RESUMO
UNC93B1 is essential for the stability and endosomal trafficking of nucleic-acid sensing Toll-like receptors (TLRs) including TLR7 and TLR8. Increased TLR7 responses are associated with lupus autoimmunity in both mice and humans. In a recent article, Al-Azab et al. demonstrate the role of a variant of UNC93B1 (p.V117L) in the induction of pediatric systemic lupus erythematosus in patients and in mice through TLR7/8 hyperresponsiveness. They also highlight a potential role for the pharmacological inhibition of interleukin-1 receptor-associated kinase (IRAK) 1 and/or 4 in ameliorating disease.
RESUMO
Systemic lupus erythematosus (SLE) is a heterogeneous autoimmune disease with a clear genetic component. While most SLE patients carry rare gene variants in lupus risk genes, little is known about their contribution to disease pathogenesis. Amongst them, SH2B3-a negative regulator of cytokine and growth factor receptor signaling-harbors rare coding variants in over 5% of SLE patients. Here, we show that unlike the variant found exclusively in healthy controls, SH2B3 rare variants found in lupus patients are predominantly hypomorphic alleles, failing to suppress IFNGR signaling via JAK2-STAT1. The generation of two mouse lines carrying patients' variants revealed that SH2B3 is important in limiting the number of immature and transitional B cells. Furthermore, hypomorphic SH2B3 was shown to impair the negative selection of immature/transitional self-reactive B cells and accelerate autoimmunity in sensitized mice, at least in part due to increased IL-4R signaling and BAFF-R expression. This work identifies a previously unappreciated role for SH2B3 in human B cell tolerance and lupus risk.
Assuntos
Autoimunidade , Lúpus Eritematoso Sistêmico , Animais , Humanos , Camundongos , Autoimunidade/genética , Fator Ativador de Células B/metabolismo , Linfócitos B , Lúpus Eritematoso Sistêmico/genética , Células Precursoras de Linfócitos BRESUMO
CD11c+ atypical B cells (ABCs) are an alternative memory B cell lineage associated with immunization, infection, and autoimmunity. However, the factors that drive the transcriptional program of ABCs have not been identified, and the function of this population remains incompletely understood. Here, we identified candidate transcription factors associated with the ABC population based on a human tonsillar B cell single-cell dataset. We identified CD11c+ B cells in mice with a similar transcriptomic signature to human ABCs, and using an optimized CRISPR-Cas9 knockdown screen, we observed that loss of zinc finger E-box binding homeobox 2 (Zeb2) impaired ABC formation. Furthermore, ZEB2 haplo-insufficient Mowat-Wilson syndrome (MWS) patients have decreased circulating ABCs in the blood. In Cd23Cre/+Zeb2fl/fl mice with impaired ABC formation, ABCs were dispensable for efficient humoral responses after Plasmodium sporozoite immunization but were required to control recrudescent blood-stage malaria. Immune phenotyping revealed that ABCs drive optimal T follicular helper (TFH) cell formation and germinal center (GC) responses and they reside at the red/white pulp border, likely permitting better access to pathogen antigens for presentation. Collectively, our study shows that ABC formation is dependent on Zeb2, and these cells can limit recrudescent infection by sustaining GC reactions.
Assuntos
Centro Germinativo , Infecção Persistente , Animais , Humanos , Camundongos , Imunização , Vacinação , Homeobox 2 de Ligação a E-box com Dedos de Zinco/genéticaRESUMO
Autosomal dominant loss-of-function (LoF) variants in cytotoxic T-lymphocyte associated protein 4 (CTLA4) cause immune dysregulation with autoimmunity, immunodeficiency and lymphoproliferation (IDAIL). Incomplete penetrance and variable expressivity are characteristic of IDAIL caused by CTLA-4 haploinsufficiency (CTLA-4h), pointing to a role for genetic modifiers. Here, we describe an IDAIL proband carrying a maternally inherited pathogenic CTLA4 variant and a paternally inherited rare LoF missense variant in CLEC7A, which encodes for the ß-glucan pattern recognition receptor DECTIN-1. The CLEC7A variant led to a loss of DECTIN-1 dimerization and surface expression. Notably, DECTIN-1 stimulation promoted human and mouse regulatory T cell (Treg) differentiation from naïve αß and γδ T cells, even in the absence of transforming growth factor-ß. Consistent with DECTIN-1's Treg-boosting ability, partial DECTIN-1 deficiency exacerbated the Treg defect conferred by CTL4-4h. DECTIN-1/CLEC7A emerges as a modifier gene in CTLA-4h, increasing expressivity of CTLA4 variants and acting in functional epistasis with CTLA-4 to maintain immune homeostasis and tolerance.
