Tissue-resident-memory T cells (TRM) populate the body’s barrier surfaces, functioning as frontline responders against reencountered pathogens. Understanding of the mechanisms by which CD8TRM achieve effective immune protection remains incomplete in a naturally recurring human disease. Using laser capture microdissection and transcriptional profiling, we investigate the impact of CD8TRM on the tissue microenvironment in skin biopsies sequentially obtained from a clinical cohort of diverse disease expression during herpes simplex virus 2 (HSV-2) reactivation. Epithelial cells neighboring CD8TRM display elevated and widespread innate and cell-intrinsic antiviral signature expression, largely related to IFNG expression. Detailed evaluation via T-cell receptor reconstruction confirms that CD8TRM recognize viral-infected cells at the specific HSV-2 peptide/HLA level. The hierarchical pattern of core IFN-γ signature expression is well-conserved in normal human skin across various anatomic sites, while elevation of IFI16, TRIM 22, IFITM2, IFITM3, MX1, MX2, STAT1, IRF7, ISG15, IFI44, CXCL10 and CCL5 expression is associated with HSV-2-affected asymptomatic tissue. In primary human cells, IFN-γ pretreatment reduces gene transcription at the immediate-early stage of virus lifecycle, enhances IFI16 restriction of wild-type HSV-2 replication and renders favorable kinetics for host protection. Thus, the adaptive immune response through antigen-specific recognition instructs innate and cell-intrinsic antiviral machinery to control herpes reactivation, a reversal of the canonical thinking of innate activating adaptive immunity in primary infection. Communication from CD8TRM to surrounding epithelial cells to activate broad innate resistance might be critical in restraining various viral diseases.

The skin at the site of HSV-2 reactivation is enriched for HSV-2 specific T cells. To evaluate whether an immunotherapeutic vaccine could elicit skin-based memory T cells we studied skin biopsies and HSV-2-reactive CD4+ T cells from peripheral blood mononuclear cells (PBMCs) by T-cell receptor (TCR) sequencing before and after vaccination with a replication-incompetent whole virus HSV-2 vaccine candidate (HSV529). The representation of HSV-2-reactive CD4+ T cell sequences from PBMCs increased from a median of 0.03% (range 0-0.09%) to 0.6% (range 0-1.3%) of the total skin TCR repertoire after the first vaccine dose. We found sustained expansion after vaccination in unique, skin-based T-cell clonotypes that were not detected in HSV-2-reactive CD4+ T cells isolated from PBMCs. While detection of skin clonotypes in the blood was related to abundance in the skin it was not related to expansion after vaccination. In one participant a switch in immunodominance was observed after vaccination with the emergence of a newly dominant TCRa/b pair in skin that was not detected in blood. We confirmed that the newly dominant clonotype was derived from an HSV-specific CD4+ T cell by creation of a synthetic TCR in a Jurkat-based cell line with a NR4A1-mNeonGreen reporter system. Our data indicate that the skin in areas of HSV-2 reactivation possesses an oligoclonal TCR repertoire that is distinct from the circulation with prominent clonotypes infrequently detected in the circulation by standard methods. Defining the influence of therapeutic vaccination on the HSV-2-specific TCR repertoire requires tissue-based evaluation.

Tissue resident memory (TRM) T cells persist in human genital mucosa long after healing of herpes simplex virus 2 (HSV-2) infection. These TRM T cells provide effective protection to rapidly contain reactivating viruses before clinical manifestation occurs, making them excellent candidates for developing novel approaches to controlling HSV2 reactivation. To understand the dynamics of TCR repertoire and the kinetics of T cell clones during disease resolution, sequential biopsies were obtained in three individuals at the time of active lesions and 2, 4, 8 weeks post-healing, and were subjected to high-throughput sequencing of the TCRβ chain hypervariable CDR3 region. Oligoclonal immune profiles were evidenced in all biopsy tissues with a few TCR sequences constituting 30–40% of the TCR repertoire. The usage pattern of various TCRVB genes was similar among tissues of active lesion and post healed biopsies of the same subject. However, the TCRVB usage differed dramatically between the TCR repertoire in tissues and those of PBMC derived HSV-2 specific T cells taken at the same time point within each individual. This difference was more extreme when evaluating the TCR sequences that selectively persisted in post-healing biopsies. The top ten predominant TCR sequences persisting in tissues during clinical quiescence 4–8 weeks post healing did not overlap with HSV-2 specific T cells isolated from PBMC. These data suggest a TCR intrinsic property might dictate the TRM repertoire, and that the antigen specificity of TRM might differ considerably from circulating HSV-2 specific T cells.