The Role Of STim-3 In Regulating Tumor Immune Response

The incidence of malignant tumors and the number of deaths is increasing sharply worldwide due to growth and aging of the global population.As a leading cause of death,malignant tumor is responsible for about 9.6 million deaths in 2018 and has posed a serious threat to human health.It is of great significance to find new targets for tumor diagnosis and therapy.Immune cells in tumor patients,especially T cells,often become exhausted.These exhausted T cells show overexpression of multiple inhibitory receptors/immune checkpoints,less effector cytokine production and reduced cytotoxic capacity to kill tumor cells.Blockade of immune checkpoints could restore function of exhausted T cells to promote tumor clearance.These checkpoint blockade strategies have gained increasing attention as a promising tumor treatment approach.Antibodies targeting CTLA-4 and PD-1 have gained US FDA approval,and have received remarkable clinical success,paving the way for a new generation of tumor immunotherapy approaches.It is reported that several inhibitory receptors could produce native soluble molecules,either by alternatively splicing or by shedding surface molecule via matrix metalloproteinase.These soluble molecules play an important role in immune regulation and related disease progression.Soluble form of lymphocyte activation gene-3(LAG3)is shed from the surface of the T cell.While LAG-3 shedding reduces surface inhibitory receptor level to enhance proliferation of the parent cell,sLAG-3 does not affect the function of the other immune cells directly.In contrast,soluble CEACAM1 can bind to the membrane-bound form to block the negative regulatory pathway in NK cells.Thus,soluble form of inhibitory receptor can regulate the function of the membrane form through various pathway.Further study is needed to elucidate the form and function of the soluble inhibitory receptor,which is of great importance for immune checkpoint blockade therapy for tumor.T-cell immunoglobulin and mucin domain containing-3(Tim-3),firstly cloned in2002,is well known as an immune checkpoint and attracts much attention as a promising target in immune therapy.Tim-3 expression is induced on T cells during chronic viral infection and tumor,which contributes to T cell exhaustion.Besides,Tim-3 also regulates the function of innate immune cells and tumor stem cell to affect the tumor progression.A soluble form of Tim-3(sTim-3)was first reported in mice as an alternative spliced form.The short alternatively spliced mRNA of Tim-3 lacks both the mucin domain and transmembrane region and directs the synthesis of soluble Tim-3 protein.Further characterization of the murine sTim-3 showed its role in promoting tumor growth,inhibiting the proliferation of T cells and the secretion of IL-2 and IFN-?.However,it has been reported that the extracellular segment of murine sTim-3 promoted the proliferation and cytokines secretion of T cells in vitro.Recent studies have reported the existence of sTim-3 in human serum,but the mechanism of human sTim-3 production and its role in tumor immunity have not been well elucidated so far.In this paper,peripheral blood was collected from patients with digestive system tumor and healthy controls.The serum level of sTim-3 was determined by ELISA.To obtain an in-depth understanding of human sTim-3,we determined the mechanism of human sTim-3 production and the role of sTim-3 in tumor immunity.The main research results are as follows:I.Serum sTim-3 level is significantly increased in patients with digestive system tumorsTo detect the level of sTim-3,peripheral blood cells and serum were collected from patients and healthy people.ELISA results showed that serum level of sTim-3 in patients with liver cancer,gastric cancer,colorectal cancer and cholangiocarcinoma was significantly higher than in healthy controls(p<0.0001).The serum level of sTim-3 in patients with chronic hepatitis B was also significantly higher than that in healthy controls(p<0.0001).Moreover,the level of sTim-3 in patients with chronic hepatitis B was positively correlated with ALT and AST,suggesting that sTim-3 may be related to HBV-related HCC process.?.Human T cells produce sTim-3 in an ADAM10/17 dependent mannner1.Human activated T cells produce sTim-3To explore the source of sTim-3,PBMCs of healthy donors were isolated and activated with T cell stimuli(anti-CD3/CD28)or monocyte stimuli(LPS).Culture supernatants were collected to detect sTim-3 level.ELISA results showed that LPS stimulation did not significantly induced sTim-3 production,while anti-CD3/CD28 stimulation significantly increased the level of sTim-3 in a dose-dependent way.These results suggested human activated T cells could produce sTim-3.2.Human sTim-3 is mainly generated by ADAM10 and ADAM17,while no alternative spliced form of human sTim-3 is detectedTo explore the mechanism of human sTim-3 production,primers were designed at different positions of