Objective T cell immunoglobulin and mucin domain-containing molecule 3 (Tim-3), a critical regulator of innate and adaptive immune responses, has been considered as a promising new target for immunotherapy. Based on an existing laboratory murine-derived anti-human Tim-3 antibody, this study aims to employ computer-aided molecular design to construct a mammalian cell-displayed antibody library targeting human Tim-3 and to screen for antibodies with optimized function. Methods The parental Tim-3 sequence was designed and optimized via a computer-assisted molecular model construction platform and mammalian cell-displayed antibody library technology. The plasmid library encoding the Tim-3-targeted mammalian antibody library was obtained by molecular biology techniques including Overlap PCR, vector cleavage, enzyme ligation and transformation. Novel antibody sequences were identified through sequencing alignment, followed by ELISA-based screening to isolate high-expression and high-affinity candidates. The function of the optimized antibody was further verified by in vitro assays (RT-PCR and CCK-8). Results Utilizing computer-aided design platform, five key amino acid substitutions were designed and introduced into the light and heavy chains of the parental murine-derived anti-human Tim-3 antibody. Three mutant light chains (L-1, L-2 and L-3) and heavy chains (H-1, H-2 and H-3) were successfully generated by Overlap PCR. Subsequent ligation into vectors and transformation into TOP10 electrocompetent cells yielded a plasmid library with substantial sequence diversity. Sequence analysis and purification of new antibodies resulted in the identification of an antibody with higher affinity, named No.1. RT-PCR showed that after blockade with antibody No.1, transcription levels of IFN-β, TNF-α, IL-1β and IL-6 were significantly upregulated in the macrophage cell line RAW264.7 cells and microglia cell line BV2 cells, and a similar upregulation was observed in levels of IL-2 and IFN-γ in JurkatTim-3-high cells. In these assays, antibody No.1 exhibited greater blocking efficacy than the parental Tim-3 antibody. CCK-8 assays demonstrated that antibody No.1 promoted JurkatTim-3-high cells proliferation more effectively than the parental Tim-3 antibody. Conclusion A novel humanized Tim-3 antibody (No.1) with enhanced affinity and capacity to upregulate multiple cytokine expressions in both human and murine cell lines was obtained via a computer-aided molecular design platform and mammalian cell antibody display library technology.

Nuclear factor 90 (NF90) is a novel virus sensor that serves to initiate antiviral innate immunity by triggering stress granule (SG) formation. However, the regulation of the NF90-SG pathway remains largely unclear. We found that Tim-3, an immune checkpoint inhibitor, promotes the ubiquitination and degradation of NF90 and inhibits NF90-SG-mediated antiviral immunity. Vesicular stomatitis virus (VSV) infection induces the up-regulation and activation of Tim-3 in macrophages, which in turn recruit the E3 ubiquitin ligase TRIM47 to the zinc finger domain of NF90 and initiate a proteasome-dependent degradation via K48-linked ubiquitination at Lys297. Targeted inactivation of Tim-3 enhances the NF90 downstream SG formation by selectively increasing the phosphorylation of protein kinase R and eukaryotic translation initiation factor 2α, the expression of SG markers G3BP1 and TIA-1, and protecting mice from VSV challenge. These findings provide insights into the crosstalk between Tim-3 and other receptors in antiviral innate immunity and its related clinical significance.

目的 构建李斯特菌脑膜炎小鼠模型,以评价T细胞免疫球蛋白和黏蛋白结构域分子3(Tim-3)在李斯特菌脑膜炎小鼠中的可能作用.方法 体内实验通过延髓池注射李斯特菌的方法构建半数致死量的李斯特菌脑膜炎小鼠模型,应用Tim-3敲除(knock out,KO)模式小鼠和Tim-3抗体评价Tim-3对李斯特菌脑膜炎的作用.体外实验通过小胶质细胞BV2与Tim-3抗体共培养,检测阻断Tim-3对BV2细胞因子表达的影响.结果 成功构建了半数致死剂量的脑膜炎小鼠模型,与对照组相比,KO小鼠及应用Tim-3抗体小鼠临床评分较高.小胶质细胞阻断Tim-3后,肿瘤坏死因子α(TNF-α)水平升高.结论 敲除及阻断Tim-3引起小鼠李斯特菌脑膜炎病情加重.

The tumor microenvironment can induce high expression of some immune checkpoint molecules to facilitate tumor cells to escape from the recognition and attack of the immune system. At present, great progress has been made in the research and development of targeted antibody drugs based on programmed cell death protein 1 (PD-1), cytotoxic T-lymphocyte associated protein 4(CTLA-4), T-cell immunoglobulin and mucin domain-containing protein 3(Tim-3). Many of the antibody drugs targeting PD-1 and CTLA-4 have been approved by the Food and Drug Administration(FDA). Although some patients have had a complete remission or prolonged survival after the treatment of drugs above, there are still many patients showing drug resistance or no remission. Recent study found that Tim-3 expression was up-regulated in PD-1-targeted drug-resistant patients, and was also considered as a potential new generation of tumor treatment target. Therefore the targeted combination therapy of Tim-3 and PD-1 is taken into consideration. At the same tiame, Tim-3 is also considered as a potential next-generation treatment target for tumor. This review will mainly introduce the research status of Tim-3 targeted drugs in tumor therapy.

