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

项目。

• 批准号: 10684029
• 负责人: Kai W Wucherpfennig
• 金额: $ 35.2万
• 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/10684029
• 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; Clonal Expansion; Clone Cells; Collaborations; Color; Combined Modality Therapy; Data; Diagnosis; Dinoprostone; Effector Cell; 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; Proteins; Relapse; Role; Sampling; Signal Transduction; Spatial Distribution; T cell infiltration; T-Cell Activation; T-Cell Receptor; T-Lymphocyte; Therapeutic; Tumor Immunity; WFDC2 gene; Work; anti-PD1 antibodies; brain tissue; cancer infiltrating T cells; cytokine; cytotoxic; cytotoxicity; effector T cell; exhaust; experimental study; genetic approach; humanized mouse; implantation; interest; mouse model; neoantigen vaccine; neoplastic cell; overexpression; participant enrollment; 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.

摘要 T细胞是保护性抗肿瘤免疫的中枢效应细胞，但目前对这些细胞的了解很少 重要的免疫细胞。我们已经生成了肿瘤浸润性T细胞的单细胞RNA-seq数据， 来自初次诊断或复发的GBM患者的人群。我们使用这些全长RNA-seq数据来鉴定 根据TCRα和β链序列克隆扩增T细胞群，然后检查 基因被这样扩增的T细胞过表达。该分析强调了KLRB 1基因，其编码 CD 161受体，以前显示抑制NK细胞介导的细胞毒性。CD 161配体， CLEC 2D在人GBM细胞的细胞表面表达。因此，我们假设CD 161- CLEC 2D通路抑制CD 8和CD 4效应T细胞群的抗肿瘤功能 GBM。初步数据显示，在原代人T细胞中KLRB 1基因的失活大大增强了人T细胞中KLRB 1的表达。 它们在GBM的人源化小鼠模型中的效应子功能。我们的初步数据还表明， 其他抑制性受体由大量GBM浸润性T细胞表达，包括CD 96 以及两种前列腺素E2受体（EP 2和EP 4）。目的1将集中在肿瘤浸润性T细胞的分析 在项目1中描述的1b期NeoVax + PD-1抗体试验中招募的GBM患者中。这些研究 将主要关注T细胞抑制性受体的表达及其肿瘤细胞和髓系配体的表达。 细胞通过16色光谱流式细胞术检查抑制性受体及其配体的表达 和单细胞RNA-seq（与Cores 1和2合作），重点是CD 161，PD-1，CD 96和 前列腺素E2受体我们将研究初始时从同一患者获得的配对肿瘤样本， 为了确定这些抑制性受体及其配体的表达如何变化， 在用NeoVax加PD-1抗体进行免疫治疗之后。与项目1合作，我们还将研究 表达CD 161和其他抑制性受体的T细胞的空间分布。目标2将调查 CD 161-CLEC 2D通路的治疗意义。我们将首先使用遗传学方法来研究这一点 通过失活原代T细胞中的KLRB 1基因来抑制抑制性受体。人CD 161特异性阻断mAb 也将被用来检查这些发现的治疗潜力。我们还将研究组合 治疗（与项目2，4和核心3合作），涉及通过单细胞识别的抑制性受体 人GBM浸润性T细胞中的RNA-seq，特别关注CD 161、PD-1、CD 96和前列腺素 E2受体。这些研究将显著推进我们对GBM中T细胞功能的理解， 表征了限制效应T细胞功能的重要抑制性受体-配体相互作用。