Dolence, Joseph J.

Abstract The production of naïve B cells in bone marrow (BM) is critical for maintaining a diverse peripheral B cell pool to fight infection and establish lifelong immunity. The generation of naïve B cells is impaired in Flt3-ligand (FL-/-) mice contributing to reductions in splenic B cells. The goal of this study was to determine the consequence of FL-deficiency on splenic B cell maturation. We found significant reductions in numbers of transitional (TS) and follicular (FO), but not marginal zone (MZ) B cells in FL-/- mice. Mixed BM chimeras revealed that the reduction in TS B cells was cell extrinsic, linking microenvironmental signals to TS B cell maturation. FL administration into FL-/- mice restored the deficiency in TS B cells that do not express Flt3, suggesting that a FL responsive component of the microenvironment is critical for TS maturation. Intracellular Ki67 revealed a significant decrease in the proliferative capacity of TS cells in FL-/- mice. Expression of a Bcl2 transgene did not rescue TS cells, uncoupling FL-deficiency to Bcl2-regulated survival pathways. BAFF-R expression and serum levels of BAFF were comparable in WT and FL-/- mice, supporting BAFF-independent regulation of TS cells. These observations support an integral indirect role for Flt3 signaling in regulation of TS B cell maturation in the spleen.

Abstract Evidence is accumulating that the molecular circuitry initiating lymphoid lineage specification correlates with expression of the receptor tyrosine kinase Flt3. Recombination activating gene-1 (RAG1) locus activation is a hallmark of lymphoid lineage specification. Previous studies established that expression of a RAG1-GFP/+ reporter begins in a subset of lineage negative (Lin-) cells that express high levels of c-kit and Sca-1 (LSK+). To investigate a regulatory connection between Flt3 signaling and RAG1 locus activation from this stage, RAG1-GFP/+ knockin mice expressing wildtype, heterozygote, and knockout Flt3-ligand (FL) alleles were generated. Mono-allelic expression of the FL gene reduced FL transcripts by half, providing an in vivo model to evaluate threshold requirements for FL in lymphocyte development. Percentages and numbers of LSK+ cells were similar in FL+/+ and FL+/- mice, but significantly decreased in FL-/- animals. Analysis of GFP expression within the LSK+ compartment revealed a FL dose dependent reduction in frequencies and numbers of GFP+ cells. The requirement for threshold levels of FL in regulation of GFP expression extended into the B220- c-kitlo IL-7R+ lymphoid progenitor and CD43+ B220+ Pro-B compartments, but not in later stage B cell precursors. These findings establish a critical requirement for FL in regulating one event in lymphoid specification, RAG1 locus activation. These studies were supported by startup funds to K.L.M. from Mayo Clinic.

AbstractB lymphopoiesis in bone marrow (BM) is critical for maintaining a diverse peripheral B cell pool to fight infection and establish lifelong immunity. The generation of immature B cells is reduced in Flt3‐ligand (FL‐/‐) mice leading to deficiencies in splenic B cells. Here, we sought to understand the cellular basis of the spleen B cell deficiency in FL‐/‐ mice. Significant reductions in transitional (TS) and follicular (FO) B cells were found in FL‐/‐ mice, and increased frequencies, but not absolute numbers, of marginal zone (MZ) B cells. BAFF‐R expression on splenic B cells and serum levels of B cell activating factor (BAFF) was comparable to wildtype (WT) mice. Mixed BM chimeras revealed that the reductions in TS and FO B cells were cell extrinsic. FL administration into FL‐/‐ mice restored the deficiency in TS B cells and normalized the MZ compartment. Ki67 analysis revealed a significant decrease in the proliferative capacity of TS B cells in FL‐/‐ mice. A Bcl2 transgene did not rescue TS cells in FL‐/‐ mice, uncoupling FL‐deficiency to Bcl2‐dependent survival pathways. Upregulation of CD1d expression and adoptive transfer experiments suggested MZ skewing in FL‐/‐ mice. These findings support an integral role for Flt3 signaling in peripheral B cell maturation.

Abstract Hoxa9 and Flt3 signaling are individually important for the generation of lymphoid lineage precursors from multipotent hematopoietic progenitors (MPP) in bone marrow. Mice deficient for Hoxa9, Flt3, or Flt3 ligand (FL) have reduced numbers of lymphoid-primed multipotential progenitors (LMPP), common lymphoid progenitors (CLP), and B/T cell precursors. Hoxa9 regulates lymphoid development, in part, through transcriptional regulation of Flt3. However, it was unclear whether Hoxa9 has functions in lymphopoiesis independent of, or alternatively, synergistically with Flt3 signaling. In this study, we show that Hoxa9−/−Flt3l−/− mice have more severe deficiencies in all B lineage cells, CLP, LMPP, and total Flt3+ MPP in bone marrow than the single knockouts. Although LMPP and Flt3+ CLP contain precursors for NK and dendritic cell lineage cells, no deficiencies in these lineages beyond that in Flt3l−/− mice was found. Thymocyte cellularity was significantly reduced in the compound knockout, although peripheral T cell numbers mirrored Flt3l−/− mice. Analysis of the hematopoietic progenitor compartment revealed elevated numbers of CD150+hiCD34−CD41+ myeloid–biased stem cells in Hoxa9−/−Flt3l−/− mice. In contrast, CD150− MPP enriched for lymphoid potential were synergistically reduced, suggesting Hoxa9 and Flt3 signaling function coordinately to regulate lymphopoiesis at a very early stage. Real-time PCR analysis of CD150−Flt3+ cells from wild-type control, Hoxa9−/−, and Flt3l−/− single knockouts revealed decreased lymphoid transcripts, corroborating the importance of these regulators in lymphoid development. Taken together, these studies reveal a very early checkpoint in lymphopoiesis dependent on the combinatorial activities of Hoxa9 function and Flt3 signaling.