RESUMO
Interferon-λ (IFN-λ) acts on mucosal epithelial cells and thereby confers direct antiviral protection. In contrast, the role of IFN-λ in adaptive immunity is far less clear. Here, we report that mice deficient in IFN-λ signaling exhibited impaired CD8+ T cell and antibody responses after infection with a live-attenuated influenza virus. Virus-induced release of IFN-λ triggered the synthesis of thymic stromal lymphopoietin (TSLP) by M cells in the upper airways that, in turn, stimulated migratory dendritic cells and boosted antigen-dependent germinal center reactions in draining lymph nodes. The IFN-λ-TSLP axis also boosted production of the immunoglobulins IgG1 and IgA after intranasal immunization with influenza virus subunit vaccines and improved survival of mice after challenge with virulent influenza viruses. IFN-λ did not influence the efficacy of vaccines applied by subcutaneous or intraperitoneal routes, indicating that IFN-λ plays a vital role in potentiating adaptive immune responses that initiate at mucosal surfaces.
Assuntos
Imunidade Adaptativa/imunologia , Citocinas/imunologia , Imunidade nas Mucosas/imunologia , Interleucinas/imunologia , Imunidade Adaptativa/efeitos dos fármacos , Imunidade Adaptativa/genética , Animais , Formação de Anticorpos/efeitos dos fármacos , Formação de Anticorpos/imunologia , Células Cultivadas , Citocinas/genética , Citocinas/metabolismo , Células Dendríticas/efeitos dos fármacos , Células Dendríticas/imunologia , Células Dendríticas/virologia , Imunidade nas Mucosas/efeitos dos fármacos , Imunidade nas Mucosas/genética , Imunização/métodos , Vírus da Influenza A/efeitos dos fármacos , Vírus da Influenza A/imunologia , Vírus da Influenza A/fisiologia , Vacinas contra Influenza/administração & dosagem , Vacinas contra Influenza/imunologia , Interleucinas/administração & dosagem , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos DBA , Camundongos Knockout , Infecções por Orthomyxoviridae/imunologia , Infecções por Orthomyxoviridae/prevenção & controle , Infecções por Orthomyxoviridae/virologia , Receptores de Interferon/genética , Receptores de Interferon/imunologia , Receptores de Interferon/metabolismo , Linfopoietina do Estroma do TimoRESUMO
BACKGROUND: Patients with asthma often suffer from frequent respiratory viral infections and reduced virus clearance. Lung resident memory T cells provide rapid protection against viral reinfections. OBJECTIVE: Because the development of resident memory T cells relies on the lung microenvironment, we investigated the impact of allergen sensitization on the development of virus-specific lung resident memory T cells and viral clearance. METHODS: Mice were sensitized with house dust mite extract followed by priming with X47 and a subsequent secondary influenza infection. Antiviral memory T-cell response and protection to viral infection was assessed before and after secondary influenza infection, respectively. Gene set variation analysis was performed on data sets from the U-BIOPRED asthma cohort using an IFN-γ-induced epithelial cell signature and a tissue resident memory T-cell signature. RESULTS: Viral loads were higher in lungs of sensitized compared with nonsensitized mice after secondary infection, indicating reduced virus clearance. X47 priming induced fewer antiviral lung resident memory CD8 T cells and resulted in lower pulmonary IFN-γ levels in the lungs of sensitized as compared with nonsensitized mice. Using data from the U-BIOPRED cohort, we found that patients with enrichment of epithelial IFN-γ-induced genes in nasal brushings and bronchial biopsies were also enriched in resident memory T-cell-associated genes, had more epithelial CD8 T cells, and reported significantly fewer exacerbations. CONCLUSIONS: The allergen-sensitized lung microenvironment interferes with the formation of antiviral resident memory CD8 T cells in lungs and virus clearance. Defective antiviral memory response might contribute to increased susceptibility of patients with asthma to viral exacerbations.
Assuntos
Influenza Humana , Células T de Memória , Camundongos , Animais , Humanos , Pulmão , Linfócitos T CD8-Positivos , AlérgenosRESUMO
The molecular mechanisms behind infection and propagation of human restricted pathogens such as human norovirus (HuNoV) have defied interrogation because they were previously unculturable. However, human intestinal enteroids (HIEs) have emerged to offer unique ex vivo models for targeted studies of intestinal biology, including inflammatory and infectious diseases. Carbohydrate-dependent histo-blood group antigens (HBGAs) are known to be critical for clinical infection. To explore whether HBGAs of glycosphingolipids contribute to HuNoV infection, we obtained HIE cultures established from stem cells isolated from jejunal biopsies of six individuals with different ABO, Lewis, and secretor genotypes. We analyzed their glycerolipid and sphingolipid compositions and quantified interaction kinetics and the affinity of HuNoV virus-like particles (VLPs) to lipid vesicles produced from the individual HIE-lipid extracts. All HIEs had a similar lipid and glycerolipid composition. Sphingolipids included HBGA-related type 1 chain glycosphingolipids (GSLs), with HBGA epitopes corresponding to the geno- and phenotypes of the different HIEs. As revealed by single-particle interaction studies of Sydney GII.4 VLPs with glycosphingolipid-containing HIE membranes, both binding kinetics and affinities explain the patterns of susceptibility toward GII.4 infection for individual HIEs. This is the first time norovirus VLPs have been shown to interact specifically with secretor gene-dependent GSLs embedded in lipid membranes of HIEs that propagate GII.4 HuNoV ex vivo, highlighting the potential of HIEs for advanced future studies of intestinal glycobiology and host-pathogen interactions.
