IntroductionThe hematopoietic system is in a constant state of self-renewal as stem cells continuously replenish short-lived blood cells. 1 All blood cells are derived from the long-term hematopoietic stem cell (LT-HSC) subset that maintains peripheral homeostasis by undergoing continual self-renewal for the life of the organism. LT-HSCs differentiate into multipotent progenitor cells (MPPs), a more mature subset that lacks the long-term ability to self-renew but retains the capacity to reconstitute all blood lineages. 1,2 Both LT-HSCs and MPPs are found in the lineage-negative, c-Kitpositive, and Sca-1-positive (LSK) populations. 3 Studying the LSK subset itself has contributed to the understanding of HSC biology because it is a population highly enriched for HSCs. Within the LSK subset, those cells positive for CD150 expression and negative for CD48 identify LT-HSCs that are capable of long-term self-renewal (CD150 ϩ CD48 Ϫ LSK), whereas those cells that are negative for both (CD150 Ϫ CD48 Ϫ LSK) identify a multipotential population that has a comparably limited contribution to long-term hematopoiesis and is enriched for MPPs. 1,4,5 Although definitive HSC experiments require a test of the functional capacity of the populations, the surface phenotype of these populations allows for an estimation of the presence of stem cells with long-term self-renewal capacity or those with more limited hematopoietic potential.Among the signal transduction pathways that have attracted considerable attention as possibly being involved in HSC selfrenewal is the phosphoinositide 3-kinase (PI3K)-AKT pathway. PI3K is a lipid kinase 6 critical for the activation of AKT, a family of serine threonine kinases essential for the control of cellular metabolism and survival in multiple tissues. 7,8 Haneline et al 9 demonstrated that HSCs with decreased PI3K activity exhibit defective hematopoietic reconstitution and a reduced proliferative capacity. Concordantly, conditional deletion of phosphatase and tensin homolog (PTEN), a phosphatase that negatively regulates PI3K, 10 promotes differentiation and proliferation at the expense of self-renewal, leading to depletion of the HSC pool. 11,12 Researchers have also examined the importance of molecules downstream of the PI3K/AKT pathway. For instance, FOXO, a family of transcription factors negatively regulated by AKT, 13 controls HSC quiescence by maintaining a low threshold of intracellular reactive oxygen species (ROS). 14 HSCs that lack multiple FOXO family members are hyperproliferative and fail to self-renew but are normalized by treatment with antioxidants. 14 A recent report by Kharas et al 15 showed that constitutive activation of AKT in hematopoietic HSCs results in a hyperproliferative state and subsequent HSC depletion, akin to the phenotype of PTEN-deleted HSCs. However, to fully appreciate the biologic role of AKT in HSC development, complementary studies in the absence of AKT are necessary. A main challenge to depleting AKT from HSCs is the expression of 3 isoforms in mammalian ...

Wiskott–Aldrich syndrome protein (WASP) is in a complex with WASP-interacting protein (WIP). WASP levels, but not mRNA levels, were severely diminished in T cells from WIP −/− mice and were increased by introduction of WIP in these cells. The WASP binding domain of WIP was shown to protect WASP from degradation by calpain in vitro . Treatment with the proteasome inhibitors MG132 and bortezomib increased WASP levels in T cells from WIP −/− mice and in T and B lymphocytes from two WAS patients with missense mutations (R86H and T45M) that disrupt WIP binding. The calpain inhibitor calpeptin increased WASP levels in activated T and B cells from the WASP patients, but not in primary T cells from the patients or from WIP −/− mice. Despite its ability to increase WASP levels proteasome inhibition did not correct the impaired IL-2 gene expression and low F-actin content in T cells from the R86H WAS patient. These results demonstrate that WIP stabilizes WASP and suggest that it may also be important for its function.

IntroductionIn adult mice most mature B cells can be divided into follicular (FO) or marginal zone (MZ) B cells. FO B cells constitute the bulk of the recirculating peripheral B-cell pool and generate both plasmablasts and memory B cells in response to a wide array of potential pathogens. In contrast, MZ B cells are actively retained in the marginal sinus of the spleen where they rapidly generate plasmablasts in response to bloodborne pathogens. 1,2 B-cell receptor (BCR)-mediated activation of naive cells within either population can be modified through coengagement of several cell surface receptors including the costimulatory receptor CD40, the CD19/ CD21 complement receptor complex, and Toll-like receptor-9 (TLR-9). 3-5 BCR and CD19 signaling also influence whether immature B cells colonize the FO or MZ B-cell pools, but how signals derived from these and additional cell surface receptors are integrated to control B-cell development and activation is unclear.Notch receptors regulate cell fate decisions in a wide variety of biologic systems including lymphocyte development and function. The mouse genome contains 4 Notch receptors (Notch1 to Notch4). Activation of Notch receptors on the cell surface requires cell-cell contact with neighboring cells expressing Notch ligands (reviewed by Maillard et al 6 ). Effective Notch-Notch ligand interactions result in proteolytic cleavage of Notch, thus allowing the intracellular portion of Notch (ICN) to translocate to the nucleus to form a transcriptional activation complex consisting of ICN, the DNAbinding transcription factor CSL (CBF1, Serrate, Lag1), and coactivators of the Mastermind-like (MAML) family. 6 Recent experiments illustrate that Notch2, CSL, MAMLs, and the Notch ligand Delta-like-1 (DL1) play critical and nonredundant roles in B-cell development. Notch2 is the primary Notch gene expressed by peripheral B cells, 7 and genetic deletion of the genes encoding Notch2, CSL, or DL1 and expression of a dominant negative form of MAML1 (DNMAML1) all result in a selective failure to generate MZ B cells. [7][8][9][10] Here we show that BCR-and CD40-mediated proliferation and production of IgG1 ϩ cells are greatly enhanced by costimulating FO B cells with DL1. Further, we demonstrate that CSL/MAML1-dependent Notch signaling contributes to the generation of IgG1 ϩ plasma cells during a T-dependent (TD) immune response. These observations suggest that Notch signaling may influence both the development and antigen-driven differentiation of B cells by amplifying BCR-and CD40-mediated signaling events. Materials and methodsMice and cell purification C57BL/6 mice, Mx1-Cre mice, and CD19-Cre were purchased from Jackson ImmunoResearch Labs (West Grove, PA). DNMAML mice have been previously described. 11 All animal procedures were approved by the University of Pennsylvania Institutional Animal Care and Use Committee.To purify CD23 ϩ cells, splenocytes were resuspended in 2 mL FACS buffer (PBS, 0.5% BSA, 1 mM EDTA) with 5 L anti-CD23-biotin (B3B4) (eBioscience, San Diego, CA) fo...

