技术资料

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

Gammaherpesviruses,including Kaposi sarcoma-associated herpesvirus (KSHV),establish latency in B cells. We hypothesized that the KSHV latency-associated nuclear antigen (LANA/orf73) provides a selective advantage to infected B cells by driving proliferation in response to antigen. To test this,we used LANA B-cell transgenic mice. Eight days after immunization with antigen without adjuvant,LANA mice had significantly more activated germinal center (GC) B cells (CD19(+) PNA(+) CD71(+)) than controls. This was dependent upon B-cell receptor since LANA did not restore the GC defect of CD19 knockout mice. However,LANA was able to restore the marginal zone defect in CD19 knockout mice. View Publication

Transfusion-related acute lung injury (TRALI) is a serious complication of transfusion and has been ranked as one of the leading causes of transfusion-related fatalities. Nonetheless,many details of the immunopathogenesis of TRALI,particularly with respect to recipient factors are unknown. We used a murine model of antibody-mediated TRALI in an attempt to understand the role that recipient lymphocytes might play in TRALI reactions. Intravenous injection of an IgG2a antimurine major histocompatibility complex class I antibody (34-1-2s) into BALB/c mice induced moderate hypothermia and pulmonary granulocyte accumulation but no pulmonary edema nor mortality. In contrast,34-1-2s injections into mice with severe combined immunodeficiency caused severe hypothermia,severe pulmonary edema,and approximately 40% mortality indicating a critical role for T and B lymphocytes in suppressing TRALI reactions. Adoptive transfer of purified CD8(+) T lymphocytes or CD4(+) T cells but not CD19(+) B cells into the severe combined immunodeficiency mice alleviated the antibody-induced hypothermia,lung damage,and mortality,suggesting that T lymphocytes were responsible for the protective effect. Taken together,these results suggest that recipient T lymphocytes play a significant role in suppressing antibody-mediated TRALI reactions. They identify a potentially new recipient mechanism that controls the severity of TRALI reactions. View Publication

The B cell-activating factor of the TNF family receptor (BAFF-R),encoded by the TNFRSF13C gene,is critically important for transitional B cell survival to maturity. Thus,ligation of BAFF-R by BAFF delivers a potent survival signal. Reports implicating the BAFF/BAFF-R signaling axis in the pathogenesis of autoimmune human diseases and B lineage malignancies have largely prompted studies focusing on BAFF expression; however,there is an equally critical need to better understand BAFF-R expression. Initial BAFF-R expression,although characterized in murine B cells,has not yet been reported in human B lymphopoiesis. In this study,we first demonstrate that BAFF-R expression is absent from early precursors and is acquired by bone marrow B cells newly expressing the BCR. We next focused on identifying the specific genomic region that controls BAFF-R expression in mature B cells (i.e.,the TNFRSF13C promoter). To accomplish this,we used in silico tools examining interspecies genomic conservation in conjunction with reporter constructs transfected into malignant B and plasma cell lines. DNase protection assays using nuclear extracts from BAFF-R-expressing cells suggested potential regulatory sites,which allowed the generation of EMSA probes that bound NFs specific to BAFF-R-expressing cells. With a more stringent analysis of interspecies homology,these assays identified a site at which a single nucleotide substitution could distinctly impact promoter activity. Finally,chromatin immunoprecipitation assays revealed the in vivo binding of the specific transcription factor c-Rel to the most proximal genomic region,and c-Rel small interfering RNA transfections in BAFF-R-expressing lines demonstrated a coincident knockdown of both c-Rel and BAFF-R mRNA. View Publication

The development of mature blood cells from hematopoietic stem cells requires coordinated activities of transcriptional networks. Transcriptional repressor growth factor independence 1 (Gfi-1) is required for the development of B cells,T cells,neutrophils,and for the maintenance of hematopoietic stem cell function. However,the mechanisms by which Gfi-1 regulates hematopoiesis and how Gfi-1 integrates into transcriptional networks remain unclear. Here,we provide evidence that Id2 is a transcriptional target of Gfi-1,and repression of Id2 by Gfi-1 is required for B-cell and myeloid development. Gfi-1 binds to 3 conserved regions in the Id2 promoter and represses Id2 promoter activity in transient reporter assays. Increased Id2 expression was observed in multipotent progenitors,myeloid progenitors,T-cell progenitors,and B-cell progenitors in Gfi-1(-/-) mice. Knockdown of Id2 expression or heterozygosity at the Id2 locus partially rescues the B-cell and myeloid development but not the T-cell development in Gfi-1(-/-) mice. These studies demonstrate a role of Id2 in mediating Gfi-1 functions in B-cell and myeloid development and provide a direct link between Gfi-1 and the B-cell transcriptional network by its ability to repress Id2 expression. View Publication

