Expression And Regulation Of HLA Class Ⅱ Molecules In Human Glioblastoma

Objective:Glioma is the most common primary central nervous system tumor in adults and children,and approximately 50%of patients present with malignant gliomas.Glioblastoma（GBM）is the most common malignant diffuse glioma.Traditional treatment methods such as surgery,temozolomide chemotherapy,radiotherapy,and corticosteroid therapy cannot significantly improve the prognosis of patients.Recent studies have found that the abnormal expression of MHC-Ⅱmolecules on tumor cells was associated with poor prognosis.HLA-DR mediated signaling plays a new role in melanoma progression by increasing the migration and invasion of melanoma cells.Recently,we found that in aflatoxin G₁（AFG₁）-induced lung adenocarcinoma（LA）,major histocompatibility complex classⅡ（MHC-Ⅱ）expression was upregulated on alveolar epithelium,and this was associated with enhanced Treg infiltration in mouse lungs.The inflammation-driven mice lung adenocarcinoma and human lung adenocarcinoma cells mainly arise from AT-Ⅱcells,which are the major source of MHC-Ⅱin the tumor microenvironment.TNF-αdependent lung inflammation upregulates MHC-Ⅱexpression on tumor cells of AT-Ⅱcellular origin to contribute to Treg expansion in IDLA.In human glioblastoma,macrophages producing TNF-αdevelop an inflammatory microenvironment that promotes tumor growth.Whether TNF-αstimulation in tumor microenvironment regulates the expression of HLA classⅡmolecules（HLA-DP,HLA-DQ and HLA-DR）in glioblastoma is not fully understood.Thus,it is necessary to explore the expression of HLA-Ⅱas well as its function involved in tumor progression in GBM,which may provide platform useful to target HLA-Ⅱmolecules to frustrate an effective anti-tumor response.In this study,we collected human glioblastoma specimens and explored the expression of HLA classⅡmolecules as well as the relation to clinical outcomes in glioblastoma,and analyzed the correlation of HLA classⅡmolecules with CD68⁺macrophage infiltration and TNF-αexpression;Additionally,we utilized human monocyte-macrophage THP-1 cells and glioblastoma cells U87 and U251 as in vitro cell models to investigate whether macrophages contribute to HLA-Ⅱupregulation by secreting TNF-α.The study will reveal the potential immune target for therapeutic intervention.Methods:1.TCGA（TCGA The Cancer Genome Atlas）was selected to analyze the m RNA expression level of HLA classⅡmolecules in glioblastoma tissues,and the relationship between the expression of HLA classⅡmolecules and the overall survival of patients were also studied.2.The fresh tumor tissues and peritumoral normal brain tissues were isolated from the Department of Neurosurgery of the Second Hospital of Hebei Medical University.Immunofluorescence staining and immunohistochemical staining were used to detect the expression of HLA classⅡmolecules and TNF-αin tumor tissues as well as the infiltration of CD68⁺macrophages.Spearman’s correlation analysis was applied to determine the relationship between HLA-Ⅱand TNF-αexpression and CD68⁺macrophage infiltration.Overall survival（OS）was measured from the date of operation to the date of death according to the Kaplan-Meier method.3.U87 and U251 cells were cultured in vitro and treated with TNF-α,and the expression of HLA classⅡmolecules in tumor cells was measured;human monocyte-macrophage THP-1 cells were cultured in vitro,and the supernatant was taken to stimulate U87 and U251 cells,with or without anti-TNF-αneutralizing antibody.The expression of HLA classⅡmolecules in tumor cells was measured to explore whether macrophages contribute to HLA-Ⅱupregulation by secreting TNF-α.Results:1.Expression of HLA classⅡmolecules in TCGA glioblastoma related databases.Statistical analysis of the m RNA expression level of HLA classⅡmolecules in glioblastoma in the TCGA database revealed that HLA-DP,HLA-DQ and HLA-DR were all highly expressed in glioblastoma,and the high expression of HLA-DP was related to the poor prognosis of patients.2.Expression of HLA classⅡmolecules in human glioblastoma tissue specimens.51 cases of human glioblastoma tissue specimens and 27 cases of peritumoral normal brain tissue specimens were selected and the expression of HLA-Ⅱand TNF-αwere detected by immunohistochemical staining.The results indicated that high expression of HLA-DP and HLA-DQ was observed in tumor tissues.The fresh tumor tissues and peritumoral normal brain tissues were collected from 21 patients with glioblastoma,and results also showed that positive expression of HLA-DP and HLA-DQ was observed in glioblastoma tissues.3.The relationship between the expression of HLA classⅡmolecules and TNF-αas well as the infiltration of CD4⁺T lymphocytes and CD68⁺macrophages in human glioblastoma tissue.51 cases of human glioblastoma tissue specimens and 27 cases of peritumoral normal brain tissue specimens were collected,the immunohistochemical staining of which revealed that HLA-DP,HLA-DQ and TNF-αwere highly expressed in tumor tissues.Increased CD68⁺macrophage infiltration was observed around tumor cells.The expression of TNF-αin tumor tissues and the infiltration of CD68⁺macrophages were closely related to the expression of HLA-DP and HLA-DQ;CD4⁺T cell infiltration was closely related to the expression of HLA-DQ.4.TNF-α⁺CD68⁺macrophages infiltration associated with the expression of HLA-DP and HLA-DQ in glioblastoma.The fresh frozen tumor tissues were collected from 30 patients with glioblastoma in the Department of Pathology of the Second Hospital of Hebei Medical University for immunofluorescence and immunohistochemical staining.TNF-αmostly localized on CD68⁺macrophage and tumor cells,and increased TNF-αand CD68 co-staining was also observed.HLA-DP and HLA-DQ were correlated with the number of TNF-α⁺CD68⁺in GBM.The results indicated that,CD68⁺macrophages expressing TNF-αmay be associated with the expression of HLA-DP and HLA-DQ in glioblastoma.5.THP-1 macrophages up-regulate the expression of HLA-DP and HLA-DQ in U87 and U251 cells by secreting TNF-α.To further verify that macrophages secrete TNF-αto regulate the expression of HLA-DP and HLA-DQ in glioblastoma cells.Human glioblastoma U87 and U251 cell lines were cultured in vitro,then stimulated with different doses of TNF-αfor 24 hours,and the expression of HLA-DP and HLA-DQ was significantly up-regulated by TNF-αstimulation.Subsequently,human monocyte THP-1 cells were cultured and differentiated into macrophages,then the supernatant was taken to stimulate U87 and U251cells,and we found that supernatant from macrophages up-regulated the expression of HLA-DP and HLA-DQ in tumor cells.The anti-TNF-αneutralizing antibody was used to neutralize the TNF-αin the supernatant,and inhibited the increased expression of HLA-DP and HLA-DQ stimulated by macrophage’s supernatant.Thus,the result indicates that macrophages were up-regulated the expression of HLA-DP and HLA-DQ in U87 and U251 cells by secreting TNF-α.Conclusions:1.HLA classⅡmolecules are highly expressed in human glioblastoma.2.The expression of TNF-αin tumor cells and the CD68⁺macrophages are significantly positively correlated with the expression of HLA classⅡmolecules.3.Macrophages upregulate the expression of HLA-DP and HLA-DQ in glioblastoma by secreting TNF-α.