Dendritic Cells Induce a Subpopulation of IL-12Rβ2-Expressing Treg that Specifically Consumes IL-12 to Control Th1 Responses

Cytokines secreted from dendritic cells (DCs) play an important role in the regulation of T helper (Th) cell differentiation and activation into effector cells. Therefore, controlling cytokine secretion from DCs may potentially regulate Th differentiation/activation. DCs also induce de-novo generation of regulatory T cells (Treg) that modulate the immune response. In the current study we used the mixed leukocyte reaction (MLR) to investigate the effect of allospecific Treg on IL-12, TNFα and IL-6 secretion by DCs. Treg cells were found to markedly down-regulate IL-12 secretion from DCs following stimulation with TLR7/8 agonist. This down-regulation of IL-12 was neither due to a direct suppression of its production by the DCs nor a result of marked DC death. We found that IL-12 was rather actively consumed by Treg cells. IL-12 consumption was mediated by a subpopulation of IL-12Rβ2-expressing Treg cells and was dependent on MHC class-II expressed on dendritic cells. Furthermore, IL-12 consumption by Tregs increased their suppressive effect on T cell proliferation and Th1 activation. These results provide a new pathway of Th1 response regulation where IL-12 secreted by DCs is consumed by a sub-population of IL-12Rβ2-expressing Treg cells. Consumption of IL-12 by Tregs not only reduces the availability of IL-12 to Th effector cells but also enhances the Treg immunosuppressive effect. This DC-induced IL-12Rβ2-expressing Treg subpopulation may have a therapeutic advantage in suppressing Th1 mediated autoimmunity.ope

New monoclonal anti-mouse DC-SIGN antibodies reactive with acetone-fixed cells

Mouse DC-SIGN CD209a is a type II transmembrane protein, one of a family of C-type lectin genes syntenic and homologous to human DC-SIGN. Current anti-mouse DC-SIGN monoclonal antibodies (MAbs) are unable to react with DC-SIGN in acetone-fixed cells, limiting the chance to visualize DC-SIGN in tissue sections. We first produced rabbit polyclonal PAb-DSCYT14 against a 14-aa peptide in the cytosolic domain of mouse DC-SIGN, and it specifically detected DC-SIGN and not the related lectins, SIGN-R1 and SIGN-R3 expressed in transfected CHO cells. MAbs were generated by immunizing rats and DC-SIGN knockout mice with the extracellular region of mouse DC-SIGN. Five rat IgG2a or IgM MAbs, named BMD10, 11, 24, 25, and 30, were selected and each MAb specifically detected DC-SIGN by FACS and Western blots, although BMD25 was cross-reactive to SIGN-R1. Two mouse IgG2c MAbs MMD2 and MMD3 interestingly bound mouse DC-SIGN but at 10 fold higher levels than the rat MAbs. When the binding epitopes of the new BMD and two other commercial rat anti-DC-SIGN MAbs, 5H10 and LWC06, were examined by competition assays, the epitopes of BMD11, 24, and LWC06 were identical or closely overlapping while BMD10, 30, and 5H10 were shown to bind different epitopes. MMD2 and MMD3 epitopes were on a 3rd noncompeting region of mouse DC-SIGN. DC-SIGN expressed on the cell surface was sensitive to collagenase treatment, as monitored by polyclonal and MAb. These new reagents should be helpful to probe the biology of DC-SIGN in vivo.ope

Yersinia pestis Interacts With SIGNR1 (CD209b) for Promoting Host Dissemination and Infection

Yersinia pestis, a Gram-negative bacterium and the etiologic agent of plague, has evolved from Yersinia pseudotuberculosis, a cause of a mild enteric disease. However, the molecular and biological mechanisms of how Y. pseudotuberculosis evolved to such a remarkably virulent pathogen, Y. pestis, are not clear. The ability to initiate a rapid bacterial dissemination is a characteristic hallmark of Y. pestis infection. A distinguishing characteristic between the two Yersinia species is that Y. pseudotuberculosis strains possess an O-antigen of lipopolysaccharide (LPS) while Y. pestis has lost the O-antigen during evolution and therefore exposes its core LPS. In this study, we showed that Y. pestis utilizes its core LPS to interact with SIGNR1 (CD209b), a C-type lectin receptor on antigen presenting cells (APCs), leading to bacterial dissemination to lymph nodes, spleen and liver, and the initiation of a systemic infection. We therefore propose that the loss of O-antigen represents a critical step in the evolution of Y. pseudotuberculosis into Y. pestis in terms of hijacking APCs, promoting bacterial dissemination and causing the plague.ope

Recognition of secretory IgA by DC-SIGN: implications for immune surveillance in the intestine