We analyzed in B-chronic lymphocytic leukemia (B-CLL) whole blood assays the activity of therapeutic mAbs alemtuzumab,rituximab,and type II glycoengineered anti-CD20 mAb GA101. Whole blood samples were treated with Abs,and death of CD19(+) B-CLL was measured by flow cytometry. Alemtuzumab efficiently lysed B-CLL targets with maximal lysis at 1-4 h (62%). In contrast,rituximab induced a more limited cell death (21%) that was maximal only at 24 h. GA101 killed B-CLL targets to a similar extent but more rapidly than rituximab,with 19.2 and 23.5% cell death at 4 and 24 h,respectively,compared with 7.9 and 21.4% for rituximab. Lysis by both rituximab and GA101 correlated directly with CD20 expression levels (r(2) = 0.88 and 0.85,respectively). Interestingly,lysis by all three Abs at high concentrations was mostly complement dependent,because it was blocked by the anti-C5 Ab eculizumab by 90% in the case of alemtuzumab and rituximab and by 64% in the case of GA101. Although GA101 caused homotypic adhesion,it induced only limited (3%) direct cell death of purified B-CLL cells. Both rituximab and GA101 showed the same efficiency in phagocytosis assays,but phagocytosis was not significant in whole blood due to excess Igs. Finally,GA101 at 1-100 μg/ml induced 2- to 3-fold more efficient NK cell degranulation than rituximab in isolated B-CLL or normal PBMCs. GA101,but not rituximab,also mediated significant NK cell degranulation in whole blood samples. Thus,complement and Ab-dependent cellular cytotoxicity are believed to be the major effector mechanisms of GA101 in whole blood assays. View Publication

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

Epstein-Barr virus (EBV) is associated with the development of malignant lymphomas and lymphoproliferative disorders in immunocompromised individuals. The LMP2A protein of EBV is thought to play a central role in this process by allowing the virus to persist in latently infected B lymphocytes. We have demonstrated that LMP2A,when expressed in B cells of transgenic mice,allows normal B-cell developmental checkpoints to be bypassed. To identify cellular genes targeted by LMP2A that are involved in this process,we have utilized DNA microarrays to compare gene transcription in B cells from wild-type versus LMP2A transgenic mice. In B cells from LMP2A transgenic mice,we observed decreased expression of many genes associated with normal B-cell development as well as reduced levels of the transcription factors that regulate their expression. In particular,expression of the transcription factor E2A was down-regulated in bone marrow and splenic B cells. Furthermore,E2A activity was inhibited in these cells as determined by decreased DNA binding and reduced expression of its target genes,including the transcription factors early B-cell factor and Pax-5. Expression of two E2A inhibitors,Id2 and SCL,was up-regulated in splenic B cells expressing LMP2A,suggesting a possible mechanism for E2A inhibition. These results indicate that LMP2A deregulates transcription factor expression and activity in developing B cells,and this likely allows for a bypass of normal signaling events required for proper B-cell development. The ability of LMP2A to interfere with B-cell transcription factor regulation has important implications regarding its role in EBV latency. View Publication

The control of uptake and utilization of necessary extracellular nutrients-glucose and glutamine-is an important aspect of B cell activation. Let-7 is a family of microRNAs known to be involved in metabolic control. Here,we employed several engineered mouse models,including B cell-specific overexpression of Lin28a or the let-7a-1/let-7d/let-7f-1 cluster (let-7adf) and knockout of individual let-7 clusters to show that let-7adf specifically inhibits T cell-independent (TI) antigen-induced immunoglobulin (Ig)M antibody production. Both overexpression and deletion of let-7 in this cluster leads to altered TI-IgM production. Mechanistically,let-7adf suppresses the acquisition and utilization of key nutrients,including glucose and glutamine,through directly targeting hexokinase 2 (Hk2) and by repressing a glutamine transporter Slc1a5 and a key degradation enzyme,glutaminase (Gls),a mechanism mediated by regulation of c-Myc. Our results suggest a novel role of let-7adf as a metabolic brake" on B cell antibody production." View Publication