Assuntos
Antígenos de Grupos Sanguíneos/metabolismo , Infecções por Caliciviridae/metabolismo , Glicoesfingolipídeos/metabolismo , Mucosa Intestinal/metabolismo , Norovirus/metabolismo , Organoides/metabolismo , Ligação Viral , Infecções por Caliciviridae/patologia , Humanos , Mucosa Intestinal/patologia , Mucosa Intestinal/virologia , Organoides/patologia , Organoides/virologiaRESUMO
Type I and type III interferons (IFNs) can promote adaptive immune responses in mice and improve vaccine-induced resistance to viral infections. The adjuvant effect of type III IFN (IFN-λ) specifically boosts mucosal immunity by an indirect mechanism, involving IFN-λ-induced production of thymic stromal lymphopoietin (TSLP), a cytokine that activates immune cells. To date, it remained unclear whether the previously described adjuvant effect of type I IFN (IFN-α/ß) would also depend on TSLP and whether type I IFN stimulates different antibody subtypes. Here, we show that after infection with a live attenuated influenza virus, mice lacking functional type I IFN receptors failed to produce normal amounts of virus-specific IgG2c and IgA antibodies. In contrast, mice lacking functional IFN-λ receptors contained normal levels of virus-specific IgG2c but had reduced IgG1 and IgA antibody levels. When applied together with protein antigen, IFN-α stimulated the production of antigen-specific IgA and IgG2c to a greater extent than IgG1, irrespective of whether the mice expressed functional TSLP receptors and irrespective of whether the vaccine was applied by the intranasal or the intraperitoneal route. Taken together, these results demonstrate that the adjuvant activities of type I and type III IFNs are mechanistically distinct.IMPORTANCE Interferons can shape antiviral immune responses, but it is not well understood how they influence vaccine efficacy. We find that type I IFN preferentially promotes the production of antigen-specific IgG2c and IgA antibodies after infection with a live attenuated influenza virus or after immunization with influenza subunit vaccines. In contrast, type III IFN specifically enhances influenza virus-specific IgG1 and IgA production. The adjuvant effect of type I IFN was not dependent on TSLP, which is essential for the adjuvant effect of type III IFN. Type I IFN boosted vaccine-induced antibody production after immunization by the intranasal or the intraperitoneal route, whereas type III IFN exhibited its adjuvant activity only when the vaccine was delivered by the mucosal route. Our findings demonstrate that type I and type III IFNs trigger distinct pathways to enhance the efficacy of vaccines. This knowledge might be used to design more efficient vaccines against infectious diseases.
Assuntos
Imunidade Adaptativa/imunologia , Adjuvantes Imunológicos , Vacinas contra Influenza/imunologia , Interferons/imunologia , Animais , Formação de Anticorpos/imunologia , Citocinas , Modelos Animais de Doenças , Feminino , Imunidade nas Mucosas/imunologia , Imunização , Imunoglobulina A/imunologia , Imunoglobulina G/imunologia , Imunoglobulinas/genética , Interferon Tipo I , Interferons/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Infecções por Orthomyxoviridae/virologia , Receptores de Citocinas/genética , Vacinação , Interferon lambda , Linfopoietina do Estroma do TimoRESUMO
Retinoic acid (RA) is a critical regulator of the intestinal adaptive immune response. However, the intrinsic impact of RA on B cell differentiation in the regulation of gut humoral immunity in vivo has never been directly shown. To address this issue, we have been able to generate a mouse model where B cells specifically express a dominant-negative receptor α for RA. In this study, we show that the silencing of RA signaling in B cells reduces the numbers of IgA(+) Ab-secreting cells both in vitro and in vivo, suggesting that RA has a direct effect on IgA plasma cell differentiation. Moreover, the lack of RA signaling in B cells abrogates Ag-specific IgA responses after oral immunization and affects the microbiota composition. In conclusion, these results suggest that RA signaling in B cells through the RA receptor α is important to generate an effective gut humoral response and to maintain a normal microbiota composition.