Secretory IgA (SIgA), the predominant class of antibody in intestinal secretions, serves as the first line of defense against enteric infections. SIgA has also been proposed to function in immune surveillance, given that both SIgA and SIgA-antigen complexes are actively transported by Peyer's patch M cells from the intestinal lumen to sub-epithelial dendritic cells (DCs). The goal of the present study was to identify the receptor(s) potentially utilized by mucosal DCs to recognize and internalize SIgA. We demonstrate that human colostral SIgA is recognized by purified recombinant human DC-specific ICAM-3 grabbing nonintegrin (DC-SIGN) in a solid phase binding assay, as well as by DC-SIGN ectopically expressed on the surface of Chinese hamster ovary (CHO-S) cells. The interaction between SIgA and DC-SIGN was specific, given that it was Ca(2+)-dependent and inhibited by mannan. Moreover, SIgA bound to, and was internalized by, endogenous DC-SIGN expressed on THP-1 cells following monocyte to macrophage-like cell differentiation by stimulation with phorbol ester and interleukin-4. These data identify DC-SIGN as a putative receptor for SIgA, and reveal a mechanism by which DCs could collaborate with M cells in immune surveillance at mucosal surfacesope

Dendritic cell targeted HIV-1 gag protein vaccine provides help to a recombinant Newcastle disease virus vectored vaccine including mobilization of protective CD8(+) T cells

INTRODUCTION: Recombinant Newcastle Disease virus (rNDV) vectored vaccines are safe mucosal applicable vaccines with intrinsic immune-modulatory properties for the induction of efficient immunity. Like all viral vectored vaccines repeated inoculation via mucosal routes invariably results to immunity against viral vaccine vectors. To obviate immunity against viral vaccine vectors and improve the ability of rNDV vectored vaccines in inducing T cell immunity in murine air way we have directed dendritic cell targeted HIV-1 gag protein (DEC-Gag) vaccine; for the induction of helper CD4(+) T cells to a Recombinant Newcastle disease virus expressing codon optimized HIV-1 Gag P55 (rNDV-L-Gag) vaccine. METHODS: We do so through successive administration of anti-DEC205-gagP24 protein plus polyICLC (DEC-Gag) vaccine and rNDV-L-Gag. First strong gag specific helper CD4(+) T cells are induced in mice by selected targeting of anti-DEC205-gagP24 protein vaccine to dendritic cells (DC) in situ together with polyICLC as adjuvant. This targeting helped T cell immunity develop to a subsequent rNDV-L-Gag vaccine and improved both systemic and mucosal gag specific immunity. RESULTS: This sequential DEC-Gag vaccine prime followed by an rNDV-L-gag boost results to improved viral vectored immunization in murine airway, including mobilization of protective CD8(+) T cells to a pathogenic virus infection site. CONCLUSION: Thus, complementary prime boost vaccination, in which prime and boost favor distinct types of T cell immunity, improves viral vectored immunization, including mobilization of protective CD8(+) T cells to a pathogenic virus infection site such as the murine airway.ope

Microbial stimulation fully differentiates monocytes to DC-SIGN/CD209(+) dendritic cells for immune T cell areas

Dendritic cells (DCs), critical antigen-presenting cells for immune control, normally derive from bone marrow precursors distinct from monocytes. It is not yet established if the large reservoir of monocytes can develop into cells with critical features of DCs in vivo. We now show that fully differentiated monocyte-derived DCs (Mo-DCs) develop in mice and DC-SIGN/CD209a marks the cells. Mo-DCs are recruited from blood monocytes into lymph nodes by lipopolysaccharide and live or dead gram-negative bacteria. Mobilization requires TLR4 and its CD14 coreceptor and Trif. When tested for antigen-presenting function, Mo-DCs are as active as classical DCs, including cross-presentation of proteins and live gram-negative bacteria on MHC I in vivo. Fully differentiated Mo-DCs acquire DC morphology and localize to T cell areas via L-selectin and CCR7. Thus the blood monocyte reservoir becomes the dominant presenting cell in response to select microbes, yielding DC-SIGN(+) cells with critical functions of DCsope

Filaria specific antibody response profiling in plasma from anti-retroviral naive Loa loa microfilaraemic HIV-1 infected people

