Phenotypic Characteristics And Function Of Exhausted NKG2A~+CD8~+ T Cells In Adenomyosis

Chapter Ⅰ:Analysis of local immune cell subsets in adenomyosisBackground:Adenomyosis(AM)is a common and refractory gynecological disease in which the endometrial glands and mesenchyme invade the myometrium to form a limited or diffuse lesion.The disease often occurs in women of childbearing age.The main clinical symptoms include severe dysmenorrhea,menorrhagia,and infertility,which seriously affect the physical and mental health of patients.Currently,treatment options for this disease are limited and there is much controversy surrounding conservative treatment.The pathogenesis of adenomyosis is complex and many theories have been proposed,but they do not yet provide a complete explanation of the heterogeneity of the clinical features of this disease.Therefore,exploring the specific pathogenesis of adenomyosis is particularly necessary for the early identification,diagnosis,and prevention of patients.Immune dysregulation has long been recognized as an important pathogenesis of adenomyosis,with multiple immune cell counts being locally abnormal in the uterus of patients with adenomyosis.However,previous studies have mostly used immunohistochemical staining techniques to identify different immune cell subpopulations,and given the limitations of quantifying cell numbers,previous studies have reported a high degree of heterogeneity and even conflicting immune cell alterations in adenomyosis.An overall objective evaluation of the localized immune cell dysregulation of the uterine microenvironment in patients with adenomyosis is still lacking.Therefore,there is a need to further refine the identification of immune cell alterations in the local uterine microenvironment of patients with adenomyosis to identify abnormal subgroups and their pathogenic functional phenotypes.Objective:To identify changes in the immune cell subsets of the local uterine microenvironment in patients with adenomyosis,to investigate the correlation between abnormal immune cell subsets and the clinicopathological features of patients with adenomyosis,and to screen for immune cell subsets closely related to the development of adenomyosis.Methods:1.Flow cytometry was applied to detect differences in the proportion and absolute number of immune cell subpopulations in the same type of tissue(eutopic endometrium vs.control endometrium,ectopic lesions vs.control myometrium)between patients with adenomyosis and control patients with uterine fibroids.2.Correlation analysis was used to clarify the correlation between alterations in immune cell subsets and the clinicopathological features of patients with adenomyosis.Results:1.Flow cytometry showed that,compared with the control endometrium and myometrium,the proportion and absolute number of CD45+total immune cells in eutopic endometrium and ectopic lesions of adenomyosis were not significantly different,but the proportion and absolute number of CD3+T cells in ectopic lesions of adenomyosis were significantly increased.2.For T cell subsets,it was found that CD8+T cells in ectopic lesions of adenomyosis accounted for an increased proportion of CD45+total immune cells and CD3+T cells,and their absolute number also increased significantly.However,there was no significant difference in CD4+T cells among different groups,and the ratio of CD4+T/CD8+T cells decreased in ectopic lesions of adenomyosis.3.For the subset of CD8+T cells,it was found that CD8+Mucosal-associated invariant T cells(CD8+MAIT)accounted for a low proportion of total CD8+T cells in uterine tissue.There was no significant difference in the proportion and absolute number of CD8+MAIT cells between adenomyosis and control group.The proportion and absolute number of CD103+CD8+T cells in ectopic lesions of adenomyosis were significantly increased compared with the control myometrium.4.Correlation analysis revealed that the number of CD3+T cells and CD8+T cells were significantly higher in patients with diffuse adenomyosis than in patients with focal adenomyosis.In addition,CD3+T and CD8+T cell counts were positively correlated with diffuse adenomyosis and serum level of CA125.5.In addition to T cells,a decrease in the proportion of B cells in the ectopic lesions of adenomyosis was found,but the absolute number was not statistically different;the proportion and number of NKT cells in the in eutopic endometrium of adenomyosis were increased.However,there was no significant difference or correlation between the number of B cells in ectopic lesions and the number of NKT cells in eutopic endometrium and clinicopathologic features.Conclusions:1.There are multiple dysregulated immune cell subsets in the local uterine microenvironment in adenomyosis,among which the increase in the number of CD8+T cells is the most important change of immune cell subsets in ectopic lesions of patients with adenomyosis.2.The increased number of CD8+T cells correlated with the clinicopathological features of the disease and had tissue-resident properties,suggesting a potential pathogenic role for CD8+T cells in the pathogenesis of uterine adenomyosis.Chapter Ⅱ:Phenotype and function of exhausted NKG2A+CD8+T cells in adenomyosis Background:In Chapter 1,we found that