技术资料

Understanding how memory B cells are induced and relate to long-lived plasma cells is important for vaccine development. Immunity to oral vaccines has been considered short-lived because of a poor ability to develop IgA B-cell memory. Here we demonstrate that long-lived mucosal IgA memory is readily achieved by oral but not systemic immunization in mouse models with NP hapten conjugated with cholera toxin and transfer of B1-8(high)/GFP(+) NP-specific B cells. Unexpectedly,memory B cells are poorly related to long-lived plasma cells and less affinity-matured. They are α4β7-integrin(+)CD73(+)PD-L2(+)CD80(+) and at systemic sites mostly IgM(+),while 80% are IgA(+) in Peyer's patches. On reactivation,most memory B cells in Peyer's patches are GL7(-),but expand in germinal centres and acquire higher affinity and more mutations,demonstrating strong clonal selection. CCR9 expression is found only in Peyer's patches and appears critical for gut homing. Thus,gut mucosal memory possesses unique features not seen after systemic immunization. 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

Primary systemic amyloidosis (AL) is a rare monoclonal plasma cell (PC) disorder characterized by the deposition of misfolded immunoglobulin (Ig) light chains (LC) in vital organs throughout the body. To our knowledge,no cell lines have ever been established from AL patients. Here we describe the establishment of the ALMC-1 and ALMC-2 cell lines from an AL patient. Both cell lines exhibit a PC phenotype and display cytokine-dependent growth. Using a comprehensive genetic approach,we established the genetic relationship between the cell lines and the primary patient cells,and we were also able to identify new genetic changes accompanying tumor progression that may explain the natural history of this patient's disease. Importantly,we demonstrate that free lambda LC secreted by both cell lines contained a beta structure and formed amyloid fibrils. Despite absolute Ig LC variable gene sequence identity,the proteins show differences in amyloid formation kinetics that are abolished by the presence of Na(2)SO(4). The formation of amyloid fibrils from these naturally secreting human LC cell lines is unprecedented. Moreover,these cell lines will provide an invaluable tool to better understand AL,from the combined perspectives of amyloidogenic protein structure and amyloid formation,genetics,and cell biology. View Publication

The tumor suppressor gene phosphatase and tensin homolog (PTEN) is inactivated in many human cancers. However,it is unknown whether PTEN functions as a tumor suppressor in human Philadelphia chromosome-positive leukemia that includes chronic myeloid leukemia (CML) and B-cell acute lymphoblastic leukemia (B-ALL) and is induced by the BCR-ABL oncogene. By using our mouse model of BCR-ABL-induced leukemias,we show that Pten is down-regulated by BCR-ABL in leukemia stem cells in CML and that PTEN deletion causes acceleration of CML development. In addition,overexpression of PTEN delays the development of CML and B-ALL and prolongs survival of leukemia mice. PTEN suppresses leukemia stem cells and induces cell-cycle arrest of leukemia cells. Moreover,PTEN suppresses B-ALL development through regulating its downstream gene Akt1. These results demonstrate a critical role of PTEN in BCR-ABL-induced leukemias and suggest a potential strategy for the treatment of Philadelphia chromosome-positive leukemia. View Publication

Germ line mutations in the Adenomatous polyposis coli tumor suppressor gene cause a hereditary form of intestinal tumorigenesis in both mice and man. Here we show that in Apc(Min/+) mice,which carry a heterozygous germ line mutation at codon 850 of Apc,there is progressive loss of immature and mature thymocytes from approximately 80 days of age with complete regression of the thymus by 120 days. In addition,Apc(Min/+) mice show parallel depletion of splenic natural killer (NK) cells,immature B cells,and B progenitor cells in bone marrow due to complete loss of interleukin 7 (IL-7)-dependent B-cell progenitors. Using bone marrow transplantation experiments into wild-type recipients,we have shown that the capacity of transplanted Apc(Min/+) bone marrow cells for T- and B-cell development appears normal. In contrast,although the Apc(Min/+) bone marrow microenvironment supported short-term reconstitution with wild-type bone marrow,Apc(Min/+) animals that received transplants subsequently underwent lymphodepletion. Fibroblast colony-forming unit (CFU-F) colony assays revealed a significant reduction in colony-forming mesenchymal progenitor cells in the bone marrow of Apc(Min/+) mice compared with wild-type animals prior to the onset of lymphodepletion. This suggests that an altered bone marrow microenvironment may account for the selective lymphocyte depletion observed in this model of familial adenomatous polyposis. View Publication

