RESUMO
Interleukin (IL)-17-producing CD8+ T (Tc17) cells have emerged as key players in host-microbiota interactions, infection, and cancer. The factors that drive their development, in contrast to interferon (IFN)-γ-producing effector CD8+ T cells, are not clear. Here we demonstrate that the transcription factor TCF-1 (Tcf7) regulates CD8+ T cell fate decisions in double-positive (DP) thymocytes through the sequential suppression of MAF and RORγt, in parallel with TCF-1-driven modulation of chromatin state. Ablation of TCF-1 resulted in enhanced Tc17 cell development and exposed a gene set signature to drive tissue repair and lipid metabolism, which was distinct from other CD8+ T cell subsets. IL-17-producing CD8+ T cells isolated from healthy humans were also distinct from CD8+IL-17- T cells and enriched in pathways driven by MAF and RORγt Overall, our study reveals how TCF-1 exerts central control of T cell differentiation in the thymus by normally repressing Tc17 differentiation and promoting an effector fate outcome.
Assuntos
Linfócitos T CD8-Positivos/metabolismo , Fator 1-alfa Nuclear de Hepatócito/metabolismo , Interleucina-17/metabolismo , Membro 3 do Grupo F da Subfamília 1 de Receptores Nucleares/metabolismo , Proteínas Proto-Oncogênicas c-maf/metabolismo , Animais , Cromatina/metabolismo , Sequenciamento de Cromatina por Imunoprecipitação , Citometria de Fluxo , Fator 1-alfa Nuclear de Hepatócito/fisiologia , Humanos , Metabolismo dos Lipídeos , Ativação Linfocitária , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Subpopulações de Linfócitos T/fisiologiaRESUMO
NKp46 (CD335) is a surface receptor shared by both human and mouse natural killer (NK) cells and innate lymphoid cells (ILCs) that transduces activating signals necessary to eliminate virus-infected cells and tumors. Here, we describe a spontaneous point mutation of cysteine to arginine (C14R) in the signal peptide of the NKp46 protein in congenic Ly5.1 mice and the newly generated NCRB6C14R strain. Ly5.1C14R NK cells expressed similar levels of Ncr1 mRNA as C57BL/6, but showed impaired surface NKp46 and reduced ability to control melanoma tumors in vivo. Expression of the mutant NKp46C14R in 293T cells showed that NKp46 protein trafficking to the cell surface was compromised. Although Ly5.1C14R mice had normal number of NK cells, they showed an increased number of early maturation stage NK cells. CD49a+ILC1s were also increased but these cells lacked the expression of TRAIL. ILC3s that expressed NKp46 were not detectable and were not apparent when examined by T-bet expression. Thus, the C14R mutation reveals that NKp46 is important for NK cell and ILC differentiation, maturation and function. Significance Innate lymphoid cells (ILCs) play important roles in immune protection. Various subsets of ILCs express the activating receptor NKp46 which is capable of recognizing pathogen derived and tumor ligands and is necessary for immune protection. Here, we describe a spontaneous point mutation in the signal peptide of the NKp46 protein in congenic Ly5.1 mice which are widely used for tracking cells in vivo. This Ncr1 C14R mutation impairs NKp46 surface expression resulting in destabilization of Ncr1 and accumulation of NKp46 in the endoplasmic reticulum. Loss of stable NKp46 expression impaired the maturation of NKp46+ ILCs and altered the expression of TRAIL and T-bet in ILC1 and ILC3, respectively.
RESUMO
E proteins and their antagonists, the Id proteins, are transcriptional regulators important for normal hematopoiesis. We found that Id2 acts as a key regulator of leukemia stem cell (LSC) potential in MLL-rearranged acute myeloid leukemia (AML). Low endogenous Id2 expression is associated with LSC enrichment while Id2 overexpression impairs MLL-AF9-leukemia initiation and growth. Importantly, MLL-AF9 itself controls the E-protein pathway by suppressing Id2 while directly activating E2-2 expression, and E2-2 depletion phenocopies Id2 overexpression in MLL-AF9-AML cells. Remarkably, Id2 tumor-suppressive function is conserved in t(8;21) AML. Low expression of Id2 and its associated gene signature are associated with poor prognosis in MLL-rearranged and t(8;21) AML patients, identifying the Id2/E-protein axis as a promising new therapeutic target in AML.
