RESUMEN
B cell activating factor (BAFF) is essential for B cells to develop and respond to Ags. Dysregulation of BAFF contributes to the development of some autoimmune diseases and malignancies. Little is known about when, where, and how BAFF is produced in vivo and about which BAFF-producing cells contribute to B cell responses. To better understand BAFF functions, we created BAFF reporter (BAFF-RFP) mice and Baff floxed (Bafffl/fl ) mice. Splenic and bone marrow neutrophils (Nphs) from BAFF-RFP mice expressed the highest constitutive levels of BAFF; other myeloid subsets, including conventional dendritic cells (cDCs) and monocyte (MO) subsets, expressed lower levels. Treatment of BAFF-RFP mice with polyinosinic:polycytidylic acid increased BAFF expression in splenic Ly6Chi inflammatory MOs, CD11bhi activated NK subset, and in bone marrow myeloid precursors. Postinfection with West Nile virus (WNV), BAFF increased in CD8- cDCs and Nphs, and BAFF+ CD11bhi NK cells expanded in draining lymph nodes. The cell- and tissue-specific increases in BAFF expression were dependent on type I IFN signaling. MAVS also was required or contributed to BAFF expression in dendritic cell and MO subsets, respectively. Mice with deletion of Baff in either cDCs or Nphs had reduced Ab responses after NP-Ficoll immunization; thus, BAFF produced by both cDCs and Nphs contributes to T cell-independent Ab responses. Conversely, mice with a cDC Baff deficiency had increased mortality after WNV infection and decreased WNV-specific IgG and neutralizing Ab responses. BAFF produced by Nphs and cDCs is regulated differently and has key roles in Ab responses and protective immunity.
Asunto(s)
Factor Activador de Células B/metabolismo , Células Dendríticas/metabolismo , Neutrófilos/metabolismo , Fiebre del Nilo Occidental/inmunología , Virus del Nilo Occidental/inmunología , Animales , Anticuerpos Antivirales/sangre , Anticuerpos Antivirales/inmunología , Anticuerpos Antivirales/metabolismo , Factor Activador de Células B/genética , Factor Activador de Células B/inmunología , Células Dendríticas/inmunología , Modelos Animales de Enfermedad , Humanos , Inmunidad Humoral , Inmunoglobulina G/sangre , Inmunoglobulina G/inmunología , Inmunoglobulina G/metabolismo , Interferón Tipo I/metabolismo , Ratones , Ratones Noqueados , Neutrófilos/inmunología , Transducción de Señal/inmunología , Fiebre del Nilo Occidental/sangre , Fiebre del Nilo Occidental/virologíaRESUMEN
Targeting Ags to the CD180 receptor activates both B cells and dendritic cells (DCs) to become potent APCs. After inoculating mice with Ag conjugated to an anti-CD180 Ab, B cell receptors were rapidly internalized. Remarkably, all B cell subsets, including even transitional 1 B cells, were programed to process, present Ag, and stimulate Ag-specific CD4+ T cells. Within 24-48 hours, Ag-specific B cells were detectable at T-B borders in the spleen; there, they proliferated in a T cell-dependent manner and induced the maturation of T follicular helper (TFH) cells. Remarkably, immature B cells were sufficient for the maturation of TFH cells after CD180 targeting: TFH cells were induced in BAFFR-/- mice (with only transitional 1 B cells) and not in µMT mice (lacking all B cells) following CD180 targeting. Unlike CD180 targeting, CD40 targeting only induced DCs but not B cells to become APCs and thus failed to efficiently induce TFH cell maturation, resulting in slower and lower-affinity IgG Ab responses. CD180 targeting induces a unique program in Ag-specific B cells and to our knowledge, is a novel strategy to induce Ag presentation in both DCs and B cells, especially immature B cells and thus has the potential to produce a broad range of Ab specificities. This study highlights the ability of immature B cells to present Ag to and induce the maturation of cognate TFH cells, providing insights toward vaccination of mature B cell-deficient individuals and implications in treating autoimmune disorders.