Assuntos
Haploinsuficiência , Lectinas Tipo C , Animais , Humanos , Camundongos , Autoimunidade , Antígeno CTLA-4/genética , Lectinas Tipo C/genéticaRESUMO
TANK-binding kinase 1 (TBK1) is a key signalling component in the production of type-I interferons, which have essential antiviral activities, including against SARS-CoV-2. TBK1, and its homologue IκB kinase-ε (IKKε), can also induce pro-inflammatory responses that contribute to pathogen clearance. While initially protective, sustained engagement of type-I interferons is associated with damaging hyper-inflammation found in severe COVID-19 patients. The contribution of TBK1/IKKε signalling to these responses is unknown. Here we find that the small molecule idronoxil inhibits TBK1/IKKε signalling through destabilisation of TBK1/IKKε protein complexes. Treatment with idronoxil, or the small molecule inhibitor MRT67307, suppresses TBK1/IKKε signalling and attenuates cellular and molecular lung inflammation in SARS-CoV-2-challenged mice. Our findings additionally demonstrate that engagement of STING is not the major driver of these inflammatory responses and establish a critical role for TBK1/IKKε signalling in SARS-CoV-2 hyper-inflammation.
Assuntos
COVID-19 , Interferon Tipo I , Animais , Camundongos , Quinase I-kappa B , Modelos Animais de Doenças , SARS-CoV-2 , InflamaçãoRESUMO
Although circumstantial evidence supports enhanced Toll-like receptor 7 (TLR7) signalling as a mechanism of human systemic autoimmune disease1-7, evidence of lupus-causing TLR7 gene variants is lacking. Here we describe human systemic lupus erythematosus caused by a TLR7 gain-of-function variant. TLR7 is a sensor of viral RNA8,9 and binds to guanosine10-12. We identified a de novo, previously undescribed missense TLR7Y264H variant in a child with severe lupus and additional variants in other patients with lupus. The TLR7Y264H variant selectively increased sensing of guanosine and 2',3'-cGMP10-12, and was sufficient to cause lupus when introduced into mice. We show that enhanced TLR7 signalling drives aberrant survival of B cell receptor (BCR)-activated B cells, and in a cell-intrinsic manner, accumulation of CD11c+ age-associated B cells and germinal centre B cells. Follicular and extrafollicular helper T cells were also increased but these phenotypes were cell-extrinsic. Deficiency of MyD88 (an adaptor protein downstream of TLR7) rescued autoimmunity, aberrant B cell survival, and all cellular and serological phenotypes. Despite prominent spontaneous germinal-centre formation in Tlr7Y264H mice, autoimmunity was not ameliorated by germinal-centre deficiency, suggesting an extrafollicular origin of pathogenic B cells. We establish the importance of TLR7 and guanosine-containing self-ligands for human lupus pathogenesis, which paves the way for therapeutic TLR7 or MyD88 inhibition.
Assuntos
Mutação com Ganho de Função , Lúpus Eritematoso Sistêmico , Receptor 7 Toll-Like , Animais , Autoimunidade/genética , Linfócitos B , GMP Cíclico/análogos & derivados , Guanosina , Humanos , Lúpus Eritematoso Sistêmico/genética , Camundongos , Fator 88 de Diferenciação Mieloide/genética , Fator 88 de Diferenciação Mieloide/metabolismo , Receptor 7 Toll-Like/genética , Receptor 7 Toll-Like/metabolismoRESUMO
Neutrophils perform critical functions in the innate response to infection, including through the production of neutrophil extracellular traps (NETs) - web-like DNA structures which are extruded from neutrophils upon activation. Elevated levels of NETs have been linked to autoimmunity but this association is poorly understood. By contrast, IL-17 producing Th17 cells are a key player in various autoimmune diseases but are also crucial for immunity against fungal and bacterial infections. Here we show that NETs, through their protein component histones, directly activate T cells and specifically enhance Th17 cell differentiation. This modulatory role of neutrophils, NETs and their histones is mediated downstream of TLR2 in T cells, resulting in phosphorylation of STAT3. The innate stimulation of a specific adaptive immune cell subset provides an additional mechanism demonstrating a direct link between neutrophils, NETs and T cell autoimmunity.
