PROJECT SUMMARY Patients with congenital immunodeficiencies, such as combined immunodeficiencies (CID) with partial recombinase activating gene (RAG) deficiency (pRD) are highly vulnerable to chronic infections and refractory autoimmune disorders. RAG1/2 are key to creating and censoring the B cell receptor diversity. In case of pRD, developing B cells that are naturally autoreactive may remain reactive to self in the periphery and be unable to mount efficient antibody responses. This results in chronic antigen exposure that can activate T and B cells. We propose to focus on two specific cell populations: hyperactive T follicular helper (Tfh) cells and innate-like extrafollicular polyreactive B cells, as markers of autoimmunity. The latter resemble age-associated B cells (ABCs), which accumulate with infections and with age. Normally, ABCs are highly sensitive to innate immune stimulation by microbes and inflammatory cytokines and play a key role in controlling viral infections by producing protective antibodies. After the infection resolves, ABC numbers contract markedly. However, with chronic infections, ABCs or ABC-like cells expand, persist and produce antibodies that are less protective against microbes and more reactive to self, especially in individuals with particular genetic immunodeficiencies. Sustained expansion of polyreactive ABCs parallels microbial/antigen load (toll-like receptor stimulation) and expansion of Tfh cells, which secrete the inflammatory cytokines, interferon gamma (IFNand interleukin 21 (IL-21). It is unclear which stimuli and cell signaling pathways are dominant in promoting ABC autoreactivity in pRD or other CID s. Our long-term goal is to understand how autoreactive ABCs emerge in CID in order to develop effective immune modulatory treatments. We hypothesize that the susceptibility to infections of patients with pRD results in increased, continual microbial/antigen presence and chronic low-grade inflammation throughout the body. In response, Tfh cells secrete inflammatory cytokines abundantly (IFNand IL-21), which together with chronic microbial stimulation induces ABC-like cells to expand and become autoantibody-secreting cells and present autoantigens to Tfh cells, which sustains them. Thus, ABC-like cell abundance and autoreactivity is perpetuated in pRD. Our specific aims are to 1) identify likely drivers of ABC- like cells in pRD patients and 2) dissect mechanisms contributing to ABC-like cell generation and persistence using pRD mouse models. Our innovative research strategies include studying an international cohort of pRD patients in parallel with using novel mouse models, exposing mice to varying levels of microbes, and correlating gene expression changes with ABC developmental characteristics and autoimmune severity of patients. Our proposed study is significant because it will likely illuminate how dysfunctional ABC-like cells develop in persons susceptible to chronic infections and lead to therapi...