Robert P. Stephan

An essential role for Daxx in the inhibition of B lymphopoiesis by type I interferons.

Interferon-alpha and -beta inhibit the interleukin-7-mediated growth and survival of T and B lymphoid progenitors via an unknown, STAT1-independent pathway. Gene expression profile analysis of interferon-beta-treated progenitor B cells revealed enhanced Daxx expression, with concomitant Daxx protein increase and nuclear body translocation. The interferon effects included downregulation of cell cycle regulating genes and cell cycle arrest, followed by Bcl-2 downregulation and apoptosis. Daxx antisense oligonucleotides rescued the interferon-treated pro-B cells from growth arrest and apoptosis in parallel with the reduction of nuclear Daxx. These findings implicate the gene repressor function of Daxx in interferon-induced apoptosis of lymphoid progenitors.

Transcription factor Pax5 (BSAP) transactivates the RAG-mediated V H -to-DJ H rearrangement of immunoglobulin genes

Immunoglobulin rearrangement from variable heavy chain (VH) to diversity (D)–joining heavy chain (JH), which occurs exclusively in B lineage cells, is impaired in mice deficient for the B lineage–specific transcription factor Pax5. Conversely, ectopic Pax5 expression in thymocytes promotes the rearrangement of DH-proximal VH7183 genes. In exploring the mechanism for Pax5 regulation of VH-to-DJH recombination, we have identified multiple Pax5 binding sites in the coding regions of human and mouse VH gene segments. Pax5 bound to those sites in vitro and occupied VH genes in early human and mouse B lineage cells. Moreover, Pax5 interacted with the recombination-activating gene 1 (RAG1)–RAG2 complex to enhance RAG-mediated VH recombination signal sequence cleavage and recombination of a VH gene substrate. These findings indicate a direct activating function for Pax5 in RAG-mediated immunoglobulin VH-to-DJH recombination.

Enforced expression of EBF in hematopoietic stem cells restricts lymphopoiesis to the B cell lineage

Mice deficient in early B cell factor (EBF) are blocked at the progenitor B cell stage prior to immunoglobulin gene rearrangement. The EBF‐dependent block in B cell development occurs near the onset of B‐lineage commitment, which raises the possibility that EBF may act instructively to specify the B cell fate from uncommitted, multipotential progenitor cells. To test this hypothesis, we transduced enriched hematopoietic progenitor cells with a retroviral vector that coexpressed EBF and the green fluorescent protein (GFP). Mice reconstituted with EBF‐expressing cells showed a near complete absence of T lymphocytes. Spleen and peripheral blood samples were >95 and 90% GFP+EBF+ mature B cells, respectively. Both NK and lymphoid‐derived dendritic cells were also significantly reduced compared with control‐transplanted mice. These data suggest that EBF can restrict lymphopoiesis to the B cell lineage by blocking development of other lymphoid‐derived cell pathways.

The transient expression of pre-B cell receptors governs B cell development.

Only a subpopulation of relatively large pre-B cells express pre-B cell receptors (preBCR) that can be seen with very sensitive immunofluorescence methods. Inefficient assembly of the multicomponent preBCR coupled with their ligand-induced endocytosis may account for the remarkably low in vivo levels of preBCR expression. Signaling initiated via the preBCR promotes cellular proliferation and RAG-1 and RAG-2 downregulation to interrupt the immunoglobulin V(D)J gene rearrangement process. Silencing of the surrogate light chain genes, VpreB and lambda5, then terminates preBCR expression to permit cell cycle exit, recombinase gene upregulation, and VJ(L) rearrangement by small pre-B cells destined to become B cells.

Resident enteric microbiota and CD8+ T cells shape the abundance of marginal zone B cells

Since enteric microbial composition is a distinctive and stable individual trait, microbial heterogeneity may confer lifelong, non‐genetic differences between individuals. Here we report that C57BL/6 mice bearing restricted flora microbiota, a distinct but diverse resident enteric microbial community, are numerically and functionally deficient in marginal zone (MZ) B cells. Surprisingly, MZ B‐cell levels are minimally affected by germ‐free conditions or null mutations of various TLR signaling molecules. In contrast, MZ B‐cell depletion is exquisitely dependent on cytolytic CD8+ T cells, and includes targeting of a cross‐reactive microbial/endogenous MHC class 1B antigen. Thus, members of certain enteric microbial communities link with CD8+ T cells as a previously unappreciated mechanism that shapes innate immunity dependent on innate‐like B cells.

Determination of lymphoid cell fate is dependent on the expression status of the IL‐7 receptor

Signaling through the IL‐7 receptor (IL‐7R) is necessary for the development of the earliest B‐ and T‐lineage cells. IL‐7R is first expressed on common lymphoid progenitor cells and is not detected on primitive common myeloid progenitors. In this study, we show that enforced expression of IL‐7R on multipotential stem cells does not influence lymphoid versus myeloid cell fate. T cell development was compatible with sustained IL‐7R expression; however, we observed a near complete block in B cell development at the onset of B‐lineage commitment. Unlike pre‐proB cells from control animals, developmentally‐arrested IL‐7R+B220+cd19−NK1.1−Ly‐6C− cells failed to express EBF and Pax5. These results suggest that transient downregulation of IL‐7R signaling is a necessary event for induction of EBF and Pax5 expression and B‐lymphocyte commitment.

