IL-7 is critical for B cell production in adult mice; however, its role in human B lymphopoiesis is controversial. One challenge was the inability to differentiate human cord blood (CB) or adult bone marrow (BM) hematopoietic stem cells (HSCs) without murine stroma. Here, we examine the role of IL-7 in human B cell development using a novel, human-only model based on coculturing human HSCs on primary human BM stroma. In this model, IL-7 increases human B cell production by >60-fold from both CB and adult BM HSCs. IL-7-induced increases are dose-dependent and specific to CD19 ؉ cells. STAT5 phosphorylation and expression of the Ki-67 proliferation Ag indicate that IL-7 acts directly on CD19 ؉ cells to increase proliferation at the CD34 ؉ and CD34؊ pro-B cell stages. Without IL-7, HSCs in CB, but not BM, give rise to a small but consistent population of CD19 lo B lineage cells that express EBF (early B cell factor) and PAX-5 and respond to subsequent IL-7 stimulation. Flt3 ligand, but not thymic stromal-derived lymhopoietin (TSLP), was required for the IL-7-independent production of human B lineage cells. As compared with CB, adult BM shows a reduction of in vitro generative capacity that is progressively more profound in developmentally sequential populations, resulting in an ϳ50-fold reduction in IL-7-dependent B lineage generative capacity. These data provide evidence that IL-7 is essential for human B cell production from adult BM and that IL-7-induced expansion of the pro-B compartment is increasingly critical for human B cell production during the progression of ontogeny. M urine studies have provided evidence that B cell development is controlled by a network of transcription factors and cytokine signaling that coordinates stagespecific expression of B lineage genes (1, 2). In mice, signaling through two cytokine receptors, Flt3 and IL-7R, are critical for B cell development. The combined loss of signaling through both of these receptors completely blocks fetal and adult B cell development (3, 4). Flt3 signaling up-regulates IL-7R␣ expression (5). In adult mice, signaling through the IL-7R up-regulates expression of early B cell factor (EBF).3 EBF in turn regulates expression of a cascade of B cell-specific genes (1, 2) required for the transition from the common lymphoid progenitor to pro-B cells (6 -8). During fetal and neonatal life, IL-7-independent expression of EBF allows early B lineage differentiation, but not proliferation, unless IL-7 is present (9). Human B cell development is thought to differ from that in mouse with respect to the requirement for IL-7. Early evidence for the importance of IL-7 in murine B cell production came from in vitro studies showing that B cell precursors derived from adult bone marrow (BM) increase by ϳ50-fold with the addition of IL-7 (10). Further evidence came from reports of blocked B cell development in the initial in vivo studies that characterized mutant mice with defects in IL-7 (11), or in components of the IL-7R (12, 13). Parallel in vitro studies of human B ce...

T hymic stromal lymphopoietin (TSLP) stimulates in vitro proliferation of human fetal B-cell precursors. However, its in vivo role during normal human B lymphopoiesis is unknown. Genetic alterations that cause overexpression of its receptor component, cytokine receptor-like factor 2 (CRLF2), lead to high-risk B-cell acute lymphoblastic leukemia implicating this signaling pathway in leukemogenesis. We show that mouse thymic stromal lymphopoietin does not stimulate the downstream pathways (JAK/STAT5 and PI3K/AKT/mTOR) activated by the human cytokine in primary high-risk leukemia with overexpression of the receptor component. Thus, the utility of classic patient-derived xenografts for in vivo studies of this pathway is limited. We engineered xenograft mice to produce human thymic stromal lymphopoietin (+T mice) by injection with stromal cells transduced to express the cytokine. Control (-T) mice were produced using stroma transduced with control vector. Normal levels of human thymic stromal lymphopoietin were achieved in sera of +T mice, but were undetectable in -T mice. Patient-derived xenografts generated from +T as compared to -T mice showed a 3-6-fold increase in normal human B-cell precursors that was maintained through later stages of B-cell development. Gene expression profiles in high-risk B-cell acute lymphoblastic leukemia expanded in +T mice indicate increased mTOR pathway activation and are more similar to the original patient sample than those from -T mice. +T/-T xenografts provide a novel pre-clinical model for understanding this pathway in B lymphopoiesis and identifying treatments for high-risk B-cell acute lymphoblastic leukemia with overexpression of cytokine-like factor receptor 2.

