Project Summary/Abstract Delayed-onset drug hypersensitivity reactions (DHR) are a significantly under-recognized and under- researched public health problem. Approximately 7% of the general population is affected by drug allergy. Skin is the most commonly affected organ and severity ranges from a mild rash to severe blistering and sloughing with internal organ involvement. The pathobiology of delayed-onset DHR is poorly understood due to a lack of both usable clinical samples and mouse models that recapitulate human disease. This negatively impacts patient care for 3 reasons. First, clinicians are ill-equipped to treat severe forms of disease. Second, there are no effective assays to identify a culprit drug, which can result in administering a less effective, more dangerous, and/or more-expensive drug in the future. Third, it can be difficult to discern drug allergy from other diagnoses with negative bearing on patient prognosis and/or treatment. It is therefore of paramount importance to elucidate the pathobiology of delayed-onset DHR to address these clinical needs. The central hypothesis of this project is that a recently identified population of T cells, termed skin resident-memory T cells (TRM), mediate delayed-onset DHR. The proposed project will directly test this hypothesis while overcoming current limitations in the field. Aim 1 employs three novel technologies to study a large bank of formalin-fixed paraffin embedded clinical specimens that until now were of little research utility. These technologies are (i) PerkinElmer’s OpalTM multiplexed immunohistochemistry and MantraTM Quantitative Pathology Workstation with inFORM® Image Analysis Software, (ii) NanoString technology for gene expression profiling and (iii) ImmunoSEQ high-throughput TCR deep sequencing to assess TCR repertoire. Aim 1 further includes laboratory investigation of prospectively collected clinical specimens, by taking advantage of the patient volume of Harvard’s tertiary care hospitals. Aim 2 tests the role of skin TRM in delayed- onset DHR ex vivo, and in-so-doing develops a much needed assay to diagnose drug allergy. Aim 3 leverages progress in humanized mouse technology to study human skin TRM in an in vivo model of delayed-onset DHR, thereby creating a mouse model of disease for future research. The data generated through this project will advance both the fields of skin T cell biology and delayed-onset DHR.