技术资料

The developmental potential of hematopoietic progenitors is restricted early on to either the erythromyeloid or lymphoid lineages. The broad developmental potential of Pax5(-/-) pro-B cells is in apparent conflict with such a strict separation,although these progenitors realize the myeloid and erythroid potential with lower efficiency compared to the lymphoid cell fates. Here we demonstrate that ectopic expression of the transcription factors C/EBPalpha,GATA1,GATA2 and GATA3 strongly promoted in vitro macrophage differentiation and myeloid colony formation of Pax5(-/-) pro-B cells. GATA2 and GATA3 expression also resulted in efficient engraftment and myeloid development of Pax5(-/-) pro-B cells in vivo. The myeloid transdifferentiation of Pax5(-/-) pro-B cells was accompanied by the rapid activation of myeloid genes and concomitant repression of B-lymphoid genes by C/EBPalpha and GATA factors. These data identify the Pax5(-/-) pro-B cells as lymphoid progenitors with a latent myeloid potential that can be efficiently activated by myeloid transcription factors. The same regulators were unable to induce a myeloid lineage switch in Pax5(+/+) pro-B cells,indicating that Pax5 dominates over myeloid transcription factors in B-lymphocytes. View Publication

The B-cell receptor (BCR) transmits life and death signals throughout B-cell development,and altered BCR signaling may be required for survival of B-lymphoma cells. We used single-cell signaling profiles to compare follicular lymphoma (FL) B cells and nonmalignant host B cells within individual patient biopsies and identified BCR-mediated signaling events specific to lymphoma B cells. Expression of CD20,Bcl-2,and BCR light chain isotype (kappa or lambda) distinguished FL tumor B-cell and nontumor host B-cell subsets within FL patient biopsies. BCR-mediated signaling via phosphorylation of Btk,Syk,Erk1/2,and p38 occurred more rapidly in tumor B cells from FL samples than in infiltrating nontumor B cells,achieved greater levels of per-cell signaling,and sustained this level of signaling for hours longer than nontumor B cells. The timing and magnitude of BCR-mediated signaling in nontumor B cells within an FL sample instead resembled that observed in mature B cells from the peripheral blood of healthy subjects. BCR signaling pathways that are potentiated specifically in lymphoma cells should provide new targets for therapeutic attention. View Publication

B-cell functions in antitumor immunity are not well understood. In this study,we evaluated the role of B cells in the development of antitumor immunity using Friend murine leukemia virus gag-expressing mouse EL-4 (EL-4 gag),D5 mouse melanoma,or MCA304 mouse sarcoma cells. To screen tumors for susceptibility to B-cell-deficient immune environments,spleen cells from naive C57BL/6 [wild-type (WT)] and B-cell knockout (BKO) mice were cultured with irradiated tumor cells in vitro. When cells were stimulated with EL-4 gag or D5 (but not MCA304 tumors),IFN-gamma production from CD8 T cells and natural killer cells was markedly decreased in WT compared with BKO cultures. IFN-gamma production was correlated with CD40 ligand expression on the tumor and inversely with interleukin-10 (IL-10) production by B cells. Sorted WT B cells produced more IL-10 than CD40 knockout (CD40KO) B cells when cocultured with EL-4 gag or D5 (but not MCA304). IFN-gamma production by BKO cells was reduced by the addition of sorted naive WT B cells (partially by CD40KO B cells) or recombinant mouse IL-10. In vivo tumor progression mirrored in vitro studies in that WT mice were unable to control tumor growth whereas EL-4 gag and D5 tumors (but not MCA304) were eliminated in BKO mice. Robust in vivo antitumor CTLs developed only in BKO tumor-challenged mice. Our studies provide the first mechanistic basis for the concept that B-cell depletion could therapeutically enhance antitumor immune responses to certain tumors by decreasing IL-10 production from B cells. View Publication

Clonal expansion of B cell chronic lymphocytic leukemia (B-CLL) occurs within lymphoid tissue pseudofollicles. IL-15,a stromal cell-associated cytokine found within spleens and lymph nodes of B-CLL patients,significantly boosts in vitro cycling of blood-derived B-CLL cells following CpG DNA priming. Both IL-15 and CpG DNA are elevated in microbe-draining lymphatic tissues,and unraveling the basis for IL-15-driven B-CLL growth could illuminate new therapeutic targets. Using CpG DNA-primed human B-CLL clones and approaches involving both immunofluorescent staining and pharmacologic inhibitors,we show that both PI3K/AKT and JAK/STAT5 pathways are activated and functionally important for IL-15→CD122/ɣc signaling in ODN-primed cells expressing activated pSTAT3. Furthermore,STAT5 activity must be sustained for continued cycling of CFSE-labeled B-CLL cells. Quantitative RT-PCR experiments with inhibitors of PI3K and STAT5 show that both contribute to IL-15-driven upregulation of mRNA for cyclin D2 and suppression of mRNA for DNA damage response mediators ATM,53BP1,and MDC1. Furthermore,protein levels of these DNA damage response molecules are reduced by IL-15,as indicated by Western blotting and immunofluorescent staining. Bioinformatics analysis of ENCODE chromatin immunoprecipitation sequencing data from cell lines provides insight into possible mechanisms for STAT5-mediated repression. Finally,pharmacologic inhibitors of JAKs and STAT5 significantly curtailed B-CLL cycling when added either early or late in a growth response. We discuss how the IL-15-induced changes in gene expression lead to rapid cycling and possibly enhanced mutagenesis. STAT5 inhibitors might be an effective modality for blocking B-CLL growth in patients. View Publication

The neutralization of integrin α4 (Itga4) is currently used as treatment in multiple sclerosis. Although most studies have focused on its function on lymphocyte migration to the CNS,we have uncovered the importance of Itga4 for the generation of regulatory B cells in peripheral immune organs and their control of pathogenic T cell response and CNS pathology. Our study underscores the importance of looking at the dual role of B cells in CNS autoimmunity and provides important perspectives regarding the efficacy and side effects associated with Itga4 neutralization and other B cell-targeting therapies. View Publication

The design of immunogens that elicit broadly reactive neutralizing antibodies (bnAbs) has been a major obstacle to HIV-1 vaccine development. One approach to assess potential immunogens is to use mice expressing precursors of human bnAbs as vaccination models. The bnAbs of the VRC01-class derive from the IGHV1-2 immunoglobulin heavy chain and neutralize a wide spectrum of HIV-1 strains via targeting the CD4 binding site of the envelope glycoprotein gp120. We now describe a mouse vaccination model that allows a germline human IGHV1-2(∗)02 segment to undergo normal V(D)J recombination and,thereby,leads to the generation of peripheral B cells that express a highly diverse repertoire of VRC01-related receptors. When sequentially immunized with modified gp120 glycoproteins designed to engage VRC01 germline and intermediate antibodies,IGHV1-2(∗)02-rearranging mice,which also express a VRC01-antibody precursor light chain,can support the affinity maturation of VRC01 precursor antibodies into HIV-neutralizing antibody lineages. View Publication