-67 among CD8⁺Foxp3⁺Treg increased compared to those from Lkb1f/f mice which indicated proliferation of CD8⁺Foxp3⁺Treg from CD11 c Cre Lkb1f/f mice was increased compared to those from Lkb1f/f mice.The results of apoptosis detection showed that CD8⁺Foxp3⁺Treg from CD11 c Cre Lkb1f/f mice displayed less prone apoptosis compared with that from Lkb1f/f mice.Thus,the increasing number of CD8⁺Foxp3⁺Treg from CD11 c Cre Lkb1f/f mice was due to the increased their proliferation ability and a decrease in apoptosis;(5)Previous research by our group showed that Lkb1 in DCs can regulate the number of CD4+Foxp3+Tregs via OX40/OX40 L signaling pathway,suggesting that Lkb1 in DCs might also regulate the number of CD8⁺Foxp3⁺Treg through the OX40/OX40 L signaling pathway.The expression of OX40 in CD8⁺Foxp3⁺Treg was the highest compared with other subgroups of CD8+T cells and the expression of OX40 L in DCs from CD11 c Cre Lkb1f/f mice was also significantly higher than that from Lkb1f/f mice;(6)As expected,we observed elevated percentage of CD8⁺Foxp3⁺Treg in Lkb1f/f mice under LPS stimulation from the lymph nodes.However,this increase was smaller than the difference between CD11 c Cre Lkb1f/f mice and Lkb1f/f mice.This is likely to be caused by Lkb1 protein still being present in the Lkb1f/f mice.Moreover,we found no significant difference in CD8⁺Foxp3⁺Treg comparing LPS-treated and non LPS-treated CD11 c Cre Lkb1f/f mice.Theses results further verified that Lkb1 in DCs can promote the proliferation and expansion of CD8⁺Foxp3⁺Treg.Conclusion:(1)Lkb1-deficient DCs can promote the proliferation of CD8⁺Foxp3⁺Treg and the increased CD8⁺Foxp3⁺Treg mainly were the natural Treg which come from the thymus;(2)Lkb1-deficient DCs promoted CD8⁺Foxp3⁺Treg exhibit a activated phenotype and can enhance the suppressive function of CD8⁺Foxp3⁺Treg.(3)The mechanism of Lkb1 in DCs regulating CD8⁺Foxp3⁺Treg and maintaining the body's immune homeostasis is partly through the OX40/OX40 L signaling pathway.(4)LPS which can specifically inhibit Lkb1 expression in DCs can also promote CD8⁺Foxp3⁺Treg expansion.Object: Objective: To explore the anti-tumor effect of Twist1 in dendritic cells(DCs)and its related mechanisms.Methods:(1)In order to study the regulatory effects of Twist1 on DCs,we first used CD11 c Cre mice and Twist1^f/f to generate Twist1 conditional deletion in DCs mice(CD11c^Cre Twist1^f/f)and Twist1^f/f mice as controls;(2)The number and phenotype of DCs in lymph nodes and spleen of CD11 c Cre Twist1^f/f mice and Twist1^f/f mice were determined by flow cytometry,and the surface markers CD80 and CD86 of DCs were detected simultaneously to determin the activation of DCs;(3)The main function of DCs is to initiate the T cell immune response or maintain the body's immune tolerance by regulating Treg pool.Thus we used flow cytometry to detect the activation status of T cells and Treg from CD11 c Cre Twist1^f/f mice and Twist1^f/f mice,respectively;(4)Melanoma is a kind of skin malignant tumor.Mouse melanoma cells B16-F12 are injected subcutaneously to construct a mouse tumor model.The length(a)and width(b)of the tumor were measured using a digital caliper,and then calculated volume using the formula V = 0.5 × a × b2.At the same time,in the tumor model,the ratio,number and activation markers of DCs in mouse lymph nodes were detected.Moreover,the T lymphocyte activation in lymph nodes also detected;(5)In order to further study the molecular mechanism of DCs function,we used magnetic bead sorting and flow sorting to sort DCs from CD11 c Cre Twist1^f/f mice and Twist1^f/f mice for RNA sequencing under steady state and tumor models,respectively.Differential genes and related pathways were analyzed using the gene enrichment analysis software GSEA;(6)In order to clarify the role of Twist1 in DCs,we analyzed the number and density of tumor infiltrating DCs and T cells in CD11 c Cre Twist1^f/f mice and Twist1^f/f mice under tumor models;(7)Furthermore,the cytokine including