RESUMO
We previously reported monophosphoryl lipid A (MPL) and synthetic cord factor trehalose-6,6'-dicorynomycolate (TDCM) significantly increase Ab responses to T cell-independent type 2 Ags (TI-2 Ags) in a manner dependent on B cell-intrinsic TLR4 expression, as well as MyD88 and TRIF proteins. Given the capacity of MPL to drive type I IFN production, we aimed to investigate the extent to which type I IFN receptor (IFNAR) signaling was required for TI-2 responses and adjuvant effects. Using Ifnar1-/- mice and IFNAR1 Ab blockade, we found that IFNAR signaling is required for optimal early B cell activation, expansion, and Ab responses to nonadjuvanted TI-2 Ags, including the pneumococcal vaccine. Further study demonstrated that B cell-intrinsic type I IFN signaling on B cells was essential for normal TI-2 Ab responses. In particular, TI-2 Ag-specific B-1b cell activation and expansion were significantly impaired in Ifnar1-/- mice; moreover, IFNAR1 Ab blockade similarly reduced activation, expansion, and differentiation of IFNAR1-sufficient B-1b cells in Ifnar1-/- recipient mice, indicating that B-1b cell-expressed IFNAR supports TI-2 Ab responses. Consistent with these findings, type I IFN significantly increased the survival of TI-2 Ag-activated B-1b cells ex vivo and promoted plasmablast differentiation. Nonetheless, MPL/TDCM adjuvant effects, which were largely carried out through innate B cells (B-1b and splenic CD23- B cells), were independent of type I IFN signaling. In summary, our study highlights an important role for B-1b cell-expressed IFNAR in promoting responses to nonadjuvanted TI-2 Ags, but it nonetheless demonstrates that adjuvants which support innate B cell responses may bypass this requirement.
Assuntos
Formação de Anticorpos , Linfócitos B , Camundongos , Animais , Antígenos , Polissacarídeos , Receptores de Antígenos de Linfócitos B , Adjuvantes Imunológicos , Camundongos Knockout , Camundongos Endogâmicos C57BLRESUMO
The inability of T cell-independent type 2 (TI-2) Ags to induce recall responses is a poorly understood facet of humoral immunity, yet critically important for improving vaccines. Using normal and VHB1-8 transgenic mice, we demonstrate that B cell-intrinsic PD-1 expression negatively regulates TI-2 memory B cell (Bmem) generation and reactivation in part through interacting with PDL1 and PDL2 on non-Ag-specific cells. We also identified a significant role for PDL2 expression on Bmems in inhibiting reactivation and Ab production, thereby revealing a novel self-regulatory mechanism exists for TI-2 Bmems This regulation impacts responses to clinically relevant vaccines, because PD-1 deficiency was associated with significantly increased Ab boosting to the pneumococcal vaccine after both vaccination and infection. Notably, we found a B cell-activating adjuvant enabled even greater boosting of protective pneumococcal polysaccharide-specific IgG responses when PD-1 inhibition was relieved. This work highlights unique self-regulation by TI-2 Bmems and reveals new opportunities for significantly improving TI-2 Ag-based vaccine responses.
Assuntos
Linfócitos B/imunologia , Infecções Pneumocócicas/imunologia , Vacinas Pneumocócicas/imunologia , Receptor de Morte Celular Programada 1/metabolismo , Linfócitos T/imunologia , Animais , Antígeno B7-H1/metabolismo , Homeostase , Imunidade Humoral , Imunogenicidade da Vacina , Memória Imunológica , Ativação Linfocitária , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteína 2 Ligante de Morte Celular Programada 1/metabolismo , Ligação Proteica , Transdução de SinaisRESUMO
The roles distinct B cell subsets play in clonal expansion, isotype switching, and memory B cell differentiation in response to T cell-independent type 2 Ags (TI-2 Ags) has been understudied. Using sorted B cells from VHB1-8 knock-in mice, we evaluated B-1b, marginal zone, and follicular B cell responses to the TI-2 Ag, NP-Ficoll. All subsets extensively divided in response to NP-Ficoll. Nonetheless, B-1b cells exhibited significantly increased IgG switching and differentiation into Ab-secreting cells (ASC)-a finding that coincided with increased AgR signaling capacity and Blimp1 expression by B-1b cells. All subsets formed memory cells and expressed markers previously identified for T cell-dependent memory B cells, including CD80, PDL2, and CD73, although B-1b cells generated the greatest number of memory cells with higher frequencies of IgG- and CD80-expressing cells. Despite memory formation, secondary immunization 4 wk after primary immunization did not increase NP-specific IgG. However, boosting occurred in B-1b cell-recipient mice when IgG levels declined. CD80+ memory B-1b cells divided, class switched, and differentiated into ASC in response to Ag in vivo, but this was inhibited in the presence of NP-specific IgG. Furthermore, CD80 blockade significantly increased memory B-1b cell division and differentiation to ASC upon Ag restimulation. Collectively, these findings demonstrate B-1b, marginal zone B, and follicular B subsets significantly contribute to the TI-2 Ag-specific memory B cell pool. In particular, we show B-1b cells generate a functional CD80-regulated memory population that can be stimulated to divide and differentiate into ASC upon Ag re-encounter when Ag-specific IgG levels decline.
