技术资料

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

Immunoglobulin (Ig) class switch DNA recombination (CSR) is the crucial mechanism diversifying the biological effector functions of antibodies. Generation of double-strand DNA breaks (DSBs),particularly staggered DSBs,in switch (S) regions of the upstream and downstream CH genes involved in the specific recombination process is an absolute requirement for CSR. Staggered DSBs would be generated through deamination of dCs on opposite DNA strands by activation-induced cytidine deaminase (AID),subsequent dU deglycosylation by uracil DNA glycosylase (Ung) and abasic site nicking by apurinic/apyrimidic endonuclease. However,consistent with the findings that significant amounts of DSBs can be detected in the IgH locus in the absence of AID or Ung,we have shown in human and mouse B cells that AID generates staggered DSBs not only by cleaving intact double-strand DNA,but also by processing blunt DSB ends generated in an AID-independent fashion. How these AID-independent DSBs are generated is still unclear. It is possible that S region DNA may undergo AID-independent cleavage by structure-specific nucleases,such as endonuclease G (EndoG). EndoG is an abundant nuclease in eukaryotic cells. It cleaves single and double-strand DNA,primarily at dG/dC residues,the preferential sites of DSBs in S region DNA. We show here that EndoG can localize to the nucleus of B cells undergoing CSR and binds to S region DNA,as shown by specific chromatin immunoprecipitation assays. Using knockout EndoG(-/-) mice and EndoG(-/-) B cells,we found that EndoG deficiency resulted in a two-fold reduction in CSR in vivo and in vitro,as demonstrated by reduced cell surface IgG1,IgG2a,IgG3 and IgA,reduced secreted IgG1,reduced circle Iγ1-Cμ,Iγ3-Cμ,Iɛ-Cμ,Iα-Cμ transcripts,post-recombination Iμ-Cγ1,Iμ-Cγ3,Iμ-Cɛ and Iμ-Cα transcripts. In addition to reduced CSR,EndoG(-/-) mice showed a significantly altered spectrum of mutations in IgH J(H)-iEμ DNA. Impaired CSR in EndoG(-/-) B cells did not stem from altered B cell proliferation or apoptosis. Rather,it was associated with significantly reduced frequency of DSBs. Thus,our findings determine a role for EndoG in the generation of S region DSBs and CSR. View Publication

Intestinal ischemia-reperfusion (IR)-induced damage requires complement receptor 2 (CR2) for generation of the appropriate natural Ab repertoire. Pathogenic Abs recognize neoantigens on the ischemic tissue,activate complement,and induce intestinal damage. Because C3 cleavage products act as ligands for CR2,we hypothesized that CR2(hi) marginal zone B cells (MZBs) require C3 for generation of the pathogenic Abs. To explore the ability of splenic CR2(+) B cells to generate the damaging Ab repertoire,we adoptively transferred either MZBs or follicular B cells (FOBs) from C57BL/6 or Cr2(-/-) mice into Rag-1(-/-) mice. Adoptive transfer of wild type CR2(hi) MZBs but not CR2(lo) FOBs induced significant damage,C3 deposition,and inflammation in response to IR. In contrast,similarly treated Rag-1(-/-) mice reconstituted with either Cr2(-/-) MZB/B1 B cells (B1Bs) or FOBs lacked significant intestinal damage and displayed limited complement activation. To determine whether C3 cleavage products are critical in CR2-dependent Ab production,we evaluated the ability of the natural Ab repertoire of C3(-/-) mice to induce damage in response to IR. Infusion of C3(-/-) serum into Cr2(-/-) mice restored IR-induced tissue damage. Furthermore,Rag-1(-/-) mice sustained significant damage after infusion of Abs from C3(-/-) but not Cr2(-/-) mice. Finally,adoptive transfer of MZBs from C3(-/-) mice into Rag-1(-/-) mice resulted in significant tissue damage and inflammation. These data indicate that CR2 expression on MZBs is sufficient to induce the appropriate Abs required for IR-induced tissue damage and that C3 is not critical for generation of the pathogenic Abs. View Publication

B1 cells differ in many ways from conventional B cells,most prominently in the production of natural immunoglobulin,which is vitally important for protection against pathogens. B1 cells have also been implicated in the pathogenesis of autoimmune dyscrasias and malignant diseases. It has been impossible to accurately study B1 cells during health and illness because the nature of human B1 cells has not been successfully defined. This has produced controversy regarding the existence of human B1 cells. Here,we determined the phenotype of human B1 cells by testing sort-purified B cell fractions for three fundamental B1 cell functions based on mouse studies: spontaneous IgM secretion,efficient T cell stimulation,and tonic intracellular signaling. We found that a small population of CD20(+)CD27(+)CD43(+) cells present in both umbilical cord and adult peripheral blood fulfilled these criteria and expressed a skewed B cell receptor repertoire. These B cells express little or no surface CD69 and CD70,both of which are markedly up-regulated after activation of CD20(+)CD27(-)CD43(-) (naive) and CD20(+)CD27(+)CD43(-) (memory) B cells. This work identifies human B1 cells as CD20(+)CD27(+)CD43(+)CD70(-). We determined that the proportion of B1 cells declines with age,which may contribute to disease susceptibility. Identification of human B1 cells provides a foundation for future studies on the nature and role of these cells in human disease. View Publication

