A Study Of Multinucleated Giant Cells And IgG4+B Cells In Esophageal Cancer Microenvironment

Background and purposeAt present,the types and functions of immune cells in tumor microenvironment have attracted more and more attention.Immune checkpoint blockade therapy based on the progress of T cell research has brought innovation to tumor treatment.While multinucleated giant cells(MGC)and B lymphocytes have not been well investigated.MGC reaction is common in granulomatous inflammation and has been reported in many epithelial tumors.However,the pathological and biological characteristics of multinucleated giant cells in tumors are still unclear.In this study,the nature and the clinical significance of MGC in esophageal cancers were investigated.Humoral immunity mediated by B cells is an important part of cancer immunity.IgG4 is the immunoglobulin G subclass with the lowest concentration in human serum secreted by mature B plasma cells.The increased infiltration of IgG4 positive plasma cells is mainly seen in IgG4 related diseases.The increase of IgG4 positive cells in tumor tissues has been reported in gastric cancer,extrahepatic cholangiocarcinoma and malignant melanoma.Until now,there is no relevant study on the expression of IgG4 in esophageal cancer.The immunopathological and clinical characteristics of IgG4 in esophageal cancer need to be answered urgently.Materials and methodsIn the first part,the occurrence and distribution of multinucleated giant cells were examined with immunohistochemistry in 107 cases of esophageal cancer.MGCs were identified by CD68 positivity and cell morphology.The polarization spectrum,cell surface markers and autocrine enzymes of multinucleated giant cells were identified by double immunofluorescence staining.The detection indexes included CD11 c,HLA-DR,CD163,cd206,CD11 b,CD64,CD32,CD16 and MMP9.We analysed the expression of multinucleated giant cell markers and clinical data of patients with Kaplan Miere survival analysis,univariate and multivariate Cox regression,and discussed whether multinucleated giant cells had a significant impact on patients' survival and prognosis.In the second part,the degrees of infiltration of B lymphocytes containing Ig G subclasses in cancer tissues,adjacent tissues and normal tissues were statistically analyzed with immunohistochemistry and immunofluorescence double staining method.The concentration of total Ig G,Ig G1 and IgG4 subclasses in cancer tissues,normal tissues and lymph nodes were detected with ELISA.Six color simultaneous immunofluorescence was used to detect the distribution of immune cells in the samples with high expression of IgG4.Ig G1 and IgG4 subclasses were extracted from patients' serum with affinity chromatography of Ig G subclasses.The extracted Ig G1 and IgG4 were labeled with biotin and reacted with corresponding tumor tissues to detect the tumor antigen reactivity of Ig G1 and IgG4.The binding ability of Fc segment of Ig G1 and IgG4 to PBMC was detected by the reaction of Ig G1 and IgG4 with PBMC.The ability of IgG4 to react with other Ig G subclasses and Ig G of other species was detected by Western blot.Ig G was cut into Fab and Fc fragments with papain,and the structural characteristics of IgG4 Fab and Fc fragments were examined.The polarization effect of Ig G1 and IgG4 on monocyte macrophages was studied by comparing Ig G1 and IgG4 in monocyte culture.ResultsIn the first part,MGCs were identified in 31.7 %(34/107)of the cases.MGCs were positive for CD11 c,CD11b,CD32,CD16,HLA-DR and MMP9,and negative for CD163,CD206 and CD64 giving a molecular profile of proinflammatory M1 but not immunosuppressive M2.MGCs were significantly related to decreased lymph node metastasis(p=0.011),low p TNM stage(p=0.044),favorable survival(p=0.04),squamous cell cancer type rather than other histopathological subtypes(p=0.020)and associated to better differentiation(p=0.063).In part two,IgG4 was found increased significantly in serum of patients with esophageal cancer(P < 0.0001),and the concentration of IgG4 and the density of IgG4 positive plasma cells in cancer tissues were significantly higher than those in normal tissues of the same patient(P <0.01,P < 0.001).Analysis of tissue samples with high expression of IgG4 showed that IL-10 cytokines were highly expressed in tumor associated lymphoid follicle center,and we further found some IgG4 positive cells are infact Breg cell.The expression of IL-10 in tissue samples was consistent with that of IgG4.Ig G1 extracted from patient's serum reacted to fresh tumor tissue,the reactivity of IgG4 to tumor was lower than Ig G1.The location of Ig G1 reaction was consistent with that of tumor cells.Results showed that the Fab terminal of IgG4 changed after class swith,which was functionally different from that of Ig G1.Comparing with Ig G1,IgG4 had high levels of mannose glycosylation and sialylation shown on western blot.The extra glycosylation of IgG4 occurred on the heavy chain,resulting in a molecular weight of one heavy chain being 2-3 k Da higher than that of the other.Subsequent enzyme digestion experiments showed that the modification was located on the Fab segment of the heavy chain.The high level of glycosylation of Fab segment of IgG4 may be one of the reasons for the difference between IgG4 and Ig G1 in the antigen recognition abilities.In the competition experiment with PBMC,IgG4 competed for the binding site of Ig G1 on PBMC indicating that although the affinity of Fc segment of IgG4 to Fc R is lower than that of Ig G1,it could still compete for the binding site of Ig G1.In addition,it was found that IgG4 could react with other Ig G subclasses through a Fc-Fc fashion.In cell experiment,Ig G1 and IgG4 were used to stimulate monocytes differentiation.It was found that Ig G1 and IgG4 could promote polarization of monocytes to M1 macrophages and M2 macrophages,respectively.Discussion and ConclusionMGCs belong to M1 type polarization and perform phagocytosis and scavenging of cancer cells and clearance of keratinized pearls in cancer microenvironment benefiting destruction of neoplastic elements and patients' survival.The presence of MGCs appears to be a common feature of esophageal squamous cell cancer and could serve as a new diagnostic indicator for the biological behavior and prognosis of cancer with implications for cancer therapy.IgG4 increased in the tissues and serum of patients with esophageal cancer,and the increase of IgG4 was related to the pathological stage of the disease.Ig G1 from the same patient has antigenic specificity for tumor tissue,but IgG4 has no anti-tumor specificity.In structure,the Fab segment of the heavy chain of IgG4 was highly glycosylated,and the Fab segment of IgG4 was different from that of Ig G1.At the same time,the Fc segment of IgG4 can react with other subclasses of Ig G and in other species(mice,rabbits,sheep).High concentration of IgG4 can also reduce the binding level of Ig G1 to PBMC.Comparing with Ig G1,the structural features of IgG4 Fab and Fc segments are the basis for different physiological functions of IgG4 and Ig G1.This also bring us new thinking about tumor immunity.When the non anti-tumor immune molecule IgG4 gradually increases in the body,would it compromise B-cell immunity against tumor antigens and the inhibit anti-tumor function in vivo.These characteristics of IgG4 provides more insights and inspiration for the development of monoclonal antibodies in tumor immunotherapy.