IntroductionMyeloid cell leukemia sequence 1 (Mcl-1) 1 has been identified as an intracellular antiapoptotic factor in a variety of hematopoietic cells, both in vitro and in vivo. [2][3][4][5][6] Human mast cells express 7,8 and Mcl-1 can promote the survival of some populations of human neoplastic mast cells in vitro. 7 Basophils, granulocytes with many characteristics and functions that partially overlap with those of tissue mast cells, [9][10][11][12] can also express Mcl-1. 13 However, it is not clear to what extent Mcl-1 is important in the development and/or survival of mast cells or basophils in vivo.Opferman et al showed that the genetic manipulation of Mcl-1 can be used to delete individual hematopoietic cell populations in mice. 4 We therefore used this approach to examine the effects of reducing expression of Mcl-1 in the mast cell lineage in vivo. To attempt to delete Mcl-1 selectively in mast cells, we used the promoter for the peptidase carboxypeptidase A3 (CPA3; originally named mast cell carboxypeptidase A 14 ). CPA3 is highly expressed in mast cells, 15 but is also expressed in basophils 16 and can be expressed in some populations of T-cell progenitors and thymic T cells 17,18 and in certain hematopoietic progenitor cells. 19 We generated C57BL/6 mice in which a segment of the Cpa3 promoter drives expression of Cre recombinase, and then mated these Cpa3-Cre transgenic mice to mice bearing a floxed allele of Mcl-1. 4 We found that C57BL/6-Cpa3-Cre; Mcl-1 fl/fl mice are severely deficient in mast cells and have a marked deficiency in basophils, and also exhibit striking impairment in mast cell-or basophil-and IgE-dependent biologic responses. Methods MiceAll animal experiments were carried out following protocols approved by the Stanford University Administrative Panel on Laboratory Animal Care. B6-Tg(Cpa3-cre)3Glli (Cpa3-Cre-transgenic mice) were generated by microinjecting the Cpa3-Cre transgene into embryonic stem cells in the B6 background (Stanford University). Gt(ROSA)26Sor tm4(ACTB-tdTomato,-EGFP)Luo /J(mT/mG) mice, obtained from The Jackson Laboratory, were crossed to Cpa3-Cre mice for Cre expression analysis. Mcl-1 ϩ/fl (B6;129-Mcl1 tm3sjk J) animals were obtained from The Jackson Laboratory. Mcl-1 ϩ/fl mice were bred to progeny from 2 Cpa3-Cre founder lines (founder lines #4 and #5) to obtain Cpa3-Cre; Mcl-1 ϩ/ϩ , Cpa3-Cre; Mcl-1 ϩ/fl , and Cpa3-Cre; Mcl-1 fl/fl animals, but only the Cpa3-Cre; Mcl-1 fl/fl mice derived from founder line #4 exhibited a substantial mast cell deficiency. Therefore, the mice used were derived from crosses between founder line #4 (subsequently referred to as Cpa3-Cre mice) and Mcl-1 fl animals, and these mice had been intercrossed a minimum of 6 generations into the C57BL/6 background. Heterozygous Cpa3-Cre mice were determined to have 5 copies of the Cpa3-Cre transgene by real-time PCR. To emphasize that Cpa3-Cre; Mcl-1 fl/fl mice have deficiencies in mast cells and basophils that are independent of mutations affecting Kit, we call them informally in our labo...

Humoral immunity is characterized by the generation of Ab-secreting plasma cells and memory B cells that can more rapidly generate specific Abs upon Ag exposure than their naive counterparts. To determine the intrinsic differences that distinguish naive and memory B cells and to identify pathways that allow germinal center B cells to differentiate into memory B cells, we compared the transcriptional profiles of highly purified populations of these three cell types along with plasma cells isolated from mice immunized with a T-dependent Ag. The transcriptional profile of memory B cells is similar to that of naive B cells, yet displays several important differences, including increased expression of activation-induced deaminase and several antiapoptotic genes, chemotactic receptors, and costimulatory molecules. Retroviral expression of either Klf2 or Ski, two transcriptional regulators specifically enriched in memory B cells relative to their germinal center precursors, imparted a competitive advantage to Ag receptor and CD40-engaged B cells in vitro. These data suggest that humoral recall responses are more rapid than primary responses due to the expression of a unique transcriptional program by memory B cells that allows them to both be maintained at high frequencies and to detect and rapidly respond to antigenic re-exposure.

