PROJECT SUMMARY Protective antibody responses depend upon secreted immunoglobulin. This is regulated in a stage-specific manner upon the differentiation of B cell blasts into plasma cells (PC) and depends on the sequential upregulation of the IRF4 and Blimp-1 transcription factors. Together, they orchestrate the plasma cell gene program that includes alternative processing of the immunoglobulin transcript and the regulation of genes important for secretion of large quantities of immunoglobulin, i.e. post-transcriptional and post-translational regulation, respectively. Despite this understanding, a gap in knowledge exists in what elaborates the observed in vivo heterogeneity of PC in regards to cell longevity, proliferative rates, tissue tropism, phenotypic markers, and secretory rates. Specifically, it is not understood how this hierarchical regulatory mechanism diversifies PC states, i.e. whether other regulators are engaged or whether the dynamics of the network control distinct PC states. We have found that halving Irf4 gene copies contributes to PC heterogeneity suggesting that the dynamics of the network diversifies PC states. This proposal aims to elucidate the mechanisms whereby the dynamic of IRF4 expression controls PC outcome. Furthermore, we will employ a newly developed reporter mouse that enables simultaneous detection of cells expressing secretory and membrane Ig by two spectrally distinct fluorescent proteins. Importantly, we expect this reporter system to function as a novel and differentiation-stage-unbiased approach to study cells undergoing changes in IgH expression levels and 3'end usage. Together, understanding the molecular basis of PC phenotypic and Ig usage heterogeneity will enable future methodologies to test their relevance in diverse settings which likely vary as a function of infection or pathologic states.