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

多形性胶质母细胞瘤（GBM）是成人最常见的脑肿瘤。尽管标准治疗，平均总生存期通常只有1年多一点。与中枢神经系统（CNS）外的其他恶性肿瘤相比，免疫治疗在GBM中的发展并不好，因为缺乏对GBM免疫过程的了解。本项目的目标是缩小对GBM中t细胞功能的理解差距。初步的单细胞rna测序数据可以根据TCR链序列鉴定克隆扩增的T细胞群。在这个分析中，我们发现CD161 （KLRB1）被这些t细胞过度表达。CD161配体CLEC2D在人GBM细胞系细胞表面表达。CD161由CD4+和CD8+ T细胞表达，其效应基因在原代人T细胞中的表达和KLRB1基因的失活大大增强了其在人源化小鼠GBM模型中的效应功能。因此，我们假设CD161 - cle2d通路抑制GBM中CD8和CD4效应T细胞群的抗肿瘤功能。目前的项目旨在更详细地研究cd161 - cle2d在人类GBM中的相互作用，以加速转化到临床。最初将采用遗传学方法通过灭活原代t细胞中的KLRB1基因或GBM细胞中的CLEC2D基因来研究这种抑制性受体-配体对。还将产生针对人类CD161和cle2d的阻断单克隆抗体，以检测抗体介导的抑制该途径的治疗潜力。体内实验将主要在人源化小鼠模型中进行，将gbm细胞立体定向植入中枢神经系统后，在免疫缺陷小鼠体内注射人t细胞（带有gbm特异性CAR或TCR）。此外，治疗潜力将在完全免疫能力的小鼠携带GBM进行测试。流式细胞术、免疫组织化学和单细胞rna测序方法将用于确定CD161 - cle2d通路被抑制时T细胞群的功能变化。此外，还将对参加临床试验（个体化新抗原肽疫苗接种加PD-1阻断剂）的GBM患者的肿瘤浸润T细胞进行分析。将对肿瘤进行初步手术和复发的研究，以确定这些抑制受体及其配体的表达在治疗后如何变化。考虑到t细胞耗竭伴随着这些受体和配体的表达，我们将讨论CD161在t细胞（以及其他抑制性受体）中的表达以及肿瘤细胞和髓细胞（以及其他抑制性配体）上的CLEC2D。假设CD161是GBM肿瘤内t细胞的重要抑制受体，抑制CD161- cle2d通路能够增强GBM中t细胞效应物的功能。

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.