Assuntos
Proteína 2 Inibidora de Diferenciação/genética , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/patologia , Proteína de Leucina Linfoide-Mieloide/genética , Proteínas de Fusão Oncogênica/genética , Proteína 2 Semelhante ao Fator 7 de Transcrição/genética , Translocação Genética , Animais , Proliferação de Células , Cromossomos Humanos Par 21/genética , Cromossomos Humanos Par 8/genética , Regulação Leucêmica da Expressão Gênica , Humanos , Proteína 2 Inibidora de Diferenciação/metabolismo , Leucemia Mieloide Aguda/metabolismo , Camundongos , Proteína de Leucina Linfoide-Mieloide/metabolismo , Neoplasias Experimentais , Proteínas de Fusão Oncogênica/metabolismo , Prognóstico , Células-Tronco/citologia , Células-Tronco/metabolismo , Análise de Sobrevida , Proteína 2 Semelhante ao Fator 7 de Transcrição/metabolismoRESUMO
Type I interferon (IFN) is crucial in host antiviral defense. Previous studies have described the pleiotropic role of type I IFNs on innate and adaptive immune cells during viral infection. Here, we demonstrate that natural killer (NK) cells from mice lacking the type I IFN-α receptor (Ifnar(-/-)) or STAT1 (which signals downstream of IFNAR) are defective in expansion and memory cell formation after mouse cytomegalovirus (MCMV) infection. Despite comparable proliferation, Ifnar(-/-) NK cells showed diminished protection against MCMV infection and exhibited more apoptosis compared with wild-type NK cells. Furthermore, we show that Ifnar(-/-) NK cells express increased levels of NK group 2 member D (NKG2D) ligands during viral infection and are susceptible to NK cell-mediated fratricide in a perforin- and NKG2D-dependent manner. Adoptive transfer of Ifnar(-/-) NK cells into NK cell-deficient mice reverses the defect in survival and expansion. Our study reveals a novel type I IFN-dependent mechanism by which NK cells evade mechanisms of cell death after viral infection.
Assuntos
Infecções por Herpesviridae/imunologia , Interferon Tipo I/metabolismo , Células Matadoras Naturais/imunologia , Muromegalovirus , Animais , Apoptose , Proliferação de Células , Infecções por Herpesviridae/metabolismo , Infecções por Herpesviridae/patologia , Células Matadoras Naturais/metabolismo , Células Matadoras Naturais/patologia , Ativação Linfocitária , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Subfamília K de Receptores Semelhantes a Lectina de Células NK/metabolismo , Perforina/deficiência , Perforina/genética , Perforina/metabolismo , Receptor de Interferon alfa e beta/deficiência , Receptor de Interferon alfa e beta/genética , Receptor de Interferon alfa e beta/metabolismo , Fator de Transcrição STAT1/deficiência , Fator de Transcrição STAT1/genética , Fator de Transcrição STAT1/metabolismoRESUMO
Norway rats (Rattus norvegicus) are globally distributed and concentrate in urban environments, where they live and feed in closer proximity to human populations than most other mammals. Despite the potential role of rats as reservoirs of zoonotic diseases, the microbial diversity present in urban rat populations remains unexplored. In this study, we used targeted molecular assays to detect known bacterial, viral, and protozoan human pathogens and unbiased high-throughput sequencing to identify novel viruses related to agents of human disease in commensal Norway rats in New York City. We found that these rats are infected with bacterial pathogens known to cause acute or mild gastroenteritis in people, including atypical enteropathogenic Escherichia coli, Clostridium difficile, and Salmonella enterica, as well as infectious agents that have been associated with undifferentiated febrile illnesses, including Bartonella spp., Streptobacillus moniliformis, Leptospira interrogans, and Seoul hantavirus. We also identified a wide range of known and novel viruses from groups that contain important human pathogens, including sapoviruses, cardioviruses, kobuviruses, parechoviruses, rotaviruses, and hepaciviruses. The two novel hepaciviruses discovered in this study replicate in the liver of Norway rats and may have utility in establishing a small animal model of human hepatitis C virus infection. The results of this study demonstrate the diversity of microbes carried by commensal rodent species and highlight the need for improved pathogen surveillance and disease monitoring in urban environments. Importance: The observation that most emerging infectious diseases of humans originate in animal reservoirs has led to wide-scale microbial surveillance and discovery programs in wildlife, particularly in the developing world. Strikingly, less attention has been focused on commensal animals like rats, despite their abundance in urban centers and close proximity to human populations. To begin to explore the zoonotic disease risk posed by urban rat populations, we trapped and surveyed Norway rats collected in New York City over a 1-year period. This analysis revealed a striking diversity of known pathogens and novel viruses in our study population, including multiple agents associated with acute gastroenteritis or febrile illnesses in people. Our findings indicate that urban rats are reservoirs for a vast diversity of microbes that may affect human health and indicate a need for increased surveillance and awareness of the disease risks associated with urban rodent infestation.
