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

项目。

• 批准号: 10210221
• 负责人: Kai W Wucherpfennig
• 金额: $ 35.92万
• 依托单位: BRIGHAM AND WOMEN'S HOSPITAL
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-07-03 至 2025-06-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10210221
• 关键词: Antibodies; Blocking Antibodies; C Type Lectin Receptors; CD8-Positive T-Lymphocytes; CD8B1 gene; Cell Line; Cell physiology; Cell surface; Cell-Mediated Cytolysis; Cells; Cellular Immunity; Clinical Trials; Clone Cells; Collaborations; Color; Combined Modality Therapy; Data; Diagnosis; Dinoprostone; Effector Cell; Enrollment; Flow Cytometry; Gene Expression; Gene Expression Profile; Genes; Glioblastoma; Glioma; Goals; Human; Immune; Immunocompetent; Immunodeficient Mouse; Immunohistochemistry; Immunosuppression; Immunotherapy; Interferon Type II; KLRB1 gene; Label; Length; Ligands; Malignant Neoplasms; Mediating; Messenger RNA; Molecular; Monoclonal Antibodies; Mus; Myeloid Cells; Natural Killer Cells; Operative Surgical Procedures; Pathway interactions; Patients; Peptide Vaccines; Peptide/MHC Complex; Phase; Population; Prostaglandin E Receptor; Proteins; Relapse; Role; Sampling; Signal Transduction; Spatial Distribution; T-Cell Receptor; T-Lymphocyte; Therapeutic; Tumor Immunity; Work; anti-PD1 antibodies; base; brain tissue; cytokine; cytotoxic; cytotoxicity; effector T cell; exhaust; experimental study; genetic approach; humanized mouse; implantation; interest; mouse model; neoantigen vaccine; neoplastic cell; overexpression; population based; programmed cell death ligand 1; programmed cell death protein 1; programs; receptor; receptor expression; response; single-cell RNA sequencing; transcriptome sequencing; tumor

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.

抽象的 T细胞是保护性抗肿瘤免疫力的中心效应细胞，但目前对此知之甚少 人GBM中的重要免疫细胞。我们已经生成了有关肿瘤浸润T细胞的单细胞RNA-seq数据 GBM患者初始诊断或缓解的人群。我们使用这些全长RNA-seq数据来识别 克隆根据其TCRα和β链序列扩展的T细胞群，然后检查哪个 这种扩展的T细胞过表达基因。该分析强调了编码的KLRB1基因 以前证明可以抑制NK细胞介导的细胞毒性的CD161受体。 CD161配体， clec2d在人类GBM细胞的细胞表面表达。因此，我们假设CD161 - CLEC2D途径抑制CD8和CD4效应T细胞种群的抗肿瘤功能 GBM。初步数据表明，原代人T细胞中KLRB1基因的失活极大地增强了 它们在GBM的人源化小鼠模型中的效应函数。我们的初步数据还表明了几个 其他抑制受体通过大量的GBM浸润T细胞（包括CD96）表达 和两个前列腺素E2受体（EP2和EP4）。 AIM 1将重点放在肿瘤浸润T细胞的分析上 在项目1中描述的1B阶段Neovax加PD-1抗体试验的GBM患者中。 将主要关注T细胞及其配体抑制受体的表达，肿瘤细胞和髓样 细胞。抑制受体及其配体的表达将通过16色光流式细胞仪检查 和单细胞RNA-Seq（与核1和2合作），重点是CD161，PD-1，CD96和 前列腺素E2受体。我们将研究来自初始患者的同一患者的配对肿瘤样品 手术和继电器以确定这些抑制受体及其配体的表达如何变化 在使用Neovax Plus PD-1抗体进行免疫疗法后。通过与项目1合作，我们还将检查 表达CD161和其他抑制受体的T细胞的空间分布。 AIM 2将调查 CD161 - CLEC2D途径的治疗意义。我们将首先使用遗传方法来研究这一点 通过在原代T细胞中灭活KLRB1基因来抑制受体。阻止特异性人类CD161的mAb 还将用于检查这些发现的治疗潜力。我们还将检查组合 疗法（与项目2、4和核心3的合作）涉及单细胞鉴定的抑制受体 人GBM浸润T细胞中的RNA-Seq，特别关注CD161，PD-1，CD96和前列腺素 E2受体。这些研究将大大提高我们对GBM中T细胞功能的理解， 表征重要的抑制受体 - 限制效应T细胞功能的配体相互作用。