Assuntos
Diferenciação Celular , Armadilhas Extracelulares/metabolismo , Histonas/metabolismo , Neutrófilos/metabolismo , Células Th17/imunologia , Receptor 2 Toll-Like/metabolismo , Adulto , Autoimunidade , DNA/metabolismo , Feminino , Humanos , Imunidade Inata , Masculino , Adulto JovemRESUMO
The SH2B family of adaptor proteins, SH2-B, APS, and LNK are key modulators of cellular signalling pathways. Whilst SH2-B and APS have been partially structurally and biochemically characterised, to date there has been no such characterisation of LNK. Here we present two crystal structures of the LNK substrate recognition domain, the SH2 domain, bound to phosphorylated motifs from JAK2 and EPOR, and biochemically define the basis for target recognition. The LNK SH2 domain adopts a canonical SH2 domain fold with an additional N-terminal helix. Targeted analysis of binding to phosphosites in signalling pathways indicated that specificity is conferred by amino acids one- and three-residues downstream of the phosphotyrosine. Several mutations in LNK showed impaired target binding in vitro and a reduced ability to inhibit signalling, allowing an understanding of the molecular basis of LNK dysfunction in variants identified in patients with myeloproliferative disease.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/química , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Motivos de Aminoácidos , Animais , Sítios de Ligação , Cristalografia por Raios X , Humanos , Janus Quinase 2/química , Janus Quinase 2/metabolismo , Janus Quinase 3/química , Janus Quinase 3/metabolismo , Camundongos , Mutação , Transtornos Mieloproliferativos/genética , Fosfotirosina , Ligação Proteica , Proteínas Proto-Oncogênicas c-kit/química , Proteínas Proto-Oncogênicas c-kit/metabolismo , Receptores da Eritropoetina/química , Receptores da Eritropoetina/metabolismo , Transdução de Sinais , Tirosina Quinase 3 Semelhante a fms/química , Tirosina Quinase 3 Semelhante a fms/metabolismo , Domínios de Homologia de srcRESUMO
Oligonucleotide-based therapeutics have the capacity to engage with nucleic acid immune sensors to activate or block their response, but a detailed understanding of these immunomodulatory effects is currently lacking. We recently showed that 2'-O-methyl (2'OMe) gapmer antisense oligonucleotides (ASOs) exhibited sequence-dependent inhibition of sensing by the RNA sensor Toll-Like Receptor (TLR) 7. Here we discovered that 2'OMe ASOs can also display sequence-dependent inhibitory effects on two major sensors of DNA, namely cyclic GMP-AMP synthase (cGAS) and TLR9. Through a screen of 80 2'OMe ASOs and sequence mutants, we characterized key features within the 20-mer ASOs regulating cGAS and TLR9 inhibition, and identified a highly potent cGAS inhibitor. Importantly, we show that the features of ASOs inhibiting TLR9 differ from those inhibiting cGAS, with only a few sequences inhibiting both pathways. Together with our previous studies, our work reveals a complex pattern of immunomodulation where 95% of the ASOs tested inhibited at least one of TLR7, TLR9 or cGAS by ≥30%, which may confound interpretation of their in vivo functions. Our studies constitute the broadest analysis of the immunomodulatory effect of 2'OMe ASOs on nucleic acid sensing to date and will support refinement of their therapeutic development.
