Equine Herpesvirus type 1 (EHV-1) continues to cause outbreaks throughout the United States despite several available vaccines. Cellular immunity, especially T-cell immunity, is a crucial mechanism to control the virus during cell-associated viremia and to prevent severe outcomes of EHV-1 infection such as abortions and equine herpesvirus myeloencephalopathy (EHM). However, only very low numbers of EHV-1 specific T-cells are circulating in peripheral blood. T-cell responses happen primarily in lymphatic tissues where T-cells reside and closely interact with other cell types to establish EHV-1 specific cellular immunity in response to infection or vaccination. It is expected that only a small surplus of EHV-1 specific cells circulates in the peripheral blood. Moreover, the lack of equine cellular immune markers has limited T-cell analysis in horses. Consequently, EHV- 1 specific T-cell immunity and how it confers protection against EHM are not completely understood. Previous work from our group has identified several immune parameters correlating with protection against EHV-1 at the viral entry site, the upper respiratory tract (URT). These include mucosal and systemic IgG antibodies and the absence of mucosal type I interferon and inflammatory markers. In addition, we identified early upregulation of multiple T-cell genes in the URT by transcriptome analysis, suggesting an important role for reactivation of preexisting T-cells in protection against EHV-1. Most recently, we developed a panel of novel equine T-cell markers to broaden the analysis of cellular immunity in horses. In this project, we will use lymphatic tissue cells from EHV-1 immune horses to further unravel T-cell immunity and its role for EHV-1 protection. Our hypothesis is that T-cell immunity against EHV-1 is primarily maintained in lymphatic tissues, and that both, systemic and mucosal T-cells are crucial in protecting against severe outcomes like EHM. In Aim 1 of this project, we will use tissues from EHV-1 protected horses with known EHV-1 infection and vaccination status to comprehensively characterize T-cell immunity. We will analyze T-cells in secondary lymphatic tissues, including different peripheral lymph nodes, spleen and bone marrow using a recently developed equine T-cell marker panel by multicolor flow cytometry. Aim 2 will evaluate local T-cell immunity in the mucosa associated lymphatic tissues (MALT) at the URT using an in vitro infection model of nasal epithelial explants. The influence of pre-existing Tcell immunity on viral entry and replication in epithelial cells, mucosal inflammation, and T-cell trafficking and activation will be evaluated in the explant tissues by immunofluorescence microscopy and in the culture supernatants by measuring secreted cytokines. The goal of this project is to advance our knowledge about different T-cell phenotypes and functions in protecting against EHV-1. The outcomes are relevant for evaluating T-cell immunity after vaccination with different existing or new vaccines to analyze their efficacy for preventing against EHM.