Assuntos
Linfócitos B/imunologia , Linfócitos B/metabolismo , Imunização , Transdução de Sinais , Tretinoína/metabolismo , Animais , Linfócitos B/citologia , Diferenciação Celular/imunologia , Trato Gastrointestinal/imunologia , Trato Gastrointestinal/microbiologia , Expressão Gênica , Imunoglobulina A/biossíntese , Imunoglobulina A/imunologia , Camundongos , Camundongos Transgênicos , Microbiota/imunologia , Plasmócitos/citologia , Plasmócitos/imunologia , Plasmócitos/metabolismo , Receptores do Ácido Retinoico/genética , Receptores do Ácido Retinoico/metabolismoRESUMO
Although much explored, oral tolerance for treatment of autoimmune diseases still awaits the establishment of novel and effective vectors. We investigated whether the tolerogenic CTA1(R7K)-COL-DD fusion protein can be expressed in edible plants, to induce oral tolerance and protect against arthritis. The fusion protein was recombinantly expressed in Arabidopsis thaliana plants, which were fed to H-2(q) -restricted DBA/1 mice to assess the preventive effect on collagen-induced arthritis (CIA). The treatment resulted in fewer mice exhibiting disease and arthritis scores were significantly reduced. Immune suppression was evident in treated mice, and serum biomarkers for inflammation as well as anticollagen IgG responses were reduced. In spleen and draining lymph nodes, CD4(+) T-cell responses were reduced. Concomitant with a reduced effector T-cell activity with lower IFNγ, IL-13 and IL-17A production, we observed an increase in IL-10 production to recall antigen stimulation in vitro, suggesting reduced Th1, Th2 and Th17 activity subsequent to up-regulated IL-10 and regulatory T-cell (Treg) functions. This study shows that edible plants expressing a tolerogen were effective at stimulating CD4 T-cell tolerance and in protecting against CIA disease. Our study conveys optimism as to the potential of using edible plants for oral treatment of rheumatoid arthritis.
Assuntos
Arabidopsis/genética , Artrite Experimental/prevenção & controle , Toxina da Cólera/genética , Toxina da Cólera/farmacologia , Plantas Geneticamente Modificadas , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/farmacologia , Animais , Anti-Inflamatórios não Esteroides/farmacologia , Artrite Experimental/dietoterapia , Linfócitos T CD4-Positivos/efeitos dos fármacos , Linfócitos T CD4-Positivos/imunologia , Regulação da Expressão Gênica de Plantas , Interleucina-10/metabolismo , Masculino , Camundongos Endogâmicos DBA , Células Th17/efeitos dos fármacos , Células Th17/imunologiaRESUMO
Most vaccines developed against Chlamydia using animal models provide partial protection against a genital tract infection. However, protection against the oviduct pathology associated with infertility is highly variable and often has no defining immunological correlate. When comparing two adjuvants (CTA1-DD and a combination of Cholera toxin plus CpG-oligodeoxynucleotide-CT/CpG) combined with the chlamydial major outer membrane protein (MOMP) antigen and delivered via the intranasal (IN), sublingual (SL) or transcutaneous (TC) routes, we identified two vaccine groups with contrasting outcomes following infection. SL immunization with MOMP/CTA1-DD induced a 70% reduction in the incidence of oviduct pathology, without significantly altering the course of infection. Conversely, IN immunization with MOMP/CT/CpG prevented an ascending infection, but not the oviduct pathology. This anomaly presented a unique opportunity to study the mechanisms by which vaccines can prevent oviduct pathology, other than by controlling the infection. The IL-17 signaling in the oviducts was found to associate with both the enhancement of immunity to infection and the development of oviduct pathology. This conflicting role of IL-17 may provide some explanation for the discordance in protection between infection and disease and suggests that controlling immunopathology, as opposed to the rapid eradication of the infection, may be essential for an effective human chlamydial vaccine that prevents infertility.
Assuntos
Infecções por Chlamydia/imunologia , Infecções por Chlamydia/patologia , Chlamydia muridarum/imunologia , Imunidade , Interleucina-17/metabolismo , Transdução de Sinais/imunologia , Animais , Anticorpos Antibacterianos/imunologia , Especificidade de Anticorpos/imunologia , Proteínas da Membrana Bacteriana Externa/imunologia , Separação Celular , Infecções por Chlamydia/microbiologia , Infecções por Chlamydia/prevenção & controle , Citocinas/biossíntese , Feminino , Regulação da Expressão Gênica , Imunidade/genética , Mediadores da Inflamação/metabolismo , Cinética , Linfonodos/patologia , Linfócitos/imunologia , Camundongos , Infiltração de Neutrófilos , Oviductos/patologia , Baço/patologia , Vacinação , Vagina/imunologia , Vagina/patologiaRESUMO
Induction and regulation of specific intestinal immunoglobulin (Ig)A responses critically depend on dendritic cell (DC) subsets and the T cells they activate in the Peyer's patches (PP). We found that oral immunization with cholera toxin (CT) as an adjuvant resulted in migration-dependent changes in the composition and localization of PP DC subsets with increased numbers of cluster of differentiation (CD)103- conventional DC (cDC)2s and lysozyme-expressing DC (LysoDCs) in the subepithelial dome and of CD103+ cDC2s that expressed CD101 in the T cell zones, while oral ovalbumin (OVA) tolerization was instead associated with greater accumulation of cDC1s and peripherally induced regulatory T cells (pTregs) in this area. Decreased IgA responses were observed after CT-adjuvanted immunization in huCD207DTA mice lacking CD103+ cDC2s, while oral OVA tolerization was inefficient in cDC1-deficient Batf3-/- mice. Using OVA transgenic T cell receptor CD4 T cell adoptive transfer models, we found that co-transferred endogenous wildtype CD4 T cells can hinder the induction of OVA-specific IgA responses through secretion of interleukin-10. CT could overcome this blocking effect, apparently through a modulating effect on pTregs while promoting an expansion of follicular helper T cells. The data support a model where cDC1-induced pTreg normally suppresses PP responses for any given antigen and where CT's oral adjuvanticity effect is dependent on promoting follicular helper T cell responses through induction of CD103+ cDC2s.