Asunto(s)
Presentación de Antígeno , Células Presentadoras de Antígenos/inmunología , Antígenos CD/inmunología , Linfocitos B/inmunología , Antígenos CD40/inmunología , Animales , Células Presentadoras de Antígenos/citología , Antígenos CD/genética , Receptor del Factor Activador de Células B/genética , Receptor del Factor Activador de Células B/inmunología , Linfocitos B/citología , Antígenos CD40/genética , Ratones , Ratones Noqueados , Ratas , Linfocitos T Colaboradores-Inductores/citología , Linfocitos T Colaboradores-Inductores/inmunologíaRESUMEN
B cell activating factor receptor (BAFFR)-/- mice have a profound reduction in mature B cells, but unlike µMT mice, they have normal numbers of newly formed, immature B cells. Using a West Nile virus (WNV) challenge model that requires antibodies (Abs) for protection, we found that unlike wild-type (WT) mice, BAFFR-/- mice were highly susceptible to WNV and succumbed to infection within 8 to 12 days after subcutaneous virus challenge. Although mature B cells were required to protect against lethal infection, infected BAFFR-/- mice had reduced WNV E-specific IgG responses and neutralizing Abs. Passive transfer of immune sera from previously infected WT mice rescued BAFFR-/- and fully B cell-deficient µMT mice, but unlike µMT mice that died around 30 days post-infection, BAFFR-/- mice survived, developed WNV-specific IgG Abs and overcame a second WNV challenge. Remarkably, protective immunity could be induced in mature B cell-deficient mice. Administration of a WNV E-anti-CD180 conjugate vaccine 30 days prior to WNV infection induced Ab responses that protected against lethal infection in BAFFR-/- mice but not in µMT mice. Thus, the immature B cells present in BAFFR-/- and not µMT mice contribute to protective antiviral immunity. A CD180-based vaccine may promote immunity in immunocompromised individuals.
Asunto(s)
Anticuerpos Antivirales/inmunología , Linfocitos B/inmunología , Fiebre del Nilo Occidental/prevención & control , Virus del Nilo Occidental/inmunología , Animales , Anticuerpos Neutralizantes/inmunología , Receptor del Factor Activador de Células B/deficiencia , Receptor del Factor Activador de Células B/genética , Femenino , Humanos , Inmunización Pasiva , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Vacunación , Fiebre del Nilo Occidental/inmunología , Fiebre del Nilo Occidental/virología , Virus del Nilo Occidental/fisiologíaRESUMEN
Whereas NO is known to regulate T cell responses, its role in regulating B cell responses remains unclear. Previous studies suggested that inducible NO synthase 2 (NOS2/iNOS) is required for normal IgA Ab responses but inhibits antiviral IgG2a Ab responses. In this study we used NOS2(-/-) mice to determine the role of NO in T cell-dependent and T cell-independent (TI)-2 Ab responses. Whereas T cell-dependent Ab responses were only modestly increased in NOS2(-/-) mice, IgM and IgG3 Ab responses as well as marginal zone B cell plasma cell numbers and peritoneal B1b B cells were significantly elevated after immunization with the TI-2 Ag 4-hydroxy-3-nitrophenyl acetyl (NP)-Ficoll. The elevated TI-2 responses in NOS2(-/-) mice were accompanied by significant increases in serum levels of BAFF/BLyS and by increases in BAFF-producing Ly6C(hi) inflammatory monocytes and monocyte-derived dendritic cells (DCs), suggesting that NO normally inhibits BAFF expression. Indeed, we found that NOS2(-/-) DCs produced more BAFF than did wild-type DCs, and addition of a NO donor to NOS2(-/-) DCs reduced BAFF production. Bone marrow chimeric mice that lack NOS2 in either nonhematopoietic or hematopoietic cells had intermediate IgM and IgG3 Ab responses after NP-Ficoll immunization, suggesting that NOS2 from both hematopoietic and nonhematopoietic sources regulates TI-2 Ab responses. Similar to NOS2(-/-) mice, depletion of Ly6C(hi) inflammatory monocytes and monocyte-derived DCs enhanced NP-specific IgM and IgG3 responses to NP-Ficoll. Thus, NO produced by inflammatory monocytes and their derivative DC subsets plays an important role in regulating BAFF production and TI-2 Ab responses.
Asunto(s)
Formación de Anticuerpos/inmunología , Factor Activador de Células B/biosíntesis , Factor Activador de Células B/genética , Óxido Nítrico Sintasa de Tipo II/fisiología , Óxido Nítrico/química , Subgrupos de Linfocitos T/inmunología , Animales , Formación de Anticuerpos/genética , Factor Activador de Células B/inmunología , Subgrupos de Linfocitos B/química , Subgrupos de Linfocitos B/inmunología , Subgrupos de Linfocitos B/metabolismo , Ficoll/química , Ficoll/inmunología , Haptenos/inmunología , Ratones , Ratones de la Cepa 129 , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Óxido Nítrico/inmunología , Óxido Nítrico Sintasa de Tipo II/deficiencia , Óxido Nítrico Sintasa de Tipo II/genética , Nitrofenoles/inmunología , Fenilacetatos/inmunología , Subgrupos de Linfocitos T/químicaRESUMEN
The C-type lectin receptor blood dendritic cell Ag 2 (BDCA2) is expressed exclusively on human plasmacytoid DCs (pDCs) and plays a role in Ag capture, internalization, and presentation to T cells. We used transgenic mice that express human BDCA2 and anti-BDCA2 mAbs to deliver Ags directly to BDCA2 on pDCs in vivo. Targeting Ag to pDCs in this manner resulted in significant suppression of Ag-specific CD4(+) T cell and Ab responses upon secondary exposure to Ag in the presence of adjuvant. Suppression of Ab responses required both a decrease in effector CD4(+) T cells and preservation of Foxp3(+) regulatory T cells (Tregs). Reduction in Treg numbers following Ag delivery to BDCA2 restored both CD4(+) T cell activation and Ab responses, demonstrating that Tregs were required for the observed tolerance. Our results demonstrate that Ag delivery to pDCs through BDCA2 is an effective method to induce immunological tolerance, which may be useful for treating autoimmune diseases or to inhibit unwanted Ab responses.
