Hematopoietic stem cells (HSC) can be harmed by disease, chemotherapy, radiation and normal aging. We now show that damage also occurs in mice repeatedly treated with very low doses of lipopolysaccharide (LPS). Overall health of the animals was good, and there were relatively minor changes in marrow hematopoietic progenitors. However, HSC were unable to maintain quiescence, and transplantation revealed them to be myeloid skewed. Moreover, HSC from treated mice were not sustained in serial transplants and produced lymphoid progenitors with low levels of the E47 transcription factor. This phenomenon was previously seen in normal aging. Screening identified monoclonal antibodies that resolve HSC subsets, and relative proportions of these HSC changed with age and/or chronic LPS treatment. For example, minor CD150Hi CD48− populations lacking CD86 or CD18 expanded. Simultaneous loss of CD150Lo/− CD48− HSC and gain of the normally rare subsets, in parallel with diminished transplantation potential would be consistent with age or Tolllike receptor (TLR) related injury. On the other hand, HSC in old mice differed from those in LPS treated animals with respect to VCAM-1 or CD41 expression, and lacked proliferation abnormalities. HSC can be exposed to endogenous and pathogen derived TLR ligands during persistent low-grade infections. This stimulation might contribute in part to HSC senescence and ultimately compromise immunity.

Expression of V(D)J recombinase activity in developing lymphocytes is absolutely required for initiation of V(D)J recombination at antigen receptor loci. However, little is known about when during hematopoietic development the V(D)J recombinase is first active, nor is it known what elements activate the recombinase in multipotent hematopoietic progenitors. Using mice that express a fluorescent transgenic V(D)J recombination reporter, we show that the V(D)J recombinase is active as early as common lymphoid progenitors (CLPs) but not in the upstream progenitors that retain myeloid lineage potential. Evidence of this recombinase activity is detectable in all four progeny lineages (B, T, and NK, and DC), and rag2 levels are the highest in progenitor subsets immediately downstream of the CLP. By single cell PCR, we demonstrate that V(D)J rearrangements are detectable at IgH loci in ∼5% of splenic natural killer cells. Finally, we show that recombinase activity in CLPs is largely controlled by the Erag enhancer. As activity of the Erag enhancer is restricted to the B cell lineage, this provides the first molecular evidence for establishment of a lineage-specific transcription program in multipotent progenitors.

Hematopoietic stem and progenitor cells were previously found to express Tolllike receptors (TLRs), suggesting that bacterial/viral products may influence blood cell formation. We now show that common lymphoid progenitors (CLPs) from mice with active HSV-1 infection are biased to dendritic cell (DC) differentiation, and the phenomenon is largely TLR9 dependent. Similarly, CLPs from mice treated with the TLR9 ligand CpG ODN had little ability to generate CD19 ؉ B lineage cells and had augmented competence to generate DCs. TNF␣ mediates the depletion of late-stage lymphoid progenitors from bone marrow in many inflammatory conditions, but redirection of lymphopoiesis occurred in TNF␣ ؊/؊ mice treated with CpG ODN. Increased numbers of DCs with a lymphoid past were identified in Ig gene recombination substrate reporter mice treated with CpG ODN. TLR9 is highly expressed on lymphoid progenitors, and culture studies revealed that those receptors, rather than inflammatory cytokines, accounted for the production of several types of functional DCs. IntroductionHematopoietic stem cells (HSCs) give rise to progenitors with potential to produce blood cell types with remarkably stable characteristics. Although this process is tightly controlled, recent findings suggest that hematopoiesis is dynamic and also responsive to environmental factors. 1 The loss of differentiation options is gradual, and T lymphocytes, natural killer (NK) cells, and dendritic cells (DCs) can each be made from multiple progenitors under experimental circumstances. 1,2 Indeed, apparently similar DCs arise from distinct myeloid or lymphoid progenitors. 3 This new perspective raises the possibility that choices are made between multiple pathways to replenish effectors of the immune system. Thus, it is important to learn what normal and disease conditions favor particular differentiation routes.Several major categories of DCs have been found in murine bone marrow (BM). Conventional DCs (cDCs) are competent to present antigens, whereas plasmacytoid dendritic cells (pDCs) are potent producers of type I interferon. 3 The pDCs are divisible into 2 subtypes (pDC1 and pDC2) on the basis of RAG-1 expression and patterns of cytokine production. 4 Under experimental conditions, DCs are produced from stem cells, as well as lymphoid and myeloid progenitors. [3][4][5] Flk-2/flt-3 ligand and the associated Stat3 signaling pathway are important for DC differentiation; consequently, efficient progenitors bear the Flk-2/flt-3 receptor. 3 In our experience, the highest yields of pDCs are obtained from the primitive Lin Ϫ c-Kit hi Sca-1 ϩ (LSK) fraction of murine BM. 4 Two recent reports identified a Lin Ϫ Flt3 ϩ c-Kit lo -CD115 ϩ pro-DC population capable of generating pDCs and at least 2 categories of DCs. 6,7 However, greater yields of DCs were produced from more primitive progenitors, and some of those are already restricted to particular DC pathways. 7 Common lymphoid progenitors (CLPs) represent the main pathway to B lineage cells and include most progenitors dest...

Immunoglobulin secretion is modulated by a competition between use of a weak promoter proximal poly(A) site and a non-consensus splice site in the last secretory-specific exon of the heavy chain pre-mRNA. RNA polymerase II transcription elongation factor ELL2, induced in plasma cells, enhanced both polyadenylation and exon skipping with the Igh gene and reporter constructs. Lowering ELL2 expression by hnRNP F transfection or siRNA reduced secretory-specific forms of IgH mRNA. ELL2 and polyadenylation factor CstF-64 co-tracked with RNA polymerase II across the Igh mu and gamma gene segments; association of both factors was blocked by ELL2 siRNA. Thus loading of ELL2 and CstF-64 on RNAP-II was linked, causative for enhanced proximal poly(A) site use and necessary for IgH mRNA processing.

The recent description of a Lin ؊ AA4.1 ؉ CD19 ؉ B220 Lo/؊ B1-specified progenitor (B1P) population in adult marrow adds support for the argument that these unique B cells arise from a distinct lineage. However, the origins of B1P were not investigated and their developmental relationships to conventional B2 cells remain unclear. We now report that B1P development is IL-7R␣-dependent, and negatively regulated by Bruton tyrosine kinase. Lymphoid characteristics of B1P were further studied with recombination activating gene (RAG)-1/GFP knock-in, RAG-1/Cre reporter, and VEX transgenic mice. Our results reveal that they are heterogeneous with respect to lymphocyte affiliation. RAG-1 ؉ early lymphoid progenitors and Lin ؊ Sca-1 ؉ cKit Lo IL-7R␣ ؉ common lymphoid progenitors from adult marrow efficiently generated CD19 ؉ CD45R/B220 Lo/؊ cells in vitro and in vivo. Moreover, early lymphoid progenitors and common lymphoid progenitors produced significant numbers of peritoneal CD11b ؉ CD5 ؉ B1a and CD11b ؉ CD5 ؊ B1b cells in vivo. Finally, 2-step transplantation experiments established a differentiation pathway between conventional lymphoid progenitors, B1P, and mature B1 lymphocytes. Thus, our findings indicate that at least some B1P can be produced in adult bone marrow from primitive B2 progenitors, and suggest a developmental relationship between the major categories of B lymphocytes.