Immune surveillance by CD8αα+ skin-resident T cells in human herpes virus infection

Abstract

Most herpes simplex virus 2 (HSV-2) reactivations in humans are subclinical and associated with rapid expansion and containment of virus. Previous studies have shown that CD8(+) T cells persist in genital skin and mucosa at the dermal-epidermal junction (DEJ)--the portal of neuronal release of reactivating virus--for prolonged time periods after herpes lesions are cleared. The phenotype and function of this persistent CD8(+) T-cell population remain unknown. Here, using cell-type-specific laser capture microdissection, transcriptional profiling and T-cell antigen receptor β-chain (TCRβ) genotyping on sequential genital skin biopsies, we show that CD8αα(+) T cells are the dominant resident population of DEJ CD8(+) T cells that persist at the site of previous HSV-2 reactivation. CD8αα(+) T cells located at the DEJ lack chemokine-receptor expression required for lymphocyte egress and recirculation, express gene signatures of T-cell activation and antiviral activity, and produce cytolytic granules during clinical and virological quiescent time periods. Sequencing of the TCR β-chain repertoire reveals that the DEJ CD8αα(+) T cells are oligoclonal with diverse usage of TCR variable-β genes, which differ from those commonly described for mucosa-associated invariant T cells and natural killer T cells. Dominant clonotypes are shown to overlap among multiple recurrences over a period of two-and-a-half years. Episodes of rapid asymptomatic HSV-2 containment were also associated with a high CD8 effector-to-target ratio and focal enrichment of CD8αα(+) T cells. These studies indicate that DEJ CD8αα(+) T cells are tissue-resident cells that seem to have a fundamental role in immune surveillance and in initial containment of HSV-2 reactivation in human peripheral tissue. Elicitation of CD8αα(+) T cells may be a critical component for developing effective vaccines against skin and mucosal infections.

Figure 1. CD8αα⁺, but not CD8αβ⁺, T cells persist at the DEJ in human HSV-2 infection

a, Average and individual expression of CD8A and CD8B genes in DEJ CD8 and BV CD8 cells laser captured from tissues of 2 (n = 8) and 8 (n = 10) week post-healed (wph) biopsies, relative to ACTB. b, Differential localization of CD8αα⁺ (red) to the DEJ and CD8αβ⁺ (yellow) to the dermis in active lesions, 2 wph and 8 wph tissue (high and low density areas). f, Quantitation of CD8αα⁺ and CD8αβ⁺ T cells at DEJ and dermis (upper) over time. Individual and average (light blue line) kinetics of DEJ CD8αα⁺ cells (middle) and dermal CD8αβ⁺ T cells (lower) over time (n = 4). Lesions: week 0.

Figure 2. Tissue residence and effector function of DEJ CD8αα⁺ T cells in post-healed tissue

a, Down-regulation of chemokine receptor expression in DEJ CD8 as compared to BV CD8 cells. Data are average signal intensities on the Illumina Beadarray (n=5). b, CCR7 and S1PR1 expression by QPCR. One subject per symbol. c, Activated effector signatures of DEJ CD8 cells. Pair-wised comparison was performed between DEJ CD8 and control CD8 cells from the same individual at the 8wph time point (n = 9). Red: up-regulated, green: down-regulated. d, QPCR analysis of antiviral gene expression (n = 9). e, Perforin protein expression in DEJ CD8 cells of active lesion (left) and 8 wph with (right) and without (middle) virus shedding. f, Perforin protein expression in DEJ CD8αα⁺ T cells but not BV CD8αβ⁺ T cells. Perforin: yellow granules (both pink and green); CD8α: red; CD8β: green. Arrows denote enlarged area of perforin expression.

Figure 3. DEJ CD8 cells in early containment during asymptomatic HSV-2 reactivation

a, HSV-2 antigen expression in keratinocytes during asymptomatic HSV-2 reactivation. Arrows denote infected cells. b, CD8 effector-to-target ratio during asymptomatic (b) and lesional (c) HSV-2 reactivation. d, High level of CD8α⁺ T cells expressing perforin granules in during asymptomatic shedding. Arrows denote enlarged area. e, Focal accumulation of CD8αα⁺ T cells (red, left) and recruitment of CD8αβ⁺ T cells (yellow, right) to the DEJ.

Figure 4. Characteristics of DEJ CD8 cell TCRβ repertoire

a, Oligoclonal distribution and dominant CDR3 sequences of the DEJ CD8 cell TCRβ repertoire. DEJ CD8 cells were laser captured from six biopsies obtained during and after multiple episodes of herpes recurrence from one individual. The pie chart illustrates the distribution of the unique CDR3 sequences detected in the DEJ CD8 TCRβ repertoire from each biopsy. Amino acid (aa) sequences of the top six clones are listed in the order of abundance. Shared aa sequences between biopsies are highlighted in the same color as pie charts. Each color represents one clone. b, Frequencies of the three most commonly shared DEJ CD8 clones. c, Venn diagram of overlapping TCRβ repertoires among 3 T-cell populations from the same individual: tissue infiltrating T cells from a healing lesion biopsy, blood PBMC-derived HSV-2 reactive CD8⁺ T cells and PBMC-derived CD8αα⁺ T cells. Clone tracking was performed at the nucleotide level.