Asunto(s)
Células Dendríticas/inmunología , Tolerancia Inmunológica/fisiología , Lectinas Tipo C/inmunología , Glicoproteínas de Membrana/inmunología , Células Plasmáticas/inmunología , Receptores Inmunológicos/inmunología , Linfocitos T Reguladores/inmunología , Animales , Células Dendríticas/citología , Humanos , Lectinas Tipo C/genética , Glicoproteínas de Membrana/genética , Ratones , Ratones Transgénicos , Células Plasmáticas/citología , Receptores Inmunológicos/genética , Linfocitos T Reguladores/citologíaRESUMEN
Purpose and methods: B cell-activating factor (BAFF) contributes to the pathogenesis of autoimmune diseases including systemic lupus erythematosus (SLE). Although several anti-BAFF Abs and derivatives have been developed for the treatment of SLE, the specific sources of BAFF that sustain autoantibody (auto-Ab) producing cells have not been definitively identified. Using BAFF-RFP reporter mice, we identified major changes in BAFF-producing cells in two mouse spontaneous lupus models (Tlr7 Tg mice and Sle1), and in a pristane-induced lupus (PIL) model. Results: First, we confirmed that similar to their wildtype Tlr7 Tg and Sle1 mice counterparts, BAFF-RFP Tlr7 Tg mice and BAFF-RFP Sle1 mice had increased BAFF serum levels, which correlated with increases in plasma cells and auto-Ab production. Next, using the RFP reporter, we defined which cells had dysregulated BAFF production. BAFF-producing neutrophils (Nphs), monocytes (MOs), cDCs, T cells and B cells were all expanded in the spleens of BAFF-RFP Tlr7 Tg mice and BAFF-RFP Sle1 mice compared to controls. Furthermore, Ly6Chi inflammatory MOs and T cells had significantly increased BAFF expression per cell in both spontaneous lupus models, while CD8- DCs up-regulated BAFF expression only in the Tlr7 Tg mice. Similarly, pristane injection of BAFF-RFP mice induced increases in serum BAFF levels, auto-Abs, and the expansion of BAFF-producing Nphs, MOs, and DCs in both the spleen and peritoneal cavity. BAFF expression in MOs and DCs, in contrast to BAFF from Nphs, was required to maintain homeostatic and pristane-induced systemic BAFF levels and to sustain mature B cell pools in spleens and BMs. Although acting through different mechanisms, Nph, MO and DC sources of BAFF were each required for the development of auto-Abs in PIL mice. Conclusions: Our findings underscore the importance of considering the relative roles of specific myeloid BAFF sources and B cell niches when developing treatments for SLE and other BAFF-associated autoimmune diseases.
Asunto(s)
Enfermedades Autoinmunes , Lupus Eritematoso Sistémico , Animales , Ratones , Autoanticuerpos , Enfermedades Autoinmunes/metabolismo , Células Dendríticas/metabolismo , Modelos Animales de Enfermedad , Interleucina-4/metabolismo , Monocitos/metabolismo , Neutrófilos , Receptor Toll-Like 7/metabolismoRESUMEN
DNA vaccines elicit antibody, T helper cell responses and CD8+ T cell responses. Currently, little is known about the mechanism that DNA vaccines employ to induce adaptive immune responses. Prior studies have demonstrated that stimulator of interferon genes (STING) and conventional dendritic cells (cDCs) play critical roles in DNA vaccine induced antibody and T cell responses. STING activation by double stranded (dsDNA) sensing proteins initiate the production of type I interferon (IFN),but the DC-intrinsic effect of STING signaling is still unclear. Here, we investigated the role of STING within cDCs on DNA vaccine induction of antibody and T cell responses. STING knockout (STING-/- ) and conditional knockout mice that lack STING in cDCs (cDC STING cKO), were immunized intramuscularly with a DNA vaccine that expressed influenza A nucleoprotein (pNP). Both STING-/- and cDC STING cKO mice had significantly lower type I T helper (Th1) type antibody (anti-NP IgG2C) responses and lower frequencies of Th1 associated T cells (NP-specific IFN-γ+CD4+ T cells) post-immunization than wild type (WT) and cDC STING littermate control mice. In contrast, all mice had similar Th2-type NP-specific (IgG1) antibody titers. STING-/- mice developed significantly lower polyfunctional CD8+ T cells than WT, cDC STING cKO and cDC STING littermate control mice. These findings suggest that STING within cDCs mediates DNA vaccine induction of type I T helper responses including IFN-γ+CD4+ T cells, and Th1-type IgG2C antibody responses. The induction of CD8+ effector cell responses also require STING, but not within cDCs. These findings are the first to show that STING is required within cDCs to mediate DNA vaccine induced Th1 immune responses and provide new insight into the mechanism whereby DNA vaccines induce Th1 responses.