Thymic stromal lymphopoietin (TSLP) and IL-7 are cytokines that signal via the IL-7 receptor alpha (IL-7Rα) to exert both overlapping and unique functions during early stages of mouse B cell development. In human B lymphopoiesis the requirement for IL-7Rα signaling is controversial and the roles of IL-7 and TSLP are less clear. Here we evaluated human B cell production using novel in vitro and xenograft models of human B cell development that provide selective IL-7 and human TSLP (hTSLP) stimulation. We show that in vitro human B cell production is almost completely blocked in the absence of IL-7Rα stimulation and that either TSLP or IL-7 can provide a signal critical for the production and proliferation of human CD19+ PAX5+ pro-B cells. Analysis of primary human bone marrow (BM) stromal cells show that they express both IL-7 and TSLP providing an in vivo source of these cytokines. Using novel xenograft models we show that the in vivo production of human pro-B cells under the influence of mouse IL-7 is reduced by anti-IL-7 neutralizing antibodies, and this loss can be restored by hTSLP at physiological levels. These data establish the importance of IL-7Rα-mediated signals for normal human B cell production.

SUMMARY To successfully colonize host cells, pathogenic bacteria must circumvent the host’s structural barrier such as the collagen-rich extracellular matrix (ECM), as a preliminary step to invasion and colonization of the periodontal tissue. Filifactor alocis possesses a putative Peptidase U32 family protein (HMPREF0389_00504) with collagenase activity that may play a significant role in colonization of host tissue during periodontitis by breaking down collagen into peptides and disruption of the host cell. Domain architecture the HMPREF0389_00504 protein predicted the presence of a characteristic PrtC-like collagenase domain, and a peptidase domain. Our study demonstrated that the recombinant F. alocis peptidase U32 protein (designated PrtFAC) can interact with, and degrade type I collagen, heat denatured collagen and gelatin in a calcium dependent manner. PrtFAC decreased viability and induced apoptosis of normal oral keratinocytes (NOKs) in a time and dose dependent manner. Transcriptome analysis of NOK cells treated with PrtFAC showed an upregulation of the genes encoding human pro-apoptotic proteins: Apoptotic Peptidase Activating Factor 1 (Apaf1) Cytochrome C, as well as Caspase 3 and 9, suggesting the involvement of the mitochondrial apoptotic pathway. There was a significant increase in caspase 3/7 activity in NOK cells treated with PrtFAC. Taken together, these findings suggest that F. alocis PrtFAC protein may play a role in the virulence and pathogenesis of F. alocis.

Expression of the IL-7 receptor alpha (IL-7R) is a distinguishing feature of common lymphoid progenitors (CLP) in mice. Human B cell development has been thought to differ from that in mouse with respect to the requirement for IL-7. Our previous studies show that IL-7 plays a critical role in human B cell production (J Immunol. 2009, 182:4255). Here we use IL-7R expression to identify a human CLP population that can be generated from hematopoietic stem cells (HSCs) in umbilical cord blood (CB). Following two weeks of co-culture with primary human bone marrow (BM) stroma, CB CD34+ cells give rise to a CD34+CD19-IL-7R+ progenitor population that is absent from fresh, uncultured CB. IL-7R+ progenitors make up ~17% of the CD19-CD34+ cells present in culture at 2 weeks. A similar IL-7R+ human progenitor population is generated in the BM of immunodeficient mice following transplant with human CD34+ CB cells. FACS-sorted CD34+CD19-IL-7R+ progenitors produced in co-culture or in the human-mouse xenograft model give rise to CD19+ B cells and CD56+ NK cells in bulk and single cell culture, but lack myeloid potential in CFU assays and when transplanted into immunodeficient mice. Ongoing experiments will evaluate the T lineage potential of IL-7R+ progenitors in the human-mouse xenograft model. These data will be important for developing therapies to accelerate and enhance lymphoid reconstitution from HSCs in CB, a hematopoietic source used increasingly for stem cell transplant.