Assuntos
Antígenos CD , Antígeno CD11b , Movimento Celular , Toxina da Cólera , Células Dendríticas , Tolerância Imunológica , Imunização , Imunoglobulina A , Cadeias alfa de Integrinas , Camundongos Knockout , Ovalbumina , Nódulos Linfáticos Agregados , Linfócitos T Reguladores , Animais , Camundongos , Nódulos Linfáticos Agregados/imunologia , Nódulos Linfáticos Agregados/metabolismo , Cadeias alfa de Integrinas/metabolismo , Antígenos CD/metabolismo , Células Dendríticas/imunologia , Imunoglobulina A/imunologia , Imunoglobulina A/metabolismo , Administração Oral , Toxina da Cólera/imunologia , Ovalbumina/imunologia , Ovalbumina/administração & dosagem , Linfócitos T Reguladores/imunologia , Antígeno CD11b/metabolismo , Fatores de Transcrição de Zíper de Leucina Básica/metabolismo , Fatores de Transcrição de Zíper de Leucina Básica/genética , Camundongos Transgênicos , Camundongos Endogâmicos C57BL , Transferência Adotiva , Proteínas RepressorasRESUMO
Adjuvants have traditionally been appreciated for their immunoenhancing effects, whereas their impact on immunological memory has largely been neglected. In this paper, we have compared three mechanistically distinct adjuvants: aluminum salts (Alum), Ribi (monophosphoryl lipid A), and the cholera toxin A1 fusion protein CTA1-DD. Their influence on long-term memory development was dramatically different. Whereas a single immunization i.p. with 4-hydroxy-3-nitrophenyl acetyl (NP)-chicken γ-globulin and adjuvant stimulated serum anti-NP IgG titers that were comparable at 5 wk, CTA1-DD-adjuvanted responses were maintained for >16 mo with a half-life of anti-NP IgG â¼36 wk, but <15 wk after Ribi or Alum. A CTA1-DD dose-dependent increase in germinal center (GC) size and numbers was found, with >60% of splenic B cell follicles hosting GC at an optimal CTA1-DD dose. Roughly 7% of these GC were NP specific. This GC-promoting effect correlated well with the persistence of long-term plasma cells in the bone marrow and memory B cells in the spleen. CTA1-DD also facilitated increased somatic hypermutation and affinity maturation of NP-specific IgG Abs in a dose-dependent fashion, hence arguing that large GC not only promotes higher Ab titers but also high-quality Ab production. Adoptive transfer of splenic CD80(+), but not CD80(-), B cells, at 1 y after immunization demonstrated functional long-term anti-NP IgG and IgM memory cells. To our knowledge, this is the first report to specifically compare and document that adjuvants can differ considerably in their support of long-term immune responses. Differential effects on the GC reaction appear to be the basis for these differences.
Assuntos
Adjuvantes Imunológicos/fisiologia , Subpopulações de Linfócitos B/citologia , Subpopulações de Linfócitos B/imunologia , Diferenciação Celular/imunologia , Toxina da Cólera/sangue , Toxina da Cólera/fisiologia , Memória Imunológica , Adjuvantes Imunológicos/sangue , Compostos de Alúmen/metabolismo , Compostos de Alúmen/farmacologia , Animais , Subpopulações de Linfócitos B/metabolismo , Esqueleto da Parede Celular/sangue , Esqueleto da Parede Celular/fisiologia , Fatores Corda/sangue , Fatores Corda/fisiologia , Relação Dose-Resposta Imunológica , Feminino , Centro Germinativo/imunologia , Centro Germinativo/metabolismo , Imunoglobulina A/biossíntese , Imunoglobulina A/sangue , Imunoglobulina G/biossíntese , Imunoglobulina G/sangue , Lipídeo A/análogos & derivados , Lipídeo A/sangue , Lipídeo A/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Dados de Sequência Molecular , Plasmócitos/citologia , Plasmócitos/imunologia , Plasmócitos/metabolismo , Proteínas Recombinantes de Fusão/sangue , Proteínas Recombinantes de Fusão/fisiologia , Fatores de TempoRESUMO
A detailed understanding of how activation of innate immunity can be exploited to generate more effective vaccines is critically required. However, little is known about how to target adjuvants to generate safer and better vaccines. In this study, we describe an adjuvant that, through complement activation and binding to follicular dendritic cells (FDC), dramatically enhances germinal center (GC) formation, which results in greatly augmented Ab responses. The nontoxic CTA1-DD adjuvant hosts the ADP-ribosylating CTA1 subunit from cholera toxin and a dimer of the D fragment from Staphylococcus aureus protein A. We found that T cell-dependent, but not -independent, responses were augmented by CTA1-DD. GC reactions and serum Ab titers were both enhanced in a dose-dependent manner. This effect required complement activation, a property of the DD moiety. Deposition of CTA1-DD to the FDC network appeared to occur via the conduit system and was dependent on complement receptors on the FDC. Hence, Cr2(-/-) mice failed to augment GC reactions and exhibited dramatically reduced Ab responses, whereas Ribi adjuvant demonstrated unperturbed adjuvant function in these mice. Noteworthy, the adjuvant effect on priming of specific CD4 T cells was found to be intact in Cr2(-/-) mice, demonstrating that the CTA1-DD host both complement-dependent and -independent adjuvant properties. This is the first demonstration, to our knowledge, of an adjuvant that directly activates complement, enabling binding of the adjuvant to the FDC, which subsequently strongly promoted the GC reaction, leading to augmented serum Ab titers and long-term memory development.
