Intracellular Cytokine Production by Dengue Virus–specific T cells Correlates with Subclinical Secondary Infection

Dengue is an important global health problem, and the dengue viruses (DENV) are the most prevalent arthropod-borne viruses in the tropics and subtropics today. There are an estimated 50–100 million DENV infections annually worldwide [1]. The spectrum of disease ranges from subclinical infection to classical dengue fever to the more severe dengue hemorrhagic fever (DHF) and dengue shock syndrome (DSS). DHF and DSS, characterized by significant plasma leakage and occasionally hemorrhage and shock, account for an estimated half million cases and >20,000 deaths annually [2]. There are 4 distinct serotypes of dengue virus (DENV 1–4); infection with 1 serotype is believed to confer lifelong immunity to that serotype, whereas protective immunity to heterologous serotypes is short-lived, probably on the order of months [3]. One of the unique features of dengue is the role of heterologous infection in disease severity. Both clinical and epidemiological studies have suggested that the immune response plays a major role in the pathogenesis of severe dengue disease. Subsequent infection with a heterologous serotype predisposes patients toward more severe disease; studies in Southeast Asia, done both in the 1980s [4, 5] and more recently [6, 7], have shown that DHF is associated with secondary infection, implying that other mechanisms in addition to viral replication could be the cause of DHF. Pre-existing heterotypic immunity is believed to increase disease severity in secondary DENV infection and, thus, the risk for DHF through 2 broad hypothetical (and not necessarily mutually exclusive) pathways. The Antibody Dependent Enhancement hypothesis proposes that pre-existing heterotypic anti-DENV IgG antibodies acquired during a previous infection can, under certain conditions, facilitate uptake of virus into macrophages and other immune cells through the binding of virus-antibody complexes to Fcγ receptors. The consequent increase in receptor-mediated endocytosis leads to higher viral loads, which in turn, triggers a host inflammatory cascade that leads to DHF [8]. The second hypothesis involves cross-reactive memory T cells, which are activated during heterologous secondary infection. This leads to altered effector responses, including the generation of cytokines that may create an immunologic imbalance in response to infection and, thus, play either a protective or harmful role in the development of sequelae of infection [9]. Our model of T cell–mediated immunopathogenesis of DENV infection postulates that the relative production of vasoactive cytokines by activated dengue-specific memory T lymphocytes influences the severity of dengue illness [10]. To our knowledge, no previous studies have looked at host-immune factors that specifically correlate with subclinical dengue infection. We hypothesized that the memory T cell response generated during primary DENV infection is a major determinant of cytokine production during secondary infection and, therefore, influences the severity of symptoms experienced during secondary infection. Specifically, we predicted that the frequencies of dengue-specific, cytokine-producing T cells in peripheral blood mononuclear cells (PBMCs) obtained before secondary infection would correlate with the severity of disease seen in secondary infection. To address this hypothesis, we tested PBMCs from Thai schoolchildren enrolled in a prospective cohort study [11]. The unique aspects of this study included the prospective scheduled collection and cryopreservation of PBMCs and the active surveillance for all febrile illnesses. This cohort provided an opportunity to study the predictive value of pre-infection cellular immune profiles with disease phenotype after subsequent DENV infection. From PBMCs obtained before secondary infection, we measured dengue-specific CD4+ and CD8+ T cell cytokine responses to dengue antigens to assess whether these responses predicted future disease severity. Because of the short interval between the collection of the baseline and follow-up blood samples, we believe that the frequencies of the DENV-specific cells at baseline reflect, as accurately as is possible in a large-scale prospective cohort, the host immune status with respect to DENV at the time of secondary infection.