Models of leukocyte trafficking include rolling adhesion, in which circulating cells interact with venular endothelium [35
]. Biopsies have shown that HSV-2–specific CD4+
T lymphocytes localize by day 2 after the onset of symptoms [9
]. Our experiments were conducted to address the mechanism(s) of CD4 localization, with the focus on CLA and ESL.
Ex vivo assays using CD154 or IFN-γ up-regulation showed that HSV-2–specific CD4+
T lymphocytes express higher levels (~20%) of CLA than do CMV-specific or bystander cells (). Ideally, we would have used HLA class II tetramers to detect HSV-2–specific CD4+
T lymphocytes ex vivo, which, unlike the CD154 and IFN-γ tests, do not require restimulation with antigen for 6 h. However, the frequency of HSV peptide–specific CD4+
T lymphocytes is quite low and approaches the background levels seen with HLA class II tetramers [36
]. Even the 6-h stimulation with antigen could initiate the expression of CLA through a cytokine-mediated or other indirect effect, as discussed below. However, several days are required to detect cytokine influences on the expression of CLA by stimulated T lymphocytes [30
The low levels of expression of CLA on HSV-2–specific CD4+
T lymphocytes contrast with the 50%–70% expression of CLA by circulating HSV-2–specific CD8+
T lymphocytes [7
]. Functional [9
] and histologic [8
] data show that CD4+
T lymphocytes traffic more quickly to HSV-2 lesions than do CD8+
T lymphocytes. The apparent dissociation between the rapidity of in vivo trafficking and the ex vivo expression of CLA requires further investigation. Some data have indicated that DCs and tissue microenvironments may influence the homing phenotype of memory cells [11
]. Recent reports, albeit in different murine models, have indicated that distinct DC subsets are involved in priming HSV-specific CD4+
T lymphocytes after peripheral inoculation [12
]. Future animal work in the HSV system may reveal mechanisms for the control of homing phenotypes of HSV-specific CD4+
T lymphocytes, but murine models, in contrast to human infection, generally involve only a single episode of lytic HSV replication, without the periodic recurrences typically observed in humans that may continue to influence T lymphocyte trafficking.
CLA has been associated with the Th2 CD4 phenotype, especially in atopic persons [39
]. Our focus on IFN-γ +
cells could bias away from the detection of Th2-like CLA+
cells. However, most researchers do not detect IL-4+
cells with CMV antigen [40
]. We excluded subjects with atopic dermatitis. In addition, CD154 up-regulation, independent of cytokine profile, showed preferential expression of CLA by HSV-2–reactive cells. CD154 is an emerging marker for antigen-specific CD4+
T lymphocytes, one that identifies cells with a physiologically important effector function, modulation of DC function [43
]. In the present study, CD154 up-regulation was associated with CD69 up-regulation (data not shown), a more widely used marker for T lymphocyte activation.
We examined a cross-sectional set of specimens from persons with chronic (>1 year) HSV-2 infection that was obtained between clinically evident outbreaks of HSV-2. If CLA+
cells preferentially traffic to skin, it is possible that the abundance of HSV-2–reactive cells, or the proportion of circulating HSV-2–reactive cells expressing CLA or ESL, could change over time. Reported low PBMC proliferative responses to HSV during reactivations could be due to exodus from blood, although the original report implicated CD8+
]. Patient-reported recurrence is poorly correlated with both objective lesions [45
] and replicating HSV-2 [46
]. Future temporal studies will use intensive serial physical examinations and sampling for virus by polymerase chain reaction and culture [47
]. The study of lesion-derived HSV-2–specific CD4+
T lymphocytes could potentially address selective trafficking of CLA+
cells. However, few lymphocytes can be isolated from human biopsy specimens [21
In agreement with the direct ex vivo data indicating that only a minority of HSV-2–reactive CD4+
T lymphocytes in PBMCs express CLA, CLA-depleted PBMCs still proliferate well in response to HSV-2 (). Our data do not address the relative replication potential of CLA+
T lymphocytes. CLA expression has been linked to the CD25+
regulatory T lymphocyte phenotype and to skin homing [33
T lymphocytes influence HSV-1 pathogenesis in animals [48
]. Therefore, the depletion of all CLA+
cells within PBMCs may have had complex effects on proliferative responses to recall antigens; we are presently addressing this possibility in depletion and reconstitution experiments.
The expression of CLA by CD4+ T lymphocytes increased during HSV-2 restimulation in vitro ( and ; – and ), to ~30%–70%. Up-regulation of CLA was not universal but was noted in most subjects (). The reason for this heterogeneity is unknown. The link between HSV-2 and CLA was maintained during restimulation, as HSV-2 stimulated higher levels of CLA than did CMV, influenza, or a mitogen. Selective expression of ESL was also documented after stimulation with HSV-2 (). Unexpectedly, on stimulation with HSV-2, cells that were initially CLA negative gained expression of CLA ().
