Soluble Form Of Tim-3 Suppresses T Cell Function And Induces Resistance To PD-1 Blockade

Exhausted T cells show consistent expression of multiple immune checkpoints,which regulate T cell activation and function.Immune checkpoint blockade(ICB)-based cancer immunotherapy,mostly accessed by anti-PD-L1/PD-1 antibodies,restore function of exhausted T cells to promote tumor clearance.Antibodies targeting PD-1 have shown remarkable clinical outcome in the treatment of a various types of tumors.However,the anti-PD-1 resistance has greatly limited the clinical application of anti-PD-1 tumor immunotherapy.Tim-3(T-cell immunoglobulin and mucin domain containing-3),as an immune checkpoint,is highly expressed on T cells during chronic viral infection and tumor progression,which contributes to T cell exhaustion.Combination of Tim-3 blockade in synergy with PD-1 blockade greatly enhances T cell function and prevents resistance to anti-PD-1 therapy.Recent work demonstrated the presence of soluble form of Tim-3(sTim-3)which is produced by different manners in mice and human.Several studies showed the correlation of sTim-3 with tumor,viral infection,and other diseases progressions.However,the role of human sTim-3 in tumor progression has not been well elucidated so far.This study aims to reveal the regulatory role and mechanisms of sTim-3 in tumor progression and PD-1 blockade resistance.The main research methods and results are as following:? sTim-3 is elevated in the serum of tumor patients and related to PD-1 blockade resistanceTo evaluate the role of sTim-3 in tumor progression,we collected serum samples from patients with NSCLC(Non-small cell lung cancer)and healthy donors.Enzyme-linked immunosorbent assay(ELISA)results showed that serum sTim-3 levels were greatly increased in NSCLC patients than that in healthy donors.The sTim-3 level correlated with the malignant behavior of lung cancer,and patients with high sTim-3 showed shorter survival than those with low sTim-3.More importantly,serum sTim-3 level was significantly higher in patients receiving PD-1 blockade therapy than those not.Notably,a higher level of serum sTim-3 was detected in patients who failed to respond to anti-PD-1 treatment than that who showed responses.In addition,compared with healthy controls,significantly higher serum level of sTim-3 was detected in patients with all detected tumors including gastric cancer,colorectal cancer,HCC and cholangiocarcinoma.? Overexpression of sTim-3 promotes tumor growth and anti-PD-1 therapy resistanceIt has been reported that shedding of human Tim-3 by ADAM 10/17 occurs at a membrane proximal site,we then prepared recombinant ectodomain of human Tim-3 as sTim-3 to treat cultured tumor cells.Results of cell growth assay showed that proliferation of all detected HCC cell lines was not affected by the sTim-3 directly in vitro.To further explore whether human sTim-3 influenced tumor growth in vivo.C57BL/6 mice were subcutaneous injected with B16M05 cells which transduced with empty vector or sTim-3 overexpression vector.Compared with control,sTim-3 overexpression significantly enhanced tumor growth in vivo and shorted survival time of mice with melanom.Futhermore,sTim-3 overexpression greatly promoted the growth of B16-M05 melanoma cells in Tim-3-/-mice,suggesting that the tumor-promoting effect of sTim-3 is independent of membrane-type Tim-3.To confirm the sTim-3 mediated tumor promotion in different tumors,we established a spontaneous intrahepatic cholangiocarcinoma(ICC)mouse model,which was conducted by Hydrodynamic injection(HDI)oncogene plasmids together with human sTim-3 expressing plasmid or control vector into hepatocytes.Compared with control,sTim-3 significantly induced faster tumor growth and shortened the survival of ICC mice.We next assessed whether sTim-3 participates in anti-PD-1 resistance in mice with ICC model.As expected,anti-PD-1 treatment significantly decreased hepatic tumor masses and prolonged survival time of ICC mice.Strikingly,in sTim-3 overexpressing ICC mice,PD-1 blocking antibodies neither affected the tumor masses nor altered the survival time.? sTim-3 suppresses effector function and anti-PD-1 response in T cellsTo explore the influence of sTim-3 on T cells,RNA-sequencing(RNA-seq)was performed with OT-I T cells treated with/without