技术资料

Mast cells (MCs) play an important role in the regulation of protective adaptive immune responses against pathogens. However,it is still unclear whether MCs promote such host defense responses via direct effects on T cells or rather by modifying the functions of antigen-presenting cells. To identify the underlying mechanisms of the immunoregulatory capacity of MCs,we investigated the impact of MCs on dendritic cell (DC) maturation and function. We found that murine peritoneal MCs underwent direct crosstalk with immature DCs that induced DC maturation as evidenced by enhanced expression of costimulatory molecules. Furthermore,the MC/DC interaction resulted in the release of the T-cell modulating cytokines IFN-γ,IL-2,IL-6 and TGF-β into coculture supernatants and increased the IL-12p70,IFN-γ,IL-6 and TGF-β secretion of LPS-matured DCs. Such MC-primed" DCs subsequently induced efficient CD4+ T-cell proliferation. Surprisingly� View Publication

Regulatory T cells (Tregs) contribute significantly to the tolerogenic nature of the liver. The mechanisms,however,underlying liver-associated Treg induction are still elusive. We recently identified the vitamin A metabolite,retinoic acid (RA),as a key controller that promotes TGF-β-dependent Foxp3(+) Treg induction but inhibits TGF-β-driven Th17 differentiation. To investigate whether the RA producing hepatic stellate cells (HSC) are part of the liver tolerance mechanism,we investigated the ability of HSC to function as regulatory APC. Different from previous reports,we found that highly purified HSC did not express costimulatory molecules and only upregulated MHC class II after in vitro culture in the presence of exogenous IFN-γ. Consistent with an insufficient APC function,HSC failed to stimulate naive OT-II TCR transgenic CD4(+) T cells and only moderately stimulated α-galactosylceramide-primed invariant NKT cells. In contrast,HSC functioned as regulatory bystanders and promoted enhanced Foxp3 induction by OT-II TCR transgenic T cells primed by spleen dendritic cells,whereas they greatly inhibited the Th17 differentiation. Furthermore,the regulatory bystander capacity of the HSC was completely dependent on their ability to produce RA. Our data thus suggest that HSC can function as regulatory bystanders,and therefore,by promoting Tregs and suppressing Th17 differentiation,they might represent key players in the mechanism that drives liver-induced tolerance. View Publication

B cell activation and Ab production in response to protein Ags requires presentation of peptides for recruitment of T cell help. We and others have recently demonstrated that B cells can also acquire innate help by presenting lipid Ags via CD1d to NKT cells. Given the newfound contribution of NKT cells to humoral immunity,we sought to identify the pathways that regulate CD1 molecule expression in human B cells. We show that ex vivo,activated and memory B cells expressed lower levels of CD1d compared with resting,naive,and marginal zone-like B cells. In vitro,CD1d was downregulated by all forms of B cell activation,leaving a narrow temporal window in which B cells could activate NKT cells. CD1c expression and function also decreased following activation by CD40L alone,whereas activation via the BCR significantly upregulated CD1c,particularly on marginal zone-like B cells. We found that the CD40L-induced downregulation of CD1d and CD1c correlated with diminished expression of retinoic acid receptor α (RARα) response genes,an effect that was reversed by RARα agonists. However,BCR-induced upregulation of CD1c was independent of the RAR pathway. Our findings that both CD1d and CD1c are upregulated by RARα signaling in human B cells is distinct from effects reported in dendritic cells,in which CD1c is inversely downregulated. One functional consequence of CD1d upregulation by retinoic acid was NKT cell cytotoxicity toward B cells. These results are central to our understanding of how CD1-restricted T cells may control humoral immunity. View Publication

New vaccines against pertussis are needed to evoke full protection and long-lasting immunological memory starting from the first administration in neonates--the major target of the life-threatening pertussis infection. A novel live attenuated Bordetella pertussis vaccine strain,BPZE1,has been developed by eliminating or detoxifying three important B. pertussis virulence factors: pertussis toxin,dermonecrotic toxin,and tracheal cytotoxin. We used a human preclinical ex vivo model based on monocyte-derived dendritic cells (MDDCs) to evaluate BPZE1 immunogenicity. We studied the effects of BPZE1 on MDDC functions,focusing on the impact of Bordetella-primed dendritic cells in the regulation of Th and suppressor T cells (Ts). BPZE1 is able to activate human MDDCs and to promote the production of a broad spectrum of proinflammatory and regulatory cytokines. Moreover,conversely to its parental wild-type counterpart BPSM,BPZE1-primed MDDCs very efficiently migrate in vitro in response to the lymphatic chemokine CCL21,due to the inactivation of pertussis toxin enzymatic activity. BPZE1-primed MDDCs drove a mixed Th1/Th17 polarization and also induced functional Ts. Experiments performed in a Transwell system showed that cell contact rather than the production of soluble factors was required for suppression activity. Overall,our findings support the potential of BPZE1 as a novel live attenuated pertussis vaccine,as BPZE1-challenged dendritic cells might migrate from the site of infection to the lymph nodes,prime Th cells,mount an adaptive immune response,and orchestrate Th1/Th17 and Ts responses. View Publication