There are 2 general explanations for this increase. First, cytokines known to increase the expression of CLA during T lymphocyte activation in vitro include IL-12, IFN-α, and transforming growth factor (TGF)–β [37
]. HSV preparations can stimulate secretion of IL-12, IFN-α, and TGF-β from PBMCs, including DCs and monocytes [52
]. Thus, cytokine effects may increase the levels of CLA and ESL in HSV-2–stimulated cultures. These cytokines are up-regulated by HSV-2 in vivo [55
] and therefore may have pathophysiologic significance. We are presently testing this possibility by stimulating memory CD4+
T lymphocytes in the presence of HSV-2 antigen in HSV-seronegative persons; preliminary results (data not shown) are consistent with a cytokine-mediated or other indirect effect. Cytokines or other factors in the HSV-2 preparation also could directly up-regulate CLA. This could be addressed with purified HSV-2 and with HSV-2 and CMV antigens from the same producer cell type.
Second, it is possible that HSV-2–specific CD4+ memory T lymphocytes are programmed to express CLA on antigenic re-stimulation. The specific expression of CLA ex vivo (see above) supports some level of specific programming even before re-stimulation. The results of our peptide experiments are consistent with this notion, because peptides, which are unlikely to drive cytokine responses or to induce expression of CLA, still result in high expression of CLA by tetramer-positive progeny. We found that CLA-negative precursor cells give rise to CLA-positive cells after stimulation through T cell receptors with HSV-2 antigen. A supportive cytokine milieu, programming for CLA expression, or both factors could be operative.
Cell culture conditions were closely regulated in the present experiments, as most lymphocytes express CLA if expanded in specific media [30
]. We used an RPMI–human serum–based medium that has been shown to minimize nonspecific expression of CLA [30
]. We confirmed [30
] that FCS-based media are permissive for CLA (data not shown). In our experiments, neither recombinant nor biologically derived IL-2 induced non-specific expression of CLA; cryopreserved PBMCs were used throughout. Strong proliferative responses were nonetheless detected, indicating that antigen presenting and responder cell populations were intact. Changes in the expression of CLA by responder cells related to freeze and thaw cannot be ruled out.
CLA and ESL expression are closely associated in inhibitor [58
] and single-cell studies [51
]. Fucosyltransferase VII (FTVII) expression up-regulates both CLA and ESL [59
]. The precise structures that bind anti-CLA and that form ESL are undefined. Anti-CLA inhibition of functional E-selectin binding is controversial [3
], and dual staining of CD4+
T lymphocytes with soluble E-selectin and anti-CLA reveals populations that are singly positive (). In the present study, using cell-cell binding and flow-cytometry assays, we have shown that HSV-2–specific T lymphocytes express ESL. In single-cell analyses, there was a correlation between a high expression of CLA and a high expression of ESL, in agreement with the results of Takahashi et al. [5
It is not known whether CLA or ESL is required for successful T lymphocyte homing to skin in vivo. Modifiers of CLA attenuate cutaneous inflammation in animals [61
]. Persons deficient in IL-12 or IL-12 receptors (who might have low levels of CLA) have not been reported to have severe HSV or cutaneous infections [62
]. Persons deficient in fucose transport (leukocyte adhesion deficiency–II) [63
] or with mutations in FTVII [64
] can have reduced CLA expression [65
] but have not been reported to have severe cutaneous infections.
The present study is the first to document the expression of CLA and ESL by pathogen-reactive CD4+
T lymphocytes. Torres et al. examined the expression of CLA with respect to erythema multiforme or Stevens-Johnson syndrome, which are generally felt to be immunopathologic reactions; there was no difference in the expression of CLA by PBMCs between the patients with virus-associated systemic dermatitis and the control subjects [66
]. HSV-related generalized syndromes such as HSV-associated erythema multiforme (HAEM) were not specifically evaluated. We used CMV and influenza as controls. Rarely do persons have rash during initial CMV infection [67
], but this is immunopathologic and is not infectious. Cutaneous disease with recurrent CMV is very unusual, even in immunocompromised persons [68
]; it would be of interest to study CLA expression during HAEM and among cells specific for other cutaneous pathogens.
In summary, circulating HSV-2–specific CD4+ T lymphocytes in persons with chronic HSV-2 infection selectively express CLA on their surface. Compared with that of circulating HSV-2–specific CD8+ T lymphocytes, the percentage of CD4+ T lymphocytes that directly express CLA ex vivo is lower, on the order of 20%. HSV-2–specific CD4+ T lymphocytes include a population of cells that circulate in a CLA-negative state but that can divide and express CLA on restimulation. This permissiveness could involve cell-resident transcriptional or chromatin mechanisms, exogenous cytokine/APC factors, or possibly both. Future studies of these issues may assist in the design of strategies to control T lymphocyte homing during vaccination or of immunotherapy for infectious, autoimmune, or malignant disorders.