One of the major questions in HSV-2 immunobiology has been the disparity between animal model data suggesting that functional host T cell responses in the ganglia have the ability to markedly limit viral reactivation at the ganglion level (25
) and human clinical studies of HSV-2 reactivation, which demonstrate that mucosal and genital skin shedding of HSV-2 in the immunocompetent person is extremely frequent (27
). With once daily sampling, HSV-2 reactivation is seen in 20–60% of days in immunocompetent persons. More recent data suggest that high titer reactivations of HSV-2 lasting only 6–12 h are common (Mark, K., personal communication). These human clinical studies indicate that control of mucosal HSV-2 reactivation at the ganglion level in humans is incomplete at best. It is likely that immune control at both the ganglion and peripheral mucosal level is needed for containment of HSV reactivation. This study was designed to define whether local host control of HSV-2 played an important role in containing viral reactivation after lesional clearing. Our data indicate that much of the control of HSV-2 reactivation in humans appears to be consolidated in the peripheral mucosal immune compartment.
Our study indicates that the peripheral mucosal immune response to HSV-2 infection is highly concentrated both spatially and functionally. The cytolytic CD8+ T cell infiltration in genital herpes lesions is very specific to the site of infection. The dramatic reduction of CD8+ T cell density 2 cm outside of the affected skin-lesion area indicates that CD8+ T cell infiltration to an HSV infection is a very focused local event. The HSV-2–specific CD8+ T cells we detected were highly concentrated in the genital lesion. Quantitatively, HSV-2–specific T cells were present at 100 times higher concentrations in lesional skin than that in circulating blood. Even after lesion resolution, the enrichment of HSV-2–specific CD8+ T cells persisted in genital skin for an extended time period. Our study extended for only 2 mo, and local persistence was documented to last at least this long. Moreover, the spatial localization of HSV-2–persistent CD8+ T cells appears to be of physiological importance. Unlike active lesions, where HSV-2–specific CD8+ T cells were distributed in all layers of skin to eliminate viral infection and prevent spreading, HSV-2–specific CD8+ T cells that persisted after healing were detected in the upper dermis and the dermal–epidermal junction, coincident with sensory nerve endings. This physical closeness between persistent CD8+ T cells and sensory nerve endings would provide a potential mechanism in which the local immune system could quickly respond to and eliminate reactivated virus before it undergoes extensive replication. We demonstrated four such instances of such containment in two patients, as well as the apparent ability of host T cell responses in the periphery to contain viral reactivation before epithelial ulceration could occur.
Estimates of the frequency of HSV-specific CD8+
T cells in genital skin are likely to be underestimates of their true prevalence in our studies. The cocktail of Qdot multimers we used was based on only a limited mapping of the breadth of the T cell response. As such, it is likely we were assaying only a portion of the CD8+
T cell response to the virus. In more recent studies using a wider breadth of peptides, we have shown that many subjects possess CD8+
T cells specific to a wider multitude of HSV proteins than we had available here (14
). As such, our quantitative assessments are likely to be underestimates of the number of HSV-specific CD8+
T cells in genital skin.
This study also provides evidence that HSV-specific CD8+ T cells play a role in immune protection against clinically symptomatic HSV-2 reactivation in genital skin. The biopsies in which we detected HSV DNA in skin tissue, without evidence of epithelial cell destruction and in association with increased localized infiltration with HSV-2–specific CD8+ T cells, suggest that resident HSV-2–specific T cells appear to be a critical component of cutaneous viral containment. The need to evolve a rapid response to viral replication in the desired region is emphasized by a clinical study, which showed that almost 40% of genital HSV-2 reactivation in humans were asymptomatic and lasted 6 h or less (Mark, K., personal communication). These short reactivations average from 103 to 104 copies of HSV-2 DNA per millimeter in swab specimens, indicating that prompt elimination of virus by the host mucosal immune responses occurred. Prospective studies investigating the relationship between in vivo persistence of HSV-2–specific T cells and the site and timing of clinical and subclinical reactivation are needed to define this association more closely. Although such studies require serial biopsies, patients with genital HSV-2 are affected greatly by this infection. The Qdot multimers and the in situ staining techniques we describe in this paper provide the tools for such studies to be undertaken.
In summary, HSV-2–specific CD8+ T cells appear to play a direct role in viral clearance in vivo. These CD8+ T cells migrate very specifically into genital lesions and persist preferentially in genital skin in the subepidermal layer contiguous to peripheral nerve endings, suggesting that they play a role in the earliest response to viral reactivation. Our studies indicate that persisting HSV-2–specific CD8+ T cells function in immune surveillance and that the peripheral T cell response influences the frequency and clinical course of HSV-2 reactivation in humans.