Interrogating Clonal Repertoire in Drug Hypersensitivity Reactions

Project Summary Delayed-type drug hypersensitivity reactions (dtDHR) are a major yet underappreciated public health problem, occurring commonly in skin-limited form, morbilliform drug eruption (MDE), and causing significant morbidity and mortality in severe systemic forms, Stevens-Johnson syndrome/toxic epidermal necrolysis (SJS/TEN) and drug reaction with eosinophilia and systemic symptoms (DRESS). dtDHR are significantly understudied. Consequently, understanding of immunopathogenesis has been limited, and subsequent to that, so too clinical care. Two major clinical limitations are the lack of a reliable laboratory test to identify culprit drug and an inability to accurately screen patients to prevent dtDHR. Our recent work in identifying T cell subsets mediating disease employed novel technologies to overcome research barriers in dtDHR and in-so-doing made several critical observations that, along with supporting publications by others, leads directly to this proposal. Herein, we propose the overarching, potentially transformative hypothesis that clonal repertoire analysis of skin could serve as a readout to identify culprit drug in dtDHR and to screen patients prior to drug administration to prevent dtDHR. Importantly, while this proposal has major implications for clinical care, it is purposefully designed to interrogate clonal repertoire and mechanisms of pathogenic T cell activation both broadly and mechanistically through innovative translational research as a critical next step. To achieve its goals, this grant builds on ample preliminary data and uniquely available patient populations along with the highly complementary expertise of its investigators to ensure feasibility and scientific rigor through three related but independent aims. First, it aims to interrogate clonal repertoire during active dtDHR using (i) T cell receptor (TCR) sequencing (seq) + single cell RNA seq on prospectively collected skin and blood and (ii) high-throughput TCR beta (HT-TCRb) sequencing on retrospectively obtained skin samples, from SJS/TEN, DRESS, MDE and healthy controls. Sample size allows breadth and depth across patient HLA alleles and culprit drugs. Second, it aims to investigate the temporality in vivo and reactivity ex vivo of pathogenic T cell clones. Specifically, using HT-TCRb sequencing, it tests whether drug- reactive T cells persist in skin and blood after disease resolution and/or pre-exist in skin prior to drug exposure. Study populations include patient cohorts and drug-tolerant controls. Third, it aims to identify the antigen specificity of pathogenic T cells in dtDHR by assaying in vivo the immunopeptidome with subsequent ex vivo mechanistic experimentation. In sum, this work has potential to profoundly impact the fields of dtDHR and more broadly adaptive immunity by generating incredible insight into in vivo mechanisms of antigen presentation and T cell activation, and concurrently clinical care by advancing toward development of much- needed diagnostic tests.