Cytokine-mediated B-cell development in lupus

Birmingham Va Medical Center, Birmingham AL

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Abstract

The overall goal of this project is to determine if the IKAROS transcription factor regulates a program that maintains IL-4 receptor (IL-4R)-mediated B-cell quiescence to ribonuclear protein (RNP) autoantigens (autoAgs). We propose that B cell activation through the type I interferon (IFN)/TLR7 activation pathway is inhibited by this B-cell quiescence program. We also propose that failure to maintain B cell quiescence is associated with the development of RNP autoantibody in systemic lupus erythematosus (SLE). Our preliminary data suggest that in both lupus mice and in human SLE, multiple immune disease phenotypes can be initiated by loss of quiescence at the earliest stage of B cell development; that is, the transitional B cell. The key forces that promote quiescence is an IKAROS (or IKZF1)-based program that is sustained through IL-4R signaling on IgD+CD23+ B cells. The major opposing program that promotes loss of quiescence is mediated through the type I interferon receptor (IFNAR) signaling, assembly and signaling competency of the TLR7 pathway, and development of B cells that can produce autoantibodies. We hypothesize that early-stage loss of quiescence leads to a series of B cell development defects after the transitional stage involving progression from a resting naïve to an activated naïve predominance and then to development of the pathogenic CD11c+Tbet+IgD−CD27− double negative 2 (DN2) or germinal center (GC) B cells that ultimately lead to the development of RNP autoantibody producing plasmablasts and plasma cells (PB/PC). In Aim 1, we will use several knockout and knock-in mouse strategies in lupus prone mice to determine if type I IFN and IL-4 act at the Tr B cells to regulate autoreactive B cell development and survival. The specific effects of Ikaros in modulating type I IFN and IL-4 signaling-mediated RNP-reactive B-cell quiescence at the Tr and naïve stage will also be analyzed. In Aim 2, we will determine if SLE patients exhibit a loss of IL-4R/IKZF1-mediated B cell quiescence program starting at the Tr stage of B-cell development. We will determine if the loss of this pathway disrupts B-cell tolerance to type I IFN and TLR7 stimulation, leading to the development of RNP- reactive DN2 B cells and PB/PC. The innovative scientific basis of this proposal will be its ability to interpret the broad spectrum of immune and disease characteristics of SLE by understanding the key initiating events of loss of B cell quiescence at the transitional stage. B cells from mouse models of lupus and from SLE subjects will be characterized by high dimension flow cytometry and transcriptomics analyses for well-established cell surface protein antigen markers that define B cell development at all major developmental stages. We have established a team of coordinators at the BVAMC to facilitate recruitment and have acquired numbers of SLE subjects necessary for definitive and statistically meaningful results. A state-of-the-art immunologic laboratory and BVAMC flow cytometry facility that includes the latest FACS analysis and sorting equipment, as well as a dedicated BVAMC 10X single-cell analysis facility, and analytical pipeline for bioinformatics analysis of the results are established. Significance: The successful conclusion of these experiments will be the development of new diagnostic and therapeutic approaches. From a diagnostic approach, the key defining phenotype of quiescent versus non-quiescent B cells can be economically and easily measured using standard clinical laboratory flow cytometry equipment with a relatively low number of cell surface (such as IL-4R or IFNAR1) or intracellular factors (such as intracellular IFNβ, TLR7, or IKAROS). This could be extended to analyze trajectories of abnormal B cell development that result from the loss of B cell quiescence at the earliest stages to understand therapeutics that may be beneficial in subjects who have acquired SLE in the past. At the very minimum, such approaches should enable the studies and types of treatments that will promote the maintenance of B cell quiescence and in effect, force SLE into a state of remission which can be maintained once the causes of the immune or environmental factors that disrupt such remission are defined.