CD4(+) T cells play a key role in host defense against Pneumocystis infection. To define the role of naïve CD4(+) T cell production through the thymopoietic response in host defense against Pneumocystis infection,Pneumocystis murina infection in the lung was induced in adult male C57BL/6 mice with and without prior thymectomy. Pneumocystis infection caused a significant increase in the number of CCR9(+) multipotent progenitor (MPP) cells in the bone marrow and peripheral circulation,an increase in populations of earliest thymic progenitors (ETPs) and double negative (DN) thymocytes in the thymus,and recruitment of naïve and total CD4(+) T cells into the alveolar space. The level of murine signal joint T cell receptor excision circles (msjTRECs) in spleen CD4(+) cells was increased at 5 weeks post-Pneumocystis infection. In thymectomized mice,the numbers of naïve,central memory,and total CD4(+) T cells in all tissues examined were markedly reduced following Pneumocystis infection. This deficiency of naïve and central memory CD4(+) T cells was associated with delayed pulmonary clearance of Pneumocystis. Extracts of Pneumocystis resulted in an increase in the number of CCR9(+) MPPs in the cultured bone marrow cells. Stimulation of cultured bone marrow cells with ligands to Toll-like receptor 2 ([TLR-2] zymosan) and TLR-9 (ODN M362) each caused a similar increase in CCR9(+) MPP cells via activation of the Jun N-terminal protein kinase (JNK) pathway. These results demonstrate that enhanced production of naïve CD4(+) T lymphocytes through the thymopoietic response and enhanced delivery of lymphopoietic precursors from the bone marrow play an important role in host defense against Pneumocystis infection. View Publication

Severe congenital neutropenia (SCN) is an inborn disorder of granulopoiesis that in many cases is caused by mutations of the ELANE gene,which encodes neutrophil elastase (NE). Recent data suggest a model in which ELANE mutations result in NE protein misfolding,induction of endoplasmic reticulum (ER) stress,activation of the unfolded protein response (UPR),and ultimately a block in granulocytic differentiation. To test this model,we generated transgenic mice carrying a targeted mutation of Elane (G193X) reproducing a mutation found in SCN. The G193X Elane allele produces a truncated NE protein that is rapidly degraded. Granulocytic precursors from G193X Elane mice,though without significant basal UPR activation,are sensitive to chemical induction of ER stress. Basal and stress granulopoiesis after myeloablative therapy are normal in these mice. Moreover,inaction of protein kinase RNA-like ER kinase (Perk),one of the major sensors of ER stress,either alone or in combination with G193X Elane,had no effect on basal granulopoiesis. However,inhibition of the ER-associated degradation (ERAD) pathway using a proteosome inhibitor resulted in marked neutropenia in G193X Elane. The selective sensitivity of G913X Elane granulocytic cells to ER stress provides new and strong support for the UPR model of disease patho-genesis in SCN. View Publication

Granulocyte colony-stimulating factor (G-CSF),the prototypical mobilizing cytokine,induces hematopoietic stem and progenitor cell (HSPC) mobilization from the bone marrow in a cell-nonautonomous fashion. This process is mediated,in part,through suppression of osteoblasts and disruption of CXCR4/CXCL12 signaling. The cellular targets of G-CSF that initiate the mobilization cascade have not been identified. We use mixed G-CSF receptor (G-CSFR)-deficient bone marrow chimeras to show that G-CSF-induced mobilization of HSPCs correlates poorly with the number of wild-type neutrophils. We generated transgenic mice in which expression of the G-CSFR is restricted to cells of the monocytic lineage. G-CSF-induced HSPC mobilization,osteoblast suppression,and inhibition of CXCL12 expression in the bone marrow of these transgenic mice are intact,demonstrating that G-CSFR signals in monocytic cells are sufficient to induce HSPC mobilization. Moreover,G-CSF treatment of wild-type mice is associated with marked loss of monocytic cells in the bone marrow. Finally,we show that bone marrow macrophages produce factors that support the growth and/or survival of osteoblasts in vitro. Together,these data suggest a model in which G-CSFR signals in bone marrow monocytic cells inhibit the production of trophic factors required for osteoblast lineage cell maintenance,ultimately leading to HSPC mobilization. View Publication

