ABSTRACT The use of checkpoint inhibitors has altered the treatment landscape in oncology, leading to cures in previously untreatable diseases. Checkpoint inhibitors block the function of the PD-1 and CTLA4 inhibitory receptors on T cells to enhance the activation of T cells, particularly tumor-specific lymphocytes. However, non-specific immune activation has no antigenic specificity and also induces immune-mediated adverse events (irAE) that resemble spontaneous autoimmunity and involve any and all tissues. IrAEs cause significant morbidity and disruption in the cancer treatment of oncologic patients and, at times, can be life-threatening. However, the occurrence of irAEs is also associated with improved tumor regression. This challenge has created a pressing need to understand the pathogenic mechanism of irAEs to allow for proper therapeutic management and better predictive algorithms to identify patients that may develop these autoimmune toxicities. However, dissecting the immunopathology of irAEs is limited by a lack of understanding of the antigenic targets of the autoimmunity. To address this gap in knowledge, we have established a prospective cohort of patients treated with PD-1 blockade. Unlike cross-sectional studies, our cohort is unique in its (1) size, (2) prospective nature (before and after PD-1 immunotherapy), and (3) longitudinal design (before and after irAE incidence). We find that almost all subjects that develop irAEs after PD-1 inhibition have an increased abundance of naïve CD4 T cells at baseline, as well as more pronounced activation of these naïve T cells following PD1 inhibition. No changes in CD8+ phenotypes correlate with irAE’s. Secondly, we utilized a whole exome autoantigen screening array to identify multiple non- tumor related targets unique to each subject’. Together, these data lead to our parallel hypotheses that 1) immune mechanisms of CD4-dependent autoimmunity are similar between subjects and independent of the target tissue except when 2) the target of autoimmunity is coexpressed in the tumor and the target tissue. In our first Aim, we will focus on the phenotype of the CD4+ T cells involved in the development of post-treatment autoimmune inflammatory arthritis. Single cell multiome assays paired with T cell receptor sequencing will identify and characterize oligoclonal expansions. In parallel, putative autoantigens identified by autoantibody screening will be used in activation-induced marker (AIM) assays to validate the presence of autoantigen-specific CD4+ T cells. We hypothesize that autoantigens are specific to the individual rather than universal; however, the phenotype of the autoreactive CD4+ T cells will be similar amongst individuals. Secondly, we will examine the antigen-specificity and CD4+ T cell responses in subjects with lung cancer that develop pneumonitis or a peripheral irAE. We hypothesize that pneumonitis will reflect preexisting target tissue damage and antigen cross- reactivity with tu...