PROJECT SUMMARY Drug-induced Stevens-Johnson syndrome (SJS) and toxic epidermal necrolysis (TEN) are the severest of immunologically-mediated adverse drug reactions (IM-ADR) associated with blistering, mucosal sloughing and epidermal necrosis. The mortality, personal disability and healthcare costs of SJS/TEN are out of proportion to its incidence of 5 cases per 1,000,000/year in the United States. Genetic screening with drug-specific HLA risk alleles has 100% negative predictive value and is cost-effective in some settings, however, only 2-8% of individuals carrying a drug-specific HLA risk allele when exposed to the relevant drug develop SJS/TEN. Strong HLA class I associations, the presence of granulysin-producing CD8+ T cells in the SJS/TEN blister fluid, and the critical need to understand what factors in addition to the HLA risk allele are necessary for development of SJS/TEN form the scientific premise and significance of this study. Using our extensive existing biobank of cryopreserved blister fluid samples from patients with rigorously phenotyped acute drug- induced SJS/TEN, and blister cells from patients with fire and scald partial thickness burns as non-antigen driven controls we will use an integrated multi-omic approach to define at a single-cell level the antigen-driven CD8+ T cells at the site of pathology of SJS/TEN and their transcriptomic signatures. In Specific Aim 1 we will use bulk and single cell T-cell receptor (TCR) sequencing approaches to quantify the TCR clonality of activated CD8+ T cells in the blister fluid of acute SJS/TEN patients. Our hypothesis is that the drug antigen drives recruitment and/or clonal expansion of activated CD8+ T cells with specific T-cell receptor(s) (TCR) that will be enriched in blister fluid during acute disease. In some cases the dominant TCR clonotype, may be shared amongst unrelated individuals with the same HLA-risk allele. Single live T cells from blister fluid will be sorted according to activated (CD3+CD8+CD137+) and non-activated (CD3+CD8+CD137-) profile and subjected to TCR repertoire sequencing. In Specific Aim 2 we will define the transcriptomic signatures of activated CD8+ T cells in the blister fluid of patients with acute SJS/TEN. RNA-seq profiling of single cells from blister fluid will be combined with index sorting data from multi-parameter flow cytometry panels that will enable us to identify transcriptomic signatures associated with potentially expanded antigen-driven TCR clonotypes. Our hypothesis is that the CD8+ T cells in blister fluid will have an expression phenotype that includes granulysin and other cytolytic mediators, and early activation markers such as CD137. We will use novel approaches to link the pathogenic T-cell receptor clonotypes and their phenotypic signatures. This will provide important insights into the immunopathogenesis of HLA class I restricted, drug- induced SJS/TEN to fuel prevention, early diagnosis and identification of rationale therapeutic targets. It will also provide a roadmap for single cell studies applicable to other severe immunologically mediated diseases.