Impact of malaria on shaping immunity to EBV in the etiology of Burkitt lymphoma

疟疾对伯基特淋巴瘤病因中 EBV 免疫力的影响

• 批准号: 10264137
• 负责人: ANN M MOORMANN
• 金额: $ 53.34万
• 依托单位: UNIV OF MASSACHUSETTS MED SCH WORCESTER
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-07-10 至 2025-06-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10264137
• 关键词: 1 year old; 6 year old; 9 year old; Activities of Daily Living; Acute; Adult; Africa; African; African Burkitt' s lymphoma; Antigens; Antiviral Agents; Area; B-Lymphocytes; Biological Assay; Blood; Burkitt Lymphoma; CD8-Positive T-Lymphocytes; CD8B1 gene; CTLA4 gene; Cell Line; Cell physiology; Cells; Child; Childhood; Chronic; Coculture Techniques; Cytotoxic T-Lymphocytes; Development; Diagnosis; EBV specific T-cells; Enrollment; Environment; Epstein-Barr Virus Infections; Etiology; Exposure to; FOXP3 gene; Falciparum Malaria; Family member; Fever; Flow Cytometry; Foxes; Frequencies; Granzyme; Herpesviridae Infections; Heterogeneity; Homeostasis; Human; Human Herpesvirus 4; IL2RA gene; Immune; Immunity; Immunocompetent; Immunologic Surveillance; Immunologics; Impairment; In Vitro; Individual; Infection; Inflammatory; Interferon Type II; Interleukin-10; Intervention; Lead; Ligands; Link; Malaria; Malignant Childhood Neoplasm; Malignant Neoplasms; Measures; Mediating; Mediator of activation protein; Memory; Mus; Natural Killer Cells; OX40; Parasites; Pathogenesis; Pathologic; Pathway interactions; Patients; Plasmodium falciparum; Recording of previous events; Regulatory T-Lymphocyte; Risk; Role; Schedule; Shapes; Signal Transduction; Site; T cell response; T memory cell; T-Lymphocyte; T-Lymphocyte Subsets; Testing; Time; Viral load measurement; Virus Replication; Visit; cancer cell; chronic infection; co-infection; cohort; cytokine; cytotoxicity; density; design; effector T cell; exhaustion; experience; experimental study; immunopathology; immunoregulation; improved; in vitro testing; infected B cell; interleukin 20; interleukin-19; interleukin-22; lymphoblastoid cell line; malaria infection; prevent; programmed cell death protein 1; receptor; receptor expression; single-cell RNA sequencing; transcriptome sequencing; transforming virus; tumor; tumorigenesis; virus related cancer

Plasmodium falciparum (Pf) malaria and Epstein-Barr Virus (EBV) co-infections in children residing in malaria holoendemic areas have been linked to an increased risk of an EBV-associated cancer called endemic Burkitt lymphoma (eBL). Most African children are infected with EBV before 1 year of age, yet this B-cell cancer does not occur until years later. It has been postulated that repeated episodes of malaria ‘suppress’ immunity to EBV, creating a permissive environment for eBL pathogenesis. However, the mechanisms responsible are not fully understood. Our prior studies found that malaria-exposed children had pathologically high EBV loads; naïve-like EBV-specific CD8+ T cells with diminished effector functions; unconventional, innate-like CD8+ T cells that expressed Granzyme B in lieu of IFN-γ; and an expansion of ‘chronic-infection induced’ CD56neg Natural Killer (NK) cells with impaired cytotoxicity. Thus, we have identified proximate immunologic alterations that allow unrestrained EBV replication and eBL tumorigenesis. In this renewal application, we will our central hypothesis that malaria-induced immunoregulatory mechanisms restrain T cell cytotoxicity against EBV-infected B cells and eBL tumors. This will be tested by the following Specific Aims. Aim 1. Determine if repeated Pf-malaria infections, known to induce EBV reactivation, lead to increased inhibitory co- receptor expression on EBV-specific CD8+ αβ T cells. Expression of TIGIT, PD1, CTLA4, LAG3, TIM3, CD160, 2B4, KLRG1, BTLA, on T cell subsets will be measured by flow cytometry. Exhaustion versus cytotoxicity signatures will be further defined with single cell RNA sequencing, and functional capacity tested in vitro by cytotoxic T lymphocyte (CTL) assays using EBV-transformed lymphoblastoid cell lines (LCLs). Aim 2. Determine if repeated Pf-malaria infections induce IL-10 producing CD4+ or CD8+ T cells that exert an immune-regulatory effect on EBV-specific T cells. The frequency of IL-10 secreting Foxp3neg regulatory CD25+, CD4+, Tr1 cells (CD49b+, LAG3+, CD226+/DNAM1+), Treg-of-B cells (LAG3+, ICOS+, PD1+, GITR+, OX40+) and CD8+ CD25neg Foxp3neg T cells will be measured by flow cytometry and RNAseq to distinguish them from classical CD4+Fox+p3+ regulatory T cells (Tregs). CTL assays will determine the impact of IL-10 cytokine family members on CD8+ T cell cytotoxicity, in vitro. Aim 3. Determine if repeated Pf-malaria infections influence the frequency of γδT to NK cell subsets and how their relative ratios impact cytotoxicity to eBL tumors. The frequency of γδT and NK cell subsets will be evaluated by flow cytometry and associated with malaria exposure. Cytotoxicity of γδ T and NK cell subsets will be quantified in vitro against BL tumors, including our newly established patient-derived eBL cell lines. Ligand-receptor blocking experiments will evaluate the relative contribution of each subset to overall cytotoxicity. Understanding how malaria influences the human immunologic landscape, especially in children, will allow us to explore interventions that modulate regulatory mechanisms while maintaining protective immunity to EBV.

