Project Summary Project 1: Evolution and Durability of allo(auto)immune B cell responses in organ transplant recipients Antibodies (Abs), which are produced by B lineage-derived Ab secreting cells (ASCs) following pathogen exposure and vaccination, play critical roles in protection from infection. However, ASCs and Abs can be pathogenic if directed against the wrong target, as is the case for autoimmune disease and organ transplantation. In the setting of kidney transplant (KT), Abs made by the recipient directed against the donor's Major Histocompatibility Complex or Human Leukocyte Antigen (HLA) proteins (donor-specific Ab, DSA) contribute to Ab-mediated rejection (AMR). Pre-formed DSA at the time of transplant can cause acute AMR and de novo DSA, which is elicited months to years after transplantation, can cause chronic AMR. Both conditions are difficult to treat and associated with organ failure. Thus, while overall transplant success rates have improved, there is still a need to better understand the fundamental mechanisms that control the development, evolution and durability of the B cell and B cell-derived DSA responses against donor (allo) HLA Class-I and Class-II. In fact, we know remarkably little about B cell responses to alloHLA proteins, which unlike pathogen-derived antigens, are very similar to the HLA proteins expressed by the patient (selfHLA). Studies using Rituximab to deplete circulating B cells in DSA+ transplant patients reveal that DSA levels remain high in many patients. These data support the idea that the DSA response is largely driven by Rituximab-resistant long-lived bone marrow ASCs (LL-ASC). However, memory B cells (Bmem), particularly those that reside in tissues, are also more resistant to Rituximab and both Bmem and LL-ASCs are formed by germinal center (GC) B cell responses that can take place in lymphoid and inflamed tissues. Bmem, which can rapidly differentiate into new cohorts of ASCs, could potentially serve as a reservoir to sustain systemic Ab responses. We found that HLA-reactive Bmem were present in the blood and kidney of a DSA+ KT patient diagnosed with AMR. These HLA-specific B cells were present in expanded B cell lineages and the BCRs expressed by these B cells were high affinity, class-switched and extensively somatically mutated, suggesting that they were generated via a TFH-dependent GC response. Moreover, we observed extensive clonal relationships between the Bmem and ASCs in both tissues and we identified Bmem and ASCs lineages that contributed to the serum DSA response. Intriguingly, many of the recombinant monoclonal Abs derived from multiple HLA-reactive B cell lineages bound both donor alloHLA and selfHLA. Therefore, we hypothesize that ongoing evolution of the alloHLA B cell response occurs in the face of a durable low affinity selfHLA response, which is not censored. The immediate goals of Project 1 are to: (i) define the B cell populations in blood and kidney that contribute to HLA reac...