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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

BACKGROUND: Successful gametogenesis requires the establishment of an appropriate epigenetic state in developing germ cells. Nevertheless,an association between abnormal spermatogenesis and epigenetic disturbances in germline-specific genes remains to be demonstrated. METHODS: In this study,the DNA methylation pattern of the promoter CpG island (CGI) of two germline regulator genes--DAZL and DAZ,was characterized by bisulphite genomic sequencing in quality-fractioned ejaculated sperm populations from normozoospermic (NZ) and oligoasthenoteratozoospermic (OAT) men. RESULTS: OAT patients display increased methylation defects in the DAZL promoter CGI when compared with NZ controls. Such differences are recorded when analyzing sperm fractions enriched either in normal or defective germ cells (Ptextless 0.001 in both cases). Significant differences in DNA methylation profiles are also observable when comparing the qualitatively distinct germ cell fractions inside the NZ and OAT groups (P= 0.003 and P= 0.007,respectively). Contrastingly,the unmethylation pattern of the DAZ promoter CGI remains correctly established in all experimental groups. CONCLUSIONS: An association between disrupted DNA methylation of a key spermatogenesis gene and abnormal human sperm is described here for the first time. These results suggest that incorrect epigenetic marks in germline genes may be correlated with male gametogenic defects. View Publication

The contribution of mammary epithelial cells (MEC) to human immunodeficiency virus type 1 (HIV-1) in breast milk remains largely unknown. While breast milk contains CD4(+) cells throughout the breast-feeding period,it is not known whether MEC directly support HIV-1 infection or facilitate infection of CD4(+) cells in the breast compartment. This study evaluated primary human MEC for direct infection with HIV-1 and for indirect transfer of infection to CD4(+) target cells. Primary human MEC were isolated and assessed for expression of HIV-1 receptors. MEC were exposed to CCR5-,CXCR4- and dual-tropic strains of HIV-1 and evaluated for viral reverse transcription and integration and productive viral infection. MEC were also tested for the ability to transfer HIV to CD4(+) target cells and to activate resting CD4(+) T cells. Our results demonstrate that MEC express HIV-1 receptor proteins CD4,CCR5,CXCR4,and galactosyl ceramide (GalCer). While no evidence for direct infection of MEC was found,HIV-1 virions were observed in MEC endosomal compartments. Coculture of HIV-exposed MEC resulted in productive infection of activated CD4(+) T cells. In addition,MEC secretions increased HIV-1 replication and proliferation of infected target cells. Overall,our results indicate that MEC are capable of endosomal uptake of HIV-1 and can facilitate virus infection and replication in CD4(+) target cells. These findings suggest that MEC may serve as a viral reservoir for HIV-1 and may enhance infection of CD4(+) T lymphocytes in vivo. View Publication

It is increasingly evident that neutrophils are able to cross-talk with other leukocytes to shape ongoing inflammatory and immune responses. In this study,we analyzed whether human NK cells may influence the survival and activation of neutrophils under co-culture conditions. We report that NK cells exposed to either IL-15 or IL-18 alone strongly protect the survival of neutrophils via the release of IFNγ and granulocyte macrophage colony-stimulating factor (GM-CSF) plus IFNγ,respectively,and cause a slight up-regulation of neutrophil CD64 and CD11b expression. In comparison,NK cells exposed to both IL-15 and IL-18 show a lesser ability to increase the survival of neutrophils but can more potently up-regulate CD64 and CD11b expression,as well as induce the de novo surface expression of CD69,in neutrophils. Analysis of the events occurring in neutrophil/NK co-cultures exposed to IL-15 plus IL-18 revealed that (i) neutrophil survival is positively affected by NK-derived GM-CSF but negatively influenced by a CD18-dependent neutrophil/NK contact,(ii) NK-derived IFNγ is almost entirely responsible for the induction of CD64,(iii) both soluble factors (primarily GM-CSF) and direct cell-cell contact up-regulate CD11b and CD69 and (iv) NK-derived GM-CSF induces the expression of biologically active heparin-binding EGF-like growth factor (HB-EGF) in neutrophils. Finally,we demonstrate that NK cells can also express HB-EGF when stimulated with either IL-2 or IL-15,yet independently of endogenous GM-CSF. Altogether,our results define a novel interaction within the innate immune system whereby NK cells,by directly modulating neutrophil functions,might contribute to the pathogenesis of inflammatory diseases. View Publication

