Molecular Mechanism Of Differentiation Of Breg-like Cells Induced By Tumor-derived Lactic Acid

Objective:Bregs are a collective term for a group of B cells with immune regulatory functions,lacking specific cell surface markers.Recent studies have found that Bregs are involved in immune regulation in autoimmune diseases,infections,tumors and other diseases.Bregs play a tumor-promoting effect through a variety of mechanisms,but the differentiation mechanism of Bregs in the tumor microenvironment is still unclear.As a metabolite of tumor cells,lactic acid plays an important role in tumor immune tolerance and immune escape by affecting a variety of immune cell functions.However,the research on the effect of lactic acid on the differentiation and function of B cells is less and unclear.Through the study of the effect of lactic acid on the immune phenotype and function of B cells and its mechanism,combined with the immunophenotype,percentage and activity of B cells,CD8⁺T cells and other immune cells in the LDHA KD D121 cells lung adenocarcinoma model in mice,we explained the role and mechanism of lactic acid in inducing B cells to differentiate into Breg-like cells and promoting tumor progression.Methods:(1)Magnetic beads were used to sort mouse spleen B lymphocytes.BAFF,BAFF combined with lactic acid were used to culture B lymphocytes for 2 days,respectively.The effect of lactic acid on the expression of Bregs related phenotypes in B cells was detected by flow cytometry.Magnetic beads were used to sort mouse spleen CD4⁺and CD8⁺T lymphocytes.B lymphocytes were co-cultured with CFSE-labeled and CD3,CD28-stimulated CD4⁺and CD8⁺T lymphocytes for 2-3 days.Flow cytometric evaluate the effects of B lymphocytes treated with lactic acid on T cell proliferation.(2)BAFF,BAFF combined with lactic acid were used to culture mouse spleen B lymphocytes for 2 days,respectively.QRT-PCR detected the m RNA expressions of IL-10?PPAR?and NADPH oxidase-related subunits in B cells of two groups.The expression differences of IL-10,TGF?and ROS in B cells of the two groups were detected by flow cytometry.Western blot detected the expression of p40phox and p47phox proteins in the two groups of B cells.(3)Magnetic beads sort mouse spleen B.B cells were cultured with BAFF,BAFF+lactic acid,BAFF+GPR81 agonist,BAFF+lactic acid+GPR81 antagonist,BAFF+lactic acid+PPAR?antagonist for 2 days,respectively.The effects of different treatment methods on the immunosuppressive function of B cells were compared.Add anti-IL-10 antibody,anti-TGF?antibody,anti-PD-L1 antibody,NIL,Nor-NOHA or catalase to the co-culture system of T cells and B cells treated with lactic acid in vitro,flow cytometric evaluate the influence of the above-mentioned molecules on the immune regulation function of lactic acid cultured B cells to determine the molecular mechanism of immunosuppressive function.(4)Use lentiviral CRISPR/cas9 technology to knock down the LDHA gene in D121cells,and subcutaneously inoculate B6 mice with NC and LDHA KD D121 cells to obtain lung adenocarcinoma mouse models.The tumor sizes were measured and recorded to evaluate the effect of LDHA KD on tumor growth.The expression of ROS in B cells and the percentages of CD8⁺INF-?⁺T cells,CD8⁺Gr B⁺T cells,CD25⁺CD81⁺B,IL-10⁺B,Tregs and MDSCs in spleen and tumor of two groups tumor-bearing mice were detected by flow cytometry.After intratumoral B cells between the two groups were sorted out,the m RNA expression levels of NADPH enzyme related subunits were compared by q PCR.(5)Flow cytometry detect the ratio of CD24^hiCD38^hiB cells and IL-10⁺B cells in the peripheral blood of lung cancer patients and healthy controls.Magnetic beads were used to sort the peripheral blood T and B lymphocytes of healthy control,and the B lymphocytes were cultured with lactic acid for 2 days.Then B lymphocytes and CFSE-labeled CD3,CD28 stimulated T lymphocytes were co-cultured for 3-5 days.Flow cytometric evaluate the effects of lactic acid on the expression of B lymphocytes IL-10,ROS,CD24,and CD38.Flow cytometric evaluate the effect of lactic acid treated B cells on the proliferation of T lymphocytes.The co-localization relationship between LDHA and ROS⁺CD19⁺B cells in lung adenocarcinoma tissue and adjacent tissues were determined by immunofluorescence staining to determine the effect of lactic acid on the differentiation of Breg-like cells.Results:(1)Lactic acid upregulated the expression of CD25 and CD81 in mouse B cells,resulting in a Breg-like phenotype of B cells.(2)Breg-like cells induced by lactic acid significantly inhibit the proliferation of T cells.The immunosuppressive function of Breg-like cells induced by lactic acid has nothing to do with IL-10,TGF?,PD-L1,arg-1,i NOS,IDO and other effector molecules.(3)Lactic acid induces high expression of ROS in mouse B cells through NADPH enzyme,and B cells cultured with lactic acid exert the immunoregulatory function of inhibiting T cell proliferation through ROS.(4)Lactic acid induces high expression of GPR81 in mouse B cells.GPR81 agonist3,5-DHBA can induce high expression of ROS in B cells,and B cells cultured with3,5-DHBA also have the function of inhibiting T cell proliferation.GPR81 inhibitor 3-OBA can reverse the immunomodulatory function of lactic acid cultured B cells,suggesting that lactic acid induces mouse B cells to exert immunomodulatory function by activating GPR81receptor.(5)Lactic acid promotes the expression of PPAR?in mouse B cells by binding to the GPR81 receptor and subsequently induces an increase in the expression level of ROS.PPAR?antagonist can reverse the expression levels of ROS and the immunoregulatory function of lactic acid-cultured B cells,suggesting that lactic acid induces differentiation of Breg-like cells through GPR81-PPAR?-ROS pathway.(6)LDHA knockdown reduced tumor volume in tumor-bearing mice,decreased Ki-67expression in tumor tissue,decreased proportion of CD25⁺CD81⁺B cells and MDSCs in spleen and tumor,and decreased the expression level of NADPH oxidase related subunit m RNA and ROS in B cells in tumor tissue.The percentage of CD8⁺INF-?⁺T cells and CD8⁺Gr B⁺T cells in draining lymph nodes,spleen and tumor increased,while the percentage of Tregs in tumor decreased.(7)Peripheral blood lactic acid levels,and the percentage of CD24^hiCD38^hiB cells and IL-10⁺B cells in patients with lung adenocarcinoma are higher than those in healthy controls.Lactic acid induces high expression of ROS,CD24,CD38 and IL-10 phenotypes in human peripheral blood B cells.Lactic acid cultured B cells inhibit the proliferation of T cells through ROS.LDHA expression level in human lung adenocarcinoma tissue is higher than that in adjacent tissues,and is co-localized with ROS⁺CD19⁺B cells.Conclusions:Lactic acid induces B cells to differentiate into Breg-like cells through GPR81/PPAR?/NADPH enzyme signals.Those Breg-like cells inhibits the proliferation of T cells by ROS,thereby promoting tumor progression.Knockdown of LDHA in tumor cells reduces the production of Breg-like cells and its expression of ROS,suggesting that targeted reduction of lactic acid in the tumor microenvironment can reduce the differentiation of Breg-like cells and play an anti-tumor therapeutic effect.This provides a new theoretical basis for the pathogenic effect of Breg cells in tumors and provides new ideas for clinical treatment of tumors.