his-tagged human sTim-3 protein.Gene set enrichment analysis(GSEA)showed that T cell activation and proliferation related signature genes were significantly enriched in control cells compared with sTim-3 treated cells.Further western blot results demonstrated the down regulation of TCR signaling in sTim-3 treated OT-I cells.Notably,sTim-3 treatment greatly reduced the secretion of effector cytokines and reduced cytotoxicity of OT-I CD8+T cells.To confirm the immune regulatory role of sTim-3 in vivo,we analyzed tumor infiltrating lymphocytes(TILs)of B16-M05 cells that were stably transduced with control vector or sTim-3 expression plasmid.Flow cytometric analysis showed that sTim-3 overexpression significantly reduced the number of tumor-infiltrating T cells and the secretion of IFN-y and TNF-?,increased levels of inhibitory receptor PD-1 and LAG-3 in T cells.Moreover,the percentage of myeloid-derived suppressor cells(MDSC)and CD4+Foxp3+T regulatory cells(Tregs)were greatly increased in mice bearing sTim-3 overexpressing B16-M05 tumor.Our GSEA anlysis showed that,sTim-3 treatment significantly enriched the anti-PD-1 unresponsive signature gene set in OT-I CD8+CTLs.In accordance,PD-1 blockade greatly increased the function of OT-I CD8+T cells,while sTim-3 treatment abrogated the enhancement of cytokine secretion by PD-1 blockade.Further ELISA results showed that sTim-3 significantly inhibited IFN-y and TNF-? secretion upon anti-CD3/CD28 stimulation in TILs from HCC patients,did not change the production of IFN-y and TNF-? in human PBMCs.Moreover,sTim-3 treatment largely dampened the anti-PD-1 induced augmented production of IFN-? in TILs from HCC patients,indicating the negative regulatory role of sTim-3 in human might be tumor microenvironment dependent.? sTim-3 impairs T cell function and anti-PD-1 response via CEACAM-1Given that sTim-3 and membrane-type Tim-3 have identical extracellular segments,we hypothesized that they can bind to the same ligand.CEACAM-1 as a binding partener of Tim3,which negatively regulates T cell mediated anti-tumor function.Based on our data showing that sTim-3 inhibited effector function of human TILs but not human PBMCs.We therefore examined the expression level of identified binding partners of Tim-3.As expected,both OT-I CD8+T cells and human tumor infiltrating T cells expressed CEACMA-1,consisting with results that sTim-3 inhibited the function of T cells.And we did not detect the expression of CEACAM-1 in human peripheral T cells.We therefore hypothesized that sTim-3 suppresses T cell function and anti-PD-1 response via CEACAM-1.In line with this,sTim-3 failed to inhibit the induction of IL-2 mRNA in Jurkat cells which do not express CEACAM-1.Interestingly,overexpression of human CEACAM-1 enabled sTim-3 to significantly inhibit the IL-2 mRNA level in Jurkat cells.This was further confirmed by knocking down Ceacam-1 in OT-I CD8+T cells with shRNA.Importantly,in OT-I CD8+T cells with Ceacam-1 silence,sTim-3 overexpression lost the ability to affect the enhancement of cytokine secretion by PD-1 blockade.? Targeting sTim-3 generation by AD AM10 inhibitor reverses anti-PD-1 resistance in human TILsPrevious study demonstrated that human sTim-3 is produced by matrix metalloproteinase ADAM 10 and ADAM 17.To assess this,ADAM 10 and ADAM 17 inhibitor was used to treated PBMCs.Both ADAM 10 and ADAM 17 inhibitor completely blocked the sTim-3 production induced by anti-CD3/CD28 stimulation in PBMCs.To tested whether targeting sTim-3 generation by ADAM 10 inhibitors could reversed anti-PD-1 resistance,TILs isolated from HCC patients who were resistant to PD-1 blockade were treated with PD-1 blocking antibodies and ADAM 10 inhibitors(GI)in vitro.As expected,GI treatment alone significantly enhanced IFN-? secretion by TILs.Strikingly,combination of anti-PD-1 with GI further promoted IFN-?production in TILs.Conclution and significance:(1)We for the first time demonstrated the correlation of sTim-3 with PD-1 blockade resistance.(2)sTim-3 inhibited T cell function and promoted PD-1 blockade resistance via CEACAM-1.(3)Targeting sTim-3 generation largely reversed the anti-PD-1 resistance in human TILs.In conclusion,our study demonstrates the important role of sTim-3 in mediating tumor immune escape and resistance to PD-1 blockade.