Assuntos
Bactérias/isolamento & purificação , Biodiversidade , Portador Sadio , Ratos , Vírus/isolamento & purificação , Animais , Animais Selvagens , Bactérias/classificação , Feminino , Masculino , Dados de Sequência Molecular , Cidade de Nova Iorque , Análise de Sequência de DNA , Vírus/classificaçãoRESUMO
The bZIP transcription factor Nfil3 (also known as E4BP4) is required for the development of natural killer (NK) cells and type 1 innate lymphoid cells (ILC1s). We find that Nfil3 plays a critical role in the development of other mucosal tissue-associated innate lymphocytes. Type 3 ILCs (ILC3s), including lymphoid tissue inducer (LTi)-like cells, are severely diminished in both numbers and function in Nfil3-deficient mice. Using mixed bone marrow chimeric mice, we demonstrate that Nfil3 is critical for normal development of gut-associated ILC3s in a cell-intrinsic manner. Furthermore, Nfil3 deficiency severely compromises intestinal innate immune defense against acute bacterial infection with Citrobacter rodentium and Clostridium difficile. Nfil3 deficiency resulted in a loss of the recently identified ILC precursor, yet conditional ablation of Nfil3 in the NKp46(+) ILC3 subset did not perturb ILC3 numbers, suggesting that Nfil3 is required early during ILC3 development but not for lineage maintenance. Lastly, a marked defect in type 2 ILCs (ILC2s) was also observed in the lungs and visceral adipose tissue of Nfil3-deficient mice, revealing a general requirement for Nfil3 in the development of all ILC lineages.
Assuntos
Fatores de Transcrição de Zíper de Leucina Básica/imunologia , Interações Hospedeiro-Patógeno/imunologia , Imunidade Inata , Subpopulações de Linfócitos/imunologia , Animais , Antígenos Ly/genética , Antígenos Ly/imunologia , Fatores de Transcrição de Zíper de Leucina Básica/deficiência , Fatores de Transcrição de Zíper de Leucina Básica/genética , Linhagem da Célula/genética , Linhagem da Célula/imunologia , Citrobacter rodentium/imunologia , Citrobacter rodentium/patogenicidade , Clostridioides difficile/imunologia , Clostridioides difficile/patogenicidade , Interações Hospedeiro-Patógeno/genética , Imunidade Inata/genética , Imunidade nas Mucosas/genética , Intestinos/imunologia , Intestinos/microbiologia , Células Matadoras Naturais/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Receptor 1 Desencadeador da Citotoxicidade Natural/genética , Receptor 1 Desencadeador da Citotoxicidade Natural/imunologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Quimeras de Transplante/imunologiaRESUMO
Development of the natural killer (NK) cell lineage is dependent on the transcription factor Nfil3 (or E4BP4), which is thought to act downstream of IL-15 signaling. Nfil3-deficient mice lack NK cells, whereas other lymphocyte lineages (B, T, and NKT cells) remain largely intact. We report the appearance of Ly49H-expressing NK cells in Nfil3(-/-) mice infected with mouse cytomegalovirus (MCMV) or recombinant viruses expressing the viral m157 glycoprotein. Nfil3(-/-) NK cells at the peak of antigen-driven expansion were functionally similar to NK cells from infected wild-type mice with respect to IFN-γ production and cytotoxicity, and could comparably produce long-lived memory NK cells that persisted in lymphoid and nonlymphoid tissues for >60 d. We demonstrate that generation and maintenance of NK cell memory is an Nfil3-independent but IL-15-dependent process. Furthermore, specific ablation of Nfil3 in either immature NK cells in the bone marrow or mature peripheral NK cells had no observable effect on NK cell lineage maintenance or homeostasis. Thus, expression of Nfil3 is crucial only early in the development of NK cells, and signals through activating receptors and proinflammatory cytokines during viral infection can bypass the requirement for Nfil3, promoting the proliferation and long-term survival of virus-specific NK cells.