PU.1 is essential for early stages of mouse T cell development but antagonizes it if expressed constitutively. Two separable mechanisms are involved: attenuation and diversion. Dysregulated PU.1 expression inhibits pro-T cell survival, proliferation, and passage through β-selection by blocking essential T cell transcription factors, signaling molecules, and Rag gene expression, which expression of a rearranged T cell antigen receptor transgene cannot rescue. However, Bcl2 transgenic cells are protected from this attenuation and may even undergo β-selection, as shown by PU.1 transduction of defined subsets of Bcl2 transgenic fetal thymocytes with differentiation in OP9-DL1 and OP9 control cultures. The outcome of PU.1 expression in these cells depends on Notch/Delta signaling. PU.1 can efficiently divert thymocytes toward a myeloid-like state with multigene regulatory changes, but Notch/Delta signaling vetoes diversion. Gene expression analysis distinguishes sets of critical T lineage regulatory genes with different combinatorial responses to PU.1 and Notch/Delta signals, suggesting particular importance for inhibition of E proteins, Myb, and/or Gfi1 (growth factor independence 1) in diversion. However, Notch signaling only protects against diversion of cells that have undergone T lineage specification after Thy-1 and CD25 up-regulation. The results imply that in T cell precursors, Notch/Delta signaling normally acts to modulate and channel PU.1 transcriptional activities during the stages from T lineage specification until commitment.

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Specification of mammalian T lymphocytes involves prolonged developmental plasticity even after lineage-specific gene expression begins. Expression of transcription factor PU.1 may maintain some myeloid-like developmental alternatives until commitment. Commitment could reflect PU.1 shutoff, resistance to PU.1 effects, and/or imposition of a suicide penalty for diversion. Here, we describe subclones from the SCID.adh murine thymic lymphoma, adh.2C2 and adh.6D4, that represent a new tool for probing these mechanisms. PU.1 can induce many adh.2C2 cells to undergo diversion to a myeloid-like phenotype, in an all-or-none fashion with multiple, coordinate gene expression changes; adh.6D4 cells resist diversion, and most die. Diversion depends on the PU.1 Ets domain but not on known interactions in the PEST or Q-rich domains, although the Q-rich domain enhances diversion frequency. Protein kinase C/MAP kinase stimulation can make adh.6D4 cells permissive for diversion without protecting from suicide. These results show distinct roles for regulated cell death and another stimulation-sensitive function that establishes a threshold for diversion competence. PU.1 also diverts normal T-cell precursors from wild type or Bcl2-transgenic mice to a myeloid-like phenotype, upon transduction in short-term culture. The adh.2C2 and adh.6D4 clones thus provide an accessible system for defining mechanisms controlling developmental plasticity in early T-cell development.

Summary The lineage restriction of prospectively isolated hematopoietic progenitors has been traditionally assessed by bulk in vitro culture and transplantation of large number of cells in vivo. These methods, however, cannot distinguish between homogenous multipotent or heterogeneous lineage-restricted populations. Using clonal assays of 1 or 5 cells in vitro, single cell quantitative gene expression analyses, and transplantation of mice with low numbers of cells, we show that the common myeloid progenitor (CMP) is Sca-1lolin-c-Kit+CD27+Flk-2- (SL-CMP; Sca-1lo CMP) and a granulocyte/macrophage progenitor (GMP) is Sca-1lolin-c-Kit+CD27+Flk-2+CD150-/lo (SL-GMP; Sca-1lo GMP). We found that mast cell progenitor potential is present in the SL-CMP fraction but not in the more differentiated SL-GMP population and is more closely related to megakaryocyte/erythrocyte specification. Our data provide criteria for the prospective isolation of SL-CMP and SL-GMP and support the conclusion that mast cells are specified during hematopoiesis earlier than and independently from granulocytes.

a4b7 integrin is a cell adhesion receptor that is crucial for the migration of hematopoietic progenitors and mature effector cells in the periphery, but its role in adult hematopoiesis is controversial. We identified a subset of hematopoietic stem cells (HSCs) in the bone marrow (BM) that expressed b7 integrin. These b7 + HSCs were capable of multilineage, long-term reconstitution and had an inherent competitive advantage over b7 -HSCs. On the other hand, HSCs that lacked b7 integrin (b7KO) had reduced engraftment potential. Interestingly, quantitative RT-PCR and flow cytometry revealed that b7KO HSCs expressed lower levels of the chemokine receptor CXCR4. Accordingly, b7KO HSCs exhibited impaired migration abilities in vitro and BM homing capabilities in vivo. Lethal irradiation induced expression of the a4b7 integrin ligand-mucosal addressin cell adhesion molecule-1 (MAdCAM-1) on BM endothelial cells. Moreover, blocking MAdCAM-1 reduced the homing of HSCs and impaired the survival of recipient mice. Altogether, these data indicate that b7 integrin, when expressed by HSCs, interacted with its endothelial ligand MAdCAM-1 in the BM microenvironment, thereby promoting HSC homing and engraftment.

In this issue of Immunity, Tsujimura et al. (2008) report that the release of platelet-activating factor by basophils stimulated with immunoglobulin G1 (IgG1)-antigen immune complexes contributes substantially to the expression of an IgG1-dependent alternative pathway of systemic anaphylaxis in mice.