Asunto(s)
Vacunas de ADN , Animales , Formación de Anticuerpos , Linfocitos T CD8-positivos , Células Dendríticas , Inmunoglobulina G/metabolismo , Ratones , Linfocitos T Colaboradores-Inductores , Vacunas de ADN/farmacologíaRESUMEN
The B cell activating factor belonging to the tumor necrosis factor family (BAFF) is required for B cell survival and maturation. The mechanisms by which BAFF mediates B cell survival are less understood. We found that BAFF and a proliferation-inducing ligand (APRIL), which are related, block B cell antigen receptor (BCR)-induced apoptosis upstream of mitochondrial damage, which is consistent with a role for Bcl-2 family proteins. BCR ligation strongly increased expression of the proapoptotic Bcl-2 homology 3-only Bcl-2 protein Bim in both WEHI-231 and splenic B cells, and increases in Bim were reversed by BAFF or APRIL. Small interfering RNA vector-mediated suppression of Bim blocked BCR-induced apoptosis. BAFF also induced Bim phosphorylation and inhibited BCR-induced association of Bim with Bcl-2. BAFF induced delayed but sustained stimulation of extracellular signal-regulated kinase (ERK) and its activators, mitogen-activated protein kinase/ERK activating kinase (MEK) and c-Raf, and MEK inhibitors promoted accumulation and dephosphorylation of Bim. These results suggest that BAFF inhibits BCR-induced death by down-regulating Bim via sustained ERK activation, demonstrating that BAFF directly regulates Bim function. Although transitional immature type 1 (T1) B cell numbers are normal in Bim(-/-) mice, T2 and follicular mature B cells are elevated and marginal zone B cells are reduced. Our results suggest that mature B cell homeostasis is maintained by BAFF-mediated regulation of Bim.
Asunto(s)
Proteínas Reguladoras de la Apoptosis/antagonistas & inhibidores , Linfocitos B/citología , Linfocitos B/fisiología , Regulación hacia Abajo/fisiología , Quinasas MAP Reguladas por Señal Extracelular/fisiología , Sistema de Señalización de MAP Quinasas/fisiología , Proteínas de la Membrana/antagonistas & inhibidores , Proteínas de la Membrana/fisiología , Proteínas Proto-Oncogénicas/antagonistas & inhibidores , Factor de Necrosis Tumoral alfa/fisiología , Animales , Proteínas Reguladoras de la Apoptosis/genética , Factor Activador de Células B , Subgrupos de Linfocitos B/citología , Subgrupos de Linfocitos B/metabolismo , Proteína 11 Similar a Bcl2 , Línea Celular Tumoral , Supervivencia Celular/fisiología , Humanos , Quinasas Quinasa Quinasa PAM/fisiología , Proteínas de la Membrana/genética , Ratones , Ratones Noqueados , Fosforilación , Proteínas Proto-Oncogénicas/genética , Receptores de Antígenos de Linfocitos B/fisiología , Bazo/citología , Miembro 13 de la Superfamilia de Ligandos de Factores de Necrosis TumoralRESUMEN
Mice lacking B cells are more susceptible to S. typhimurium infection. How B cells contribute to protective immunity against Salmonella and what signals drive their activation are still unclear. Neutrophils (Nphs), monocytes (MOs), and dendritic cells (DCs) are involved in early immune responses to control the initial replication of S. typhimurium. These cells can produce B cell activating factor (BAFF) required for mature B cell survival and may help regulate B cell responses during Salmonella infection. Using BAFF reporter mice (BAFF-RFP+/-), we discovered that an i.p. infection with a virulent strain of S. typhimurium increased BAFF expression in splenic conventional DCs (cDC) and inflammatory Ly6Chi MOs/DCs four days post-infection. S. typhimurium infection induced the release of BAFF from Nphs, a decrease of BAFF-RFP expression and expansion of BAFF-RFP+ Nphs in the spleen and peritoneal cavity. After S. typhimurium infection, serum BAFF levels and immature and mature B cell subsets and plasma cells increased substantially. Conditional knockout (cKO) mice lacking BAFF in either Nphs or cDCs compared to control Bafffl/fl mice had reduced up-regulation of systemic BAFF levels and reduced expansion of mature and germinal center B cell subsets after infection. Importantly, the cKO mice lacking BAFF from either Nphs or cDCs had impaired induction of Salmonella-specific IgM Abs, and were more susceptible to S. typhimurium infection. Thus, Nphs and cDCs are major cellular sources of BAFF driving B cell responses, required for mounting optimal protective immunity against lethal Salmonella infection.
