Study On The Co-expression Characteristics And Effects Of LAG3 And PD-1 On The T Cells Of Breast Cancer Patients

Objective:1.To investigate the co-expression characteristics of negative costimulatory molecules LAG3 and PD-1 on the T cells of breast cancer patients.2.To analyze the effect of co-expression of LAG3 and PD-1 on T cell function.3.To investigate the effects of different blocking treatment strategies of LAG3 and PD-1 on the development of breast cancer.Methods:1.According to inclusion and exclusion criteria,peripheral blood and tumor tissues of patients with breast cancer were collected.The co-expressions of LAG3 and PD-1 on the T cells in peripheral blood,tumor infiltrating and pericarcinomatous tissue infiltrating ones were analyzed by flow cytometry,and the co-expression of LAG3 and PD-1 on T cells were analyzed by stratification according to clinical stages and molecular subtypes;2.To detect the proliferation and secretion functions of T cells with different expressions of LAG3 and PD-1,the CFSE staining,BrdU incorporation and ELISA method were used respectively;3.Immunohistochemical method was used to analyze the different expression of MHC classⅡmolecule,FGL1 and PD-L1 in breast cancer tissue and pericarcinomatous tissue,which are the main ligand of LAG3and PD-1,respectively,and then their expression difference in different clinical stages and different molecular subtypes were analyzed;4.Through the breast cancer modeling of EMT6 and E0771 mice,single or combined monoclonal antibody blocking treatment of LAG3 and PD-1 was conducted,respectively,to observe the tumor growth of the mice,which would reversely verify the effect of co-expression of LAG3 and PD-1 on the development of breast cancer.Results:1.After screening by informed consent,inclusion and exclusion criteria,a total of 112 breast cancer patients were enrolled,including 82cases of peripheral blood samples,47 cases of patients with cancer tissues and pericarcinomatous tissue,and 18 cases of healthy controls.There was no significant difference in age among the three groups(P>0.05).2.There was no significant difference in the percentage of LAG3 and PD-1 double positive T lymphocytes in peripheral blood lymphocytes(PBL),tumor infiltrating lymphocytes(TIL)and pericarcinomatous tissue infiltrating lymphocytes(PIL)in patients with different clinical stages(P>0.05).However,in PIL and TIL of patients with different clinical stages,the proportion of LAG3 and PD-1 double positive CD8~+T cells was higher than that of CD4~+T cells(P<0.05).3.The percentage of LAG3 and PD-1 double positive T lymphocytes in PBL,PIL and TIL of patients with different molecular subtypes of breast cancer was significantly different(P<0.01),which had the highest proportion in the triple negative breast cancer group(TNBC),and the lowest percentage in the estrogen/progesterone receptor-positive group(ER~+/PR~+)(P<0.01).In the same molecular subtyping,the percentages of LAG3 and PD-1 double positive CD8~+T cells were higher than that of CD4~+T cells(P<0.05).4.Are found by CFSE staining and BrdU incorporation method,the proliferation ratio of LAG3 and PD-1 double positive T cells after stimulation is lowest,about 80%were still in the parent generation which were not divided,and the proliferation of LAG3 and PD-1 double negative T cells were most active,only about 10%of which were not divided,while LAG3 or PD-1 single positive T cells were about 50%of the cells were not divided.Through comparative analysis,it was found that the proliferation function of these T cells with different phenotypes was significantly different(P<0.01).5.Enzyme-linked immunosorbent assay(ELISA)was used to detect cytokine secretion capacity of T cells with different phenotypes.After the stimulation,the levels of IL-2 and IL-6 secreted by CD4~+T cells with LAG3 and PD-1 double positive and the levels of IFN-γ,TNF-αsecreted by CD8~+T cells with LAG3 and PD-1 double positive were significantly lower than other groups(P<0.01).6.Immunohistochemical analysis showed that ligands of LAG3,including MHC class II molecule and FGL1,and the ligand of PD-1(PD-L1),had different expression levels in cancer tissues and pericarcinomatous tissue(P<0.05).The expression levels of MHC class II molecule in pericarcinomatous tissues were higher than those in cancer tissues(P<0.05),while the expression levels of FGL1 and PD-L1 in pericarcinomatous tissues were lower than those in cancer tissues(P<0.05).7.In breast cancer tissues,the expression levels of MHC class II molecule,FGL1 and PD-L1 were different in different clinical stages.There was no statistical difference between clinical stage I and II(P>0.05),but there was significant difference compared with clinical stage III(P<0.05).8.In breast cancer tissues,the expression levels of MHC class II molecules,FGL1 and PD-L1 were also different in different molecular subtypes of tumors(P<0.05).Except for MHC class II molecules,the expression levels of FGL1 and PD-L1 were the highest in triple negative breast cancer(P<0.05).9.In the mouse models of triple negative breast cancer,the tumor volume and tumor weight of mice treated with anti-PD-1 and anti-LAG3combined blocking treatment were significantly smaller and lower,respectively,than those of mice treated with anti-PD-1 or anti-LAG3 alone blocking treatment(P<0.05),and also significantly smaller than those of control group(P<0.05).Conclusion:1.The proportion of LAG3 and PD-1 double positive T lymphocytes in breast cancer presented a trend of gradual increase from peripheral blood to pericarcinomatous tissues and then to cancer tissues,and there was no difference in peripheral blood.However,there were differences between the cancer tissues and the pericarcinomatous tissues,which were related to the molecular subtypes,the highest percentage in the three negative breast cancer,and the lowest percentage in the ER~+/PR~+breast cancer.2.Compared with those of PD-1 or LAG3 positive T cells,the function of LAG3 and PD-1 double positive T cells were significantly exhausted,showing synergistic inhibitory effect between PD-1 and LAG3.3.The expression levels of FGL1 and PD-L1,the ligand of LAG3 and PD-1 respectively,were different in different clinical stages and molecular subtyping cancer tissues.The later the overall staging was,the higher the expression level was.In the molecular subtyping,the expression level of TNBC was the highest and the lowest in ER~+/PR~+breast cancer,while MHC class II molecular showed the opposite expression.4.For triple-negative breast cancer,the combined use of anti-LAG3and anti-PD-1 has a better therapeutic effect than the use of the two alone,which can be used as an important reference strategy for future immunoregulatory treatment of triple-negative breast cancer.