This is the first description of the selective expression of a putative tissue-specific homing molecule by circulating microbe-specific CD8+
T cells. The cell-surface expression and functional data in this report are consistent with a role for a CLA-associated E-selectin ligand in the trafficking of circulating HSV-2–specific memory CD8+
T cells to skin during recurrent genital herpes. Because many patients with genital herpes have lesions on keratinized epithelial surfaces of the external genitalia, perineum, back, or legs (34
), CLA expression by HSV-2–specific T cells is anatomically appropriate.
In common with HSV-2, EBV and CMV undergo intermittent reactivations and are episodically shed in infectious form by most immunocompetent, infected individuals (35
). In contrast to HSV-2, neither primary nor recurrent infection with EBV or CMV is associated with cutaneous infection. The most common site of EBV shedding is the oropharynx, while the most common sites of CMV shedding are the uterine cervix, the urinary tract, and the oropharynx. Reactivations of EBV and CMV in immunocompetent persons are usually asymptomatic (35
). We did not study the presence or absence of EBV or CMV reactivation at mucosal or skin sites in our subjects. Reactivations of EBV and CMV are intermittent, brief, and anatomically unpredictable, complicating the assessment of the possible influence of reactivation status on homing receptor expression at the time of phlebotomy.
We found that expression by CD8+
T cells specific for EBV and CMV was similar to the background level of 5–10% (37
) observed for circulating CD8+
lymphocytes. The possibility that expression levels of CLA might change during reactivations could be addressed by combining intensive virologic monitoring with flow cytometry. Study of additional subjects and epitopes, and T cells specific for a variety of skin-tropic and non–skin-tropic pathogens, will help to determine to what extent CLA expression is tightly associated with infection in the skin. Given the anatomic loci of EBV and CMV infection, measurement of the expression of CD103 (αE
integrin), a putative homing molecule for musocal epithelium (4
), by T cells specific for these viruses would also be of interest.
T cells are functionally important in containing HSV-2 infection. Levels of CD8+
CTLs correlate inversely with the severity of HSV-2 in men coinfected with HIV and HSV-2 (38
), and correlate temporally with the local clearance of HSV-2 in lesions (8
CTLs are also important in the control of ganglionic infection, maintenance of neuronal latency, and in protection against infectious challenge in murine models (39
). HSV evades CD8+
T cells by inhibiting the transporter associated with antigen processing (TAP) and degrading host mRNA (43
). The tetramer-based measurements in this report reveal higher levels of circulating HSV-2–specific CD8+
T cells than were previously observed with limiting dilution CTL assays (38
). In particular, high levels of VP22-specific CD8+
T cells were detected. VP22 may be recognized efficiently due to its delivery into the class I antigen processing pathway before TAP inhibition can occur. Our previous data show that virion input VP22 can be processed and that endogenous synthesis is not required (11
). VP22 also has efficient intercellular spread and has CTL adjuvant activity (44
) although it is not known if this is related to its antigenicity.
In the present report, we studied HSV-2–specific T cells before and after trafficking from the circulation to HSV-2–infected skin, extending previous research concerning the role of CLA in pathogenesis of autoimmune and atopic disorders. MelanA-specific CD8+
T cells in PBMCs from subjects with vitiligo express higher levels of CLA than do similar cells from normal subjects (46
). In atopic subjects, proliferative responses to allergy-associated antigens are enriched among CLA+
T cells (47
). Few reports have examined homing receptor expression by circulating virus-specific T cells. Circulating memory rotavirus-specific CD4+
cells preferentially express the adhesion molecule α4
). Our data indicate that memory CD8+
T cells specific for the skin-tropic herpesvirus HSV-2 express CLA prior to leaving the circulation.
E-selectin is expressed at low basal levels in noninflamed skin, and is increased in diverse inflammatory skin conditions. We observed apparent upregulation of E-selectin in HSV-2–infected tissue. This is not surprising, since IFN-γ, IL-1β, and TNF-α, which are upregulated in HSV lesions (49
), cooperate to increase E-selectin expression by endothelial cells (52
). Additional work is required to document the magnitude and time course of upregulation. Lymphocytes infiltrating the dermis commonly express CLA (35
). The influx of HSV-2–specific CD4+
cells and NK cells into recurrent HSV-2 lesions precedes the inflow of HSV-2–specific CD8+
T cells (10
), and additional work is required to define the molecules involved in the trafficking of early responder cells. The proportion of HSV-2–specific CD8+
T cells that express CLA appears to be higher in the skin than in the blood (Figure ). The approximately 50–80% of circulating HSV-2–specific cells that express CLA (Figure ) may preferentially migrate to skin, or the local microenvironment may further promote CLA expression.
Our finding that circulating HSV-2–specific memory cells express CLA implies that expression of this antigen is upregulated during the priming of naive HSV-2–specific CD8+
T cells or at a subsequent stage of conditioning. The control of CLA expression is incompletely understood. Expression of α(1
)-fucosyltransferase VII is a probable key regulator of CLA expression, although control over other glycosyltransferases (56
) and the primary polypeptide backbone, P-selectin glycoprotein ligand-1 (2
), may also be important. In vivo, CLA is expressed by cells coexpressing CD45RA and CD45RO in skin-draining lymph nodes, consistent with upregulation during the priming of naive T cells (57
). In a murine model, molecules associated with skin-homing are upregulated during cutaneous priming (58
). It is rational to hypothesize that inflammatory cytokines and local antigen-presenting cells could influence CLA expression during priming.
