One of the hallmarks of the adaptive immune system is its ability to provide long-term protection against infection by otherwise lethal pathogens. Great efforts have been placed into understanding the effector mechanisms that are capable of preventing diseases caused by infection. For viral infections, a vast majority of studies have focused on the role of CTL and neutralizing antibody (Ab) responses. A critical importance of these antiviral effectors in eliminating viral pathogens has been manifested by the large number of evasion strategies used by viruses to subvert detection by CTLs and Abs. In fact, some viruses are so efficient at preventing detection by CTLs and Abs that these effectors are rendered incapable of providing protection in an immunized host (1
), which is exemplified by infection with HIV-1 and γ-herpesvirus (2
). Alternative means of providing antiviral protection are required to combat infection by such viruses.
HSV-2, one of the most common sexually transmitted infections, causes primary infection in the genital mucosal epithelial layer and establishes latency in the sacral ganglia. In the mouse model of genital herpes, priming of the host with an attenuated thymidine kinase (TK) mutant HSV-2 via the intravaginal (ivag) route provides lifelong protection against challenge with virulent WT HSV-2. Such protection is mediated in a CD4 T cell–dependent manner (4
). In contrast, mice deficient in immunoglobulin or CD8 T cells are protected from virulent HSV-2 challenge after ivag immunization with TK−
HSV-2 virus (4
), suggesting that the protection requires CD4 T cells but not CTL or Ab responses. However, the precise mechanism by which the memory Th1 cells provide immune protection in the vaginal mucosa is unknown.
The importance of Th1 effector cells in defense against intracellular bacterial and protozoan pathogens has been well characterized (8
). This process primarily involves the activation of infected phagocytes through IFN-γ, resulting in enhanced phagocytosis and intracellular degradation of bacterial and protozoan pathogens. In contrast, the mechanisms by which Th1 memory cells provide protection against viruses remain much less clear (10
). There are at least three distinct mechanisms that can account for the ability of Th1 cells to mediate antiviral responses. The first is an indirect mechanism where Th1 cells are required for providing help to sustain effector CTL and B cells but do not themselves play a direct role in clearance of virus in vivo. Examples of this type of Th1 function has been seen in West Nile virus (12
) and influenza virus infections (13
). The second is the direct lysis of virally infected cells by Th1 killer cells. A recent study revealed the importance of antiviral Th1 cells in directly recognizing and killing influenza virus–infected cells through perforin-dependent pathways (14
). In this study, it was shown that IFN-γ secretion by CD4 T cells was not required for their antiviral effector function. Direct recognition and lysis of infected B cells by CD4 T cells also plays an important role in control of Epstein Barr virus infection (15
). A third mechanism involves antiviral function mediated by secreted factors. CD4 T cells secrete cytokines such as IFN-γ and TNF, which are known to control viral replication. Such a mechanism was shown to mediate viral clearance after the transfer of in vitro–derived Th1 cell against vesicular stomatitis virus (16
) and in hepatitis B virus transgenic (Tg) mice (10
). In the case of genital herpes infection, neutralization of IFN-γ (5
) or genetic deficiencies in IFN-γ (4
) render mice incapable of suppressing viral replication. However, the precise mechanism by which Th1 cells are elicited to secrete IFN-γ during the recall response is unknown.
A key question in this regard is whether Th1 cells are stimulated to secrete antiviral cytokines by direct recognition of virally infected cells through viral antigenic peptides presented on MHC class II or by an indirect mechanism through recognition of local APCs that have taken up viral antigens from infected cells. This issue is particularly relevant for infection by viruses, including HSV-2, that specifically replicate within non-APCs, namely, the mucosal epithelial cells (19
). Most viruses target cells that do not normally express MHC class II. Previous studies have shown that in HSV-2–primed mice, up-regulation of MHC class II on vaginal epithelial cells occurs after secondary infection in an IFN-γ–dependent manner (7
), raising the possibility that nonprofessional APCs could present viral peptides to CD4 T cells directly and become the target of lysis by CD4 killer cells. Because one of the hallmarks of IFN-γ is its ability to induce MHC class II molecules in professional and nonprofessional APCs, it has been assumed that this enables infected cells to directly present viral antigens on MHC class II for recognition by Th1 cells. In fact, HSV infection leads to inhibition of MHC class II processing pathways, suggesting that direct recognition of infected cells by CD4 T cells is detrimental to viral spread in the host (21
In this study, we examine the mechanism by which memory Th1 cells provide protection against genital HSV-2 infection in mice previously immunized with an attenuated TK−HSV-2. Specifically, we investigate the importance of the localization of memory Th1 cells, and the effector molecules and cell types involved in mediating Th1 antiviral protection in the mucosa in response to HSV-2 challenge.