Although the 3 isoforms of Akt regulate cell growth, proliferation, and survival in a wide variety of cell types, their role in B-cell development is unknown. We assessed B-cell maturation in the bone marrow (BM) and periphery in chimeras established with fetal liver progenitors lacking Akt1 and/or Akt2. We found that the gen- IntroductionIn adults, mature B cells derive from a series of precursors in the bone marrow (BM) and periphery. B-lineage committed pro-B cells in the BM undergo V-DJ recombination at the immunoglobulin (Ig) heavy chain locus, and cells possessing functional heavy chains are selected via the pre-B cell receptor (pre-BCR) to generate pre-B cells. 1 The majority of Ig light chain rearrangements occur in pre-B cells, and cells with productive light chain rearrangements yield immature B-cell receptor-positive (BCR ϩ ) B cells. 2 Newly formed BCR ϩ cells in the BM either die or mature further after entering peripheral lymphoid tissues such as the spleen. 3 One outcome of peripheral B-cell maturation is the selection of recently formed "transitional" B cells into functionally distinct B-cell subpopulations. Whereas the bulk of surviving transitional B cells yield follicular B cells, so named because of their enrichment in B cell-rich follicles in the spleen and lymph nodes, small numbers of transitional B cells differentiate into marginal zone (MZ) or B1 B cells, the main sources of antibody to T cell-independent bacterial pathogens. 4,5 The Akt family of serine/threonine kinases is expressed in 3 distinctly coded isoforms termed Akt-1, Akt-2, and Akt-3. 6 All 3 proteins share similar functions and structures, 7 and are known to enhance cellular metabolism and positively regulate cell survival and proliferation by activating numerous downstream biochemical pathways. 8 Activation of Akt requires phosphatidylinositol-3 kinase (PI-3K), a heterodimeric lipid kinase recruited to the plasma membrane upon ligation of a variety of surface receptors. 9 In B cells, PI-3K and Akt are activated upon ligation of a BCR coreceptor complex consisting of CD19, CD81, or CD21. [10][11][12] Interestingly, mice lacking CD19 or the catalytic subunit of PI-3K fail to develop MZ or B1 B cells, [13][14][15] suggesting that the CD19/ PI-3K pathway optimizes BCR-mediated peripheral B-cell maturation and selection. Consistent with this model, aggregation of the BCR leads to CD19-dependent phosphorylation and activation of Akt. 11 However, the CD19/PI-3K pathway activates several additional signaling pathways with important roles in BCR-mediated B-cell activation. Therefore, whether the Akt pathway plays an essential role in peripheral B-cell maturation and selection remains unclear.Recent observations show that Akt activity is required for early stages of T-cell development in the thymus. Specifically, thymocyte progenitors lacking Akt1 and Akt2 were unable to effectively transit the DN3 to double-positive transition, 16,17 which is characterized by a robust proliferative burst mediated by the pre-T cell receptor. 18 Beca...

Conditional knock-out (KO) of Polycomb Group (PcG) protein YY1 results in pro-B cell arrest and reduced immunoglobulin locus contraction needed for distal variable gene rearrangement. The mechanisms that control these crucial functions are unknown. We deleted the 25 amino-acid YY1 REPO domain necessary for YY1 PcG function, and used this mutant (YY1ΔREPO), to transduce bone marrow from YY1 conditional KO mice. While wild-type YY1 rescued B-cell development, YY1ΔREPO failed to rescue the B-cell lineage yielding reduced numbers of B lineage cells. Although the IgH rearrangement pattern was normal, there was a selective impact at the Igκ locus that showed a dramatic skewing of the expressed Igκ repertoire. We found that the REPO domain interacts with proteins from the condensin and cohesin complexes, and that YY1, EZH2 and condensin proteins co-localize at numerous sites across the Ig kappa locus. Knock-down of a condensin subunit protein or YY1 reduced rearrangement of Igκ Vκ genes suggesting a direct role for YY1-condensin complexes in Igκ locus structure and rearrangement.