Previously we showed that CD8+
T cells persist at the dermal-epidermal junction at a high density for several months upon complete healing of herpes lesions (29
). In this report, we provide evidence that the CD8+
T cells that are adjacent to the neuronal nerve endings are not quiescent but exhibit the metabolic and functional pattern of effector T cells, despite the lack of HSV-2 DNA or inflammation in these areas. The transcriptional induction of genes for T-cell activation in this cell population is consistent with an immunosurveillance function, as many of the signature transcripts identified have half-lives of <48 h. Thus, the uniform and consistent pattern of a wide variety of RNA transcripts in biopsy specimens from areas of known HSV-2 reactivation compared to areas with infrequent or undetectable reactivation during periods of clinical quiescence suggests that these T cells likely see HSV-2 antigen frequently without evidence of clinical reactivation.
One salient feature of our experimental approach was the lack of any in vitro manipulation of cells from human skin biopsy specimens, which allowed direct analysis of T-cell gene expression profiles indicative of the cells' in vivo physiological state. In addition, by comparing JC_CD8 cells to three types of controls (CL_CD8 cells, axillary cells, and CD1a+ cells) from the same patients, we were able to define the distinctive transcriptional response of T cells persisting at the site of previous herpes lesions.
During lymphocyte activation and proliferation, transcriptional regulation of a variety of cellular processes, including cell cycle initiation and glucose metabolism, adapts in response to antigenic and inflammatory stimuli. GLUT1, the primary glucose transporter in T lymphocytes, is transcriptionally expressed at low levels in resting T cells and both transcriptional induction and posttranslational trafficking are necessary for GLUT1-dependent glucose uptake during T-cell activation (6
), a process largely dependent on T-cell receptor (TCR) stimulation (19
). In 89% of patients (8/9), GLUT1
was expressed at low levels in CL_CD8 cells (), suggesting that this cell population was largely quiescent. In contrast, JC_CD8 cells in these patients expressed high levels of the transporter, indicative of an activated phenotype, with values ranging from 10- to 270-fold over control samples. Due to the short half-life of GLUT1
mRNA (between 10 and 12 h) (4
), its detection indicates active transcription of the gene and suggests that resident CD8+
T cells likely encounter HSV-2 during periods in which HSV-2 DNA can be detected. Similarly, the half-life of IFNG
mRNA is approximately 3 h in plasma T cells (7
), and as with GLUT1
, the expression pattern of IFNG
was low in CL_CD8 cells but much higher in JC_CD8 cells. The detection of these short-lived transcripts is consistent with the model that HSV-2 frequently leaks from sacral neurons into the genital tract, where it subsequently stimulates resident T cells (2
JC_CD8 cells express a variety of antiviral cytokines and chemokines, and the expression pattern we observed corresponds to known antiviral mechanisms of the cytokines IFN-γ, IL-32, and TNF-α on virus DNA replication of HSV-2 and vaccinia virus (3
). In addition, IFN-γ can act synergistically with type I IFN or TNF-α in the production of antiviral immune responses. The IL-32-mediated antiviral cellular pathway effects only partially overlap those of type I IFN; it will be interesting to investigate whether IFN-γ, IL-32, and TNF-α can have cooperative effects to inhibit HSV-2 replication at peripheral sites.
One of the interesting observations of our study was that CD8+
T cells at the junction site express a substantial amount of CCL5
. These data suggest a positive feedback mechanism for the recruitment of CD8+
T cells to this region during the clinically quiescent phase of HSV-2 reactivation. It is not clear whether keratinocytes in 8-week-posthealing tissue express high levels of CCL5
, although these cells can secret large quantities of this chemokine in response to Th1 cytokine (including IFN-γ and TNF-α) stimulation in vitro
). Expression of CCL5
as measured by qRT-PCR was significantly higher in JC_CD8 cells relative to CL_CD8 cells in five of the nine patients, similarly high in three patients in both cell populations, and modestly higher in JC_CD8 cells in one patient (). Expression of CCR1
(by qRT-PCR) was higher in JC_CD8 cells than in CL_CD8 cells in six of the nine patients, slightly lower in JC_CD8 cells in two patients, and significantly downregulated in JC_CD8 cells in one patient (). Interestingly, the patients with increased levels of CCL5
also had increased levels of CCR1
, which suggests that this pathway may be an important chemotactic mechanism in these patients. These results are partially consistent with numerous studies that have shown that CCL5 is responsible for chemotaxis of CCR1+
). The correlation of upregulation of both genes in the same patient may be due to the location of the biopsy. Studies have shown that microenvironments of the genital dermis and DEJ separated by just millimeters can have diverse immune cell density. Therefore, we hypothesize that the CCL5-CCR1 interaction may play a role in mediating the migration of CD8 cells to the dermal-epidermal junction area.
One limitation of this study is variance in the specificity and sensitivity of both microarray and qRT-PCR techniques for each gene. In most cases, both methods produced similar expression patterns. However, there were instances in which expression was inconsistent between the two methods. For example, expression of CCL2 was upregulated >3-fold in JC_CD8 cells in five of nine patients and >10-fold in four of nine patients as measured by microarray (). In contrast to array data, CCL2 was downregulated in three of nine patients as measured by qRT-PCR (patients 3, 6, and 8) (). Expression of CCL2 in patient 6 was near basal level and the limit of detection in microarray studies; qPCR detected modest expression of CCL2 in both cell types and a downregulation in JC_CD8 relative to CL_CD8 cells. The low level of CCL2 expression in patients 3 and 8 can be potentially explained by experimental design: cDNA synthesized from laser captured cells (approximately 100 cells) has a mean size of 300 to 500 bp, and the Illumina single 50-bp probes and the PCR product (about 60 bp) detected by ABI TaqMan probes do not cover the same area of the cDNA.
Thus, while validation of gene expression studies via microarray and qRT-PCR is essential for making conclusions regarding expression patterns, the dynamic range and similarities in detection sensitivity and specificity may be complex. It should be noted that we observed concurrence in the two methods with a large number of genes.
In summary, transcriptional analysis of resident CD8+ T cells of the genital mucosal junction epithelium uncovered highly expressed genes in the functional categories of activation, proliferation, antiviral activity, and metabolism. High expression of chemokines and their receptors in these cells, as well as mucosal tissue, provides possible evidence for a positive regulatory loop in the homing of T cells to the sites of herpes lesions. This study will help further elucidate the complicated dynamic interplay between the adaptive host immune system and HSV-2 during frequent reactivation episodes over the course of this chronic infection.