Background: T-cell immunoglobulin and mucin protein 3 (Tim-3) is an immune checkpoint inhibitor that has therapeutic implications for many tumors and infectious diseases. However, the mechanisms by which Tim-3 promotes immune evasion remain unclear. Methods: In this study, we demonstrated that Tim-3 inhibits the expression of major histocompatibility complex class I (MHC-I) in macrophages at both the messenger ribonucleic acid and protein levels by inhibiting the STAT1-NLRC5 signaling pathway. Results: As a result, MHC-I-restricted antigen presentation by macrophages was inhibited by Tim-3 both in vitro and in a Listeria monocytogenes infection model in vivo. Systemic overexpression of Tim-3 or specific knockout of Tim-3 in macrophages significantly attenuated or enhanced CD8(+) T-cell activation and infection damage in L monocytogenes-infected mice, respectively. Conclusions: Thus, we identified a new mechanism by which Tim-3 promotes L monocytogenes immune evasion. Further studies on this pathway might shed new light on the physio-pathological roles of Tim-3 and suggest new approaches for intervention.

T cell immunoglobulin and mucin protein 3 (Tim-3) is an immune checkpoint inhibitor that has therapeutic implications for many tumors and infectious diseases. However, the mechanisms by which Tim-3 promotes immune evasion remain unclear. Here, we demonstrated that Tim-3 inhibits the expression of major histocompatibility complex class I (MHC-I) in macrophages at both the mRNA and protein levels by inhibiting the STAT1-NLRC5 signaling pathway. As a result, MHC-I-restricted antigen presentation by macrophages was inhibited by Tim-3 both in vitro and in L. monocytogenes infection model in vivo. Systemic overexpression of Tim-3 or specific knockout of Tim-3 in macrophages significantly attenuated or enhanced CD8+ T cell activation and infection damage in L. monocytogenes-infected mice, respectively. We thus identified a new mechanism by which Tim-3 promotes Lister Monocytogenes immune evasion. Further studies on this pathway might shed new light on the physio-pathological roles of Tim-3 and suggest new approaches for intervention.

目的 探究T细胞免疫球蛋白黏蛋白-3(Tim-3)对白细胞介素-2(IL-2)的调控作用及机制.方法 通过酶联免疫吸附实验(ELISA)、流式细胞术(FACS)以及实时定量荧光聚合酶链反应(RT-PCR)方法,检测T细胞活化后,Jurkat高表达Tim-3(JurkatTim-3-high)与低表达Tim-3(JurkatTim-3-low)细胞系中IL-2的基因转录及蛋白表达水平;使用中和性抗人Tim-3抗体阻断Jurkat细胞系中Tim-3功能,ELISA及RT-PCR方法检测IL-2蛋白分泌及转录水平变化;通过Western蛋白免疫印迹法检测Tim-3对IL-2表达调控相关分子[包括蛋白激酶C(PKC)β1及核因子(NF)-κB亚基P65]的磷酸化的影响.结果 ELISA、FACS和RT-PCR结果均表明,Tim-3抑制IL-2的表达,使用抗人Tim-3抗体阻断后,IL-2的表达上调;Western印迹结果显示,与JurkatTim-3-low细胞相比,JurkatTim-3-high细胞中蛋白激酶PKCβ1及NF-κB亚基P65的磷酸化受到明显抑制.使用中和性抗人Tim-3抗体阻断后,PKCβ1及P65的磷酸化明显增强.结论 Tim-3对IL-2的基因转录及蛋白表达起负性调控作用,并且可能是通过抑制NF-κB(NF-κB/p65)及PKCβ1的磷酸化,从而抑制IL-2的产生.

目的 利用假病毒方法评价T细胞免疫球蛋白和黏蛋白结构域分子1(Tim-1)抗体在防治埃博拉病毒(EBOV)和马尔堡病毒(MARV)感染中的可能作用.方法 通过转染EBOV及MARV糖蛋白质粒,制备埃博拉及马尔堡假病毒,以HEK-293T细胞为靶细胞,利用假病毒评价抗人Tim-1(hTim-1)抗体对EBOV及MARV的防治作用.结果 成功制备了能感染HEK-293T细胞的埃博拉、马尔堡假病毒,发现Tim-1在HEK-293T细胞上的表达水平影响假病毒感染效率.同时,成功表达了抗hTim-1抗体,ELISA及FACS实验证实了其结合特异性,发现制备的Tim-1抗体能中和埃博拉、马尔堡假病毒的感染,并显示出与埃博拉及马尔堡假病毒抗体的协同作用.结论制备的Tim-1抗体显示了良好的中和活性,为埃博拉、马尔堡假病毒感染的防治提供了新的手段,认为假病毒是可行的抗体功能评价方法.

目的 探究T细胞免疫球蛋白和黏蛋白结构域分子-3(Tim-3)对小胶质细胞极化的调控作用及机制.方法 借助流式细胞术检测神经小胶质细胞BV-2上Tim-3及其配体的表达情况;利用ELISA、实时定量PCR方法检测阻断Tim-3功能对BV-2细胞因子表达的影响,进而明确Tim-3对小胶质细胞M1/M2极化的调控作用;通过Western印迹阐明Tim-3调控小胶质细胞极化的分子机制.结果 Tim-3及其配体分子半乳糖凝集素-9(Gal-9)表达于BV-2细胞;阻断Tim-3信号显著增强了BV-2细胞M1极化相关的促炎性细胞因子,包括白细胞介素6(IL-6)、IL-1β、IL-12、肿瘤坏死因子α(TNF-α)的表达,抑制了M2极化抗炎性细胞因子,如转化生长因子β (TGF-β)和IL-10的表达,提示Tim-3参与了小胶质细胞极化的调控.同时,分子机制研究发现,Tim-3可能通过抑制小胶质细胞中视黄酸诱导基因蛋白Ⅰ (RIG-Ⅰ)和核因子κB(NF-κB)的表达及活化发挥极化调控功能.结论 免疫检查点分子Tim-3参与了BV-2细胞的极化调控.该发现为通过Tim-3探索脑损伤及脑功能障碍性疾病的治疗策略提供了新的实验依据.