the number of CD8+ T cells increased in ectopic lesions of adenomyosis,which was correlated with the clinicopathological features of the disease,suggesting that CD8+T cells may be involved in the occurrence and development of adenomyosis.As a typical killer cell,CD8+T cells increased could not prevent abnormal accumulation and retention of ectopic endometrial cells in the pathological site,the exact mechanism of which is not clear.Previous studies have suggested that in addition to alterations in cell numbers,the functional status of immune cells is also closely related to disease development.However,whether the dysfunction of CD8+T cells may lead to abnormal aggregation and retention of ectopic endometrial cells at pathological sites remains unclear,so it is necessary to further explore the pathogenic phenotype of CD8+T cells in adenomyosis.CD8+T-cell function deteriorates in response to sustained exposure to antigenic and/or inflammatory signals,which is referred to as T-cell exhaustion.During T-cell exhaustion,CD8+T cells upregulate a variety of negative immune checkpoint(IC)molecules and downregulate cytokine production.Recently,natural killer group protein 2A(NKG2A)has been identified as a novel IC molecule that is selectively expressed on NK and CD8+T cells and forms a heterodimeric receptor with CD94;binding of CD94/NKG2A to its ligand HLA-E induces NK and T cell exhaustion,thereby promote tumor progression.Uterine adenomyosis is also a chronic inflammatory disease with dysregulation of multiple inflammatory signals.Therefore,we suspect that prolonged dysregulation of inflammatory signaling leading to CD8+T-cell exhaustion may be associated with the development of adenomyosis.Whether dysregulation of the HLA-E/NKG2A axis leads to CD8+T-cell exhaustion in patients with adenomyosis remains to be further explored.Objective:To determine whether exhausted NKG2A+CD8+T cells exist in adenomyosis,investigate their relationship with the clinicopathological features of the disease,and explore the related immune microenvironment changes leading to CD8+T-cell exhaustion.Methods:1.Flow cytometry was applied to detect the difference in expression of CD94/NKG2A on NK and T cells in peripheral blood,eutopic endometrium,and ectopic lesions of patients with adenomyosis;to detect differences in NKG2A expression on CD8+T cells in uterine tissues of patients with adenomyosis and controls;and to analyze the phenotypic characteristics and functions of NKG2A+CD8+T cell subsets.2.Correlation analysis was used to clarify the correlation between the proportion and number of NKG2A+CD8+T cells and the clinicopathological characteristics of patients with adenomyosis.3.Immunohistochemistry was used to detect the expression levels of HLA-E,IL-15,and TGF-β in eutopic endometrium,ectopic lesions of adenomyosis and control endometrial tissue.4.CD8+ T cells were sorted from peripheral blood using a flow sorting technique,cultured in vitro using cytokines IL-15 and TGF-β,and flow cytometry was applied to detect changes in NKG2A expression on CD8+T cells.5.Immunofluorescence was used to detect the expression and localization of CD8,NKG2A,HLA-E,and IL-15 in ectopic lesions of adenomyosis.Results:1.Flow cytometry showed that the expression of NKG2A was positively correlated with that of CD94.Heterodimeric CD94/NKG2A was expressed enriched on tissue-resident CD8+T cells in adenomyosis lesions.2.Analysis of changes in the number of NKG2A+CD8+T cells revealed that the proportion and the absolute number of this cell subpopulation were significantly increased in both eutopic endometrium and ectopic lesions in adenomyosis compared to control endometrium and myometrium.3.Analysis of their phenotypic characteristics and function revealed that NKG2A+CD8+T cells expressed significantly higher levels of the negative checkpoint molecules PD-1,LAG-3,TIM-3,and key regulator of exhaustion TOX,but had decreased production of perforin,granzyme B and CD 107a.4.Correlation analysis showed that the proportion and number of NKG2A+CD8+T cells were significantly higher in patients with diffuse adenomyosis than in patients with focal adenomyosis.In addition,the proportion and number of NKG2A+CD8+T cells were positively correlated with diffuse adenomyosis and serum level of CA125.5.Immunohistochemical results showed that HLA-E,IL-15,and TGF-β were significantly more expressed in eutopic endometrium and ectopic lesions in patients with adenomyosis compared to control endometrium.Moreover,HLA-E,IL-15,and TGF-β were expressed at higher levels in the ectopic lesions than in their paired eutopic endometrium.6.Results after in vitro culture showed that exogenous rhIL-15 significantly upregulated NKG2A expression on CD8+T cells,while rhTGF-β treatment did not significantly alter NKG2A expression.7.Multiple immunofluorescence staining revealed that glandular epithelial cells from ectopic lesions of adenomyosis co-expressed IL-15 and HLA-E,and NKG2A+CD8+T cells were closely distributed around ectopic glands co-expressing IL-15 and HLA-E.Conclusion:1.NKG2A was mainly expressed on tissue-resident CD8+T cells in adenomyosis lesions,and the expression of NKG2A increased on CD8+T cells in ectopic lesions,which was correlated with the clinicopathological