Characterization of the roles of STAT5 and TAK1 in memory B cell generation and homeostasis

Memory B cells are a mature population of B cells which emerge from selection following antigen challenge. Memory B cells prevent repeated infections by rapidly recognizing and responding to those pathogens that have previously been encountered by the body. They are identified by their high affinity surface immunoglobulin and extended life span. Very little is known about the mechanisms that regulate the selection and homeostasis of memory B cells during an immune response. Signals originating from both the B cell receptor (BCR) and cytokine receptors have been shown to play important roles in the regulation of memory B cells.;Memory B cells are a unique population of cells that can be maintained over many decades. The signaling requirements for the generation and homeostasis of these cells have not yet been defined. Cytokine signaling through the Signal Transducers and Activators of Transcription 5 (STAT5) is involved in the homeostasis of memory T cell in vivo. STAT5 is also up regulated in human memory B cells in vitro and suggested to play a role in human memory B cell differentiation. The function for STAT5 in memory B cells in vivo is still unclear. We hypothesized the cytokine signaling through STAT5 is important for the generation and homeostasis of the memory B cell population. To address this, we used mice conditionally deficient of STAT5 in the B cell lineage. These mice were immunized with a T-dependent antigen and analyzed for memory B cell formation and homeostasis. Memory B cell and plasma cell populations were unaffected by the loss of STAT5 suggesting cytokine signaling through STAT5 is not required for memory B cells. We also examined STAT5 signaling during earlier time points after immunization using inducible deleted STAT5 mice. These mice had an increase in germinal center B cells which was a result of increased numbers of T follicular helper cells (TFH). Along with our collaborators, we discovered STAT5 was a negative regulator in TFH cells and STAT5 deficiency in CD4 cells resulted in increased TFH and germinal center B cell populations. These findings demonstrated an extrinsic and important role of STAT5 through TFH cells in regulating germinal center B cell formation.;Phospholipase C γ2 (PLCγ2), an important signaling molecule for the BCR, is critical for the formation and maintenance of memory B cells. However, the signals downstream of PLCγ2 that are required for memory B cell formation and homeostasis are largely unknown. A molecule known as TGF-beta activated kinase 1 (Tak1) acts downstream of PLCγ2 during BCR signaling. Tak1 plays a critical role in the maturation and homeostasis of naive B cells through nuclear factor κB (NF-κB) activation. Of note, NF-κB activation is required for TCR-mediated immune response in naive mature T cells but not memory T cells. It is highly possible that the signaling requirements for regulating memory B cells are different from those for naive mature B cells. Therefore, questions of whether Tak1 functions downstream of PLCγ2 in regulating memory B cell formation and homeostasis remain unanswered. We hypothesized that Tak1 is critical for BCR-mediated formation and homeostasis of memory B cells by providing the signals necessary for selection and survival. To address these questions we generated mice in which Tak1 was conditionally deleted during germinal center B cell formation. In these mice we observed normal B cell development in the bone marrow and normal maturation in the spleen.;After immunization, we found that Tak1 deficiency reduced production of germinal center B cells and IgG1-switched B cells. Tak1-deficient germinal center B cells showed an increase in apoptosis and a reduction in proliferation, demonstrating the requirement of Tak1 in the formation of germinal center B cells. We also generated mice in which Tak1 could be inducibly deleted through the injection of tamoxifen. In these mice, a normal number of memory B cells could be generated following immunization. Interestingly, the inducible deletion of Tak1 failed to affect the number of preformed memory B cells, demonstrating that memory B cell homeostasis was unaffected by Tak1 deficiency. Taken together, we have demonstrated that the Tak1 pathway plays a critical role in germinal center and memory B cell formation, but is not required for memory B cell homeostasis.