Hematopoietic stem cells (HSCs) are first found in the aorta-gonad-mesonephros region and vitelline and umbilical arteries of the midgestation mouse embryo. Runx1 (AML1), the DNA binding subunit of a core binding factor, is required for the emergence and/or subsequent function of HSCs. We show that all HSCs in the embryo express Runx1. Furthermore, HSCs in Runx1(+/-) embryos are heterogeneous and include CD45(+) cells, endothelial cells, and mesenchymal cells. Comparison with wild-type embryos showed that the distribution of HSCs among these various cell populations is sensitive to Runx1 dosage. These data provide the first morphological description of embryonic HSCs and contribute new insight into their cellular origin.

Cystic fibrosis transmembrane conductance regulator (CFTR)-mediated Cl؊ secretion across fluid-transporting epithelia is regu-

PDZ domains are ubiquitous peptide-binding modules that mediate protein-protein interactions in a wide variety of intracellular trafficking and localization processes. These include the pathways that regulate the membrane trafficking and endocytic recycling of the cystic fibrosis transmembrane conductance regulator (CFTR), an epithelial chloride channel mutated in patients with cystic fibrosis. Correspondingly, a number of PDZ proteins have now been identified that directly or indirectly interact with the C terminus of CFTR. One of these is CAL, whose overexpression in heterologous cells directs the lysosomal degradation of WT-CFTR in a dose-dependent fashion and reduces the amount of CFTR found at the cell surface. Here, we show that RNA interference targeting endogenous CAL specifically increases cell-surface expression of the disease-associated ⌬F508-CFTR mutant and thus enhances transepithelial chloride currents in a polarized human patient bronchial epithelial cell line. We have reconstituted the CAL-CFTR interaction in vitro from purified components, demonstrating for the first time that the binding is direct and allowing us to characterize its components biochemically and biophysically. To test the hypothesis that inhibition of the binding site could also reverse CAL-mediated suppression of CFTR, a three-dimensional homology model of the CAL⅐CFTR complex was constructed and used to generate a CAL mutant whose binding pocket is correctly folded but has lost its ability to bind CFTR. Although produced at the same levels as wild-type protein, the mutant does not affect CFTR expression levels. Taken together, our data establish CAL as a candidate therapeutic target for correction of post-maturational trafficking defects in cystic fibrosis.

The family of core-binding factors includes the DNA-binding subunits Runx1-3 and their common non-DNA-binding partner CBF␤. We examined the collective role of core-binding factors in hematopoiesis with a hypomorphic Cbfb allelic series. Reducing CBF␤ levels by 3-or 6-fold caused abnormalities in bone development, megakaryocytes, granulocytes, and T cells. T-cell development was very sensitive to an incremental reduction of CBF␤ levels: mature thymocytes were decreased in number upon a 3-fold reduction in CBF␤ levels, and were virtually absent when CBF␤ levels were 6-fold lower. Partially penetrant consecutive differentiation blocks were found among early T-lineage progenitors within the CD4 ؊ CD8 ؊ double-negative 1 and downstream double-negative 2 thymocyte subsets. Our data define a critical CBF␤ threshold for normal T-cell development, and situate an essential role for corebinding factors during the earliest stages of T-cell development. IntroductionHypomorphic alleles have long been known to cause developmental disorders in model organisms and in humans, and can reveal additional functions for genes in pathways that are completely obliterated when the gene's function is eliminated. For example, the many different spontaneous, chemically induced, and targeted mutant alleles of the Kit gene have illuminated c-kit's multiple roles in gametogenesis, melanogenesis, and hematopoiesis, and in the interstitial cells of Cajal. [1][2][3] Hypomorphic alleles can reveal differences in the requirements of certain developmental pathways for a protein's concentration, and help pinpoint lineage decisions that are influenced by that protein. Many mutations in cancercausing genes may cause a functional dosage reduction as part of their overall activity, and the study of hypomorphic alleles may allow an assessment of this contribution.In this study, we used a hypomorphic allele of the core-binding factor ␤ (Cbfb) gene to unveil new developmental requirements for all 3 core-binding factors. Core-binding factors (CBFs) are a small family of transcription factors consisting of a DNA-binding subunit encoded by the Runx1, Runx2, or Runx3 genes, and a common non-DNA-binding CBF␤ subunit. Runx1 is required for hematopoietic stem cell (HSC) emergence in the fetus, 4 and during postnatal hematopoiesis for megakaryocyte, B-, and T-lymphocyte development. [5][6][7] Runx1 participates in CD4 silencing during the CD4 Ϫ CD8 Ϫ double-negative (DN) and CD8 ϩ stages of T-cell development and is necessary at the DN2 to DN3 and DN3 to DN4 transitions. [5][6][7][8] Runx2 is required for bone formation, both for osteoblast differentiation and chondrocyte hypertrophy. [9][10][11][12] Runx3 contributes to the maturation of chondrocytes during bone formation 13 and is necessary for CD4 silencing at the CD8 ϩ stage of T-cell development, for Langerhans-cell development, and its deletion accelerates the maturation of dendritic cells resulting in an allergic airway inflammation. 7,8,14 We used a hypomorphic Cbfb allele in conjunction with a nonfunction...

