This is the first comprehensive study of EBV-specific CD8+ T-cell immunity in individuals, including children, who reside in a region of malaria endemicity that imparts a significantly elevated risk for the development of eBL. Accordingly, the results presented here reveal previously unappreciated characteristics of the human immune response to this common, chronic viral infection and the potential role of altered immunity in susceptibility to cancer.
For epitopes presented by HLA A*0201, we found that EBV-specific CD8+ T cells directed against lytic and latent antigens were equally represented across the study population as a whole, even though the proteins are presumably expressed with different kinetics and under different conditions in vivo. We also found that the signatures of EBV-specific T-cell immunity are largely set following primary infection and appear to change little over time, as no phenotypic differences were linked to age. Furthermore, although the immune system is thought to “see” latent antigens constantly, CD8+ T cells directed against these epitopes generally retained homeostatic potential and were rarely exhausted/senescent. In contrast, CD8+ T cells directed against lytic antigens, the expression of which is thought to be episodic and infrequent, were more likely to display an exhausted/senescent phenotype lacking homeostatic potential. Given the prevalent hypothesis that such differences are related to antigen exposure, these observations suggest the occurrence of frequent EBV reactivations in the overall study population.
T cells directed against the latent epitope LLD (EBNA3C) displayed phenotypic characteristics similar to those observed for the corresponding lytic antigen-specific populations; this finding is in keeping with previous studies that detail the typical immunodominance of EBNA3C (13
) and suggests quantitatively enhanced or qualitatively distinct presentation of this protein among the latent antigens studied.
An intriguing observation from our data is the identification of EBV-specific CD8+
T cells with a naïve-like (CD45RO−
) phenotype. Within the context of this study, the functional capacity of these EBV-experienced naïve-like CD8+
T cells is as yet unknown. It is likely that such cells populate a very early differentiated memory pool (41
) and perhaps even constitute an antigen-specific “stem cell-like memory” population (42
). However, further work is required to clarify the biological significance of this observation.
The features of EBV-specific CD8+
T-cell immunity described above were independent of malaria exposure yet were observed in individuals who typically acquire the virus during early childhood (25
). Nonetheless, malaria coinfections were associated with additional phenotypic differences between CD8+
T-cell populations specific for latent epitopes and the lytic epitope GLC (BMLF1), which displayed characteristics of a more differentiated stage in individuals from regions of malaria holoendemicity. Thus, despite the fact that all study participants were healthy, distinct malaria-associated irregularities in EBV-specific CD8+
T-cell immunity were apparent. Overall, these results support a combined role for early primary EBV infection and cumulative malaria exposure in EBV-specific T-cell immune dysregulation.
Due to the cross-sectional nature of this study and the relatively low incidence of eBL, we were not able to address directly the causal relationship between EBV, P. falciparum malaria, and eBL. However, our data offer new evidence that addresses two long-standing questions in the field.
First, as with any immunologic study in humans that attempts to inform pathogenic mechanisms, it is critical to understand whether the observed deficits represent cause or effect in the disease process. Our previous study revealed a deficiency in IFN-γ production by EBV-specific CD8+
T cells in children residing in a region with holoendemic malaria (23
). However, the events that precede this immune deficiency are unclear. On that basis, we postulated that more detailed studies of EBV-specific CD8+
T-cell immunity in areas with divergent malaria endemicity might provide a better window into the complex etiology of eBL. Given the remarkable diversity of T cells, simple immunophenotypic analyses could miss fine subsets critical to the understanding of this relationship. In addition, although EBNA1 is the only EBV-derived protein expressed in eBL (45
), the risk for disease may be set when T cells specific for a wide variety of lytic and latent antigens are generated. For these reasons, we used a sophisticated flow cytometric approach optimized for rare event analysis to demonstrate that a loss of central memory-like EBV-specific CD8+
T cells is associated with malaria exposure. Such immunologic perturbations within the EBV-specific CD8+
T-cell compartment could predispose to the functional deficits that accompany eBL (46
Second, our study addresses the long-standing question of whether eBL is associated with a generalized, malaria-induced suppression of T-cell immunity (21
). Our data demonstrate that EBV-specific, but not CMV-specific or total, CD8+
T-cell populations show significant differences associated with malaria exposure. These findings are consistent with previous studies showing that T-cell responses to malaria antigens and nonspecific mitogens are equally robust across study populations and age groups (46
). Thus, malaria exposure uniquely impacts the EBV-specific CD8+
T-cell compartment, which argues against a role for generalized immune suppression in the pathogenesis of eBL.
In the context of a cross-sectional study, however, it is not possible to discern how the observed immunologic differences arise. Recently, a malarial antigen capable of inducing the reactivation of latent EBV was identified (48
), possibly providing a mechanistic explanation for our findings. In this scenario, repeated malaria infections induce EBV reactivation, thereby seeding new, potentially highly activated and dividing B cells (27
), which consequently increase the risk of B-cell transformation. As EBV-specific CD8+
T cells attempt to control these frequent reactivations, their differentiation is accelerated and the pool of central memory cells for such specificities is selectively depleted. This possibility is supported by seroprofiling studies, which demonstrate that elevated antibody titers to viral capsid antigen (VCA) and the Z Epstein-Barr replication activator (ZEBRA) protein are associated with holoendemic malaria exposure (49
). An elevation in these antibody titers signifies viral reactivation (50
), and higher VCA antibody titers were observed prior to the development of eBL in a study of Ugandan children (51
). In addition, malaria infection in concert with EBV reactivation may modulate elements of the innate immune system that influence T-cell maturation and differentiation (53
In summary, we have demonstrated that a select set of EBV-derived antigen-specific CD8+ T cells is altered in individuals coinfected with P. falciparum malaria and at increased risk for eBL. These findings inform our studies of eBL pathogenesis and suggest that EBV-targeted immune interventions in the setting of holoendemic malaria might hold promise for the prevention of this devastating pediatric malignancy.