Role of CD8 T cell-mediated Pathology in Globoid Cell Leukodystrophy

CD8 T 细胞介导的病理学在球状细胞脑白质营养不良中的作用

• 批准号: 10634808
• 负责人: Stephen J Crocker
• 金额: $ 51.91万
• 依托单位: UNIVERSITY OF CONNECTICUT SCH OF MED/DNT
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-03-01 至 2028-02-29
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10634808
• 关键词: 5 year old; Address; Affect; Antibodies; Antigen-Presenting Cells; Antigens; Automobile Driving; Behavioral; Biochemical; Blood; Body Weight decreased; Brain; CD4 Positive T Lymphocytes; CD8-Positive T-Lymphocytes; CD8B1 gene; Cell Therapy; Cells; Cellular Immunity; Central Nervous System; Central Nervous System Diseases; Cessation of life; Child; Clinical; Cognitive deficits; Complex; Cytometry; Cytotoxic T-Lymphocytes; Data; Demyelinations; Development; Disease; Effectiveness; Flow Cytometry; Future; Genes; Genetic; Genetic Diseases; Globoid cell leukodystrophy; Histologic; Human; Image; Immune; Immunity; Inflammation; Inflammatory; Investigational Therapies; Knockout Mice; Knowledge; Lesion; Life Expectancy; Lipids; Live Birth; Location; Longevity; Maps; Mediating; Meningeal; Modeling; Molecular; Motor; Mus; Mutation; Nature; Nerve Degeneration; Nerve Tissue; Nonsense Codon; Oligodendroglia; Onset of illness; Outcome; Outcome Measure; Outcome Study; Paralysed; Pathogenesis; Pathogenicity; Pathologic; Pathology; Phenotype; Play; Population; Process; Psychosine; Reporter Genes; Role; T cell clonality; T-Cell Depletion; T-Lymphocyte; Technology; Testing; Therapeutic; Therapeutic Intervention; Time; Tissues; Transgenic Animals; Transgenic Organisms; adaptive immunity; brain tissue; cell type; central nervous system demyelinating disorder; cytokine; cytotoxic CD8 T cells; cytotoxicity; defined contribution; disability; galactosylceramidase; gene therapy; genetic approach; genetic profiling; improved; insight; loss of function; loss of function mutation; mouse model; multidisciplinary; neuroinflammation; neuropathology; novel; pharmacologic; postnatal; preservation; prevent; response; single-cell RNA sequencing; spatial relationship; success; tool; transcriptome sequencing; transcriptomics; white matter

Abstract Globoid cell leukodystrophy (GLD) or Krabbe's disease is a fatal genetic demyelinating disease of the central nervous system affecting 1 in 100,000 live births with no cure or effective long-term treatment. GLD is caused by loss-of-function mutations in the galactosylceramidase (galc) gene, where loss of GALC enzymatic function results in toxic accumulation of its substrate, a lipid called galactosylsphingosine or `psychosine'. Psychosine cytotoxicity is considered the basis of several key pathologies in GLD. Neuropathology in GLD is marked by profound demyelination and inflammation. However, molecular details of these processes are limited, leaving few therapeutic options. Early histological evidence for CD8+ T cells within demyelinated lesions in both the twi mouse brain and human GLD brain had suggested a role for adaptive immunity in this disease. However, the function of CD8+ T cells in GLD has not been previously determined. To address this gap in our knowledge, we analyzed the timing of T cell population changes by flow cytometry in CNS tissues from the twitcher mouse model of GLD and compared with wt littermates. We identified a rapid and protracted elevation of T cells in the twi CNS that was coincident with the onset of clinical disease at postnatal day 21 (P21). These data were confirmed using single-cell RNA sequencing on twi and wt littermate brain tissues from which identified a 9-fold increase in CD8+ T cells in twi mouse brains at P21. Our transcriptomic data also defined the CD8+ population as cytotoxic T lymphocytes (CTLs). To test the function of CD8+ CTLs in GLD-like disease in twi mice, we depleted CD8+ T cells by administering anti-CD8 antibody to twi mice and found that this treatment effectively prevented disease onset, preserved wellness and completely prevented CNS demyelination while also attenuating pro-inflammatory cytokine levels in brain and blood. These novel and highly translational data portend a pathogenic role for CD8+ T cells in GLD and underlie the basis for our overall hypothesis that CD8+ T cells are pathogenic in GLD and directly contribute to disease. Accordingly, Aim 1 will characterize the spatial and temporal development of CD8+ T cells in twi mice and challenge the contribution of CD8+ T cells using knockout mouse lines to then assess clinical, biochemical, and pathological outcomes. Aim 2 will define the overall nature of adaptive immunity by evaluating contribution of CD4+ T cells to CD8+ T cell-mediated immunity in twi mice using targeted depletion and genetic strategies. Aim 3 will identify and profile the timing, and location T cells responding to authentic antigen, the clonality of the T cells using T cell β chain VDJ phenotyping, and then identify the nature of the antigen presenting cell types using transgenic reporter mice. These multi-disciplinary studies will interrogate a previously unrecognized CD8+ T cell neuroinflammatory response in GLD. Outcomes of these studies are expected to fill an important gap in our understanding on the fundamental cellular pathological mechanisms underlying the development CD8+ T cell mediated neuropathology and disease in GLD.