BACKGROUND: In West and Central Africa areas of endemic Loa loa infections overlap with regions of high prevalence of human immunodeficiency virus type 1 (HIV-1) infections. Because individuals in this region are exposed to filarial parasites from birth, most HIV-1 infected individuals invariably also have a history of filarial parasite infection. Since HIV-1 infection both depletes immune system and maintains it in perpetual inflammation, this can hamper Loa loa filarial parasite mediated immune modulation, leading to enhanced loaisis. METHODS: In this study we have assessed in plasma from asymptomatic anti-retroviral (ARV) naive Loa loa microfilaraemic HIV-1 infected people the filarial antibody responses specific to a filariasis composite antigen consisting of Wbgp29-BmR1-BmM14-WbSXP. The antibody responses specific to the filariasis composite antigen was determined by enzyme linked immunosorbent assay (ELISA) in plasma from ARV naive Loa loa microfilaraemic HIV-1 infected participants. In addition the filarial antigen specific IgG antibody subclass profiles were also determined for both HIV-1 positive and negative people. RESULTS: Both Loa loa microfilaraemic HIV-1 positive and negative individuals showed significantly higher plasma levels of IgG1 (P < 0.0001), IgG2 (P < 0.0001) and IgM (P < 0.0001) relative to amicrofilaraemic participants. A significant increase in IgE (P < 0.0001) was observed exclusively in Loa loa microfilaraemic HIV-1 infected people. In contrast there was a significant reduction in the level of IgG4 (p < 0.0001) and IgG3 (P < 0.0001) in Loa loa microfilaraemic HIV-1 infected individuals. CONCLUSIONS: Loa loa microfilaraemia in ARV naive HIV-1 infected people through differential reduction of plasma levels of filarial antigen specific IgG3, IgG4 and a significant increase in plasma levels of filarial antigen specific IgE could diminish Loa loa mediated immune-regulation. This in effect can result to increase loaisis mediated immunopathology in antiretroviral naive HIV-1 infected people.ope

Dendritic Cell-Specific Intercellular Adhesion Molecule 3-Grabbing Nonintegrin/CD209 Is Abundant on Macrophages in the Normal Human Lymph Node and Is Not Required for Dendritic Cell Stimulation of the Mixed Leukocyte Reaction

The C-type lectin dendritic cell-specific ICAM 3-grabbing nonintegrin (DC-SIGN)/CD209 efficiently binds several pathogens, including HIV-1. DC-SIGN is expressed on monocyte-derived DCs in culture, and importantly, it is able to sequester HIV-1 within cells and facilitate transmission of virus to CD4+ T cells. To investigate DC-SIGN function, we have generated new mAbs. We report in this study that these and prior anti-DC-SIGN mAbs primarily label macrophages in the medullary sinuses of noninflamed human lymph node. In contrast, expression is not detected on most DCs in the T cell area, except for occasional cells. We also noted that IL-4 alone can induce expression of DC-SIGN in CD14+ monocytes and circulating blood DCs. However, blockade of DC-SIGN with Abs and DC-SIGN small interfering RNA did not result in a major reduction in the capacity of these DCs to transfer HIV to T cells, confirming significant DC-SIGN-independent mechanisms. The blocking approaches did reduce HIV-1 transmission by DC-SIGN-transfected cells by >90%. DC-SIGN blockade also did not reduce the ability of DCs to stimulate T cell proliferation in the MLR. These results indicate that DC-SIGN has the potential to contribute to macrophage function in normal human lymph node, and that DCs do not require DC-SIGN to transmit HIV or to initiate T cell responses.ope

Extended Culture of Bone Marrow with Granulocyte Macrophage-Colony Stimulating Factor Generates Immunosuppressive Cells

Bone marrow-derived dendritic cells (BM-DCs) are generated from bone marrow (BM) cells cultured with granulocyte macrophage-colony stimulating factor (GM-CSF) for a week. In this study we investigated the effect of duration on the BM culture with GM-CSF. Within several months, the cells in the BM culture gradually expressed homogeneous levels of CD11c and major histocompatibility complex II on surface, and they became unable to stimulate allogeneic naive T cells in mixed lymphocyte reaction (MLR). In addition, when the BM culture were sustained for 32 wk or longer, the BM cells acquired ability to suppress the proliferation of allogeneic T cells in MLR as well as the response of ovalbumin-specific OT-I transgenic T cells in antigen-dependent manner. We found that, except for programmed death-ligand 1, most cell surface molecules were expressed lower in the BM cells cultured with GM-CSF for the extended duration. These results indicate that BM cells in the extended culture with GM-CSF undergo 2 distinct steps of functional change; first, they lose the immunostimulatory capacity; and next, they gain the immunosuppressive ability.ope

Vaccine strategies utilizing C-type lectin receptors on dendritic cells in vivo

Dendritic cells (DCs) are professional antigen-presenting cells capable of initiating and regulating innate and adaptive immunity. The development of effective ways to produce a large number of DCs in laboratories made the use of DCs available in various vaccine approaches. Compared to conventional vaccines, focused on protective antibody responses, DC vaccines emphasize protective T cell immunity but might elicit strong antibody responses as well. In addition, the recent discoveries of functionally distinct DC subsets in various organs and tissues are likely to increase the potential of exploiting DCs in vaccines and immunotherapy. Vaccines composed of DCs generated ex vivo, pulsed with antigens, and matured prior to being re-infused to the body have been widely tried clinically but resulted in limited success due to various obstacles. In this review, new approaches that protein vaccines are selectively targeted to the endocytic C-type lectin receptors on surface of DCs in vivo are discussed.ope