The inhibition of the CD161-CLEC2D interaction as immune checkpoint blockade in glioblastoma multiforme

多形性胶质母细胞瘤中 CD161-CLEC2D 相互作用的抑制作为免疫检查点阻断

• 批准号: 419826340
• 负责人: Dr. Sascha Marx
• 金额: --
• 依托单位: Department of Cancer Immunology and Virology
• 依托单位国家: 德国
• 项目类别: Research Fellowships
• 财政年份: 2019
• 资助国家: 德国
• 起止时间: 2018-12-31 至 2022-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/419826340?language=en
• 关键词: inhibition; CD161; CLEC2D; interaction; immune

Glioblastoma multiforme (GBM) is the most common brain tumor in adults. Despite standard treatment the mean overall survival is frequently only a little over 1 year. Immunotherapy is not as well developed in GBM compared to other malignancies outside the central nervous system (CNS), because there is lack of knowledge about immunological processes in GBM. The goal of the present project is to close gaps in the understanding of T-cell function in GBM. Preliminary single cell RNA-sequencing data could identify clonally expanded T cell populations based on their TCR chain sequences. In this analysis it was seen that the CD161 (KLRB1) is overexpressed by such T-cells. The CD161 ligand, CLEC2D, is expressed at the cell surface of human GBM cell lines. CD161 is expressed by CD4+ und CD8+ T-cells with an effector gene expression and inactivation of the KLRB1 gene in primary human T cells greatly enhances their effector function in a humanized mouse model of GBM. It is therefore hypothesized that the CD161 – CLEC2D pathway inhibits the anti-tumor function of both CD8 and CD4 effector T cell populations in GBM.The current project aims to study the CD161-CLEC2D interaction in human GBM in more detail to accelerate a translation into the clinic. Initially a genetic approach will be used to study this inhibitory receptor – ligand pair by inactivating the KLRB1 gene in primary T-cells or the CLEC2D gene in GBM cells. Blocking monoclonal antibodies specific for human CD161 and CLEC2D will also be generated to examine the therapeutic potential of antibody-mediated inhibition of this pathway. In vivo experiments will be conducted mainly in humanized mouse models, in which human T-cells (with GBM-specific CAR or TCR) will be injected in immunodeficient mice after stereotactic implantation of GBM-cells into the CNS. Furthermore, the therapeutic potential will be tested in fully immunocompetent mice harboring GBM. Flow cytometry, immunohistochemistry and single-cell RNA-sequencing approaches will be used to define the functional changes in T cell populations when the CD161 – CLEC2D pathway is inhibited.Furthermore analysis of tumor-infiltrating T cells in GBM patients enrolled in a clinical trial (personalized neoantigen peptide vaccination plus PD-1 blockade) will be done. Tumors both at initial surgery and relapse will be investigated in order to determine how expression of these inhibitory receptors and their ligands changes following therapy. CD161 expression in T-cells (as well as other inhibitory receptors) and CLEC2D on tumor cells and myeloid cells (as well as other inhibitory ligands) will be addressed, given that T-cell exhaustion is accompanied with expression of these receptors and ligands. It is hypothesized that CD161 is an important inhibitory receptor for intra-tumoral T-cells in GBM and that inhibition of the CD161-CLEC2D pathway is able to enhance T-cell effector function in GBM.

多形性胶质母细胞瘤(GBM)是成人最常见的脑肿瘤。尽管接受标准治疗，平均总存活率通常只有1年多一点。与中枢神经系统(CNS)以外的其他恶性肿瘤相比，GBM的免疫治疗没有得到很好的发展，因为GBM缺乏对免疫过程的了解。本项目的目标是弥合对GBM中T细胞功能的理解上的差距。初步的单细胞RNA测序数据可以根据它们的TCR链序列识别克隆性扩增的T细胞群体。在这个分析中可以看到CD161(KLRB1)是由这样的T细胞过度表达的。CD161配体CLEC2D表达于人GBM细胞系的细胞表面。CD161是由CD4+和CD8+T细胞表达的，在人源化的GBM小鼠模型中，KLRB1基因在原代人类T细胞中的表达和失活极大地增强了它们的效应功能。因此，我们假设CD161-CLEC2D通路抑制了GBM中CD8和CD4效应T细胞群的抗肿瘤功能。本项目旨在更详细地研究CD161-CLEC2D在人GBM中的相互作用，以加速将其转化为临床。最初，将使用遗传学方法研究这种抑制性受体-配体对，方法是使原代T细胞中的KLRB1基因或GBM细胞中的CLEC2D基因失活。还将产生针对人类CD161和CLEC2D的封闭性单抗，以检测抗体介导的抑制这一途径的治疗潜力。体内实验将主要在人源化的小鼠模型中进行，在此模型中，将GBM-细胞立体定向植入中枢神经系统后，将人T细胞(带有GBM特异性CAR或TCR)注射到免疫缺陷小鼠体内。此外，治疗潜力将在携带GBM的完全免疫活性小鼠身上进行测试。我们将利用流式细胞术、免疫组织化学和单细胞RNA测序的方法来确定CD161-CLEC2D途径被抑制时T细胞群的功能变化。此外，还将对参加临床试验(个性化新抗原肽疫苗加PD-1阻断)的GBM患者的肿瘤浸润性T细胞进行分析。将对初次手术和复发的肿瘤进行调查，以确定这些抑制受体及其配体的表达在治疗后如何变化。鉴于T细胞耗竭伴随着这些受体和配体的表达，CD161在T细胞(以及其他抑制性受体)和CLEC2D在肿瘤细胞和髓系细胞(以及其他抑制性配体)的表达将得到解决。推测CD161是GBM瘤内T细胞的重要抑制性受体，抑制CD161-CLEC2D通路可增强GBM的T细胞效应功能。