Assuntos
Nucleotidiltransferases/antagonistas & inibidores , Oligonucleotídeos Antissenso/química , Receptor Toll-Like 9/antagonistas & inibidores , Adulto , Animais , Sequência de Bases , Células Cultivadas , DNA , Humanos , Camundongos , Transdução de Sinais , Receptor 3 Toll-Like/antagonistas & inibidores , Receptor 7 Toll-Like/antagonistas & inibidoresRESUMO
Genetic primary immunodeficiency diseases are increasingly recognized, with pathogenic mutations changing the composition of circulating leukocyte subsets measured by flow cytometry (FCM). Discerning changes in multiple subpopulations is challenging, and subtle trends might be missed if traditional reference ranges derived from a control population are applied. We developed an algorithm where centiles were allocated using non-parametric comparison to controls, generating multiparameter heat maps to simultaneously represent all leukocyte subpopulations for inspection of trends within a cohort or segregation with a putative genetic mutation. To illustrate this method, we analyzed patients with Primary Antibody Deficiency (PAD) and kindreds harboring mutations in TNFRSF13B (encoding TACI), CTLA4, and CARD11. In PAD, loss of switched memory B cells (B-SM) was readily demonstrated, but as a continuous, not dichotomous, variable. Expansion of CXCR5+/CD45RA- CD4+ T cells (X5-Th cells) was a prominent feature in PAD, particularly in TACI mutants, and patients with expansion in CD21-lo B cells or transitional B cells were readily apparent. We observed differences between unaffected and affected TACI mutants (increased B cells and CD8+ T-effector memory cells, loss of B-SM cells and non-classical monocytes), cellular signatures that distinguished CTLA4 haploinsufficiency itself (expansion of plasmablasts, activated CD4+ T cells, regulatory T cells, and X5-Th cells) from its clinical expression (B-cell depletion), and those that were associated with CARD11 gain-of-function mutation (decreased CD8+ T effector memory cells, B cells, CD21-lo B cells, B-SM cells, and NK cells). Co-efficients of variation exceeded 30% for 36/54 FCM parameters, but by comparing inter-assay variation with disease-related variation, we ranked each parameter in terms of laboratory precision vs. disease variability, identifying X5-Th cells (and derivatives), naïve, activated, and central memory CD8+ T cells, transitional B cells, memory and SM-B cells, plasmablasts, activated CD4 cells, and total T cells as the 10 most useful cellular parameters. Applying these to cluster analysis of our PAD cohort, we could detect subgroups with the potential to reflect underlying genotypes. Heat mapping of normalized FCM data reveals cellular trends missed by standard reference ranges, identifies changes associating with a phenotype or genotype, and could inform hypotheses regarding pathogenesis of genetic immunodeficiency.
Assuntos
Citometria de Fluxo , Doenças Genéticas Inatas/imunologia , Temperatura Alta , Síndromes de Imunodeficiência/imunologia , Mutação , Adulto , Idoso , Linfócitos B/imunologia , Linfócitos B/patologia , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/patologia , Feminino , Doenças Genéticas Inatas/genética , Doenças Genéticas Inatas/patologia , Humanos , Síndromes de Imunodeficiência/genética , Síndromes de Imunodeficiência/patologia , Memória Imunológica , Masculino , Pessoa de Meia-Idade , Linfócitos T Auxiliares-Indutores/imunologia , Linfócitos T Auxiliares-Indutores/patologiaRESUMO
Systemic lupus erythematosus (SLE) is the prototypic systemic autoimmune disease. It is thought that many common variant gene loci of weak effect act additively to predispose to common autoimmune diseases, while the contribution of rare variants remains unclear. Here we describe that rare coding variants in lupus-risk genes are present in most SLE patients and healthy controls. We demonstrate the functional consequences of rare and low frequency missense variants in the interacting proteins BLK and BANK1, which are present alone, or in combination, in a substantial proportion of lupus patients. The rare variants found in patients, but not those found exclusively in controls, impair suppression of IRF5 and type-I IFN in human B cell lines and increase pathogenic lymphocytes in lupus-prone mice. Thus, rare gene variants are common in SLE and likely contribute to genetic risk.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/genética , Predisposição Genética para Doença , Lúpus Eritematoso Sistêmico/genética , Proteínas de Membrana/genética , Quinases da Família src/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Adolescente , Adulto , Animais , Linfócitos B/citologia , Linfócitos B/imunologia , Linfócitos B/metabolismo , Estudos de Casos e Controles , Linhagem Celular , Núcleo Celular/imunologia , Núcleo Celular/metabolismo , Criança , Modelos Animais de Doenças , Feminino , Frequência do Gene , Células HEK293 , Voluntários Saudáveis , Humanos , Fatores Reguladores de Interferon/imunologia , Fatores Reguladores de Interferon/metabolismo , Interferon Tipo I/imunologia , Interferon Tipo I/metabolismo , Lúpus Eritematoso Sistêmico/sangue , Lúpus Eritematoso Sistêmico/imunologia , Masculino , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Mutação de Sentido Incorreto , Sequenciamento do Exoma , Quinases da Família src/metabolismoRESUMO
