Proj. 3: Immunosuppressive circuits in T cells and other immune cells in GBM patients enrolled in clinical trials

摘要 T细胞是保护性抗肿瘤免疫的中枢效应细胞，但目前对此知之甚少 人肾小球基底膜的重要免疫细胞。我们已经生成了关于肿瘤浸润性T细胞的单细胞RNA-SEQ数据 初诊或复发的GBM患者的人群。我们使用这些全长rna-seq数据来识别 根据它们的TCRα和β链序列克隆扩增的T细胞群体，然后检查 这些扩增的T细胞过度表达了基因。这一分析突出了编码KLRB1基因 CD161受体先前被证明能抑制NK细胞介导的细胞毒作用。CD161配体， CLEC2D，表达于人肾小球系膜细胞表面。因此，我们假设CD161- CLEC2D途径抑制CD8和CD4效应T细胞群的抗肿瘤功能 GBM。初步数据显示，原代人类T细胞中KLRB1基因的失活大大增强 它们的效应器在人源化的GBM小鼠模型中发挥作用。我们的初步数据还表明，有几个 其他抑制性受体由大量的GBM浸润性T细胞表达，包括CD96 和两个前列腺素E2受体(EP2和EP4)。目标1将重点分析肿瘤浸润性T细胞 在项目1中描述的参加1b期NeoVax加PD-1抗体试验的GBM患者中。这些研究 将主要关注T细胞及其配体在肿瘤细胞和髓系细胞中表达抑制性受体 细胞。抑制性受体及其配体的表达将用16色光谱流式细胞术进行检测 和单细胞RNA-seq(与核心1和2合作)，重点是CD161、PD-1、CD96和 前列腺素E2受体。我们将调查来自同一患者的配对肿瘤样本 手术和复发，以确定这些抑制性受体及其配体的表达如何变化 用NeoVax加PD-1抗体进行免疫治疗。与项目1合作，我们还将研究 表达CD161和其他抑制性受体的T细胞的空间分布。目标2将调查 CD161-CLEC2D通路的治疗意义我们将首先使用遗传方法来研究这一点 通过灭活原代T细胞中的KLRB1基因来抑制受体。阻断人CD161特异性单抗 也将被用来检查这些发现的治疗潜力。我们还将检查组合 涉及单细胞识别的抑制性受体的疗法(与项目2、4和核心3合作) CD161、PD-1、CD96和前列腺素在人GBM浸润性T细胞中的表达 E2受体。这些研究将极大地促进我们对GBM和 描述限制效应器T细胞功能的重要的抑制性受体-配体相互作用。

Abstract T cells are central effector cells of protective anti-tumor immunity, but little is currently known about these important immune cells in human GBM. We have generated single-cell RNA-seq data on tumor-infiltrating T cell populations from GBM patients at initial diagnosis or relapse. We used these full-length RNA-seq data to identify clonally expanded T cell populations based on their TCRα and β chain sequences and then examined which genes were overexpressed by such expanded T cells. This analysis highlighted the KLRB1 gene which encodes the CD161 receptor that was previously shown to inhibit NK cell-mediated cytotoxicity. The CD161 ligand, CLEC2D, is expressed at the cell surface of human GBM cells. We therefore hypothesize that the CD161 – CLEC2D pathway inhibits the anti-tumor function of both CD8 and CD4 effector T cell populations in GBM. Preliminary data show that inactivation of the KLRB1 gene in primary human T cells greatly enhances their effector function in a humanized mouse model of GBM. Our preliminary data also demonstrate that several other inhibitory receptors are expressed by substantial populations of GBM-infiltrating T cells, including CD96 and two prostaglandin E2 receptors (EP2 and EP4). Aim 1 will focus on the analysis of tumor-infiltrating T cells in GBM patients enrolled in the phase 1b NeoVax plus PD-1 antibody trial described in Project 1. These studies will primarily focus on the expression of inhibitory receptors by T cells and their ligands by tumor cells and myeloid cells. Expression of inhibitory receptors and their ligands will be examined by 16-color spectral flow cytometry and single-cell RNA-seq (in collaboration with Cores 1 and 2), with an emphasis on CD161, PD-1, CD96 and prostaglandin E2 receptors. We will investigate paired tumor samples from the same patient obtained at initial surgery and relapse in order to determine how expression of these inhibitory receptors and their ligands changes following immunotherapy with NeoVax plus PD-1 antibody. In collaboration with Project 1, we will also examine the spatial distribution of T cells that express CD161 and other inhibitory receptors. Aim 2 will investigate the therapeutic significance of the CD161 – CLEC2D pathway. We will first use a genetic approach to study this inhibitory receptor by inactivating the KLRB1 gene in primary T cells. Blocking mAbs specific for human CD161 will also be used to examine the therapeutic potential of these findings. We will also examine combination therapies (collaboration with Projects 2, 4 and Core 3) involving the inhibitory receptors identified by single-cell RNA-seq in human GBM infiltrating T cells, with a particular focus on CD161, PD-1, CD96 and the prostaglandin E2 receptors. These studies will significantly advance our understanding of T cell function in GBM and characterize important inhibitory receptor – ligand interactions that constrain effector T cell function.