Stat-1 Is Not Essential for Inhibition of B Lymphopoiesis by Type I IFNs

Type I IFNs, IFN-α, -β, and -ω, are cytokine family members with multiple immune response roles, including the promotion of cell growth and differentiation. Conversely, the type I IFNs are potent inhibitors of IL-7-dependent growth of early B lineage progenitors, effectively aborting further B lineage differentiation at the pro-B cell stage. Type I IFNs α and β function via receptor-mediated activation of a Jak/Stat signaling pathway in which Stat-1 is functionally important, because many IFN-induced responses are abrogated in Stat-1-deficient mice. To the contrary, we show here that the inhibition of IL-7-dependent B lymphopoiesis by IFN-αβ is unaffected in Stat-1-deficient mice. The present data indicate that the type I IFNs can activate an alternative signaling pathway in which neither Stat-1 nor phosphatidylinositol 3′-kinase are essential components.

Expression of CD28 by Bone Marrow Stromal Cells and Its Involvement in B Lymphopoiesis

Young mice lacking CD28 have normal numbers of peripheral B cells; however, abnormalities exist in the humoral immune response that may result from an intrinsic defect in the B cells. The goal of this study was to assess whether CD28 could be involved in the development of B cells. CD28 mRNA was detected preferentially in the fraction of bone marrow enriched for stromal cells. Flow cytometry and RT-PCR analysis demonstrated that CD28 was also expressed by primary-cultured stromal cells that supported B lymphopoiesis. Confocal microscopy revealed that in the presence of B-lineage cells, CD28 was localized at the contact interface between B cell precursors and stromal cells. In addition, CD80 was detected on 2–6% of freshly isolated pro- and pre-B cells, and IL-7 stimulation led to induction of CD86 on 15–20% of pro- and pre-B cells. We also observed that stromal cell-dependent production of B-lineage cells in vitro was greater on stromal cells that lacked CD28. Finally, the frequencies of B-lineage precursors in the marrow from young (4- to 8-wk-old) CD28−/− mice were similar to those in wild-type mice; however, older CD28−/− mice (15–19 mo old) exhibited a 30% decrease in pro-B cells and a 50% decrease in pre-B cells vs age-matched controls. Our results suggest that CD28 on bone marrow stromal cells participates in stromal-dependent regulation of B-lineage cells in the bone marrow. The localization of CD28 at the stromal cell:B cell precursor interface suggests that molecules important for T cell:B cell interactions in the periphery may also participate in stromal cell:B cell precursor interactions in the bone marrow.

Role of stromal cells and their products in protecting young and aged B-lineage precursors from dexamethasone-induced apoptosis

Bone marrow stromal cells are potent providers of stimuli that induce proliferation of B-cell precursors. We proposed that stromal cells play a role in protecting B-lineage cells from corticosteroid-induced apoptosis. We found that stromal cells protected B-cell precursors from dexamethasone-induced apoptosis, but this did not strictly correlate with interleukin-7 (IL-7) production. To determine if stromal-derived factors were involved in protection of B-cell precursors from apoptosis, we examined the activity of three lymphopoietic growth factors: IL-7, stem cell factor (SCF), and insulin-like growth factor-1 (IGF-1). Either IL-7 or IGF-1 alone protected B-cell precursors from dexamethasone-induced apoptosis. The combined activities of IGF-1 and IL-7 were additive rather than synergistic. SCF did not protect B-cell precursors from apoptosis. Aging altered the ability of B-cell precursors to respond to protective stimuli induced by IL-7 and IGF-1. Precursors from aged animals were deficient in ability to modulate expression of apoptosis regulatory genes Bax, Bcl-2, and Bcl-x in comparison to B-cell precursors from young animals. Taken together, these results suggest that stromal cells can protect B-lineage precursors from a corticosteroid-induced apoptotic signal, protection is mediated by stromal-derived cytokines, and aging decreases the ability of B-cell precursors to respond efficiently to protective stimuli.

Analysis of VpreB Expression During B Lineage Differentiation in λ5-Deficient Mice

The VpreB/λ5 surrogate L chain complex is an essential component of the pre-B cell receptor, the expression of which serves as an important checkpoint in B cell development. Surrogate L chains also may serve as components of murine pro-B cell receptors whose function is unknown. We have produced two new mAbs, R3 and R5, that recognize a different VpreB epitope than the one recognized by the previously described VP245 anti-mouse VpreB Ab. These Abs were used to confirm the expression of surrogate L chains on wild-type pro-B and pre-B cell lines. Although undetectable on the cell surface, VpreB was found to be normally expressed within B lineage cells of λ5-deficient mice. Nevertheless, VpreB expression was extinguished at the B cell stage of differentiation in these mice. The normal pattern of VpreB expression in λ5-deficient mice excludes an essential role for pro-B and pre-B cell receptors in VpreB regulation.