Enumeration and functional evaluation of virus-specific CD4+ and CD8+ T cells in lymphoid and peripheral sites of coxsackievirus B3 infection

Previous studies have suggested that coxsackievirus B (CVB) activates CD8⁺ T cells in vivo, but the extent of this activation and the antigen specificity of the CD8⁺ T cells remain uncertain. Furthermore, CVB-induced CD4⁺ T-cell responses have not been carefully investigated. Herein, we evaluate CD8⁺ and CD4⁺ T-cell responses both in a secondary lymphoid organ (spleen) and in peripheral tissues (heart and pancreas), using a recombinant CVB3 (rCVB3.6) that encodes well-characterized CD8⁺ and CD4⁺ T-cell epitopes. Despite reaching high levels in vivo, rCVB3.6 failed to trigger a marked expansion of CD8⁺ or CD4⁺ T cells, and T-cell activation was surprisingly limited. Furthermore, epitope-specific effector functions could not be detected using highly sensitive in vivo and ex vivo assays. Moreover, major histocompatibility complex (MHC) class I tetramer analysis indicated that our inability to detect CVB3-specific CD8⁺ T-cell responses could not be explained by the cells being dysfunctional. In contrast to naïve T cells, epitope-specific memory CD8⁺ and CD4⁺ T cells proliferated markedly, indicating that both of the rCVB3.6-encoded epitopes were presented by their respective MHC molecules in vivo. These data are consistent with the observation that several CVB3 proteins can limit the presentation of viral epitopes on the surface of infected cells and suggest that the level of MHC/peptide complex is sufficient to trigger memory but not naïve T cells. Finally, our findings have implications for the biological significance of cross-priming, a process thought by some to be important for the induction of antiviral CD8⁺ T-cell responses.Coxsackieviruses are members of the picornavirus family and enterovirus genus, which includes type A and B coxsackieviruses, polioviruses, echoviruses, and other unclassified enteroviruses. Although the majority of type B coxsackievirus (CVB) infections in humans are subclinical or cause relatively mild disease (including rash, myalgia, or upper respiratory complications), CVB are important human pathogens, and a substantial proportion of infections can lead to severe—even lethal—acute and chronic diseases. In particular, CVB is the most common infectious cause of myocarditis, which can lead to dilated cardiomyopathy and cardiac failure (38, 44, 45). CVB also targets cells of the central nervous system and the pancreas, frequently leading to aseptic meningitis and pancreatitis (7, 12, 33, 35, 40). Overall, CVB infection can cause considerable morbidity and mortality, particularly in newborns and in young or immunocompromised individuals (35, 52).The murine model of CVB3 infection is a valuable system for studying CVB pathogenesis and immunity, as mice infected with CVB develop diseases similar to those observed in humans (52, 53). Intraperitoneal inoculation of adult C57BL/6 mice with CVB3 results in systemic acute infection; viremia peaks on day 2 to 3 postinfection (p.i.), and infectious virus is cleared by 2 weeks p.i. (33, 34). Control of CVB3 infection depends on both cell-mediated and humoral components of the immune response. Agammaglobulinemic individuals are particularly susceptible to CVB3-associated encephalitis (15, 18), and mice lacking B cells develop a chronic infection and remain viremic for at least 2 months; viremia can be alleviated by the adoptive transfer of B cells from CVB3-immune wild-type mice (34). CD8⁺ T cells also play an important role in controlling virus replication. T cells are present in the inflammatory infiltrates associated with myocarditis and pancreatitis (17, 20, 41), and CD8⁺ T-cell depletion of CVB3-infected mice simultaneously increases viral titers and reduces myocarditis, suggesting that T-cell-mediated protection is associated with elevated immunopathology (17). This immunopathology can be uncoupled from antiviral efficacy; mice lacking perforin control cardiac infection just as well as wild-type mice but show markedly diminished myocarditis (14).Many—probably most—acute viral infections trigger extensive CD8⁺ T-cell activation and division; these responses can readily be detected directly ex vivo, without any need for extensive restimulation. The convincing evidence that CD8⁺ T cells can contribute to control of CVB3 in mice, together with the fact that CVB3 replicates to high titers in many mouse tissues, led us to surmise that CVB3—like most other viruses—would induce readily detectable CD8⁺ T-cell responses in mice. Indeed, early studies had identified cytolytic T-cell activity in CVB3-infected mice, although the precise antigen specificity of the cells was unknown (16, 21, 22). Subsequent elegant work showed that synthetic peptides representing CVB3 VP1 sequences could drive in vitro T-cell proliferation, but neither the phenotype of the proliferating T cells (CD4⁺ or CD8⁺) nor the precise epitope specificity was determined (19). Therefore, we undertook a preliminary analysis of epitope-specific CD8⁺ T-cell responses against CVB3; contrary to our expectations, we found that CVB3-induced epitope-specific CD8⁺ T-cell responses were difficult to detect (42). However, those studies were incomplete: they relied on ex vivo detection methods of rather limited sensitivity, and they were limited to cells from the spleen. Furthermore, those studies focused only on CD8⁺ T cells, and it is clear that regulation of antiviral CD8⁺ T cells differs from that of CD4⁺ T cells. Therefore, herein we have extended our previous analysis in five ways: first, we evaluate general T-cell activation in CVB3-infected mice; second, we use more sensitive in vivo approaches to detect epitope-specific T-cell responses; third, we investigate the possibility that the virus induces the expansion of dysfunctional T cells; fourth, we extend our analyses of CVB3 epitope-specific T-cell responses to major targets of infection, such as the heart, where CD8⁺ T cells are present in the virus-induced infiltrate; and, fifth, we investigate CD4⁺ T-cell responses induced by CVB3. Our studies employ a new recombinant CVB3 (rCVB3) that encodes both a CD8 and CD4 T-cell epitope derived from lymphocytic choriomeningitis virus (LCMV). Our data are not only relevant to understanding the T-cell responses induced by coxsackievirus in particular but also have broader implications for the mechanism(s) by which CD4⁺ and CD8⁺ T cells are induced by viruses in general.