Asunto(s)
Factor Activador de Células B/metabolismo , Células Dendríticas/inmunología , Neutrófilos/inmunología , Infecciones por Salmonella/inmunología , Animales , Factor Activador de Células B/genética , Células Cultivadas , Ratones , Ratones Endogámicos C57BL , Infecciones por Salmonella/microbiología , Salmonella typhimurium/patogenicidad , Bazo/citología , Bazo/inmunologíaRESUMEN
Positive selection of T-cell precursors is the process by which a diverse T-cell repertoire is established. Positive selection begins at the CD4(+)CD8(+) double positive (DP) stage of development and involves at least two steps. First, DP thymocytes down-regulate CD8 to become transitional single positive (TSP) CD4(+) thymocytes. Then, cells are selected to become either mature single positive CD4(+) or mature single positive CD8(+) thymocytes. We sought to define the function of Gads during the two steps of positive selection by analyzing a Gads-deficient mouse line. In Gads(+/+) mice, most TSP CD4(+) thymocytes are TCR(hi)Bcl-2(hi)CD69(+), suggesting that essential steps in positive selection occurred in the DP stage. Despite that Gads(-/-) mice could readily generate TSP CD4(+) thymocytes, many Gads(-/-) TSP CD4(+) cells were TCR(lo)Bcl-2(lo)CD69(-), suggesting that Gads(-/-) cells proceeded to the TSP CD4(+) stage prior to being positively selected. These data suggest that positive selection is not a prerequisite for the differentiation of DP thymocytes into TSP CD4(+) thymocytes. We propose a model in which positive selection and differentiation into the TSP CD4(+) stage are separable events and Gads is only required for positive selection.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/inmunología , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD8-positivos/inmunología , Diferenciación Celular/inmunología , Animales , Antígenos CD/inmunología , Linfocitos T CD4-Positivos/citología , Linfocitos T CD8-positivos/citología , Citometría de Flujo , Inmunofenotipificación , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Receptores de Antígenos de Linfocitos T alfa-beta/inmunología , Organismos Libres de Patógenos EspecíficosRESUMEN
The ongoing COVID-19 pandemic, caused by infection with SARS-CoV-2, is having a dramatic and deleterious impact on health services and the global economy. Grim public health statistics highlight the need for vaccines that can rapidly confer protection after a single dose and be manufactured using components suitable for scale-up and efficient distribution. In response, we have rapidly developed repRNA-CoV2S, a stable and highly immunogenic vaccine candidate comprised of an RNA replicon formulated with a novel Lipid InOrganic Nanoparticle (LION) designed to enhance vaccine stability, delivery and immunogenicity. We show that intramuscular injection of LION/repRNA-CoV2S elicits robust anti-SARS-CoV-2 spike protein IgG antibody isotypes indicative of a Type 1 T helper response as well as potent T cell responses in mice. Importantly, a single-dose administration in nonhuman primates elicited antibody responses that potently neutralized SARS-CoV-2. These data support further development of LION/repRNA-CoV2S as a vaccine candidate for prophylactic protection from SARS-CoV-2 infection.
RESUMEN
The coronavirus disease 2019 (COVID-19) pandemic, caused by infection with the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), is having a deleterious impact on health services and the global economy, highlighting the urgent need for an effective vaccine. Such a vaccine would need to rapidly confer protection after one or two doses and would need to be manufactured using components suitable for scale up. Here, we developed an Alphavirus-derived replicon RNA vaccine candidate, repRNA-CoV2S, encoding the SARS-CoV-2 spike (S) protein. The RNA replicons were formulated with lipid inorganic nanoparticles (LIONs) that were designed to enhance vaccine stability, delivery, and immunogenicity. We show that a single intramuscular injection of the LION/repRNA-CoV2S vaccine in mice elicited robust production of anti-SARS-CoV-2 S protein IgG antibody isotypes indicative of a type 1 T helper cell response. A prime/boost regimen induced potent T cell responses in mice including antigen-specific responses in the lung and spleen. Prime-only immunization of aged (17 months old) mice induced smaller immune responses compared to young mice, but this difference was abrogated by booster immunization. In nonhuman primates, prime-only immunization in one intramuscular injection site or prime/boost immunizations in five intramuscular injection sites elicited modest T cell responses and robust antibody responses. The antibody responses persisted for at least 70 days and neutralized SARS-CoV-2 at titers comparable to those in human serum samples collected from individuals convalescing from COVID-19. These data support further development of LION/repRNA-CoV2S as a vaccine candidate for prophylactic protection against SARS-CoV-2 infection.
