| Glioma,as the most prevalent and lethal primary brain tumor in adults,features a high rate of recurrence,mortality and disability.The median survival period of high-grade glioma(Glioblastoma multiforme,GBM)is only about 14-17 months post-diagnosis,which is severely harmful to human health.Although patients with glioma undergo aggressive and multimodality treatment such as standard-of-care surgery combined with chemoradiotherapy,the prognosis is still poor,which is closely related to the biological characteristics of glioma such as unlimited proliferation enhanced invasion,resistance to radiotherapy and chemotherapy drugs.Therefore,it is imperative to search for more effective therapeutic strategies for glioma.With the recent progresses in neurobiology and neuroimmunology,the tumor immunotherapy has made great progress,which act on lymphocytes to boost their endogenous anti-tumor activity.Immunotherapy has garnered increasing attention as an attractive treatment modality for tumors,owing to the precision and memory of anti-tumor immunologic cytotoxicity.Immune checkpoints are a series of membrane receptor molecules,which keep the balance of costimulatory signals and co-inhibitory signals,leading the immunoregulation.However,cancers could utilize the negative immune checkpoint molecules to inhibit the effective functions of immune cells,impair the immune reactive,and eventually lead to immune escape.Immune checkpoint may also play a vital role in myeloid-derived suppressor cell(MDSC),T helper cells(TH),and regulatory cells(Tregs)development to involve in the formation of tumor immunosuppression microenvironment.Therefore,the immune checkpoint receptors and their corresponding ligands become the best druggable targets for immune regulation.Immunotherapy,especially immune checkpoint inhibitors,recently achieved great success in treating several human malignant tumors.These antibodies boost the body's anti-cancer immune responses via blocking the checkpoint molecules and resuming the function of T cells.T-cell immunoglobulin and mucin domain 3(Tim-3)is a member of Tim family.Growing evidence has further characterized Tim-3 as a key checkpoint regulator responsible for the exhaustion and dysfunction of T cells that arises in chronic infections and malignant tumors.Tim-3–expressing T cells fail to proliferate and produce cytokines in response to antigens,which mediates immunologic tolerance and immune evasion of tumors.Expression level of Tim-3 on CD4+/CD8+ T cells is elevated in peripheral and tumor-infiltrating T cells in patients with glioma,and further increased on C D8+T cells in high-grade glioma,suggesting that Tim-3 may participate in the progression and development of glioma through their negative regulatory effects on T cells.C EACAM1 is considered as a crucial molecule in downregulating immune responses.In addition to Galectin-9(Gal-9),CEAC AM1 is the novel specific ligand of Tim-3.The up-regulation expression of C EAC AM1 could strengthen the negative immune regulation function of Tim-3.The latest studies proved that Tim-3/C EAC AM1 play a key role in development and immune escape of malignant tumors.Based on the above data,the present study focused on immune checkpoint molecules Tim-3/C EAC AM1,observe the expression pattern of C EACAM1 in glioma.Further,we analyzed the correlation between CEACAM1 expression and multiple clinicopathological characteristics in patients with glioma,dicussed the effect on malignant biological behavior and antitumor immune characteristics.We observed the effects of target blocking the Tim-3/CEACAM1 signals in restoring the immune functions and immunologic memory.Our studies will further improve our understanding of Tim-3/CEACAM1,reveal the dual-acting mechanism in promoting the proliferation and immune escape of glioma,and provide the novel treatment strategies for malignant glioma.The current study was performed from three aspects as follows:1.Clinical significance of expression of circulating and tumor-infiltrating CEACAM1 in patients with glioma.Objective:To investigate expression level of CEACAM1 on CD4+,CD8+T cells in PBMCs and TILs of glioma,and analyze the correlation between CEACAM1 expression and multiple clinicopathological characteristics in patients with glioma.Methods: We selected 51 cases of patients with glioma and 30 healthy control subjects,collected the PBMCs and TILs.We assessed the expression levels of CEACAM1 on CD4+?CD8+T cells in PBMCs and TILs of patients with glioma by flow cytometry,investigated the expression levels of CEACAM1 in glioma tissues and normal tissues by immunohistochemical analysis.Further,we analyzed the correlation between CEACAM1 expression and multiple clinicopathological characteristics in patients with glioma.Results:(1)Significant increases in the percentage of CEACAM1+ cells were observed among CD4+ T cells(P<0.0001)and among CD8+ T cells(P<0.0001)in PBMCs of patients with glioma and this percentage was further elevated in TILs of patients with glioma.Similar results were observed when mean fluorescence intensity(MFI)of CEACAM1 on C D4+T cells(P<0.0001)and CD8+T cells(P<0.0001)was analyzed.(2)Expression of CEACAM1 on the C D4+ T cells(P<0.0001)and CD8+ T cells(P<0.0001)in PBMCs was markedly higher in high-grade gliomas than in low-grade gliomas;the expression of C EAC AM1 on CD4+ T cells(P<0.0001)and C D8+ T cells(P<0.0001)was further increased in TILs.(3)The analyses revealed that the frequencies of CEACAM1 expressed on CD4+ T cells(P<0.05)and CD8+ T cells(P<0.05)in TILs and PBMCs were inversely correlated with the Karnofsky scores of patients with glioma.