抽象的 球状细胞脑白质营养不良（GLD）或克拉伯氏病是一种致命的中枢神经系统脱髓鞘疾病。 神经系统影响着十万分之一的活产儿，无法治愈或有效的长期治疗。 GLD引起 通过半乳糖神经酰胺酶 (galc) 基因的功能丧失突变，其中 GALC 酶功能丧失 导致其底物（一种称为半乳糖基鞘氨醇或“精神鞘氨醇”的脂质）的毒性积累。心神 细胞毒性被认为是 GLD 中几种关键病理的基础。 GLD 的神经病理学特点是 严重脱髓鞘和炎症。然而，这些过程的分子细节是有限的，使得 治疗选择很少。两组患者脱髓鞘病变内 CD8+ T 细胞的早期组织学证据 小鼠大脑和人类 GLD 大脑表明适应性免疫在这种疾病中发挥着作用。然而， CD8+ T 细胞在 GLD 中的功能先前尚未确定。为了弥补我们的知识差距，我们 通过流式细胞术分析了 twitcher 小鼠中枢神经系统组织中 T 细胞群变化的时间 GLD 模型并与 wt 同窝小鼠进行比较。我们发现 T 细胞快速且持久地升高 twi CNS 与出生后第 21 天临床疾病的发作同时发生 (P21)。这些数据是 使用单细胞 RNA 测序对 twi 和 wt 同窝小鼠脑组织进行证实，从中鉴定出 9 倍 P21 时双胞胎小鼠大脑中 CD8+ T 细胞增加。我们的转录组数据还定义了 CD8+ 群体 作为细胞毒性 T 淋巴细胞 (CTL)。为了测试 CD8+ CTL 在双胞胎小鼠 GLD 样疾病中的功能，我们 通过给双胞胎小鼠施用抗 CD8 抗体来耗尽 CD8+ T 细胞，并发现这种治疗有效 预防疾病发作，保持健康并完全预防中枢神经系统脱髓鞘，同时还 减弱大脑和血液中促炎细胞因子的水平。这些新颖且高度转化的数据 预示着 CD8+ T 细胞在 GLD 中的致病作用，并且是我们总体假设的基础：CD8+ T 细胞在 GLD 中具有致病性，并直接导致疾病。因此，目标 1 将描述 双胞胎小鼠 CD8+ T 细胞的空间和时间发育并挑战 CD8+ T 细胞的贡献 使用基因敲除小鼠系来评估临床、生化和病理结果。目标 2 将定义 通过评估 CD4+ T 细胞对 CD8+ T 细胞介导的贡献来评估适应性免疫的整体性质 使用靶向消除和遗传策略对 Twi 小鼠进行免疫。目标 3 将确定并分析时间， 并定位对真实抗原有反应的 T 细胞，使用 T 细胞 β 链 VDJ 确定 T 细胞的克隆性 表型分析，然后使用转基因报告小鼠鉴定抗原呈递细胞类型的性质。 这些多学科研究将探究以前未被识别的 CD8+ T 细胞神经炎症 GLD 中的响应。这些研究的结果预计将填补我们对这一问题理解的一个重要空白。 CD8+ T 细胞介导的发育的基本细胞病理机制 GLD 中的神经病理学和疾病。