T follicular helper cells (Tfh) are critical for the longevity and quality of antibody-mediated protection against infection. Yet few signaling pathways have been identified to be unique solely to Tfh development. ROQUIN is a post-transcriptional repressor of T cells, acting through its ROQ domain to destabilize mRNA targets important for Th1, Th17, and Tfh biology. Here, we report that ROQUIN has a paradoxical function on Tfh differentiation mediated by its RING domain: mice with a T cell-specific deletion of the ROQUIN RING domain have unchanged Th1, Th2, Th17, and Tregs during a T-dependent response but show a profoundly defective antigen-specific Tfh compartment. ROQUIN RING signaling directly antagonized the catalytic α1 subunit of adenosine monophosphate-activated protein kinase (AMPK), a central stress-responsive regulator of cellular metabolism and mTOR signaling, which is known to facilitate T-dependent humoral immunity. We therefore unexpectedly uncover a ROQUIN-AMPK metabolic signaling nexus essential for selectively promoting Tfh responses.
Assuntos
Proteínas Quinases Ativadas por AMP/metabolismo , Diferenciação Celular , Transdução de Sinais , Linfócitos T Auxiliares-Indutores/fisiologia , Ubiquitina-Proteína Ligases/metabolismo , Animais , Camundongos , Deleção de Sequência , Ubiquitina-Proteína Ligases/genéticaRESUMO
Objective. Systemic lupus erythematosus (SLE) isa chronic and heterogeneous autoimmune disease. Both twin and sibling studies indicate a strong genetic contribution to lupus, but in the majority of cases the pathogenic variant remains to be identified. The genetic contribution to disease is likely to be greatest in cases with early onset and severe phenotypes. Whole-exome sequencing now offers the possibility of identifying rare alleles responsible for disease in such cases. This study was undertaken to identify genetic causes of SLE using whole-exome sequencing.Methods. We performed whole-exome sequencing in a 4-year-old girl with early-onset SLE and conducted biochemical analysis of the putative defect.Results. Whole-exome sequencing in a 4-year-old girl with cerebral lupus identified a rare, homozygous mutation in the three prime repair exonuclease 1 gene(TREX1) that was predicted to be highly deleterious.The TREX1 R97H mutant protein had a 20-fold reduction in exonuclease activity and was associated with an elevated interferon-alpha signature in the patient.The discovery and characterization of a pathogenic TREX1 variant in our proband has therapeutic implications.The patient is now a candidate for therapy. Conclusion. Our study is the first to demonstrate that whole-exome sequencing can be used to identify rare or novel deleterious variants as genetic causes of SLE and, through a personalized approach, improve therapeutic options.
Assuntos
Exodesoxirribonucleases/genética , Exoma/genética , Homozigoto , Interferon-alfa/análise , Vasculite Associada ao Lúpus do Sistema Nervoso Central/genética , Fosfoproteínas/genética , Pré-Escolar , Feminino , Humanos , LinhagemRESUMO
Angioimmunoblastic T-cell lymphoma (AITL) is the second most common peripheral T-cell lymphoma with unusual clinical and pathologic features and a poor prognosis despite intensive chemotherapy. Recent studies have suggested AITL derives from follicular helper T (T(FH)) cells, but the causative molecular pathways remain largely unknown. Here we show that approximately 50% of mice heterozygous for the "san" allele of Roquin develop tumors accompanied by hypergammaglobulinemia by 6 months of age. Affected lymph nodes displayed the histologic features diagnostic of AITL, except for the presence of expanded FDC networks. Accumulation of T(FH) cells preceded tumor development, and clonal rearrangements in the TCR-ß genes were present in most tumors. Furthermore, T(FH) cells exhibited increased clonality compared with non-T(FH) cells from the same lymph nodes, even in the absence of tumors. Genetic manipulations that prevent T(FH) development, such as deletion of ICOS, CD28, and SAP, partially or completely abrogated tumor development, confirming a T(FH)-derived origin. Roquin(san/+) mice emerge as a useful model to investigate the molecular pathogenesis of AITL and for preclinical testing of therapies aimed at targeting dysregulated T(FH) cells or their consequences.