features,suggesting that NKG2A+CD8+T cells may be involved in the development of adenomyosis.2.The exhaustion of NKG2A+CD8+ T cells in ectopic lesions of adenomyosis may be the reason that the endometrial cells infiltrating into the myometrium cannot be cleared.3.Increased expression of IL-15 and HLA-E in glandular epithelial cells in the adenomyotic microenvironment may lead to CD8+T cell exhaustion by promoting NKG2A expression on CD8+ T cells or inhibiting the effector function of these cells.Chapter Ⅲ:Exploring the therapeutic strategies for targeting NKG2A blocking in a mouse model of adenomyosisBackground:The studies in the previous two chapters identified abnormally increased HLA-E/NKG2A axis signaling in adenomyosis,which may be responsible for the exhaustion of CD8+T cells and consequently the inability to prevent abnormal aggregation and retention of ectopic endometrial cells in pathological sites.In recent years,the use of checkpoint inhibitors has revolutionized cancer immunotherapy,and NKG2A as a new immune checkpoint molecule has received much attention.Studies have shown that blocking the binding of NKG2A to its ligand using antibodies can promote the anti-tumor effects of NK and T cells.In recent years,a number of studies blocking the HLA-E/NKG2A axis have yielded good results,and this has provided research directions for immunotherapeutic strategies for adenomyosis.As it is difficult to obtain CD8+T cells from uterine tissues,animal models were used to initially investigate the therapeutic effects of blocking the HLA-E/NKG2A axis in adenomyosis.Objective:To investigate the therapeutic effect of blocking the HLA-E/NKG2A axis using an anti-NKG2A antibody in a mouse model of adenomyosis.Methods:1.The mouse model of ICR adenomyosis was established by oral tamoxifen method,and the model mice were divided into two groups,which were intraperitoneally injected with anti-NKG2A(anti-NKG2A group)and PBS(PBS group),respectively.2.The general morphology of the mouse uterus was used to identify the model construction and to evaluate the effectiveness of the treatment.3.H&E staining was applied to identify model construction as well as to detect the score of the degree of uterine infiltration.4.Masson trichrome staining was applied to detect collagen volume fraction in the myometrium.5.Flow cytometry was applied to detect alterations in immune cell subsets in the uterus of adenomyosis mice and normal mice;to detect differences in NKG2A expression on CD8+T cells in the uterus of adenomyosis mice and normal mice;to analyze the phenotypic characteristics of NKG2A+CD8+T cell subsets.6.Correlation analysis was used to clarify the correlation between the proportion of NKG2A+CD8+T cells and the severity of adenomyosis in mice.7.Immunohistochemistry was used to detect differences in the expression of Qa-1 and IL-15 in uterine tissue of adenomyosis mice and normal mice.Results:1.Compared with the control group,the uterus weight and uterus/body weight ratio of mice in the model group were significantly increased;and different degrees of uterine congestion,thickening,local enlargement,and nodularity was visible on the surface.2.The H&E staining results showed that all mice in the model group showed different degrees of endometrial glandular or mesenchymal infiltration into the myometrium,and the modeling success rate was as high as 100%;the score of the degree of uterine infiltration in the model group was significantly higher than that in the control group.3.Masson’s trichrome staining showed that the collagen volume fraction in the myometrium of the mice in the model group was significantly higher than that in the control group.4.The flow cytometry results showed no significant difference in the proportion of infiltrated NK,CD3+T,CD4+T,and CD8+T cells in the uterus of adenomyosis mice compared to controls,but there was a significant increase in the expression of NKG2A on CD8+T cells in the uterus of adenomyosis mice;and the expression of PD-1,LAG-3,and TIM-3 on NKG2A+CD8+T cells was significantly increased.5.Correlation analysis revealed that the proportion of uterine NKG2A+CD8+T cells in adenomyosis mice was positively correlated with the score of the degree of uterine infiltration and tended to be positively correlated with the collagen volume fraction of the myometrium.6.Immunohistochemical results showed that the expression of Qa-1 and IL-15 was significantly increased in the uterine glandular epithelial cells of mice with adenomyosis compared to the uterus of control mice.7.After anti-NKGA antibody treatment,the results showed that compared with the PBS group,the mice in the anti-NKGA group had a reduced uterine weight and a decreasing trend in the uterine/body weight ratio;the nodules and thickening of the uterus were improved;the collagen volume fraction of the myometrium was significantly reduced and there was a decreasing trend in the uterine infiltration degree score.Conclusion:1.The oral tamoxifen method allows for the convenient and efficient establishment of a mouse model of adenomyosis in which there is an abnormally increased HLA-E(Qa-1)/NKG2A axis signaling leading to exhaustion of CD8+T cells.2.Blocking the interaction between HLA-E(Qa-1)and NKG2A reduces the severity of adenomyosis in mice.