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 landsc...