Assuntos
Adjuvantes Imunológicos/farmacologia , Toxina da Cólera/imunologia , Ativação do Complemento/imunologia , Células Dendríticas Foliculares/imunologia , Receptores de Complemento 3d/imunologia , Proteínas Recombinantes de Fusão/imunologia , Adjuvantes Imunológicos/síntese química , Animais , Separação Celular , Toxina da Cólera/síntese química , Toxina da Cólera/farmacologia , Células Dendríticas Foliculares/metabolismo , Ensaio de Imunoadsorção Enzimática , Citometria de Fluxo , Imunofluorescência , Centro Germinativo/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Knockout , Receptores de Complemento/imunologia , Proteínas Recombinantes de Fusão/síntese química , Proteínas Recombinantes de Fusão/farmacologiaRESUMO
Curative therapies against autoimmune diseases are lacking. Indeed, most of the currently available treatments are only targeting symptoms. We have developed a novel strategy for a therapeutic vaccine against autoimmune diseases based on intranasal administration of a fusion protein tolerogen, which consists of a mutant, enzymatically inactive, cholera toxin A1 (CTA1)-subunit genetically fused to disease-relevant high-affinity peptides and a dimer of D-fragments from protein A (DD). The CTA1 R7K mutant - myelin oligodendrocyte glycoprotein (MOG), or proteolipid protein (PLP) - DD (CTA1R7K-MOG/PLP-DD) fusion proteins effectively reduced clinical symptoms in the experimental autoimmune encephalitis model of multiple sclerosis. The treatment induced Tr1 cells, in the draining lymph node, which produced interleukin (IL)-10 and suppressed effector clusters of differentiation 4+ T-cell responses. This effect was dependent on IL-27 signaling because treatment was ineffective in bone marrow chimeras lacking IL-27Ra within their hematopoietic compartment. Single-cell RNA sequencing of dendritic cells in draining lymph nodes demonstrated distinct gene transcriptional changes of classic dendritic cells 1, including enhanced lipid metabolic pathways, induced by the tolerogenic fusion protein. Thus, our results with the tolerogenic fusion protein demonstrate the possibility to vaccinate and protect against disease progression by reinstating tolerance in multiple sclerosis and other autoimmune diseases.
Assuntos
Esclerose Múltipla , Linfócitos T Reguladores , Humanos , Administração Intranasal , Toxina da Cólera , Linfócitos T CD4-Positivos , Esclerose Múltipla/tratamento farmacológicoRESUMO
Germinal center (GC) formation and antibody production in lymph node follicles require coordinated interactions between B-cells, T-cells and dendritic cells (DCs), orchestrated by the extracellular matrix-rich reticular fiber (RF) network. We describe a unique laminin 523-containing RF network around and between follicles that associates with PDGFrecßhighCCL19lowgp38low fibroblastic reticular cells (FRC). In the absence of FRC expression of laminin α5 (pdgfrb-cre:Lama5fl/fl), pre-Tfh-cells, B-cells and DCs are displaced from follicle borders, correlating with fewer Tfh-cells and GC B-cells. Total DCs are not altered in pdgfrb-cre:Lama5fl/fl mice, but cDC2s, which localize to laminin α5 in RFs at follicle borders, are reduced. In addition, PDGFrecßhighCCL19lowgp38low FRCs show lower Ch25h expression, required for 7α,25-dihydroxycholesterol synthesis that attracts pre-Tfh-cells, B-cells and DCs to follicle borders. We propose that RF basement membrane components represent a type of tissue memory that guides the localization and differentiation of both specialized FRC and DC populations, required for normal lymph node function.
RESUMO
Most cases of gastric cancer are caused by chronic Helicobacter pylori infection, but the lack of early onco-diagnostics and a high risk for antibiotic resistance hampers early intervention through eradication of H. pylori infection by antibiotics. We reported on a protective mechanism where H. pylori gastric mucosal attachment can be reduced by natural antibodies that block the binding of its attachment protein BabA. Here we show that challenge infection with H. pylori induced response of such blocking antibodies in both human volunteers and in rhesus macaques, that mucosal vaccination with BabA protein antigen induced blocking antibodies in rhesus macaques, and that vaccination in a mouse model induced blocking antibodies that reduced gastric mucosal inflammation, preserved the gastric juice acidity, and fully protected the mice from gastric cancer caused by H. pylori.