Assuntos
Subpopulações de Linfócitos B/imunologia , Memória Imunológica/imunologia , Linfócitos T/imunologia , Animais , Antígenos T-Independentes/imunologia , Antígeno B7-1/imunologia , Diferenciação Celular/imunologia , Divisão Celular/imunologia , Switching de Imunoglobulina/imunologia , Imunoglobulina G/imunologia , Ativação Linfocitária/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Transdução de Sinais/imunologiaAssuntos
Carcinógenos/toxicidade , Adutos de DNA , Ocratoxinas/toxicidade , Efeitos Tardios da Exposição Pré-Natal , Testículo/efeitos dos fármacos , Animais , Animais Recém-Nascidos , Feminino , Rim/efeitos dos fármacos , Rim/metabolismo , Masculino , Troca Materno-Fetal , Camundongos , Camundongos Endogâmicos BALB C , Gravidez , Testículo/metabolismoRESUMO
Fetal mice are more sensitive to chemical carcinogens than are adults. We previously demonstrated that resistant offspring of a DBA/2 x (C57BL/6 x DBA2) backcross exhibited a high incidence of lung tumors 12-13 mo after transplacental exposure to 3-methylcholanthrene (MC). We compared the effects of in utero treatment with MC on lung tumor incidence in the offspring of intermediately susceptible BALB/c (C), resistant C57BL/6 (B6), and reciprocal crosses between these strains. Pregnant mice were treated with 45 mg/kg of MC on day 17 of gestation and tumor incidence, multiplicity, and the Ki-ras mutational spectrum determined in the offspring 12-18 mo after birth. Tumor incidences in C mice and reciprocal crosses were 86% and 100%, respectively, while B6 mice demonstrated resistance to tumorigenesis, with a tumor incidence of 11%. Tumor multiplicities in C, B6C, CB6, and B6 mice were 3.3 +/- 3.2, 5.8 +/- 3.2, 5.0 +/- 2.7, and <0.1, respectively. Ki-ras mutations, which occurred chiefly in the K(s) allele (96%), were found in 79-81% of reciprocally crossed F1 mice, 64% of C mice, and 50% of B6 mice, with the Val(12), Asp(12), and Arg(13) mutations associated with more aggressive tumors. A subset of these mice was used to demonstrate the utility of computer tomography (CT) for the visualization and measurement of lung tumors in the submillimeter range in vivo. Based on known genetic differences in murine strains for lung cancer, our results suggest the presence of a previously unidentified genetic factor(s) which appears to specifically influence lung tumorigenesis following exposure to carcinogens during fetal development.
Assuntos
Neoplasias Pulmonares/induzido quimicamente , Neoplasias Pulmonares/genética , Lesões Pré-Natais/genética , Animais , Feminino , Desenvolvimento Fetal/efeitos dos fármacos , Feto/efeitos dos fármacos , Genes ras/genética , Endogamia , Neoplasias Pulmonares/diagnóstico por imagem , Metilcolantreno/toxicidade , Camundongos , Tomografia Computadorizada por Raios X , ÚteroRESUMO
Adenovirus E1A sensitizes cells to the cytotoxic action of tumor necrosis factor alpha (TNF-alpha). This effect has been attributed to direct blockade of NF-kappaB activation, as well as to increased activation of components of the apoptotic pathway and decreases in inhibitors of apoptosis. In this report we evaluated the mechanism by which E1A modulates the expression of the cytokine-inducible cytoprotective genes manganese superoxide dismutase (MnSOD), interleukin-6 (IL-6), and ferritin heavy chain (FH). We observed that E1A blocks induction of MnSOD, IL-6, and FH by TNF-alpha or IL-1alpha. Because NF-kappaB plays a role in cytokine-dependent induction of MnSOD, IL-6, and FH, we assessed the effect of E1A on NF-kappaB in cells treated with TNF. IkappaB, the inhibitor of NF-kappaB, was degraded similarly in the presence and absence of E1A. TNF induced a quantitatively and temporally equivalent activation of NF-kappaB in control and E1A-transfected cells. However, TNF-dependent acetylation of NF-kappaB was diminished in cells expressing E1A. E1A mutants unable to bind p400 or the Rb family proteins were still capable of repressing TNF-dependent induction of FH. However, mutants of E1A that abrogated binding of p300/CBP blocked the ability of E1A to repress TNF-dependent induction of FH. These results suggest that p300/CBP is a critical control point in NF-kappaB-dependent transcriptional regulation of cytoprotective genes by cytokines.