Asunto(s)
Alphavirus/genética , Anticuerpos Neutralizantes/inmunología , Betacoronavirus/inmunología , Infecciones por Coronavirus/inmunología , Neumonía Viral/inmunología , ARN Viral/genética , Replicón/genética , Linfocitos T/inmunología , Vacunas Virales/inmunología , Animales , Formación de Anticuerpos/inmunología , COVID-19 , Vacunas contra la COVID-19 , Infecciones por Coronavirus/prevención & control , Compuestos Inorgánicos/química , Lípidos/química , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Nanopartículas/química , Pandemias , Primates , SARS-CoV-2RESUMEN
Dendritic cell-associated lectin-1 (DCAL-1), also known as C-type lectin-like-1 (CLECL1), is a novel C-type lectin-like molecule expressed by antigen presenting cells including dendritic cells (DCs). Here we report that incubation of immature DCs (iDCs) with an anti-DCAL-1 monoclonal antibody (mAb) induced downstream signaling, including phosphorylation of c-Jun N-terminal kinase (JNK) and p44/42 MAP kinase. Furthermore, ligation of DCAL-1 expressed by iDCs specifically enhanced HLA-DR expression, whereas the expression of other co-stimulatory molecules remained unchanged and minimal cytokine secretion was detected. DCs that express high levels of major histocompatibility complex (MHC) class II in the absence of high levels of other co-stimulatory molecules and inflammatory cytokine secretion may play an important role in the maintenance of immune tolerance. Therefore, our data suggests an important role for DCAL-1 in the regulation of the immune response.
Asunto(s)
Células Dendríticas/inmunología , Lectinas Tipo C/inmunología , Proteínas de la Membrana/inmunología , Monocitos/inmunología , Anticuerpos Monoclonales/inmunología , Células Cultivadas , Citocinas/metabolismo , Células Dendríticas/citología , Células Dendríticas/metabolismo , Antígenos HLA-DR/metabolismo , Humanos , Proteínas Quinasas JNK Activadas por Mitógenos/metabolismo , Activación de Linfocitos , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteína Quinasa 3 Activada por Mitógenos/metabolismo , Monocitos/citología , Monocitos/metabolismo , FosforilaciónRESUMEN
Mitochondrial antiviral signaling protein (MAVS) is a critical innate immune signaling protein that directs the actions of the RIG-I-like receptor (RLR) signaling pathway of RNA virus recognition and initiation of anti-viral immunity against West Nile virus (WNV). In the absence of MAVS, mice die more rapidly after infection with the pathogenic WNV-Texas (TX) strain, but also produce elevated WNV-specific IgG concomitant with increased viral burden. Here we investigated whether there was a B cell intrinsic role for MAVS during the development of protective humoral immunity following WNV infection. MAVS-/- mice survived infection from the non-pathogenic WNV-Madagascar (MAD) strain, with limited signs of disease. Compared to wildtype (WT) controls, WNV-MAD-infected MAVS-/- mice had elevated serum neutralizing antibodies, splenic germinal center B cells, plasma cells and effector T cells. We found that when rechallenged with the normally lethal WNV-TX, MAVS-/- mice previously infected with WNV-MAD were protected from disease. Thus, protective humoral and cellular immune responses can be generated in absence of MAVS. Mice with a conditional deletion of MAVS only in CD11c+ dendritic cells phenocopied MAVS whole body knockout mice in their humoral responses to WNV-MAD, displaying elevated virus titers and neutralizing antibodies. Conversely, a B cell-specific deletion of MAVS had no effect on immune responses to WNV-MAD compared to WT controls. Thus, MAVS in dendritic cells is required to control WNV replication and thereby regulate downstream humoral immune responses.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/inmunología , Células Dendríticas/inmunología , Inmunidad Humoral/inmunología , Replicación Viral/inmunología , Virus del Nilo Occidental/inmunología , Animales , Anticuerpos Neutralizantes/inmunología , Anticuerpos Antivirales/inmunología , Linfocitos B/inmunología , Inmunidad Celular/inmunología , Inmunidad Innata/inmunología , Madagascar , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Transducción de Señal/inmunología , Linfocitos T/inmunología , Texas , Fiebre del Nilo Occidental/inmunologíaRESUMEN
Although the spleen is a major site for West Nile virus (WNV) replication and spread, relatively little is known about which innate cells in the spleen replicate WNV, control viral dissemination, and/or prime innate and adaptive immune responses. Here we tested if splenic macrophages (MΦs) were necessary for control of WNV infection. We selectively depleted splenic MΦs, but not draining lymph node MΦs, by injecting mice intravenously with clodronate liposomes several days prior to infecting them with WNV. Mice missing splenic MΦs succumbed to WNV infection after an increased and accelerated spread of virus to the spleen and the brain. WNV-specific Ab and CTL responses were normal in splenic MΦ-depleted mice; however, numbers of NK cells and CD4 and CD8 T cells were significantly increased in the brains of infected mice. Splenic MΦ deficiency led to increased WNV in other splenic innate immune cells including CD11b- DCs, newly formed MΦs and monocytes. Unlike other splenic myeloid subsets, splenic MΦs express high levels of mRNAs encoding the complement protein C1q, the apoptotic cell clearance protein Mertk, the IL-18 cytokine and the FcγR1 receptor. Splenic MΦ-deficient mice may be highly susceptible to WNV infection in part to a deficiency in C1q, Mertk, IL-18 or Caspase 12 expression.