RESUMO
Immunizations via the i.n. and intravaginal (ivag) routes effectively generate strong genital tract antibody-mediated immunity. To what extent the same is true for T-cell responses is incompletely known. Therefore, we set out to investigate optimal conditions for stimulation of genital tract CD4(+) T-cell responses, using adoptive transfer of mouse DO11.10 TCR transgenic T cells specific for OVA and OVA conjugated to cholera toxin (CT) as an immunogen. We observed that progesterone was required for a T-cell response following ivag immunization, whereas estradiol prevented a response. Although i.n. immunization stimulated OVA-specific CD4(+) T-cell responses in the draining LNs, it was substantially less effective compared to ivag. More importantly, an ivag booster immunization was absolutely required to attract T cells to the genital tract mucosa itself. While clinical use of CT is precluded because of its toxicity, we developed a combined adjuvant vector based on a non-toxic derivative of CT and immune-stimulating complexes. The CTA1-DD/immune-stimulating complexes (ISCOMs) adjuvant together with major outer membrane protein was effective at stimulating genital tract CD4(+) T-cell immunity and protection against a live chlamydial infection, which holds promise for the development of mucosal vaccines against sexually transmitted infections.
Assuntos
Linfócitos T CD4-Positivos/metabolismo , Genitália Feminina/patologia , Imunidade nas Mucosas , Administração Intravaginal , Transferência Adotiva , Animais , Antígenos/imunologia , Linfócitos T CD4-Positivos/efeitos dos fármacos , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/patologia , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Toxina da Cólera/administração & dosagem , Estradiol/administração & dosagem , Feminino , Humanos , Imunidade nas Mucosas/efeitos dos fármacos , Imunidade nas Mucosas/genética , Imunização Secundária , Ativação Linfocitária/efeitos dos fármacos , Ativação Linfocitária/genética , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Transgênicos , Ovalbumina/administração & dosagem , Ovalbumina/imunologia , Progesterona/administração & dosagemRESUMO
While a primary genital tract infection with C. trachomatis stimulates partial-protection against re-infection, it may also result in severe inflammation and tissue destruction. Here we have dissected whether functional compartments exist in the genital tract that restrict Th1-mediated protective immunity. Apart from the Th1-subset, little is known about the role of other CD4(+) T cell subsets in response to a genital tract chlamydial infection. Therefore, we investigated CD4(+) T cell subset differentiation in the genital tract using RT-PCR for expression of critical transcription factors and cytokines in the upper (UGT) and lower genital tract (LGT) of female C57BL/6 mice in response to C. trachomatis serovar D infection. We found that the Th1 subset dominated the UGT, as IFN-γ and T-bet mRNA expression were high, while GATA-3 was low following genital infection with C. trachomatis serovar D. By contrast, IL-10 and GATA-3 mRNA dominated the LGT, suggesting the presence of Th2 cells. These functional compartments also attracted regulatory T cells (Tregs) differently as increased FoxP3 mRNA expression was seen primarily in the UGT. Although IL-17A mRNA was somewhat up-regulated in the LGT, no significant change in RORγ-t mRNA expression was observed, suggesting no involvement of Th17 cells. The dichotomy between the LGT and UGT was maintained during infection by IL-10 because in IL-10-deficient mice the distinction between the two compartments was completely lost and a dramatic shift to the predominance of Th1 cells in the LGT occurred. Unexpectedly, the major source of IL-10 was CD11c(+) CD11b(+) DC, probably creating an anti-inflammatory privileged site in the LGT.