Assuntos
Hipergamaglobulinemia/etiologia , Linfadenopatia Imunoblástica/etiologia , Perda de Heterozigosidade , Linfonodos/patologia , Linfoma Folicular/etiologia , Linfoma de Células T/etiologia , Ubiquitina-Proteína Ligases/fisiologia , Animais , Antígenos CD28/fisiologia , Ensaio de Imunoadsorção Enzimática , Citometria de Fluxo , Hipergamaglobulinemia/patologia , Linfadenopatia Imunoblástica/patologia , Técnicas Imunoenzimáticas , Proteína Coestimuladora de Linfócitos T Induzíveis/fisiologia , Linfoma Folicular/patologia , Linfoma de Células T/patologia , Camundongos , Camundongos Knockout , Receptores de Antígenos de Linfócitos T alfa-beta/metabolismo , Linfócitos T Auxiliares-Indutores/metabolismo , Linfócitos T Auxiliares-Indutores/patologiaRESUMO
The transcription factor BATF directly regulates key components of the formation and function of follicular helper T cells and antibody class switching in B cells.
Assuntos
Linfócitos B/imunologia , Fatores de Transcrição de Zíper de Leucina Básica/imunologia , Linfócitos T/imunologia , Proteínas Adaptadoras de Transdução de Sinal , Animais , Linfócitos B/metabolismo , Fatores de Transcrição de Zíper de Leucina Básica/genética , Fatores de Transcrição de Zíper de Leucina Básica/metabolismo , Ligante de CD40/genética , Ligante de CD40/imunologia , Ligante de CD40/metabolismo , Proteínas de Transporte/genética , Proteínas de Transporte/imunologia , Proteínas de Transporte/metabolismo , Células Cultivadas , Citometria de Fluxo , Perfilação da Expressão Gênica , Centro Germinativo/imunologia , Centro Germinativo/metabolismo , Camundongos , Camundongos Knockout , Modelos Imunológicos , Proteínas Proto-Oncogênicas c-bcl-6/genética , Proteínas Proto-Oncogênicas c-bcl-6/imunologia , Proteínas Proto-Oncogênicas c-bcl-6/metabolismo , Linfócitos T/metabolismo , Linfócitos T Auxiliares-Indutores/imunologia , Linfócitos T Auxiliares-Indutores/metabolismoRESUMO
Activation-induced deaminase (AID) is a B lymphocyte-specific DNA deaminase that triggers Ig class-switch recombination (CSR) and somatic hypermutation. It shuttles between cytoplasm and nucleus, containing a nuclear export sequence (NES) at its carboxyterminus. Intriguingly, the precise nature of this NES is critical to AID's function in CSR, though not in somatic hypermutation. Many alterations to the NES, while preserving its nuclear export function, destroy CSR ability. We have previously speculated that AID's ability to potentiate CSR may critically depend on the affinity of interaction between its NES and Crm1 exportin. Here, however, by comparing multiple AID NES mutants, we find that - beyond a requirement for threshold Crm1 binding - there is little correlation between CSR and Crm1 binding affinity. The results suggest that CSR, as well as the stabilisation of AID, depend on an interaction between the AID C-terminal decapeptide and factor(s) additional to Crm1.