IFN-y from tumor infiltrating DCs and T cells were determined,simultaneously.Results: The appearance of CD11 c Cre Twist1^f/f mice was similar to that from Twist1^f/f mice.There was no significantl difference in body weight between them and CD11 c Cre Twist1^f/f mice have no obvious autoimmune disease.The results of flow cytometry showed that there was no significant difference in the proportion and number of DCs between CD11 c Cre Twist1^f/f mice and Twist1^f/f mice under steady-state conditions,and there was no abvious difference in the expression of activation markers such as CD80 and CD86.Moreover,T cell activation and Treg ratios displayed no difference between CD11 c Cre Twist1^f/f mice and Twist1^f/f mice.Interestingly,the ratio of alveolar macrophages in the lungs of CD11 c Cre Twist1^f/f mice was significantly increased.The results of the mouse melanoma tumor model exhibited that the tumor in CD11 c Cre Twist1^f/f mice is larger than that in Twist1^f/f mice,indicating that Twist-defecient DCs have anti-tumor defects.However,there was still no significant difference in the proportion and number of T cells and Treg between CD11 c Cre Twist1^f/f mice and Twist1^f/f mice.,and there was no significant difference in the activation status of T cells.DCs play an antigen presentation role in the anti-tumor immune response,and the number of DCs is related to the antigen presentation ability.We found that the numbers and proportions of DCs from draining lymph nodes in CD11 c Cre Twist1^f/f mice were significantly lower than those from Twist1^f/f mice,but the expressions of their activation markers such as CD80 and CD86 had no difference,indicating that the absence of Twist1 caused DCs antitumor defects due to the decreased number of DCs rather than the activation of DCs.At the same time,cytokine staining experiments showed that DCs and T in lymph node from CD11 c Cre Twist1^f/f mice and Twist1^f/f mice displayed no significant difference.Tumor infiltrating immune cells is one of the important factor to measure the anti-tumor ability.Both the tumor infiltrating DCs and T cells in CD11 c Cre Twist1^f/f mice were significantly reduced compared that in Twist1^f/f mice under tumor model.CD103+DCs are the most powerful DCs subpopulation for tumor antigen presentation.We found that the density of CD103+DCs in intra-tumors of CD11 c Cre Twist1^f/f mice was significantly lower than that in Twist1^f/f mice.Treg is an important factor that suppresses the body's anti-tumor immune response but there is no significant difference in the ratio of the number of Treg cells in tumors between CD11 c Cre Twist1^f/f mice and Twist1^f/f mice.INF-? is an important cytokine for T cells to exert anti-tumor immunity.We found that the expression of INF-? in T cells within intral-tumor in CD11 c Cre Twist1^f/f mice decreased significantly,indicating that the anti-tumor immune function of tumor-infiltrating T cells was impaired.The result of transcription profile analysis and gene enrichment software in steady state and tumor model exhibit that DCs from CD11 c Cre Twist1^f/f mice had more reactive oxygen radical(ROS)production and the related with signal pathway was activated,however,there was no significant difference in the signal pathway of ROS production in Twist1^f/f mice under steady-state conditions.In tumor models,genes such as Fas,Asns,Nurp1,Wnt11,and ROS were significantly increased,but there was no significant difference in steady state.Conclusion:1)The absence of Twist1 in DCs does not affect the number and activation of DCs uder steady state2)The absence of Twist1 in DCs does not affect the activation of T cells and Treg pool hematastasis.3)Twist1-defecient DCs exhibits impaired anti-tumor immunity,mainly due to the reduction numbers DCs in lymph nodes and within tumors4)Twist1-defecient DCs leads to the reduced tumor infiltrating T cells and impaired T cell function.5)The absence of Twist1 in DCs increased ROS production which promotes DCs apoptosis.