(4)That plasma IFN-? levels were inversely correlated with the expression of CEACAM1 on CD4+ T cells in TILs and PBMCs(P<0.05).CEACAM1+ CD8+ T cells in TILs were also negatively correlated with the plasma IFN-? levels of patients with glioma(P<0.05),whereas those in PBMCs did not show any significant correlation with IFN-? levels in serum(P>0.05).(5)We observed negligible or no staining of C EAC AM1 in the brain tissues of control;however,an increased positive expression of CEACAM1 was ob served in the tumor tissues.The expression levels of CEAC AM1 in tumor tissues were associated with the grades of the glioma.Compared with brain tissues,the C EAC AM1 expressions in grade I–II gliomas were markedly increased and in grade III–IV gliomas were further increased(P<0.05).Furthermore,CEACAM1 expression in tumor tissues was correlated with the proportion of CEACAM1+ T cells(P<0.05).2.Mechanism and key molecules of Tim-3 in glioma immune escape.Objective:To investigate the mechanisms and key molecules of Tim-3 in glioma.Methods: We Enriched and separated the CD8+T cells in PBMCs with immunomagnetic beads,and assessed the purity with flow cytometry.Up-regulate the expression of Tim-3 on collected CD8+T cells with IL-12.Targeted block the Tim-3 on Tim-3+C D8+T cells with monoclonal antibody.Then,we co-cultured each group of C D8+T cells with U87 and U251 human glioma cell line.We observed the effect on proliferation of CD8+ T cells by CFSE,and evaluated the production of IFN-?with Realtime-PCR and flow cytometry.Further,we investigate the proliferation and apoptosis of glioma cells,and assessed the cytokines level in cell supernatant of co-culture system by ELISA.Results:(1)The purity of CD8+ T cells,which are all above 95%,conformed to the requirements of the experimental.(2)IL-12 markedly increased the expression level of Tim-3 on CD8+ T cells.(3)CFSE data showed that after targeted blocking the pathway of Tim-3,the proliferation of CD8+ T cells is increased(P<0.05).(4)Realtime-PCR and flow cytometry results indicated that the production of IFN-?is increased(P<0.05).(5)After targeted blocking the pathway of Tim-3,the proliferation of glioma cells is reduced and the apoptosis is increased(P<0.05).(6)ELISA data suggested that IFN-?level is increased(P<0.05).3.Combined blockade of Tim-3 and CEACAM1 in mice with intracranial glioma.Objective:To observe the effects and mechanisms on growth of tumor,survival,anti-tumor immunity,and immunologic memory in mice with intracranial glioma after combined blockade of Tim-3 and CEACAM1.Methods: Utilizing the slow virus with luciferase gene and puromycin resistance to transfect GL261 mice glioma cell line,with stereotactic technique to C57BL/6 mice implanted glioma cells on the right side of the caudate nucleus,construct the intracranial glioma model in mice.Then we utilizing small living animal imaging technology to observe the Tumor growth in mice.The mice were averagely divided into 4 groups: control group,anti-Tim-3 group,anti-CEACAM1 group,and anti-Tim-3+anti-CEACAM1 group,which given intraperitoneal injection of Tim-3/C EAC AM1 monoclonal antibody or equivalent isotype control antibody.Then we observed the growth of intracranial glioma,recorded survival,analyzed survival curve.We determined the pathological type of intracranial tumor by HE staining.The CD4+?CD8+T cells and CD4+C D25+FoxP3+ Tregs in brain infiltrating lymphocytes were analyzed using flow cytometry,and the effector function of T cells was assessed using ELISA.The survival benefit of the combination therapy was observed again after the dependent on the CD4+ and CD8+ T cells.We performed a rechallenge by subcutaneous injection of GL261-luc cells in the ?cured?(surviving for more than 90 days post-orthotopic tumor implantation)and na?ve mice.Results:(1)C57BL/6-GL261 intracranial glioma models is constructed successfully,and the rate of tumor formation is up to 80%.(2)The mean survival time in the control,anti-Tim-3,anti-CEACAM1,and combined treatment groups was 29.8,43.4,42.3,and 86.0 days,respectively.(3)80% of the mice in the combined group were becoming long-term survivors(>90 days post-orthotopic tumor implantation)that showed immune memory against glioma cells.(4)Infiltrating C D4+ and CD8+ T cells increased and immunosuppressive Tregs were decreased with the combined therapy(P<0.05),which resulted in a markedly elevated ratio of CD4+ and CD8+ cells to Tregs.(5)plasma IFN-? and TGF-? levels were up-regulated and down-regulated,respectively(P<0.05).(6)Mice with CD4 or CD8 depleted had a marginal survival benefit,but the treatment effect on the mice with both CD4 and CD8 cells depleted was completely abrogated.(7)Long-term survivors receiving the combination therapy showed immunologic memory.Conclusion:(1)Expression levels of CEACAM1 in glioma were elevated,and is correlated with multiple clinicopathological characteristics in patients with glioma,indicating that homophilic interactions of C EAC AM1 may participate in the progression and immune escape of glioma through their negative regulatory effects on T cells.(2)Targeted blockade of Tim-3 could reverse the ?exhaust? of CD8+ T cell,and enhance immune elimination of glioma cells,which may be related to the increasing of proliferation rate and production of IFN-?,and effect on related cytokines.(3)Combined blockade of Tim-3 and C EAC AM1 generates robust therapeutic efficacy in mice with intracranial tumor,and provides a promising option for glioma immunotherapy. |
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