PROJECT SUMMARY (ABSTRACT) Humoral immunity forms the basis for vaccine-induced immune protection. Following infection and vaccination, a fraction of activated B cells differentiates into long-lived plasma cells (LLPCs) or memory B cells to provide longitudinal protection against pathogens. Whereas LLPCs constitutively produce circulating antibodies, memory B cells in circulation and tissues are reactivated following antigenic reencounter through rapid expansion and antibody production. Existing forms of vaccination rely upon generation of neutralizing antibodies by LLPCs. However, evolving pathogens harboring frequent mutations at sites of immune recognition might be better targeted by heterogeneous memory B cells, with their broader distribution across tissues and enhanced cross-reactivity. Understanding these aspects of memory B cells is thus crucial for leveraging the additional layer of protection offered by the heterogeneous population in vaccine development. Co-expression of Tbet and CD11c (Tbet+CD11c+) marks a subset of such heterogeneous, antigen-experienced B cells that arises in aging, infection, and autoimmunity, respectively, termed age-associated, atypical, and double-negative (in humans, IgD- CD27-) B cells which mediate protection from pathogens in mice and humans. Their contribution towards protection is likely in part due to their unique localization as a resident memory B cell subset in tissue, most notably the splenic marginal zone which via its exposure to circulation provides front-line defense against bloodborne pathogens upon rechallenge. Tbet+CD11c+ B cells also infiltrate non-lymphoid tissues, including the liver, with residency establishment at non-lymphoid tissue sites positioning them for early detection of invading pathogens. Confounding their investigation, however, is lack of knowledge of the relationship between the non-lymphoid and lymphoid populations. Tbet+CD11c+ memory B cells also contain autoreactive clones and appears to be a major source of autoantibodies, which may be pathogenic. Although the ability of Tbet+CD11c+ B cells to mount robust antibody responses at tissue sites of infection makes them an attractive candidate to target in vaccination, we need to understand the relationship between the lymphoid and non-lymphoid pools, and how the balance between their protective and autoreactive functions is regulated. Are these cellular sites, and protective and autoreactive functions, a consequence of two pools of Tbet+CD11c+ B cells? Do cross-reactive Tbet+CD11c+ B cells, recognizing both self and non-self, rely on environmental signals to mediate protection or pathology? To address these knowledge gaps, in this revised application, we will identify the mechanism by which Tbet+CD11c+ B cells infiltrate non-lymphoid organs and determine if they maintained therein and elucidate the nature of their autoreactivity. Both goals fit the R21 Exploratory/ Developmental Grant mechanism. We are at “early and conceptual ...