Asunto(s)
Inmunidad Innata , Macrófagos/inmunología , Bazo/citología , Virus del Nilo Occidental/inmunología , Animales , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD8-positivos/inmunología , Femenino , Masculino , Ratones , Ratones Endogámicos C57BL , Bazo/inmunología , Linfocitos T Citotóxicos/inmunología , Carga Viral , Virus del Nilo Occidental/aislamiento & purificaciónRESUMEN
CD22 is a BCR co-receptor that regulates B cell signaling, proliferation and survival and is required for T cell-independent Ab responses. To investigate the role of CD22 during T cell-dependent (TD) Ab responses and memory B cell formation, we analyzed Ag-specific B cell responses generated by wild-type (WT) or CD22-/- B cells following immunization with a TD Ag. CD22-/- B cells mounted normal early Ab responses yet failed to generate either memory B cells or long-lived plasma cells, whereas WT B cells formed both populations. Surprisingly, B cell expansion and germinal center (GC) differentiation were comparable between WT and CD22-/- B cells. CD22-/- B cells, however, were significantly less capable of generating a population of CXCR4hiCD38hi GC B cells, which we propose represent memory B cell precursors within GCs. These results demonstrate a novel role for CD22 during TD humoral responses evident during primary GC formation and underscore that CD22 functions not only during B cell maturation but also during responses to both TD and T cell-independent antigens.
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Linfocitos B/inmunología , Diferenciación Celular/inmunología , Centro Germinal/inmunología , Células Precursoras de Linfocitos B/inmunología , Lectina 2 Similar a Ig de Unión al Ácido Siálico/metabolismo , Animales , Linfocitos B/metabolismo , Centro Germinal/metabolismo , Ratones , Ratones Endogámicos C57BL , Células Precursoras de Linfocitos B/metabolismoRESUMEN
Dendritic cells (DCs) are best known for their ability to activate naive T cells, and emerging evidence suggests that distinct DC subsets induce specialized T cell responses. However, little is known concerning the role of DC subsets in the initiation of B cell responses. We report that antigen (Ag) delivery to DC-inhibitory receptor 2 (DCIR2) found on marginal zone (MZ)-associated CD8α(-) DCs in mice leads to robust class-switched antibody (Ab) responses to a T cell-dependent (TD) Ag. DCIR2(+) DCs induced rapid up-regulation of multiple B cell activation markers and changes in chemokine receptor expression, resulting in accumulation of Ag-specific B cells within extrafollicular splenic bridging channels as early as 24 h after immunization. Ag-specific B cells primed by DCIR2(+) DCs were remarkably efficient at driving naive CD4 T cell proliferation, yet DCIR2-induced responses failed to form germinal centers or undergo affinity maturation of serum Ab unless toll-like receptor (TLR) 7 or TLR9 agonists were included at the time of immunization. These results demonstrate DCIR2(+) DCs have a unique capacity to initiate extrafollicular B cell responses to TD Ag, and thus define a novel division of labor among splenic DC subsets for B cell activation during humoral immune responses.
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Formación de Anticuerpos/inmunología , Linfocitos B/inmunología , Células Dendríticas/inmunología , Activación de Linfocitos/inmunología , Linfocitos T/inmunología , Animales , Células Presentadoras de Antígenos/inmunología , Antígenos/inmunología , Antígenos/metabolismo , Linfocitos B/metabolismo , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD4-Positivos/metabolismo , Comunicación Celular/inmunología , Células Dendríticas/metabolismo , Epítopos/inmunología , Centro Germinal/inmunología , Centro Germinal/metabolismo , Inmunoglobulina G/inmunología , Inmunoglobulina G/metabolismo , Ratones , Ratones Endogámicos C57BL , Receptor Toll-Like 7/agonistas , Receptor Toll-Like 9/agonistasRESUMEN
Using NOS2 KO mice, we investigated the hypothesis that NO modulation of BM-DC contributes to the NO-mediated control of Th1 immune responses. BM-DCs from NOS2 KO mice, compared with WT BM-DCs, have enhanced survival and responsiveness to TLR agonists, develop more Ly6C(hi)PDCA1(+) DCs that resemble inflammatory DCs and produce high levels of inflammatory cytokines. Also, compared with WT-infected mice, NOS2 KO mice infected with WNV showed enhanced expansion of a similar inflammatory Ly6C(hi)PDCA1(+) DC subset. Furthermore, in contrast to WT DCs, OVA-loaded NOS2 KO BM-DCs promoted increased IFN-γ production by OTII CD4(+) T cells in vitro and when adoptively transferred in vivo. The addition of a NO donor to NOS2 KO BM-DCs prior to OTII T cells priming in vivo was sufficient to revert Th1 immune responses to levels induced by WT BM-DCs. Thus, autocrine NO effects on maturation of inflammatory DCs and on DC programming of T cells may contribute to the protective role of NO in autoimmune diseases and infections. Regulating NO levels may be a useful tool to shape beneficial immune responses for DC-based immunotherapy.