TOX is a DNA-binding factor required for development of CD4(+) T cells,natural killer T cells and regulatory T cells. Here we document that both natural killer (NK) cell development and lymphoid tissue organogenesis were also inhibited in the absence of TOX. We found that the development of lymphoid tissue-inducer cells,a rare subset of specialized cells that has an integral role in lymphoid tissue organogenesis,required TOX. Tox was upregulated considerably in immature NK cells in the bone marrow,consistent with the loss of mature NK cells in the absence of this nuclear protein. Thus,many cell lineages of the immune system share a TOX-dependent step for development. View Publication

The viral infection of higher vertebrates elicits potent innate and adaptive host immunity. However,an excessive or inappropriate immune response also may lead to host pathology that often is more severe than the direct effects of viral replication. Therefore,several mechanisms exist that regulate the magnitude and class of the immune response. Here,we have examined the potential involvement of regulatory T (Treg) cells in limiting pathology induced by influenza A virus (IAV) infection. Using lymphocyte-deficient mice as hosts,we showed that Treg cell reconstitution resulted in a significant delay in weight loss and prolonged survival following infection. The adoptively transferred Treg cells did not affect the high rate of IAV replication in the lungs of lymphocyte-deficient hosts,and therefore their disease-ameliorating effect was mediated through the suppression of innate immune pathology. Mechanistically,Treg cells reduced the accumulation and altered the distribution of monocytes/macrophages in the lungs of IAV-infected hosts. This reduction in lung monocytosis was associated with a specific delay in monocyte chemotactic protein-2 (MCP-2) induction in the infected lungs. Nevertheless,Treg cells failed to prevent the eventual development of severe disease in lymphocyte-deficient hosts,which likely was caused by the ongoing IAV replication. Indeed,using T-cell-deficient mice,which mounted a T-cell-independent B cell response to IAV,we further showed that the combination of virus-neutralizing antibodies and transferred Treg cells led to the complete prevention of clinical disease following IAV infection. Taken together,these results suggested that innate immune pathology and virus-induced pathology are the two main contributors to pathogenesis during IAV infection. View Publication

EBV infects most of the human population and is associated with a number of human diseases including cancers. Moreover,evasion of the immune system and chronic infection is an essential step for EBV-associated diseases. In this paper,we show that EBV can alter the regulation and expression of TLRs,the key effector molecules of the innate immune response. EBV infection of human primary B cells resulted in the inhibition of TLR9 functionality. Stimulation of TLR9 on primary B cells led to the production of IL-6,TNF-α,and IgG,which was inhibited in cells infected with EBV. The virus exerts its inhibitory function by decreasing TLR9 mRNA and protein levels. This event was observed at early time points after EBV infection of primary cells,as well as in an immortalized lymphoblastoid cell line. We determined that the EBV oncoprotein latent membrane protein 1 (LMP1) is a strong inhibitor of TLR9 transcription. Overexpression of LMP1 in B cells reduced TLR9 promoter activity,mRNA,and protein levels. LMP1 mutants altered in activating the NF-κB pathway prevented TLR9 promoter deregulation. Blocking the NF-κB pathway recovered TLR9 promoter activity. Mutating the NF-κB cis element on the TLR9 promoter restored luciferase transcription in the presence of LMP1. Finally,deletion of the LMP1 gene in the EBV genome abolished the ability of the virus to induce TLR9 downregulation. Our study describes a mechanism used by EBV to suppress the host immune response by deregulating the TLR9 transcript through LMP1-mediated NF-κB activation. View Publication

Information on natural killer (NK)-cell receptor-ligand interactions involved in the response to human cytomegalovirus (HCMV) is limited and essentially based on the study of infected fibroblasts. Experimental conditions were set up to characterize the NK response to HCMV-infected myeloid dendritic cells (DCs). Monocyte-derived DCs (moDCs) infected by the TB40/E HCMV strain down-regulated the expression of human leukocyte antigen class I molecules and specifically activated autologous NK-cell populations. NKG2D ligands appeared virtually undetectable in infected moDCs,reflecting the efficiency of immune evasion mechanisms,and explained the lack of antagonistic effects of NKG2D-specific monoclonal antibody. By contrast,DNAM-1 and DNAM-1 ligands (DNAM-1L)-specific monoclonal antibodies inhibited the NK response at 48 hours after infection,although the impact of HCMV-dependent down-regulation of DNAM-1L in infected moDCs was perceived at later stages. moDCs constitutively expressed ligands for NKp46 and NKp30 natural cytotoxicity receptors,which were partially reduced on HCMV infection; yet,only NKp46 appeared involved in the NK response. In contrast to previous reports in fibroblasts,human leukocyte antigen-E expression was not preserved in HCMV-infected moDCs,which triggered CD94/NKG2A(+) NK-cell activation. The results provide an insight on key receptor-ligand interactions involved in the NK-cell response against HCMV-infected moDCs,stressing the importance of the dynamics of viral immune evasion mechanisms. View Publication