human Tim-3 gene.PBMCs from patients and healthy people were collected.RT-PCR results showed that only about 1000bp of full-length Tim-3 mRNA molecules were amplified in all samples,and no short spliced Tim-3 mRNA was detected.PBMCs activated by anti-CD3/CD28 stimuli were also collected to amplify Tim-3 gene,and the same results were obtained.The above results suggest that there might be no alternative spliced form of sTim-3 in human,which is consistent with previous studies.To investigate the role of ADAM10/17 in the production of human sTim-3,PBMCs stimulated by anti-CD3/CD28 were treated with ADAM 10 and ADAM 17 inhibitor respectively.sTim-3 level in supernatant was detected by ELISA.The results showed that human sTim-3 production can be mediated by ADAM 10 and ADAM 17.Further qPCR results showed ADAM 10 expression in patients with chronic hepatitis B and liver cancer were significantly higher than that in healthy people(p<0.05),while ADAM 17 expression showed no significant difference between the three groups,suggesting that ADAM10 mediated sTim-3 production may play a more important role in the development of chronic hepatitis B and liver cancer.III.sTim-3 suppresses T cells mediated anti-tumor response1.sTim-3 does not affect tumor cell proliferationTo investigate the effect of sTim-3 on tumor cells,HepG2,Huh7,hepa1-6 and B16F10 were treated with recombinant sTim-3 protein.The proliferation of tumor cells was detected by CCK8 assay.The results showed that sTim-3 protein did not affect the proliferation of tumor cells in vitro.To verify the above results,a lentiviral plasmid was constructed to overexpress sTim-3 in tumor cells.CCK8 assay and EdU cell proliferation assay showed that overexpression of sTim-3 did not affect the proliferation of B16F10-OVA cells.An AAV virus plasmid was also constructed to overexpress sTim-3 in tumor cells,and CCK8 assay showed same results.2.sTim-3 significantly inhibits the secretion of cytokines by T cellsTo explore the influence of sTim-3 for immune cells,we treated human PBMCs and T cells from OT-I mice with sTim-3 in vitro.(1)sTim-3 significantly inhibits the cytokines secretion of human peripheral T cells:Fresh isolated human PBMCs were pretreated with sTim-3 protein,then were stimulated with anti-human CD3/CD28 antibody.Effector cytokines in the culture supernatant,such as TNF-? and IFN-?,were detected by ELISA.The results showed that sTim-3 protein significantly inhibited the secretion of TNF-? and IFN-?by T cells.(2)sTim-3.significantly inhibits the cytokines secretion of OT-I TCR specific T cells:OT-I CD8+ cytotoxic T cell were generated with or without sTim-3 proteins,then cytokines secretion was assessed by flow cytometric analysis.The results showed the sTim-3 protein treatment in vitro suppressed production of TNF-?,IFN-? and IL-2 by CD8+T cells.3.sTim-3 significantly promotes tumor growth and reduces the survival period of miceTo further explore the function of sTim-3 in vivo,stable B16F10-OVA cells expressing of sTim-3 was constructed by lentivirus infection.B16F10-OVA cells with or without sTim-3 overexpression were subcutaneously injected into C57BL6 mice separately.Tumor size and survival were monitored every other day.The results showed that sTim-3 significantly promoted tumor growth in vivo and reduced the survival period.Tumor infiltrating lymphocytes of above mice were isolated,and we found that sTim-3 overexpression significantly reduced the number of tumor infiltrating CD8+T cells and CD4-T cells by flow cytometric analysis.Tumor infiltrating lymphocytes were stimulated by PMA and ionomycin to detect the cytokines secretion of T cells and NK cells by flow cytometry.The results showed that sTim-3 overexpression in tumor environment significantly suppressed the cytokines secretion capacity of tumor infiltrating T cells and NK cells.?.sTim-3 inhibits immune response in membrane type Tim-3 independent mannerIn order to explore whether the effect of sTim-3 is dependent on membrane type Tim-3,the tumor-bearing experiment was conducted in Tim-3 KO mice.The results showed that sTim-3 overexpression promoted the growth of melanoma in Tim-3 KO mice,and significantly reduced the survival period,suggesting that the tumor-promoting effect of sTim-3 is independent of membrane type Tim-3.In summary.this study reports that sTim-3 level is significantly increased in patients with digestive system tumors for the first time,and finds that human sTim-3 is mainly generated by ADAM 10 and ADAM 17.Study also finds that sTim-3 significantly suppresses effector function of T cells,and promotes the growth of tumor cells in tumor-bearing mice by affecting the tumor microenvironment.How sTim-3 interacts with ligands remains further investigation.In conclusion,this study finds the role of sTim-3 in promoting immune suppression and mediating tumor immune escape,which provides a new idea for revealing the tumorigenesis mechanism and a new mechanism for immune checkpoint treatment.