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Antígenos Ly/metabolismo , Antígenos de Superficie/metabolismo , Proteínas Reguladoras de la Apoptosis/metabolismo , Células Dendríticas/inmunología , Células Dendríticas/metabolismo , Inmunidad/inmunología , Inflamación/inmunología , Óxido Nítrico/metabolismo , Células TH1/inmunología , Animales , Antígeno B7-2/metabolismo , Biomarcadores/metabolismo , Linfocitos T CD4-Positivos/inmunología , Proliferación Celular , Supervivencia Celular , Células Cultivadas , Citocinas/biosíntesis , Células Dendríticas/enzimología , Regulación hacia Abajo , Epítopos/inmunología , Inflamación/patología , Mediadores de Inflamación/metabolismo , Interferón gamma/biosíntesis , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Óxido Nítrico Sintasa de Tipo II/deficiencia , Óxido Nítrico Sintasa de Tipo II/metabolismo , Receptor de Muerte Celular Programada 1 , Receptores Toll-Like/inmunología , Regulación hacia Arriba , Fiebre del Nilo Occidental/inmunología , Fiebre del Nilo Occidental/virología , Virus del Nilo Occidental/fisiologíaRESUMEN
The TNF family ligand, RANKL, and its two TNFR family receptors, RANK and OPG, enable coordinated regulation between the skeletal and immune systems. Relatively little is known about how OPG influences RANKL-RANK interactions for the regulation of DCs. Here, we show that OPG KO bone marrow-derived DCs survive better and produce more TNF-alpha, IL-12p40, and IL-23 in response to Escherichia coli LPS than WT DCs. RANKL is induced on DCs within 24 h after LPS stimulation. OPG limits RANKL-RANK interactions between DCs, which can promote DC survival and elevated expression of proinflammatory cytokines. Survival of and cytokine production by OPG KO DCs are inhibited by soluble OPG; conversely, anti-OPG enhances survival and cytokine production by WT DCs. Bim KO DCs, like OPG KO, also survive longer and produce more TNF-alpha than WT DCs; however, unlike OPG KO, Bim KO DCs do not produce more IL-23. In addition, after inoculation with LPS, OPG KO mice produce more TNF-alpha and IL-12p40 than WT mice but not more IL-6. Thus, OPG regulates not only DC survival but also the nature of DC-dependent inflammatory responses.
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Citocinas/biosíntesis , Células Dendríticas/metabolismo , Osteoprotegerina/metabolismo , Animales , Proteínas Reguladoras de la Apoptosis/genética , Proteínas Reguladoras de la Apoptosis/inmunología , Proteínas Reguladoras de la Apoptosis/metabolismo , Proteína 11 Similar a Bcl2 , Supervivencia Celular/fisiología , Células Cultivadas , Citocinas/inmunología , Células Dendríticas/citología , Células Dendríticas/inmunología , Lipopolisacáridos/farmacología , Proteínas de la Membrana/genética , Proteínas de la Membrana/inmunología , Proteínas de la Membrana/metabolismo , Ratones , Ratones Noqueados , Osteoprotegerina/genética , Osteoprotegerina/inmunología , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas/inmunología , Proteínas Proto-Oncogénicas/metabolismo , Ligando RANK/genética , Ligando RANK/inmunología , Ligando RANK/metabolismo , Receptor Activador del Factor Nuclear kappa-B/genética , Receptor Activador del Factor Nuclear kappa-B/inmunología , Receptor Activador del Factor Nuclear kappa-B/metabolismo , Factores de TiempoRESUMEN
Recent studies have shown that dendritic cells (DCs) regulate B cell functions. In this study, we report that bone marrow (BM)-derived immature DCs, but not mature DCs, can inhibit BCR-induced proliferation of B cells in a contact-dependent manner. This inhibition is overcome by treatment with BAFF and is dependent on the BCR coreceptor CD22; however, it is not dependent on expression of the CD22 glycan ligand(s) produced by ST6Gal-I sialyltransferase. We found that a second CD22 ligand (CD22L) is expressed on CD11c(+) splenic and BM-derived DCs, which does not contain ST6Gal-I-generated sialic acids and which, unlike the B cell-associated CD22L, is resistant to neuraminidase treatment and sodium metaperiodate oxidation. Examination of splenic and BM B cell subsets in CD22 and ST6Gal-I knockout mice revealed that ST6Gal-I-generated B cell CD22L plays a role in splenic B cell development, whereas the maintenance of long-lived mature BM B cells depends only on CD22 and not on alpha2,6-sialic acids produced by ST6Gal-I. We propose that the two distinct CD22L have different functions. The alpha2,6-sialic acid-containing glycoprotein is important for splenic B cell subset development, whereas the DC-associated ST6Gal-I-independent CD22L may be required for the maintenance of long-lived mature B cells in the BM.