Assuntos
Fatores de Transcrição de Zíper de Leucina Básica/genética , Células Matadoras Naturais/citologia , Animais , Fatores de Transcrição de Zíper de Leucina Básica/metabolismo , Linhagem da Célula , Separação Celular , Sobrevivência Celular , Citocinas/metabolismo , Citometria de Fluxo , Regulação da Expressão Gênica , Homeostase , Inflamação , Interferon gama/metabolismo , Interleucina-15/metabolismo , Células Matadoras Naturais/virologia , Ligantes , Ativação Linfocitária , Camundongos , Camundongos Endogâmicos C57BL , Muromegalovirus/imunologia , Subfamília A de Receptores Semelhantes a Lectina de Células NK/metabolismo , Baço/citologia , Tamoxifeno/químicaRESUMO
The emergence of the adaptive immune system took a toll in the form of pathologies mediated by self-reactive cells. Regulatory T cells (T reg cells) exert a critical brake on responses of T and B lymphocytes to self- and foreign antigens. Here, we asked whether T reg cells are required to restrain NK cells, the third lymphocyte lineage, whose features combine innate and adaptive immune cell properties. Although depletion of T reg cells led to systemic fatal autoimmunity, NK cell tolerance and reactivity to strong activating self- and non-self-ligands remained largely intact. In contrast, missing-self responses were increased in the absence of T reg cells as the result of heightened IL-2 availability. We found that IL-2 rapidly boosted the capacity of NK cells to productively engage target cells and enabled NK cell responses to weak stimulation. Our results suggest that IL-2-dependent adaptive-innate lymphocyte cross talk tunes NK cell reactivity and that T reg cells restrain NK cell cytotoxicity by limiting the availability of IL-2.
Assuntos
Interleucina-2/imunologia , Células Matadoras Naturais/imunologia , Linfócitos T Reguladores/imunologia , Animais , Autoimunidade/imunologia , Adesão Celular/imunologia , Células Cultivadas , Tolerância Imunológica/imunologia , Interleucina-2/metabolismo , Células Matadoras Naturais/metabolismo , Ativação Linfocitária/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Linfócitos T Reguladores/metabolismoRESUMO
In lymphocytes, stimulation of cell surface activating receptors induces the formation of protein microclusters at the plasma membrane that contain the receptor itself, along with other signaling molecules. Although these microclusters are generally thought to be crucial for promoting downstream cellular responses, evidence that specifically links clustering potential to signaling output is lacking. We found that protein kinase C-θ (PKCθ), a key signaling molecule in multiple lymphocyte subsets, formed microclusters in activated NK cells. These microclusters coalesced within the immunological synapse between the NK cell and its target cell. Clustering was mediated by the regulatory region of PKCθ and specifically required a putative phosphotyrosine-binding site within its N-terminal C2 domain. Whereas expression of wild-type PKCθ rescued the cytokine production defect displayed by PKCθ-deficient NK cells, expression of a PKCθ point-mutant incapable of forming microclusters had little to no effect. Hence, PKCθ clustering was necessary for optimal effector function. Notably, only receptors containing ITAMs induced PKCθ microclusters on their own, explaining previous observations that ITAM-coupled receptors promote stronger activating signals and effector responses than do receptors lacking these motifs. Taken together, our results provide a cell biological basis for the role of PKCθ clustering during NK cell activation, and highlight the importance of subcellular compartmentalization for lymphocyte signal transduction.
