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

居住在疟疾地区的儿童中恶性疟和EB病毒的混合感染 全流行区与EBV相关癌症的风险增加有关，这种癌症称为地方性Burkitt 淋巴瘤(EBL)。大多数非洲儿童在1岁之前就感染了EBV，但这种B细胞癌确实感染了EBV 直到几年后才会发生。据推测，反复发作的疟疾抑制了对 EBV，为EBL的发病创造了一个允许的环境。然而，负责的机制并不是 完全理解。我们先前的研究发现，接触疟疾的儿童具有病理性的高EBV载量； 幼稚的EBV特异性CD8T细胞，其效应功能减弱；非传统的先天的CD8T细胞 表达颗粒酶B而不是干扰素-γ的细胞；慢性感染诱导的CD56neg的扩增 细胞毒性受损的自然杀伤细胞(NK细胞)。因此，我们已经确定了最近的免疫学改变。 允许不受限制的EBV复制和EBL肿瘤发生。在这次续期申请中，我们将在我们的中央 假设疟疾诱导的免疫调节机制抑制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 如果反复感染PF-疟疾，已知会导致EBV重新激活，则会导致抑制协同作用增加 EB病毒特异性CD8CD8αβT细胞的受体表达TIGIT、PD1、CTLA4、LAG3、TIM3、 采用流式细胞仪检测T细胞亚群CD160、2B4、KLRG1、BTLA。精疲力竭与 将通过单细胞RNA测序进一步定义细胞毒性特征，并在 体外用EBV转化的淋巴母细胞系(LCLS)进行细胞毒性T淋巴细胞(CTL)检测。 目的2.确定反复感染肺吸虫病是否会诱导产生IL-10的CD4或CD8 T细胞 对EBV特异性T细胞发挥免疫调节作用。IL-10分泌Foxp3neg的频率 调节性CD25、CD4、TR1细胞(CD49b、LAG3、CD226/DNAM1)、Treg of-B细胞(LAG3、ICOS、PD1、 GITR，OX40)和CD8 CD25neg Foxp3neg T细胞将通过流式细胞仪和RNAseq进行检测 将它们与经典的CD4Fox p3调节性T细胞(Treg)区分开来。CTL检测将确定 IL-10细胞因子家族成员对CD8T细胞体外杀伤作用的研究。目标3.确定反复发生的PF-疟疾 感染影响γδT/NK细胞亚群的频率及其相对比例如何影响 对EBL肿瘤的细胞毒作用。用流式细胞仪检测γδT细胞亚群和NK细胞亚群 并与疟疾暴露有关。γδT和NK细胞亚群的细胞毒作用将在体外进行量化 抗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 实验将评估每个亚群对整体细胞毒性的相对贡献。了解如何 疟疾影响人类的免疫状况，特别是在儿童中，这将使我们能够探索 在维持对EBV的保护性免疫的同时调节调节机制的干预措施。

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.