居住在疟疾地区的儿童中的恶性疟原虫（Pf）疟疾和EB病毒（EBV）合并感染 全流行区与一种称为地方性伯基特的EB病毒相关癌症的风险增加有关 淋巴瘤（eBL）。大多数非洲儿童在1岁之前感染EBV，但这种B细胞癌确实如此 直到几年后才发生。据推测，疟疾的反复发作“抑制”了对 EBV，为eBL发病机制创造了一个允许的环境。然而，负责的机制不是 完全理解我们先前的研究发现，接触疟疾的儿童有病理性高EBV载量; 效应子功能减弱的幼稚样EBV特异性CD 8 + T细胞;非常规先天性样CD 8 + T细胞 表达颗粒酶B代替IFN-γ的细胞;以及“慢性感染诱导的”CD 56阴性扩增。 具有受损细胞毒性的自然杀伤（NK）细胞。因此，我们已经确定了近似的免疫学改变， 允许EBV无限制复制和eBL肿瘤发生。在此更新申请中，我们将以 疟疾诱导免疫调节机制抑制T细胞对 EBV感染的B细胞和eBL肿瘤。这将通过以下具体目标进行检验。目标1。确定 如果已知可诱导EBV再活化的反复Pf-malaria感染导致抑制性共刺激增加， EBV特异性CD 8 + αβ T细胞上的受体表达。TIGIT、PD1、CTLA4、LAG3、TIM3、 将通过流式细胞术测量T细胞亚群上的CD 160、2B 4、KLRG 1、BTLA。疲劳与 细胞毒性特征将通过单细胞RNA测序进一步确定，并在 通过使用EBV转化的淋巴母细胞样细胞系（LCL）的细胞毒性T淋巴细胞（CTL）测定法进行体外研究。 目标二。确定反复的Pf-malaria感染是否诱导产生IL-10的CD 4+或CD 8 + T细胞， 对EBV特异性T细胞发挥免疫调节作用。IL-10分泌型Foxp 3阴性的频率 调节性CD 25+、CD 4+、Tr 1细胞（CD 49 B+、LAG 3+、CD 226 +/DNAM 1+）、Treg-of-B细胞（LAG 3+、ICOS+、PD 1+、 GITR+、0X 40+）和⑶ 8 + ⑶ 25-Foxp 3-T细胞将通过流式细胞术和RNAseq测量， 将它们与经典的CD 4 +Fox+p3+调节性T细胞（Tcells）区分开。CTL检测将确定 体外IL-10细胞因子家族成员对CD 8 + T细胞细胞毒性的影响。目标3.确定是否重复PF疟疾 感染影响γδT与NK细胞亚群的频率以及它们的相对比例如何影响 对eBL肿瘤的细胞毒性。将通过流式细胞术评价γδT和NK细胞亚群的频率 与疟疾暴露有关。将在体外定量γδ T和NK细胞亚群的细胞毒性 针对BL肿瘤，包括我们新建立的患者来源的eBL细胞系。配体受体阻断 实验将评估每个子集对总体细胞毒性的相对贡献。了解如何 疟疾对人类免疫系统的影响，特别是对儿童的影响，将使我们能够探索 调节调节机制，同时保持对EBV的保护性免疫的干预。