Assuntos
Sinapses Imunológicas/imunologia , Isoenzimas/imunologia , Células Matadoras Naturais/imunologia , Ativação Linfocitária , Proteína Quinase C/imunologia , Transdução de Sinais/fisiologia , Motivos de Aminoácidos , Animais , Humanos , Sinapses Imunológicas/enzimologia , Sinapses Imunológicas/genética , Isoenzimas/genética , Isoenzimas/metabolismo , Células Matadoras Naturais/citologia , Células Matadoras Naturais/enzimologia , Camundongos , Mutação Puntual , Proteína Quinase C/genética , Proteína Quinase C/metabolismo , Proteína Quinase C-theta , Estrutura Terciária de Proteína , Transdução de Sinais/genéticaRESUMO
C3d is a sub-fragment of the C3 component of the complement system. Covalent binding of multiple C3ds to antigen reduces the activation threshold of cognate B lymphocytes by one thousand fold through co-ligation of the B cell antigen receptor (BCR) and complement receptor 2 (CR2/CD21). Reverse transcriptase polymerase chain reaction (RT-PCR) revealed that, in cattle, four distinct complement receptors are produced from the Cr2 gene by alternative splicing. Cattle express two major variants of the Cr2 gene representing homologues of murine CR1 and CR2, each of which is expressed in both a long and a short form. Expression of CR1 and CR2 was detected in IgM(+) cells from both the spleen and peripheral blood. Additionally, the coding sequence of CD19, the CR2 co-signaling molecule, was determined. CD19 was confirmed to be expressed by IgM(+) cells from the spleen and peripheral blood.
Assuntos
Antígenos CD19/metabolismo , Expressão Gênica , Receptores de Complemento 3b/metabolismo , Receptores de Complemento 3d/metabolismo , Sequência de Aminoácidos , Animais , Antígenos CD19/genética , Bovinos , Células Cultivadas , Sequência Consenso , Leucócitos Mononucleares/metabolismo , Masculino , Dados de Sequência Molecular , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Receptores de Complemento 3b/genética , Receptores de Complemento 3d/genética , Análise de Sequência de DNA , Homologia de Sequência de Aminoácidos , Baço/citologiaRESUMO
AIMS: Radiation-guided sentinel lymph node (SLN) biopsy is a well-established procedure in many countries. However, histopathology protocols in different centres specify widely differing sample handling delays of between 0 and 72 h. Introducing a delay reduces the radiation exposure of pathologists, but has a detrimental effect on the quality and validity of histology. This study aims to show that a sample handling delay is not justified by the radiation doses to pathologists handling samples received directly from surgery. METHODS AND RESULTS: Radiation doses to the body and hands of pathologists handling samples delivered directly from theatres were measured using personal dose meters. These measurements were supplemented by dose assessments undertaken using dose-rate measurements at 1 cm and 30 cm from Tc-99m sources to simulate the processing of samples. The study has shown that radiation doses arising from a zero delay in sample handling represent a negligible radiation risk to pathologists and are well within relevant limits specified in the Ionising Radiations Regulations 1999. CONCLUSIONS: This study supports adoption of a zero-delay SLN histopathology protocol. Centres must, however, complete a risk assessment that accounts for local practice and adopt simple precautions to keep doses to pathologists as low as reasonably achievable.