Runx1 is expressed in skeletal elements, but its role in fracture repair has not been analyzed. We created mice with a hypomorphic Runx1 allele (Runx1L148A) and generated Runx1L148A/− mice in which >50% of Runx1 activity was abrogated. Runx1L148A/− mice were viable but runted. Their growth plates had extended proliferating and hypertrophic zones, and the percentages of Sox9‐, Runx2‐, and Runx3‐positive cells were decreased. Femoral fracture experiments revealed delayed cartilaginous callus formation, and the expression of chondrogenic markers was decreased. Conditional ablation of Runx1 in the mesenchymal progenitor cells of the limb with Prx1‐Cre conferred no obvious limb phenotype; however, cartilaginous callus formation was delayed following fracture. Embryonic limb bud–derived mesenchymal cells showed delayed chondrogenesis when the Runx1 allele was deleted ex vivo with adenoviral‐expressed Cre. Collectively, our data suggest that Runx1 is required for commitment and differentiation of chondroprogenitor cells into the chondrogenic lineage. © 2012 American Society for Bone and Mineral Research.

Background: P. aeruginosa chronically colonizes the lung in CF patients and elicits a proinflammatory response. Excessive secretion of IL-6 and IL-8 by CF airway cells in response to P. aeruginosa infection in the CF airway is though to contribute to lung injury. Accordingly, the goal of this study was to test the hypothesis that Corr4a and VRT325, investigational compounds that increase ΔF508-CFTR mediated Cl^- secretion in human CF airway cells, reduce the pro-inflammatory response to P. aeruginosa. Methods: IL-6 and IL-8 secretion by polarized CF human airway epithelial cells (CFBE41o-) were measured by multiplex analysis, and ΔF508-CFTR Cl- secretion was measured in Ussing chambers. Airway cells were exposed to P. aeruginosa (PAO1 or PA14) and Corr4a or VRT325. Results: Corr4a and VRT325 increased ΔF508-CFTR Cl^- secretion but did not reduce either constitutive IL-6 or IL-8 secretion, or IL-6 and IL-8 secretion stimulated by P. aeruginosa (PA14 or PAO1). Conclusions: Corr4a and VRT325 do not reduce the inflammatory response to P. aeruginosa in human cystic fibrosis airway epithelial cells.

The number of circulating lymphocytes on day 15 after transplantation correlates with improved survival in patients with myeloma, but the lymphocyte subset responsible is unknown. NKG2D is a natural killer (NK) cell activating receptor that mediates non-MHC restricted and TCR-independent cell lysis. Our preliminary results indicate that CD3+CD8+ T cells expressing NKG2D may be a critical lymphocyte population. A phase II trial examined the feasibility of infusing ex vivo-expanded cells enriched for NKG2D+CD3+CD8+ T cells at weeks 1, 2, 4, and 8 after an autologous transplantation. In addition, low-dose IL-2 (6 × 105 IU/m2/day) was administered for 4 weeks, beginning on the day of transplantation. Twenty-three patients were accrued and 19 patients are evaluable. There were no treatment-related deaths. All patients completed their course of IL-2 and demonstrated normal engraftment. When compared with patients with myeloma who underwent transplantation not receiving posttransplantation immune therapy, the treated patients demonstrated an increase in the number of circulating NKG2D+CD3+CD8+ T cells/μL (P < .004), CD3+CD8+ T cells/μL (P < .04), CD3+CD8+CD56+ T cells/μL (P < .004), and NKG2D+CD3−CD56+ T cells/μL (P < .003). Myeloma cell-directed cytotoxicity by the circulating mononuclear cells increased after transplantation (P < .002). When compared to posttransplantation IL-2 therapy alone in this patient population, the addition of cells enriched for NKG2D+CD3+CD8+ T cells increased tumor-specific immunity, as demonstrated by enhanced lysis of autologous myeloma cells (P = .02). We postulate that this regimen that increased the number and function of the NKG2D+CD3+CD8+ T cells after transplantation may improve clinical outcomes by eliminating residual malignant cells in vivo.

Background aims A phase I trial examined the ability of immunotherapy to mobilize progenitor and activated T cells. Methods Interleukin (IL)-2 was administered subcutaneously for 11 days, with granulocyte (G)-colony-stimulating factor (CSF) (5 mcg/kg/day) and granulocyte–macrophage (GM)-CSF (7.5 mcg/kg/day) added for the last 5 days. Leukapheresis was initiated on day 11. Thirteen patients were treated (myeloma n = 11, non-Hodgkin’s lymphoma n = 2). Results Toxicities were minimal. IL-2 was stopped in two patients because of capillary leak (n = 1) and diarrhea (n = 1). Each patient required 2.5 leukaphereses (median; range 1–3) to collect 3.2 × 106 CD34+ cells/kg (median; range 1.9–6.6 × 106/kg). Immune mobilization increased the number of CD3+ CD8+ T cells (P = 0.002), CD56+ natural killer (NK) cells (P = 0.0001), CD8+ CD56+ T cells (P = 0.002) and CD4+ CD25+ cells (P = 0.0001) compared with cancer patients mobilized with G-CSF alone. There was increased lysis of myeloma cells after 7 days (P = 0.03) or 11 days (P = 0.02). The maximum tolerated dose of IL-2 was 1 × 106 IU/m2/day. Conclusions Immune mobilization is well tolerated with normal subsequent marrow engraftment. As cells within the graft influence lymphocyte recovery, an increased number of functional lymphocytes may result in more rapid immune reconstitution.