Assuntos
Infecções por Chlamydia/imunologia , Chlamydia trachomatis/imunologia , Células Dendríticas/imunologia , Genitália Feminina/imunologia , Interleucina-10/metabolismo , Células Th1/imunologia , Animais , Western Blotting , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/microbiologia , Linfócitos T CD4-Positivos/patologia , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/microbiologia , Linfócitos T CD8-Positivos/patologia , Linhagem Celular , Infecções por Chlamydia/microbiologia , Infecções por Chlamydia/patologia , Citocinas/metabolismo , Células Dendríticas/microbiologia , Células Dendríticas/patologia , Ensaio de Imunoadsorção Enzimática , Feminino , Humanos , Imunidade Celular , Técnicas Imunoenzimáticas , Rim/citologia , Rim/virologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , RNA Mensageiro/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Linfócitos T Reguladores/imunologia , Linfócitos T Reguladores/microbiologia , Linfócitos T Reguladores/patologia , Células Th1/microbiologia , Células Th1/patologia , Células Th17/imunologia , Células Th17/microbiologia , Células Th17/patologia , Células Th2/imunologia , Células Th2/microbiologia , Células Th2/patologiaRESUMO
Recently, we reported that CD40(-/-) mice, exhibiting exclusively T cell-independent IgA class switch recombination (CSR), demonstrated near normal levels of IgA plasma cells in the gut lamina propria (LP), despite the complete lack of germinal centers (GCs). In this study, we have extended our analysis focusing on how to reconcile these findings using flow cytometry and molecular markers for IgA CSR. In agreement with our previous results with small intestinal LP, the colon LP was found to host IgA CSR only when lymphoid follicles were present. Thus, no IgA CSR was observed in the nonorganized colon LP. By contrast, the Peyer's patch (PP) was the dominant IgA CSR site in both CD40(-/-) and wild type (WT) mice, and they both hosted similar levels of mRNA expression for B cell activating factor of the TNF family, a proliferation inducing ligand, and inducible NO synthase, potential switch-factors for IgA. Unexpectedly, we found that PP B cells undergoing IgA CSR were GL7-intermediate. These cells had not undergone somatic hypermutations (SHMs), whereas GL7-high cells in WT PP, which exhibited GCs, were heavily mutated. Moreover, IgA plasma cells in the LP of CD40(-/-) mice demonstrated few mutations in their Ig V regions, whereas WT LP B cells from different sites showed extensive SHMs, which were also clonally related. Therefore, IgA CSR can occur in PP at a stage preceding manifest GC (GL7-intermediate), whereas SHM require GC formations (GL7-high). These findings reconcile that IgA CSR can occur in PP in the absence of GC with the fact that CD40(-/-) mice host near normal levels of IgA plasma cells in the LP.
Assuntos
Centro Germinativo/imunologia , Imunoglobulina A/imunologia , Switching de Imunoglobulina/imunologia , Intestinos/imunologia , Nódulos Linfáticos Agregados/imunologia , Animais , Antígenos CD40/deficiência , Antígenos CD40/genética , Antígenos CD40/imunologia , Separação Celular , Citometria de Fluxo , Imunidade nas Mucosas/imunologia , Imunoglobulina A/genética , Imuno-Histoquímica , Intestinos/citologia , Camundongos , Camundongos Knockout , Mucosa/imunologia , Plasmócitos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Hipermutação Somática de Imunoglobulina/imunologia , Linfócitos T/imunologiaRESUMO
Mast cell activation is one of the most dramatic immune-mediated responses the body can encounter. In the worst scenario (i.e., anaphylaxis), this response is fatal. However, the importance of mast cells as initiators and effectors of both innate and adaptive immunity in healthy individuals has recently been appreciated. It was reported that mast cell activation can be used as an adjuvant to promote Ag-specific humoral immune responses upon vaccination. In this study, we have used a clinically relevant mucosal adjuvant, cholera toxin A1 subunit (CTA1)-DD, which is a fusion protein composed of CTA1, the ADP-ribosylating part of cholera toxin, and DD, two Ig-binding domains derived from Staphylococcus aureus protein A. CTA1-DD in combination with polyclonal IgG induced degranulation and production of TNF-alpha from mouse mast cells. Furthermore, CTA1-DD and polyclonal IgG complex induced mast cell degranulation in mouse skin tissue and nasal mucosa. We also found that intranasal immunization with hapten (4-hydroxy-3-nitrophenyl) acetyl (NP) coupled to chicken gammaglobulin admixed with CTA1-DD complexed with polyclonal IgG greatly enhanced serum IgG anti-NP Ab responses and stimulated higher numbers of NP-specific plasma cells in the bone marrow as compared with that observed in mice immunized with NP-chicken gammaglobulin with CTA1-DD alone. This CTA1-DD/IgG complex-mediated enhancement was mast cell dependent because it was absent in mast cell-deficient Kit(W-sh/W-sh) mice. In conclusion, our data suggest that a clinically relevant adjuvant, CTA1-DD, exerts additional augmenting effects through activation of mucosal mast cells, clearly demonstrating that mast cells could be further exploited for improving the efficacy of mucosal vaccines.