Genetic factors influence susceptibility to systemic lupus erythematosus (SLE). A recent family-based analysis in Caucasian and Chinese populations provided evidence for association of single-nucleotide polymorphisms (SNPs) in the complement receptor 2 (CR2/CD21) gene with SLE. Here we confirmed this result in a case-control analysis of an independent European-derived population including 2084 patients with SLE and 2853 healthy controls. A haplotype formed by the minor alleles of three CR2 SNPs (rs1048971,rs17615,rs4308977) showed significant association with decreased risk of SLE (30.4% in cases vs 32.6% in controls,P=0.016,OR=0.90 (0.82-0.98)). Two of these SNPs are in exon 10,directly 5' of an alternatively spliced exon preferentially expressed in follicular dendritic cells (FDC),and the third is in the alternatively spliced exon. Effects of these SNPs and a fourth SNP in exon 11 (rs17616) on alternative splicing were evaluated. We found that the minor alleles of these SNPs decreased splicing efficiency of exon 11 both in vitro and ex vivo. These findings further implicate CR2 in the pathogenesis of SLE and suggest that CR2 variants alter the maintenance of tolerance and autoantibody production in the secondary lymphoid tissues where B cells and FDCs interact. View Publication

CXCL12-induced chemotaxis and adhesion to VCAM-1 decrease as B cells differentiate in the bone marrow. However,the mechanisms that regulate CXCL12/CXCR4-mediated signaling are poorly understood. We report that after CXCL12 stimulation of progenitor B cells,focal adhesion kinase (FAK) and PI3K are inducibly recruited to raft-associated membrane domains. After CXCL12 stimulation,phosphorylated FAK is also localized in membrane domains. The CXCL12/CXCR4-FAK pathway is membrane cholesterol dependent and impaired by metabolic inhibitors of G(i),Src family,and the GTPase-activating protein,regulator of G protein signaling 1 (RGS1). In the bone marrow,RGS1 mRNA expression is low in progenitor B cells and high in mature B cells,implying developmental regulation of CXCL12/CXCR4 signaling by RGS1. CXCL12-induced chemotaxis and adhesion are impaired when FAK recruitment and phosphorylation are inhibited by either membrane cholesterol depletion or overexpression of RGS1 in progenitor B cells. We conclude that the recruitment of signaling molecules to specific membrane domains plays an important role in CXCL12/CXCR4-induced cellular responses. View Publication

Purine nucleoside phosphorylase (PNP) deficiency in humans results in T lymphocytopenia. Forodesine,a potent inhibitor of PNP,was designed based on the transition-state structure stabilized by the enzyme. Previous studies established that forodesine in the presence of deoxyguanosine (dGuo) inhibits the proliferation of T lymphocytes. A phase 1 clinical trial of forodesine in T-cell malignancies demonstrated significant antileukemic activity with an increase in intracellular dGuo triphosphate (dGTP). High accumulation of dGTP in T cells may be dependent on the levels of deoxynucleoside kinases. Because B-cell chronic lymphocytic leukemia (B-CLL) cells have high activity of deoxycytidine kinase (dCK),we hypothesized that these lymphocytes would respond to forodesine. This postulate was tested in primary lymphocytes during in vitro investigations. Lymphocytes from 12 patients with CLL were incubated with forodesine and dGuo. These CLL cells showed a wide variation in the accumulation of intracellular dGTP without any effect on other deoxynucleotides. This was associated with DNA damage-induced p53 stabilization,phosphorylation of p53 at Ser15,and activation of p21. The dGTP accumulation was related to induction of apoptosis measured by caspase activation,changes in mitochondrial membrane potential,and PARP cleavage. Based on these data,a phase 2 clinical trial of forodesine has been initiated for CLL patients. View Publication

We evaluated the effects of ectopic granulocyte/macrophage colony-stimulating factor (GM-CSF) signals on hematopoietic commitment and differentiation. Lineage-restricted progenitors purified from mice with the ubiquitous transgenic human GM-CSF receptor (hGM-CSFR) were used for the analysis. In cultures with hGM-CSF alone,hGM-CSFR-expressing (hGM-CSFR+) granulocyte/monocyte progenitors (GMPs) and megakaryocyte/erythrocyte progenitors (MEPs) exclusively gave rise to granulocyte/monocyte (GM) and megakaryocyte/erythroid (MegE) colonies,respectively,providing formal proof that GM-CSF signals support the GM and MegE lineage differentiation without affecting the physiological myeloid fate. hGM-CSFR transgenic mice were crossed with mice deficient in interleukin (IL)-7,an essential cytokine for T and B cell development. Administration of hGM-CSF in these mice could not restore T or B lymphopoiesis,indicating that enforced GM-CSF signals cannot substitute for IL-7 to promote lymphopoiesis. Strikingly,textgreater50% hGM-CSFR+ common lymphoid progenitors (CLPs) and textgreater20% hGM-CSFR+ pro-T cells gave rise to granulocyte,monocyte,and/or myeloid dendritic cells,but not MegE lineage cells in the presence of hGM-CSF. Injection of hGM-CSF into mice transplanted with hGM-CSFR+ CLPs blocked their lymphoid differentiation,but induced development of GM cells in vivo. Thus,hGM-CSF transduces permissive signals for myeloerythroid differentiation,whereas it transmits potent instructive signals for the GM differentiation to CLPs and early T cell progenitors. These data suggest that a majority of CLPs and a fraction of pro-T cells possess plasticity for myelomonocytic differentiation that can be activated by ectopic GM-CSF signals,supporting the hypothesis that the down-regulation of GM-CSFR is a critical event in producing cells with a lymphoid-restricted lineage potential. View Publication