To characterize mechanisms of CTL inhibition within an ocular tumor microenvironment,tumor-specific CTLs were transferred into mice with tumors developing within the anterior chamber of the eye or skin. Ocular tumors were resistant to CTL transfer therapy whereas skin tumors were sensitive. CTLs infiltrated ocular tumors at higher CTL/tumor ratios than in skin tumors and demonstrated comparable ex vivo effector function to CTLs within skin tumors indicating that ocular tumor progression was not due to decreased CTL accumulation or inhibited CTL function within the eye. CD11b(+)Gr-1(+)F4/80(-) cells predominated within ocular tumors,whereas skin tumors were primarily infiltrated by CD11b(+)Gr-1(-)F4/80(+) macrophages (Ms),suggesting that myeloid derived suppressor cells may contribute to ocular tumor growth. However,CD11b(+) myeloid cells isolated from either tumor site suppressed CTL activity in vitro via NO production. Paradoxically,the regression of skin tumors by CTL transfer therapy required NO production by intratumoral Ms indicating that NO-producing intratumoral myeloid cells did not suppress the effector phase of CTL. Upon CTL transfer,tumoricidal concentrations of NO were only produced by skin tumor-associated Ms though ocular tumor-associated Ms demonstrated comparable expression of inducible NO synthase protein suggesting that NO synthase enzymatic activity was compromised within the eye. Correspondingly,in vitro-activated Ms limited tumor growth when co-injected with tumor cells in the skin but not in the eye. In conclusion,the decreased capacity of Ms to produce NO within the ocular microenvironment limits CTL tumoricidal activity allowing ocular tumors to progress. 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

Among different cancer immunotherapy approaches,bispecific antibodies (BsAbs) are of great interest due to their ability to recruit immune cells to kill tumor cells directly. Various BsAbs against Her2 tumor cells have been proposed with potent cytotoxic activities. However,most of these formats require extensive processing to obtain heterodimeric bispecific antibodies. In this study,we describe a bispecific antibody,BiHC (bispecific Her2-CD3 antibody),constructed with a single-domain anti-Her2 and a single-chain Fv (variable fragment) of anti-CD3 in an IgG-like format. In contrast to most IgG-like BsAbs,the two arms in BiHC have different molecular weights,making it easier to separate hetero- or homodimers. BiHC can be expressed in Escherichia coli and purified via Protein A affinity chromatography. The purified BiHC can recruit T cells and induce specific cytotoxicity of Her2-expressing tumor cells in vitro. The BiHC can also efficiently inhibit the tumor growth in vivo. Thus,BiHC is a promising candidate for the treatment of Her2-positive cancers. View Publication

Lung macrophage subpopulations have been identified based on size. We investigated characteristics of small and large macrophages in the alveolar spaces and lung interstitium of COPD patients and controls. Alveolar and interstitial cells were isolated from lung resection tissue from 88 patients. Macrophage subpopulation cell-surface expression of immunological markers and phagocytic ability were assessed by flow cytometry. Inflammatory related gene expression was measured. Alveolar and interstitial macrophages had subpopulations of small and large macrophages based on size and granularity. Alveolar macrophages had similar numbers of small and large cells; interstitial macrophages were mainly small. Small macrophages expressed significantly higher cell surface HLA-DR,CD14,CD38 and CD36 and lower CD206 compared to large macrophages. Large alveolar macrophages showed lower marker expression in COPD current compared to ex-smokers. Small interstitial macrophages had the highest pro-inflammatory gene expression levels,while large alveolar macrophages had the lowest. Small alveolar macrophages had the highest phagocytic ability. Small alveolar macrophage CD206 expression was lower in COPD patients compared to smokers. COPD lung macrophages include distinct subpopulations; Small interstitial and small alveolar macrophages with more pro-inflammatory and phagocytic function respectively,and large alveolar macrophages with low pro-inflammatory and phagocytic ability. View Publication