Summary: Prior alloimmunization places patients at risk for anamnestic alloantibody formation following alloantigen re-exposure that can result in accelerated RBC clearance and lead to a potentially fatal delayed hemolytic transfusion reaction (DHTR). The inability to prevent DHTRs largely stems from a fundamental lack of understanding regarding key immune pathways that govern anamnestic RBC alloantibody responses capable of driving DHTRs. Our long-term goal is to identify and then target critical pathways that regulate the development of anamnestic alloantibody formation. Our central hypothesis is that anamnestic RBC alloantibody formation occurs through a distinct toll-like receptor (TLR), bridging channel dendritic cell (DC) and follicular (FO) B cell- dependent pathway that fundamentally differs from primary RBC alloimmunization. Our hypothesis is formulated on the basis of our discovery that unlike initial RBC alloimmunization, which requires marginal zone (MZ) B cells, MZ B cells are not required for anamnestic alloantibody formation, but are required for early priming events that occur following initial RBC alloantigen exposure. In addition to differences in MZ B cell requirements, while initial alloantibody formation is CD4 T cell independent (TI), increased alloantibody levels observed following RBC re- exposure is entirely CD4 T cell dependent (TD). Furthermore, while T follicular helper cell (TFH) and follicular (FO) B cells are not required for initial alloantibody formation, initial RBC transfusion does increase TFH and FO B cell numbers. Re-transfusion also increases 33D1+ DC activation and chemokine receptor expression. These results suggest that initial RBC transfusion primes recipients by generating distinct alloantigen-specific TFH and FO B cell populations that can, in turn, be activated by 33D1+ DCs following RBC alloantigen re-exposure independent of MZ B cells. In addition, while type I interferons (IFNab) are required for primary alloantibody formation, TLR signaling is dispensable for initial alloantibody development, yet is required for anamnestic alloantibody formation. Given the ability of MZ B cells to directly activate CD4 T cells and traffic antigen to the B cell follicle, our data suggest that initial priming events require MZ B cell-mediated CD4 T cell and FO B cell development through an IFNab-dependent process. However, as anamnestic alloantibody formation occurs through a MZ B cell-independent pathway and DCs can also traffic antigen to B cells and directly activate CD4 T cells, DCs likely orchestrate anamnestic alloantibody formation through a TLR-dependent pathway. To test this hypothesis, we will pursue the following specific aims: Specific Aim 1: Define the role of MZ B cells and IFNab in the development of CD4 T cells and FO B cells required for a subsequent anamnestic alloantibody response. Specific Aim 2: Define the role of TLRs and DCs in the development of an anamnestic alloantibody response following R...