In this small cross-sectional study, the proportion of HSV-2 VP22-specific CD8+ T cells that expressed CLA was relatively constant in our set of six subjects (Figure ). We recently studied two more HLA B7–bearing subjects (not included in Table ) who are HSV-2 seropositive but have no clinical history of genital herpes. Staining of unmanipulated PBMCs with tetramer B7-RPR showed that 54.5% and 62.1% of tetramer-high, CD8α-high cells expressed CLA, similar to the six subjects shown in Figure . We do not know whether CLA expression is influenced by recurrences of HSV-2 infection. While the number of subjects that we studied with frequently recurrent versus asymptomatic HSV-2 infection were too small for statistical comparisons, we observed no obvious segregation of CLA expression by HSV-2–specific CD8+ T cells by the clinical or virologic (shedding) severity of HSV-2 infection (Table and Figure ). Similarly, we do not yet know whether CLA expression by HSV-2–specific cells in the periphery fluctuates temporally in association with symptomatic or asymptomatic recurrences of HSV-2.
Epitope-specific heterogeneity in CLA expression by HSV-2–specific CD8+ T cells cannot be excluded with the available data. CLA expression by VP13/14-specific cells was somewhat lower than for VP22-specific cells, both before and after peptide restimulation, but was still clearly above background levels for tetramer-negative cells. In sorting experiments using unmanipulated PBMCs, based on CLA and CD8 expression, we have successfully enriched HSV-2–specific CTLs from several subjects at the bulk and clonal levels. Expression cloning to determine fine specificity, using the methods described in this report, has yielded five additional novel epitopes recognized by HLA class I–restricted CD8+ CTLs (Koelle et al., unpublished observations). CLA does appear to be expressed above background levels by CD8+ CTLs specific for a diverse set of HSV-2 epitopes.
TGF-β and IL-12 (57
) upregulate CLA expression in vitro, while IL-4 may downregulate CLA (56
). Each of these cytokines is upregulated at the protein and/or mRNA levels in HSV-associated lesions (50
). Secretion of TGF-β and IL-12 in response to HSV infection has been demonstrated in vitro (51
). Draining lymph nodes in animals display expression of IL-12, TGF-β, IL-4, and other cytokines (65
). While a cytokine milieu appropriate for CLA induction is associated with HSV infection, the key factor or factors controlling CLA expression by CD8+
T cells responsive to HSV-2 remain to be determined.
Memory cells that acquire tissue-specific homing molecules may be specialized for extravasation to sites of antigen challenge. They may also retain a capacity for self-renewal and recirculation through lymphoid tissue. We found that most circulating VP22-specific CD8+
T cells expressed CD28 (Figure ), an important molecule for the delivery of costimulatory signals to memory T cells. HSV-2–reactive T cells specific for each of three different viral epitopes were able to proliferate in vitro in response to peptide, sometimes to very high levels (Figure ). We also found that the majority, but not all, circulating VP22-specific CD8+
T cells expressed CD62L and CCR7, adhesion and chemokine receptor molecules, respectively, associated with recirculation to lymphoid tissue and a substantial capacity for self-renewal (68
). As CD62L ligands are expressed in skin (69
) as well as lymph nodes, it is possible that CD62L on HSV-specific T cells may participate in homing to this site. There are conflicting data on the expression of CCR7 by CLA+
cells in the circulation, with published findings concentrating on CD4+
). Our findings for CCR7 are similar to those noted for CD8+
T cells specific for some EBV epitopes, but contrast with findings for CMV- and HIV-specific CD8+
T cells (71
). Taken together, our data indicate that virus-specific CD8+
T cells with the central memory phenotype may also be capable of trafficking to sites of antigenic challenge.
Rolling adhesion to the vessel wall is only the initial stage of lymphocyte trafficking into tissue. Both integrin maturation and chemotaxis have been associated with chemokines and their receptors. Several candidate chemokine receptors have been implicated in homing to skin, including CCR4 and CCR10 (74
). Most of the published data have concerned CD4+
T cells. Future flow cytometric and functional studies will examine the expression of chemokine receptors by HSV-2–specific CD8+
In summary, subjects with recurrent, symptomatic HSV-2 infection have readily detectable circulating VP22-specific CD8+
T cells that express CLA. CLA is tightly associated with functional E-selectin binding activity, which is an anatomically appropriate property for HSV-2–specific T cells. HSV-specific CD8+
T cells in PBMCs expressed functional E-selectin binding activity. Neither CLA expression nor E-selectin binding was noted among the responses to CMV or EBV, two non–skin-tropic herpesviruses. We propose that vaccines and immunotherapies for HSV (77
) should not only elicit specific T cells but also guide these T cells to express appropriate homing molecules. More broadly, preventative and therapeutic T cell–based treatments may be optimized if the identity, mechanisms of action, and control mechanisms for homing molecules can be understood and manipulated.