Assuntos
Adjuvantes Imunológicos/administração & dosagem , Complexo Antígeno-Anticorpo/metabolismo , Toxina da Cólera/administração & dosagem , Imunidade nas Mucosas , Imunoglobulina G/metabolismo , Mastócitos/imunologia , Mastócitos/metabolismo , Proteínas Recombinantes de Fusão/administração & dosagem , Adjuvantes Imunológicos/fisiologia , Animais , Complexo Antígeno-Anticorpo/biossíntese , Células Cultivadas , Toxina da Cólera/fisiologia , Imunidade nas Mucosas/genética , Imunoglobulina G/biossíntese , Mucosa Intestinal/imunologia , Mucosa Intestinal/metabolismo , Mucosa Intestinal/patologia , Mastócitos/transplante , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C3H , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Mutantes , Mutagênese Insercional/imunologia , Proteínas Proto-Oncogênicas c-kit/deficiência , Proteínas Proto-Oncogênicas c-kit/genética , Proteínas Recombinantes de Fusão/fisiologiaRESUMO
Accumulating evidence suggests that the dichotomy between tolerance and active IgA immunity in mucosal immune responses is regulated at the APC level. Therefore, immunomodulation of the APC could be an effective mechanism to control the two response patterns. In this study, we demonstrate that ADP-ribosylation controls the outcome of tolerance or active effector T cell immunity to an internal peptide p323-339 from OVA inserted into the cholera toxin (CT)-derived CTA1-OVA-DD adjuvant. We found that a single point mutation, CTA1R7K-OVA-DD, resulting in lack of enzymatic activity, promoted peptide-specific tolerance in TCR transgenic CD4(+) T cells following a single intranasal (i.n.) treatment. The CTA1R7K-OVA-DD-induced tolerance was strong, long-lasting, and impaired the ability of adoptively transferred naive peptide-specific CD4(+) T cells to respond to Ag-challenge, irrespective if this was given i.p or i.n. The tolerance correlated with induction of regulatory T cells of the regulatory T type 1 characterized by CD25(-)Foxp3(-)CD4(+) T cells producing IL-10. In contrast, in IL-10-deficient mice, no peptide-specific tolerance was observed, and these mice exhibited unimpaired CD4(+) T cell responsiveness to recall Ag irrespective of if they were untreated (PBS) or treated i.n. with CTA1R7K-OVA-DD. Thus, for the first time, we can provide unequivocal proof that ADP-ribosylation can control the outcome of mucosal Ag exposure from tolerance to an enhanced effector CD4(+) T cell response. The exploitation of this system for clinical treatment of autoimmune diseases is discussed.
Assuntos
ADP Ribose Transferases/metabolismo , Tolerância Imunológica , Imunidade nas Mucosas , Mucosa Nasal/imunologia , ADP Ribose Transferases/fisiologia , Administração Intranasal , Animais , Células Cultivadas , Feminino , Tolerância Imunológica/genética , Imunidade nas Mucosas/genética , Interleucina-10/biossíntese , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Nus , Camundongos Transgênicos , Ovalbumina/administração & dosagem , Ovalbumina/genética , Ovalbumina/imunologia , Fragmentos de Peptídeos/administração & dosagem , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/imunologia , Proteínas Recombinantes de Fusão/administração & dosagem , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/imunologia , Linfócitos T Reguladores/imunologia , Linfócitos T Reguladores/metabolismoRESUMO
T helper 17 (TH17) cells located at the Peyer's patch (PP) inductive site and at the lamina propria effector site of the intestinal immune system are responsive to both pathogenic and commensal bacteria. Their plasticity to convert into follicular helper T (TFH) cells has been proposed to be central to gut immunoglobulin A (IgA) responses. Here, we used an IL-17A fate reporter mouse and an MHC-II tetramer to analyze antigen-specific CD4+ T cell subsets and isolate them for single-cell RNA sequencing after oral immunization with cholera toxin and ovalbumin. We found a TFH-dominated response with only rare antigen-specific TH17 cells (<8%) in the PP. A clonotypic analysis provided little support that clonotypes were shared between TFH and TH17 cells, arguing against TH17 plasticity as a major contributor to TFH differentiation. Two mouse models of TH17 deficiency confirmed that gut IgA responses to oral immunization do not require TH17 cells, with CD4CreRorcfl/fl mice exhibiting normal germinal centers in PP and unperturbed total IgA production in the intestine.
Assuntos
Imunoglobulina A , Nódulos Linfáticos Agregados , Células Th17 , Animais , Antígenos/imunologia , Toxina da Cólera , Imunização , Imunoglobulina A/imunologia , Camundongos , Nódulos Linfáticos Agregados/citologia , Nódulos Linfáticos Agregados/imunologia , Células Th17/imunologia , VacinaçãoRESUMO
Type 1 conventional dendritic cells (cDC1) efficiently cross-present antigens that prime cytotoxic CD8+ T cells. cDC1 therefore constitute conceivable targets in cancer vaccine development. We generated recombinant fusion cancer vaccines that aimed to concomitantly deliver tumor antigen and adjuvant to CD103+ migratory cDC1, following intranasal administration. The fusion vaccine constructs comprised a cDC1-targeting anti-CD103 single chain antibody (aCD103) and a cholera toxin A1 (CTA1) subunit adjuvant, fused with MHC class I and II- or class II-restricted tumor cell antigens to generate a CTA1-I/II-aCD103 vaccine and a CTA1-II-aCD103 vaccine. The immunostimulatory and anti-tumor efficacy of these vaccines was evaluated in murine B16F1-ovalbumin (OVA) melanoma models in C57BL/6 J mice. The CTA1-I/II-aCD103 vaccine was most efficacious and triggered robust tumor antigen-specific CD8+ T cell responses along with a Th17-polarized CD4+ T cell response. This vaccine construct reduced the local growth of implanted B16F1-OVA melanomas and efficiently prevented hematogenous lung metastasis after prophylactic and therapeutic vaccination. Anti-tumor effects of the CTA1-I/II-aCD103 vaccine were antigen-specific and long-lasting. These results imply that adjuvant-containing recombinant fusion vaccines that target and activate cDC1 trigger effective anti-tumor immunity to control tumor growth and metastasis.