ABSTRACT In this renewal, we continue efforts to develop agents that will make allogeneic hematopoietic stem cell transplantation (HCT) less toxic for patients. Our major objective is to develop strategies that target chronic graft versus host disease (cGVHD)-related immune pathology without blocking the critical immunotherapeutic effects of HCT. B cells have a substantiated pathobiological role in cGVHD, but distinguishing disease-mediating B cells subsets remains elusive. Targeting only aberrant B cells is important because we have found that global abrogation of peripheral B cells with rituximab perpetuates so-called altered immune homeostasis and cGVHD in patients unable to recover a comprehensive B-cell compartment. In the previous funding period, we elucidated how pathological B Cell Activating Factor (BAFF) and NOTCH2 promote aberrant B Cell Receptor (BCR)- signaling after HCT. Based on known BCR signaling events during B cell maturation, we hypothesized that antigen-hyperresponsive and SYK-reliant B cells can be eliminated without affecting immune homeostasis. In preclinical studies and in the clinical trial, we showed that inhibition of the BCR-proximal protein, SYK, eliminated aberrantly activated B cells and afforded recovery of B-cell homeostasis. We have also begun to dissect novel mechanisms underpinning BCR hyper-responsiveness. Published evidence and our additional preliminary data, including a single cell RNA-Seq dataset, form the solid scientific basis for this proposal and afford our current overarching hypothesis that aberrant activation and effector function pathways in B cells can be effectively targeted in cGVHD. In the current proposal, we address major gaps in our knowledge regarding human B-cell programming that confer a propensity for ongoing loss of B cell tolerance in cGVHD. Our experimental approach entails study of cGVHD patient samples in our ex vivo B-cell assay system and parallel experiments in mice using B-cell conditional and inducible knockout mice as donors to verify molecular mechanisms. Specifically, we will delineate molecular pathways that promote pathological B cells and determine the distinct disease-mediating functions of cGVHD B cells. We will pursue three specific aims: 1) persistence of SYK protein after BCR engagement, 2) the ability to block SYK and restore humoral immunity in cGVHD patients in a Phase II clinical trial 3) molecular underpinnings of antibody and inflammatory cytokine production. We will define targetable signaling pathways in B cell subsets that we will show have pathological B-effector cell functions in cGVHD. Our proposal relies on a team science concept and is made possible by co-investigators on the Duke clinical trial team (Drs. Horwitz and Rizzieri) and Duke Cancer Institute statistical group (Drs. Li and Owzar). Our group remains at the forefront of efforts elucidating aberrant human B-cell signaling and testing small molecule inhibitors in chronic GVHD patients.