PROJECT SUMMARY/ABSTRACT Keratinocytes, the major building block of skin epidermis, are the primary target of herpes simplex virus (HSV) infection as well as the mediator of local environmental cues, including those directed by CD8 tissue resident memory T cells (TRM) at the dermal epidermal junction (DEJ). The ability of keratinocytes to relay and amplify TRM immune signals could dictate the epithelium resistance or susceptibility to HSV infection and ultimately affect the outcome of HSV manifestation. Genital HSV-2 is a disease of global importance. There are an estimated 4 million incident HSV-2 infections yearly, with 572,000 in the US. HSV-2 is the underlying attributable risk in 30% of the incident 1.5 million HIV infections yearly. HSV-2 disease varies widely among individuals, ranging from disease free to infrequent and short-lasting recurrences to frequent and severe ulcerations. Subclinical shedding occurs commonly and frequently. The molecular mechanisms underpinning varied host responses to HSV-2 reactivation in genital skin are poorly defined. Understanding host-viral interactions, with the goal of enhancing responses to rapidly eliminate viral infection at the site of HSV-2 release before local viral dissemination, is the key to developing novel interventions and a successful vaccine. We have shown that CD8 TRM constitute the frontline of defense at the barrier surface. Our recent studies indicate CD8 TRM at the DEJ communicate with surrounding keratinocytes via IFN-γ to enhance their cell- intrinsic and innate antiviral responses through IFN-stimulated gene pathways. These observations alter the canonical belief that communication only goes from innate to adaptive immunity and suggest the reverse also occurs. This proposal is designed to dissect the cross talk between TRM and the surrounding epithelium and the microenvironmental signals that dictate the recruitment and function of CD8 TRM using sequential genital skin biopsy tissue in humans. By applying state-of-the-art single-cell RNA sequencing, spatial transcriptome analysis and highly multiplexed imaging technology, we aim to identify cellular and immunological characteristics that distinguish between effective vs defective antiviral responses and can be targeted for modification and manipulation to enhance immunity at the peripheral tissue. Findings generated in this study will provide rationale for eliciting optimal local immune responses and for development of a functional cure for HSV-2 recurrent disease in humans.