Cellular and molecular analysis of B lymphocyte development and selection

Yale University, New Haven CT

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Abstract

Summary. The bone marrow (BM) produces myeloid and lymphoid cells at a rate that ensures hematopoietic cell homeostasis. How hematopoietic cell production is regulated to control the size of the peripheral immune cell compartment is not understood. Immune cells develop from a very small population of hematopoietic stem cells (HSCs). HSC numbers are kept relatively constant due to a balance between dormancy and division, and between self-renewal and differentiation. Although each of these processes has been extensively studied over the past several decades, major gaps remain, perhaps the biggest of all being "the stem cell paradox". The cytokines Stem Cell Factor (SCF) and Thrombopoietin (THPO) are critical signals produced by niches that control HSC self-renewal and survival through the activation of receptor tyrosine kinases (RTK) cKit and cMPL, respectively. The stem cell paradox arises from the fact that cKit and cMPL strongly activate Pi3K/AKT/mTOR signaling and are involved in hematopoietic progenitor differentiation, and yet HSCs remain undifferentiated and dormant. To address this long-standing conceptual gap, we hypothesized that undefined signals must counteract the pro-proliferative and pro-differentiation effects of RTK signaling to ensure the homeostasis of the HSC compartment in vivo. G-protein coupled receptors (GPCRs) signal through heterotrimeric G proteins composed of a, b and g subunits, with the a subunit conferring pathway specificity. GPCR signaling via Ga12/13 proteins potently inhibits PI3K signaling such that genetic deficiency in Ga12 or Ga13 results in lymphoproliferative disease and lymphomagenesis. Ga12/13 protein signaling also activate Rho kinases which in turn antagonize the functions of the small GTPases Rac1 and Rac2 and inhibit cell migration. In recent and unpublished studies, we uncovered a critical role for the Ga12/13-coupled GPCRs GPR56 and GPR97 in HSCs. Specifically, we demonstrate that deficiency in both receptors is lethal due to a complete loss of HSCs during embryogenesis, while induced deficiency in both receptors in HSCs of adult mice leads to the complete loss of HSCs and adult hematopoiesis. Our findings provide the foundation for a new paradigm in which HSC homeostasis is controlled by a balance between stimulatory and inhibitory pathways. The central goal of this grant proposal is to precisely define the role of GPR56 and GPR97 in HSCs. In Aim 1 we will study whether and how GPR56 and GPR97 control HSC quiescence, self-renewal, survival, differentiation, and niche localization. In addition, we will visualize the impact of GPR56 and GPR97 in restricting HSC movement and confining HSC localization in the HSC niche by intravital 2-photon microcopy and in situ confocal microscopy. In Aim 2, we will use selective gene deletion exclusively in HSCs to study the signal transduction pathways and transcriptional hubs controlled by GPR56 and GPR97 in HSCs. Together, the experiments described in these aims will reveal novel mechanisms and fundamental new principles of HSC homeostasis and multilineage differentiation.