Assuntos
Neoplasias da Mama/diagnóstico por imagem , Neoplasias da Mama/diagnóstico , Biópsia Guiada por Imagem/métodos , Biópsia de Linfonodo Sentinela/métodos , Protocolos Clínicos , Feminino , Humanos , Metástase Linfática/diagnóstico , Metástase Linfática/diagnóstico por imagem , Exposição Ocupacional , Doses de Radiação , Proteção Radiológica , Cintilografia , Compostos Radiofarmacêuticos , Medição de Risco , Tecnécio , Fatores de TempoRESUMO
Limited active antibody responses in neonates following vaccination have been attributed to immaturity of the immune system and to the suppressive effects of maternal antibodies. The activating receptor CD21 (CR2), when co-ligated with membrane IgM (mIgM) by complement-bound antigen lowers the threshold for activation of B lymphocytes. The inhibitory receptor CD32 (FcgammaRII) when co-ligated with mIgM by antigen-antibody complexes raises the threshold for activation. Expression of these receptors, which potentially play roles in regulation of B cell responses in the presence of maternal antibodies in neonates, has been recently characterized in blood lymphocytes in neonatal calves. Little is known however about expression of these receptors in the lymphoid tissues, where immune responses are initiated. In this study, expression of CD21, mIgM and CD32 receptors by B lymphocytes was studied in a range of lymphoid tissues including spleen, lymph nodes and bone marrow from newborn and 7-week-old calves using flow cytometry. The proportion of naïve B lymphocytes in the lymphocyte gate was significantly lower in blood and spleen of newborn calves compared to 7-week-old calves. Over 90% of B lymphocytes expressed CD21 in the lymphoid tissues. In the lymph nodes and spleen, a lower proportion of mIgM(+) B lymphocytes expressed CD32 compared to blood. In addition, intensity of expression of CD32 on B cells in lymph nodes was significantly lower compared to that in blood, suggesting a lower potential for inhibitory signalling in B cells in the lymphoid microenvironment. Investigation of the CD5(+) B cell population (as an indicator of B1 B cells) suggested an increase in the proportion of IgM(+)CD5(+) cells with age in calves, in both blood and lymphoid tissue, in contrast to the situation in humans and mice. Overall, the majority of naïve B lymphocytes in lymphoid tissues in neonatal calves expressed both activating (CD21, mIgM) and inhibitory (CD32) receptors. These receptors may provide targets for novel adjuvants, to lower the threshold for activation of B cells in neonates, and enhance antibody responses.
Assuntos
Animais Recém-Nascidos/imunologia , Bovinos/imunologia , Imunoglobulina M/análise , Tecido Linfoide/imunologia , Receptores de Complemento 3d/análise , Receptores de IgG/análise , Animais , Linfócitos B/imunologia , Medula Óssea/imunologia , Antígenos CD5/análise , Linfonodos/imunologia , Linfócitos/imunologia , Baço/imunologia , Membro 7 da Superfamília de Receptores de Fatores de Necrose Tumoral/análiseRESUMO
Typically, neonatal calves have poor active antibody responses to vaccination, attributed to immaturity of the neonatal immune system and suppressive effects of maternal (colostral) antibodies. Responses of naïve B cells are regulated by ligation of opposing activating (CD21, membrane IgM [mIgM]) and inhibitory (CD32) receptors. Expression of these receptors on blood lymphocytes of 15 calves, from birth to 6 months of age, was investigated by three-colour flow cytometry. Although the absolute number of mIgM(+) B lymphocytes was low in calves under 6 weeks, the intensity of mIgM expression per cell was significantly higher than for adults and >90% expressed both CD21 and CD32. The intensity of CD21 expression in calves did not differ significantly from adults, whereas CD32 expression was lower. Paradoxically, these findings suggest that responses of neonates should bias toward activation at the B cell level, warranting further investigation to reveal strategies for development of vaccines that are efficacious at an early age.