In human B-acute lymphoblastic leukemia (B-ALL),RAG1-induced genomic alterations are important for disease progression. However,given that biallelic loss of the RAG1 locus is observed in a subset of cases,RAG1's role in the development of B-ALL remains unclear. We chose a p19Arf(-/-)Rag1(-/-) mouse model to confirm the previously published results concerning the contribution of CDKN2A (p19ARF /INK4a) and RAG1 copy number alterations in precursor B cells to the initiation and/or progression to B-acute lymphoblastic leukemia (B-ALL). In this murine model,we identified a new,Rag1-independent leukemia-initiating mechanism originating from a Sca1(+)CD19(+) precursor cell population and showed that Notch1 expression accelerates the cells' self-renewal capacity in vitro. In human RAG1-deficient BM,a similar CD34(+)CD19(+) population expressed p19ARF. These findings suggest that combined loss of p19Arf and Rag1 results in B-cell precursor leukemia in mice and may contribute to the progression of precursor B-ALL in humans. View Publication

Innate lymphoid cells (ILCs) are a new family of immune cells that play important roles in innate immunity in mucosal tissues,and in the maintenance of tissue and metabolic homeostasis. Recently,group 2 ILCs (ILC2s) were found to promote the development and effector functions of Th2-type CD4(+) T cells by interacting directly with T cells or by activating dendritic cells,suggesting a role for ILC2s in regulating adaptive immunity. However,our current knowledge on the role of ILCs in humoral immunity is limited. In this study,we found that ILC2s isolated from the lungs of naive BALB/c mice enhanced the proliferation of B1- as well as B2-type B cells and promoted the production of IgM,IgG1,IgA,and IgE by these cells in vitro. Soluble factors secreted by ILC2s were sufficient to enhance B cell Ig production. By using blocking Abs and ILC2s isolated from IL-5-deficient mice,we found that ILC2-derived IL-5 is critically involved in the enhanced production of IgM. Furthermore,when adoptively transferred to Il7r(-/-) mice,which lack ILC2s and mature T cells,lung ILC2s promoted the production of IgM Abs to a polysaccharide Ag,4-hydroxy-3-nitrophenylacetyl Ficoll,within 7 d of airway exposure in vivo. These findings add to the growing body of literature regarding the regulatory functions of ILCs in adaptive immunity,and suggest that lung ILC2s promote B cell production of early Abs to a respiratory Ag even in the absence of T cells. View Publication

Pentraxin 3 (PTX3) is a fluid-phase pattern recognition receptor of the humoral innate immune system with ancestral antibody-like properties but unknown antibody-inducing function. In this study,we found binding of PTX3 to splenic marginal zone (MZ) B cells,an innate-like subset of antibody-producing lymphocytes strategically positioned at the interface between the circulation and the adaptive immune system. PTX3 was released by a subset of neutrophils that surrounded the splenic MZ and expressed an immune activation-related gene signature distinct from that of circulating neutrophils. Binding of PTX3 promoted homeostatic production of IgM and class-switched IgG antibodies to microbial capsular polysaccharides,which decreased in PTX3-deficient mice and humans. In addition,PTX3 increased IgM and IgG production after infection with blood-borne encapsulated bacteria or immunization with bacterial carbohydrates. This immunogenic effect stemmed from the activation of MZ B cells through a neutrophil-regulated pathway that elicited class switching and plasmablast expansion via a combination of T cell-independent and T cell-dependent signals. Thus,PTX3 may bridge the humoral arms of the innate and adaptive immune systems by serving as an endogenous adjuvant for MZ B cells. This property could be harnessed to develop more effective vaccines against encapsulated pathogens. View Publication

The extrafollicular (EF) plasmablast response to self-antigens that contain Toll-like receptor (TLR) ligands is prominent in murine lupus models and some bacterial infections,but the inhibitors and activators involved have not been fully delineated. Here,we used two conventional dendritic cell (cDC) depletion systems to investigate the role of cDCs on a classical TLR-dependent autoreactive EF response elicited in rheumatoid-factor B cells by DNA-containing immune complexes. Contrary to our hypothesis,cDC depletion amplified rather than dampened the EF response in Fas-intact but not Fas-deficient mice. Further,we demonstrated that cDC-dependent regulation requires Fas and Fas ligand (FasL) expression by T cells,but not Fas expression by B cells. Thus,cDCs activate FasL-expressing T cells that regulate Fas-expressing extrafollicular helper T (Tefh) cells. These studies reveal a regulatory role for cDCs in B cell plasmablast responses and provide a mechanistic explanation for the excess autoantibody production observed in Fas deficiency. View Publication