Polymorphonuclear neutrophils (PMNs) are critical for the formation,maintenance,and resolution of bacterial abscesses. However,the mechanisms that regulate PMN survival and proliferation during the evolution of an abscess are not well defined. Using a mouse model of Staphylococcus aureus abscess formation within a cutaneous wound,combined with real-time imaging of genetically tagged PMNs,we observed that a high bacterial burden elicited a sustained mobilization of PMNs from the bone marrow to the infected wound,where their lifespan was markedly extended. A continuous rise in wound PMN number,which was not accounted for by trafficking from the bone marrow or by prolonged survival,was correlated with the homing of c-kit(+)-progenitor cells from the blood to the wound,where they proliferated and formed mature PMNs. Furthermore,by blocking their recruitment with an antibody to c-kit,which severely limited the proliferation of mature PMNs in the wound and shortened mouse survival,we confirmed that progenitor cells are not only important contributors to PMN expansion in the wound,but are also functionally important for immune protection. We conclude that the abscess environment provides a niche capable of regulating PMN survival and local proliferation of bone marrow-derived c-kit(+)-progenitor cells. View Publication

The immune system is replenished by self-renewing hematopoietic stem cells (HSCs) that produce multipotent progenitors (MPPs) with little renewal capacity. E-proteins,the widely expressed basic helix-loop-helix transcription factors,contribute to HSC and MPP activity,but their specific functions remain undefined. Using quantitative in vivo and in vitro approaches,we show that E47 is dispensable for the short-term myeloid differentiation of HSCs but regulates their long-term capabilities. E47-deficient progenitors show competent myeloid production in short-term assays in vitro and in vivo. However,long-term myeloid and lymphoid differentiation is compromised because of a progressive loss of HSC self-renewal that is associated with diminished p21 expression and hyperproliferation. The activity of E47 is shown to be cell-intrinsic. Moreover,E47-deficient HSCs and MPPs have altered expression of genes associated with cellular energy metabolism,and the size of the MPP pool but not downstream lymphoid precursors in bone marrow or thymus is rescued in vivo by antioxidant. Together,these observations suggest a role for E47 in the tight control of HSC proliferation and energy metabolism,and demonstrate that E47 is not required for short-term myeloid differentiation. View Publication

P-selectin glycoprotein ligand-1 (PSGL-1) is a homodimeric transmembrane mucin on leukocytes. During inflammation,reversible interactions of PSGL-1 with selectins mediate leukocyte rolling on vascular surfaces. The transmembrane domain of PSGL-1 is required for dimerization,and the cytoplasmic domain propagates signals that activate β(2) integrins to slow rolling on integrin ligands. Leukocytes from knock-in ΔCD" mice express a truncated PSGL-1 that lacks the cytoplasmic domain. Unexpectedly� View Publication

Bright/Arid3a has been characterized both as an activator of immunoglobulin heavy-chain transcription and as a proto-oncogene. Although Bright expression is highly B lineage stage restricted in adult mice,its expression in the earliest identifiable hematopoietic stem cell (HSC) population suggests that Bright might have additional functions. We showed that textgreater99% of Bright(-/-) embryos die at midgestation from failed hematopoiesis. Bright(-/-) embryonic day 12.5 (E12.5) fetal livers showed an increase in the expression of immature markers. Colony-forming assays indicated that the hematopoietic potential of Bright(-/-) mice is markedly reduced. Rare survivors of lethality,which were not compensated by the closely related paralogue Bright-derived protein (Bdp)/Arid3b,suffered HSC deficits in their bone marrow as well as B lineage-intrinsic developmental and functional deficiencies in their peripheries. These include a reduction in a natural antibody,B-1 responses to phosphocholine,and selective T-dependent impairment of IgG1 class switching. Our results place Bright/Arid3a on a select list of transcriptional regulators required to program both HSC and lineage-specific differentiation. View Publication

Dendritic cell (DC)-based immunotherapy has potential for treating infections and malignant tumors,but the functional capacity of DC must be assessed in detail,especially maturation and Ag-specific CTL priming. Recent reports suggest that DC that are provided with continuous maturation signals in vivo after transfer into patients are required to elicit the full DC functions. We demonstrate in this study that the rSendai virus vector (SeV) is a novel and ideal stimulant,providing DC with a continuous maturation signal via viral RNA synthesis in the cytosol,resulting in full maturation of monocyte-derived DC(s). Both RIG-I-dependent cytokine production and CD4 T cell responses to SeV-derived helper Ags are indispensable for overcoming regulatory T cell suppression to prime melanoma Ag recognized by T cell-1-specific CTL in the regulatory T cell abundant setting. DC stimulated via cytokine receptors,or TLRs,do not show these functional features. Therefore,SeV-infected DC have the potential for DC-directed immunotherapy. View Publication