Assuntos
Envelhecimento/imunologia , Linfócitos B/metabolismo , Regulação da Expressão Gênica no Desenvolvimento/imunologia , Subpopulações de Linfócitos/metabolismo , Receptores de Complemento 3d/metabolismo , Receptores de IgG/metabolismo , Fatores Etários , Animais , Animais Recém-Nascidos , Linfócitos B/citologia , Linfócitos B/imunologia , Bovinos , Contagem de Células , Imunidade Humoral , Imunoglobulina M/biossíntese , Estudos Longitudinais , Subpopulações de Linfócitos/citologia , Subpopulações de Linfócitos/imunologia , Receptores de Complemento 3d/genética , Receptores de Complemento 3d/imunologia , Receptores de IgG/genética , Receptores de IgG/imunologia , VacinaçãoRESUMO
Receptors for the Fc portion of immunoglobulin molecules (FcR) provide an important and vital link between circulating antibody and cellular effector functions. These receptors have been well characterized in human and murine species, however few of these receptors have been investigated in livestock. FcgammaRII (CD32) is an FcR previously shown in mice and humans to exist in multiple isoforms, both activating (FcgammaRIIa, FcgammaRIIc) and inhibitory (FcgammaRIIb), on a wide variety of cells including B cells, T cells, dendritic cells, monocytes, macrophages and platelets. On B cells, FcgammaRIIb acts to suppress cell activation and immunoglobulin production by means of an intracellular immunoreceptor tyrosine-based inhibitory motif signaling domain. Two sub-isoforms of FcgammaRIIb, designated b1 and b2, distinguished by the inclusion of an additional cytoplasmic exon in the b1 form, have been demonstrated in humans and mice, whereas only one sequence corresponding to the human and mouse b2 isoform has been identified in cattle. In this study, the expression profile of FcgammaRIIb in bovine blood mononuclear cells was characterized by collecting blood samples from mature cattle of dairy and beef breeds, and determining their FcgammaRIIb mRNA expression profile by RT-PCR. Analysis revealed the presence of two uncharacterized bovine FcgammaRIIb transcripts in addition to the single previously published transcript. Analysis of the first unknown transcript revealed high homology with published human and murine FcgammaRIIb1 sequences. This transcript was present in all cell types examined, with little variation in primary sequence between individuals or among breeds. The second unknown sequence was found to be homologous to the murine FcgammaRIIb3 (IgG-binding protein or soluble FcgammaR in humans) sequence. This transcript appears to have a much more limited expression profile, which may indicate that expression varies with the cellular activation-state of the cell. These results indicate that cattle, like humans and mice, express multiple sub-isoforms of FcgammaRIIb. These findings add further complexity to the regulation of IgG-mediated immunity and provide new insight into the role Fc receptors play in antigen acquisition and presentation in cattle.
Assuntos
Bovinos/imunologia , Receptores de IgG/biossíntese , Processamento Alternativo/genética , Processamento Alternativo/imunologia , Animais , Bovinos/genética , Bovinos/metabolismo , Linhagem Celular Tumoral , Citometria de Fluxo/veterinária , Leucócitos/imunologia , Leucócitos/metabolismo , Linfócitos/imunologia , Linfócitos/metabolismo , Fases de Leitura Aberta/genética , Filogenia , Isoformas de Proteínas/biossíntese , Isoformas de Proteínas/genética , Receptores de IgG/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa/veterinária , Alinhamento de Sequência , Análise de Sequência de DNARESUMO
It is difficult to induce active immune responses in neonatal calves, partly due to limited functional ability of the immune system and partly due to immune inhibitory effects of maternal antibodies. CD21 (complement receptor 2), an activating receptor, and CD32 (Fc gamma receptor II), an inhibitory receptor, can both be expressed by mature B lymphocytes. Studies of the signalling pathways regulating B cell activation suggest that these receptors have mutually antagonistic effects, that may determine immune responsiveness in the first months of life. In a cross-sectional study, blood was collected from 41 Holstein calves, 1-90 days of age, and 12 mature cows. The absolute number of CD21 and CD32 positive cells increased from birth until 90 days of age, with CD21+ cells showing a greater relative increase compared to CD32+ cells. Approximately 89% of CD21+ cells also expressed CD32. In calves, CD32+ cells consisted of two distinct populations, characterized to be CD14+CD32+IgM(-) (44%) and CD14(-)CD32+IgM+ (53%) cells, consistent with monocytes and B cells respectively. Mean fluorescence intensity (MFI) for CD21 did not change appreciably up to 90 days of age, and mean values were slightly lower than for adults. MFI for CD32 on CD21+ lymphocytes increased slightly over the first 3 months of life, but values were lower than for adults. Over 92% of calf membrane immunoglobulin M+ (mIgM) B cells expressed CD21, however only 60-80% of CD21+ cells were mIgM positive, regardless of age. Although the CD21+IgM(-) cells were not characterized further, it was evident that CD21 is not a suitable surrogate marker for B lymphocytes in calves. Most importantly this study showed that from birth, the majority of circulating B cells express both CD21 and CD32. This suggests that these cells are subject to activating and inhibiting influences mediated by these receptors from birth. Expression of CD21 does not appear to be a limiting factor in neonatal B cell responses.
