PROJECT SUMMARY Patients receiving organ transplants have the risk of organ rejection due to mismatched human leukocyte antigens (HLA). Though organ recipients are screened for pre-formed anti-HLA antibodies (HLA-Abs) and are cross-matched using donor cells and recipient serum prior to transplantation, up to 25% of kidney transplant recipients will develop a new donor-specific HLA-Ab following transplant. Within that patient group, there is a 40% decline in 10-year graft survival. At the heart of this negative outcome are antibody-secreting cells (ASCs), which produce donor-specific Abs, and memory B cells (Bmems), which may provide help to alloreactive T cells and differentiate into ASCs. Current immunosuppressive therapies aimed at depleting ASCs and Bmems are largely ineffective. Thus, Ab-mediated rejection remains a significant clinical problem. The overall objective of this U19 Program is to define the factors that govern the evolution of memory lymphocyte responses over time and to assess the durability of these responses. Two Projects in this U19 will specifically focus on characterizing the evolution and durability of the Bmem and ASC subsets responsible for alloimmune responses elicited toward donor-specific HLA Class-I and Class-II alleles. To perform these studies, Projects will require antigen-specific reagents to aide in identification of antigen-specific B cells, clone B cell receptors, and further characterize antigen to Ab interactions. One objective of the Immuno-Reagent Production, Validation, and Biophysical Analysis Core (Core C) is to provide these reagents, which will include B cell tetramers (specific to HLA alleles and respiratory viral antigens), Abs, Fabs, antigen-specific polyclonal antibodies (pAbs), and proteins for structural studies. By centralizing the production and validation of proteins for distribution within the Program, the expertise of Core C will allow Projects to focus on their own Aims, unimpeded by the time and effort to develop these reagents on their own. A second objective of Core C is to function as a multidisciplinary biophysical analysis center. In this capacity, we will coordinate with Projects to analyze antibody to antigen interactions. Using surface plasmon resonance, we will develop an understanding of these interactions at the kinetic level. Using X-ray crystallography and cryo-electron microscopy, we will observe epitope-paratope interfaces at near atomic resolution. Lastly, we will perform proteomics experiments using mass spectrometry with pAbs to aide in assigning pools of Abs to lineages. In summary, Core C will provide reagents to the exceptional group of Immunologists in the Program to expedite their discoveries. Biophysical analyses will provide new innovation and synergy to these groups. Structural biology will be a transformative tool for the Projects that will lead to assignment of rules regarding HLA engagement by alloAbs. Filling this knowledge gap could lead to improved donor-to...