Assuntos
Proteínas E1A de Adenovirus/metabolismo , Citocinas/metabolismo , Ferritinas/química , Interleucina-6/metabolismo , Superóxido Dismutase/metabolismo , Animais , Ferritinas/metabolismo , Camundongos , Mutação , NF-kappa B/metabolismo , Células NIH 3T3 , Estrutura Terciária de Proteína , Transfecção , Fatores de Transcrição de p300-CBP/metabolismoRESUMO
Mutations in Ki-ras occur in approximately 30-50% of patients with adenocarcinoma (AC) of the lung. We previously reported the development of a bitransgenic mouse model that expressed the human Ki-ras(G12C) allele in a lung-specific, tetracycline-inducible manner and gave rise to benign lung tumors. In the current study, these benign tumors, which represent relatively early lesions in neoplastic progression, were analyzed for molecular alterations secondary to mutant Ki-ras expression to determine the gene(s) that contribute to adenoma (AD) development. Tumors were removed following doxycycline (DOX) treatment for 9 and 12 mo and examined for alterations in cell-cycle regulatory genes. Quantification of mRNA expression for cyclin D1, retinoblastoma, p16(Ink4a), p19(Arf), and survivin was carried out by real-time PCR. All of the tumors examined exhibited a mean reduction of approximately fivefold for the retinoblastoma gene (P < 0.02). Increased expression of both p19(Arf) and survivin were detected in a majority of the tumors examined (P < 0.01 and 0.001, respectively), but no change in cyclin D1 RNA expression was observed. A subset of the lung tumors (8/28) displayed reduced levels of p16(Ink4a) expression (P = 0.02). Immunohistochemical analysis confirmed the upregulation of p19(Arf) and survivin in all 10 of the lung tumors examined. However, increased staining for cyclin D1 was observed in the tumor tissue. In addition, increased levels of activated p53 were found in lung tumor tissues stained with an anti-phospho-p53 antibody, while an absence of staining was observed with an anti-phospho-pRb antibody in both normal control and tumor tissue. Analysis of the methylation status of p16(Ink4a) by methylation-specific PCR (MSP) demonstrated that seven of eight tumors exhibiting decreased expression of p16(Ink4a) had at least partial methylation of the promoter region. Single stranded conformational polymorphism (SSCP) analysis demonstrated that neither exons 1 or 2 of p16(Ink4a) nor exons 5-8 of p53 exhibited mutations. These data thus identify alterations in specific genes and pathways that combine with the mutation in Ki-ras to promote the formation of benign lung tumors and suggest potential targets for the development of novel chemotherapeutic and chemopreventive agents during the early stages of lung tumor progression.
Assuntos
Genes ras/genética , Neoplasias Pulmonares/genética , Polimorfismo de Nucleotídeo Único , Proteínas ras/genética , Animais , Sequência de Bases , Ciclo Celular/genética , Ciclina D1/genética , Inibidor p16 de Quinase Dependente de Ciclina/genética , Primers do DNA , DNA de Neoplasias/genética , DNA de Neoplasias/isolamento & purificação , Modelos Animais de Doenças , Humanos , Neoplasias Pulmonares/patologia , Camundongos , Camundongos Transgênicos , Proteína do Retinoblastoma/genéticaRESUMO
Lymphoma and leukemia are the most common cancers in children and young adults; in utero carcinogen exposure may contribute to the etiology of these cancers. A polycyclic aromatic hydrocarbon (PAH), dibenzo[a,l]pyrene (DBP), was given to pregnant mice (15 mg/kg body weight, gavage) on gestation day 17. Significant mortalities in offspring, beginning at 12 weeks of age, were observed due to an aggressive T-cell lymphoblastic lymphoma. Lymphocytes invaded numerous tissues. All mice surviving 10 months, exposed in utero to DBP, exhibited lung tumors; some mice also had liver tumors. To assess the role of the aryl hydrocarbon receptor (AHR) in DBP transplacental cancer, B6129SF1/J (AHR(b-1/d), responsive) mice were crossed with strain 129S1/SvIm (AHR(d/d), nonresponsive) to determine the effect of maternal and fetal AHR status on carcinogenesis. Offspring born to nonresponsive mothers had greater susceptibility to lymphoma, irrespective of offspring phenotype. However, when the mother was responsive, an AHR-responsive phenotype in offspring increased mortality by 2-fold. In DBP-induced lymphomas, no evidence was found for TP53, beta-catenin, or Ki-ras mutations but lung adenomas of mice surviving to 10 months of age had mutations in Ki-ras codons 12 and 13. Lung adenomas exhibited a 50% decrease and a 35-fold increase in expression of Rb and p19/ARF mRNA, respectively. This is the first demonstration that transplacental exposure to an environmental PAH can induce a highly aggressive lymphoma in mice and raises the possibility that PAH exposures to pregnant women could contribute to similar cancers in children and young adults.