Assuntos
Linfócitos B/imunologia , Bovinos/imunologia , Receptores de Complemento 3d/biossíntese , Receptores de IgG/biossíntese , Fatores Etários , Animais , Animais Recém-Nascidos , Linfócitos B/citologia , Bovinos/sangue , Estudos Transversais , Feminino , Citometria de Fluxo/veterinária , Imunoglobulina M/sangue , Imunoglobulina M/imunologia , Contagem de Leucócitos/veterinária , Receptores de Complemento 3d/sangue , Receptores de Complemento 3d/imunologia , Receptores de IgG/sangue , Receptores de IgG/imunologia , Estatísticas não ParamétricasRESUMO
The gene fragment coding for bovine C3d gene (boC3d) was cloned and expressed as a component of fusion proteins destined for use in vaccine studies in cattle, and for in vitro experiments. This fragment of complement protein C3 (C3d) has been shown to enhance B cell responses when complexed with antigen. Three potential vaccine constructs were engineered to contain one, two or three boC3d units linked to a fragment of the leukotoxin of Mannheimia haemolytica A1, an economically important pathogen of cattle that causes a fibrinous pneumonia in calves. A recombinant biotinylated boC3d protein (for use in in vitro studies) was generated by endogenous biotinylation in Escherichia coli by means of the BirA holoenzyme synthetase. All recombinant proteins incorporated polyhistidine tags and were purified by nickel-agarose chromatography, then analyzed by SDS-PAGE and Western immunoblot. The identity of boC3d was confirmed by mass spectrometry, since monoclonal antibodies to boC3d were not available. To date, published research into the adjuvant activities of C3d has been limited to experiments in mice and rabbits, using antigens unrelated to diseases occurring naturally in these species. The boC3d fusion proteins expressed in this study will provide the basis for immunization trials in cattle and studies of receptor binding and cell activation of bovine lymphocytes.
Assuntos
Bovinos/genética , Bovinos/imunologia , Complemento C3d/genética , Complemento C3d/imunologia , Sequência de Aminoácidos , Animais , Proteínas de Bactérias/genética , Proteínas de Bactérias/imunologia , Vacinas Bacterianas/genética , Vacinas Bacterianas/imunologia , Sequência de Bases , Western Blotting/veterinária , Clonagem Molecular , Complemento C3d/biossíntese , Feminino , Proteínas Hemolisinas/genética , Proteínas Hemolisinas/imunologia , Dados de Sequência Molecular , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/imunologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa/veterinária , Análise de Sequência de DNARESUMO
Innate immune defenses are crucial for survival in the first days and weeks of life. At birth, newborns are confronted with a vast array of potentially pathogenic microorganisms that were not encountered in utero. At this age, cellular components of the adaptive immune system are in a naive state and are slow to respond. Antibodies received from the dam are essential for defense, but represent a finite and dwindling resource. Innate components of the immune system detect pathogen-associated molecular patterns (PAMPs) on microorganisms (and their products) by means of pattern-recognition receptors (PRRs). Soluble mediators of the innate system such as complement proteins, pentraxins, collectins, ficolins, defensins, lactoferrin, lysozyme etc. can bind to structures on pathogens, leading to agglutination, interference with receptor binding, opsonization, neutralization, direct membrane damage and recruitment of additional soluble and cellular elements through inflammation. Cell-associated receptors such as the Toll-like receptors (TLRs) can activate cells and coordinate responses (both innate and adaptive). In this paper, accumulated knowledge of the receptors, soluble and cellular elements that contribute to innate defenses of young animals is reviewed. Research interest in this area has been intermittent, and the literature varies in quantity and quality. It is hoped that documentation of the limitations of our knowledge base will lead to more extensive and enlightening studies.