Fbxo7 is an unusual F-box protein because most of its interacting proteins are not substrates for ubiquitin-mediated degradation. Fbxo7 directly binds p27 and Cdk6,enhances the level of cyclin D-Cdk6 complexes,and its overexpression causes Cdk6-dependent transformation of immortalised fibroblasts. Here,we test the ability of Fbxo7 to transform haematopoietic pro-B (Ba/F3) cells which,unexpectedly,it was unable to do despite high levels of Cdk6. Instead,reduction of Fbxo7 expression increased proliferation,decreased cell size and shortened G1 phase. Analysis of cell cycle regulators showed that cells had decreased levels of p27,and increased levels of S phase cyclins and Cdk2 activity. Also,Fbxo7 protein levels correlated inversely with those of CD43,suggesting direct regulation of its expression and,therefore,of B cell maturation. Alterations to Cdk6 protein levels did not affect the cell cycle,indicating that Cdk6 is neither rate-limiting nor essential in Ba/F3 cells; however,decreased expression of Cdk6 also enhanced levels of CD43,indicating that expression of CD43 is independent of cell cycle regulation. The physiological effect of reduced levels of Fbxo7 was assessed by creating a transgenic mouse with a LacZ insertion into the Fbxo7 locus. Homozygous Fbxo7(LacZ) mice showed significantly increased pro-B cell and pro-erythroblast populations,consistent with Fbxo7 having an anti-proliferative function and/or a role in promoting maturation of precursor cells. View Publication

It is unknown how dendritic cells (DCs) become specialized as mucosal DCs and maintain intestinal homeostasis. We report that a subset of bone marrow cells freshly isolated from C57BL/6 mice express the retinoic acid (RA)-synthesizing enzyme aldehyde dehydrogenase family 1,subfamily A2 (ALDH1a2) and are capable of providing RA to DC precursors in the bone marrow microenvironment. RA induced bone marrow-derived DCs to express CCR9 and ALDH1a2 and conferred upon them mucosal DC functions,including induction of Foxp3(+) regulatory T cells,IgA-secreting B cells,and gut-homing molecules. This response of DCs to RA was dependent on a narrow time window and stringent dose effect. RA promoted bone marrow-derived DC production of bioactive TGF-β by inhibiting suppressor of cytokine signaling 3 expression and thereby enhancing STAT3 activation. These RA effects were evident in vivo,in that mucosal DCs from vitamin A-deficient mice had reduced mucosal DC function,namely failure to induce Foxp3(+) regulatory T cells. Furthermore,MyD88 signaling enhanced RA-educated DC ALDH1a2 expression and was required for optimal TGF-β production. These data indicate that RA plays a critical role in the generation of mucosal DCs from bone marrow and in their functional activity. View Publication

The contribution of B cells to the pathology of Omenn syndrome and leaky severe combined immunodeficiency (SCID) has not been previously investigated. We have studied a mut/mut mouse model of leaky SCID with a homozygous Rag1 S723C mutation that impairs,but does not abrogate,V(D)J recombination activity. In spite of a severe block at the pro-B cell stage and profound B cell lymphopenia,significant serum levels of immunoglobulin (Ig) G,IgM,IgA,and IgE and a high proportion of Ig-secreting cells were detected in mut/mut mice. Antibody responses to trinitrophenyl (TNP)-Ficoll and production of high-affinity antibodies to TNP-keyhole limpet hemocyanin were severely impaired,even after adoptive transfer of wild-type CD4(+) T cells. Mut/mut mice produced high amounts of low-affinity self-reactive antibodies and showed significant lymphocytic infiltrates in peripheral tissues. Autoantibody production was associated with impaired receptor editing and increased serum B cell-activating factor (BAFF) concentrations. Autoantibodies and elevated BAFF levels were also identified in patients with Omenn syndrome and leaky SCID as a result of hypomorphic RAG mutations. These data indicate that the stochastic generation of an autoreactive B cell repertoire,which is associated with defects in central and peripheral checkpoints of B cell tolerance,is an important,previously unrecognized,aspect of immunodeficiencies associated with hypomorphic RAG mutations. View Publication