Assuntos
Citidina Desaminase/metabolismo , Switching de Imunoglobulina/fisiologia , Carioferinas/metabolismo , Sinais de Exportação Nuclear/fisiologia , Receptores Citoplasmáticos e Nucleares/metabolismo , Substituição de Aminoácidos/fisiologia , Animais , Linfócitos B/imunologia , Linfócitos B/metabolismo , Citidina Desaminase/genética , Estabilidade Enzimática/fisiologia , Humanos , Carioferinas/genética , Camundongos , Camundongos Knockout , Ligação Proteica/fisiologia , Domínios e Motivos de Interação entre Proteínas/fisiologia , Receptores Citoplasmáticos e Nucleares/genética , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteína Exportina 1RESUMO
Earlier studies have shown that the adoptive transfer of Th2-polarized CD4 T cells can clear established tumors from mice in an antigen-specific manner. Although eosinophils were implicated in this process, the exact mechanism of tumor clearance and which immune effector cells were involved, remain to be defined. Consequently, experiments were undertaken to elucidate the mechanism of Th2-mediated destruction of B16-F1 melanoma cells by examining the in vitro antitumor activity of leukocytes within a type-2 inflammatory infiltrate. The experimental data show that activation of alternatively activated macrophages (aaMacs) within type-2 infiltrates by IL-4 or IL-13 can inhibit B16-F1 melanoma cell proliferation through a mechanism that is dependent on arginase-1 depletion of L-arginine within the tumor cell microenvironment. Interestingly, whilst at higher E:T ratios aaMac exhibited antitumor activity, at lower E:T ratios aaMacs were observed to enhance rather than inhibit B16-F1 melanoma cell growth. This highlights the fine balance between stimulating the antitumorigenic and protumorigenic properties of aaMacs in tumor immunotherapy protocols.
Assuntos
Arginase/metabolismo , Imunoterapia Adotiva , Interleucina-4/imunologia , Macrófagos Peritoneais/metabolismo , Melanoma Experimental/imunologia , Animais , Proliferação de Células , Técnicas de Cocultura , Citotoxicidade Imunológica , Genes Codificadores dos Receptores de Linfócitos T/genética , Interleucina-4/metabolismo , Ativação de Macrófagos , Macrófagos Peritoneais/imunologia , Macrófagos Peritoneais/patologia , Masculino , Melanoma Experimental/patologia , Melanoma Experimental/terapia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Ovalbumina/imunologia , Células Th2/imunologiaRESUMO
Follicular helper T (Tfh) cells provide selection signals to germinal center B cells, which is essential for long-lived antibody responses. High CXCR5 and low CCR7 expression facilitates their homing to B cell follicles and distinguishes them from T helper 1 (Th1), Th2, and Th17 cells. Here, we showed that Bcl-6 directs Tfh cell differentiation: Bcl-6-deficient T cells failed to develop into Tfh cells and could not sustain germinal center responses, whereas forced expression of Bcl-6 in CD4(+) T cells promoted expression of the hallmark Tfh cell molecules CXCR5, CXCR4, and PD-1. Bcl-6 bound to the promoters of the Th1 and Th17 cell transcriptional regulators T-bet and RORgammat and repressed IFN-gamma and IL-17 production. Bcl-6 also repressed expression of many microRNAs (miRNAs) predicted to control the Tfh cell signature, including miR-17-92, which repressed CXCR5 expression. Thus, Bcl-6 positively directs Tfh cell differentiation, through combined repression of miRNAs and transcription factors.
Assuntos
Linhagem da Célula , Proteínas de Ligação a DNA/metabolismo , Linfócitos T Auxiliares-Indutores/imunologia , Linfócitos T Auxiliares-Indutores/metabolismo , Fatores de Transcrição/metabolismo , Animais , Diferenciação Celular , Células Cultivadas , Citocinas/biossíntese , Proteínas de Ligação a DNA/deficiência , Proteínas de Ligação a DNA/genética , Humanos , Camundongos , Camundongos Knockout , MicroRNAs/genética , Família Multigênica , Ligação Proteica , Proteínas Proto-Oncogênicas c-bcl-6 , Linfócitos T Auxiliares-Indutores/citologia , Fatores de Transcrição/deficiência , Fatores de Transcrição/genética , Regulação para CimaRESUMO
An important feature of chemokines is their ability to bind to the glycosaminoglycan (GAG) side chains of proteoglycans, predominately heparin and heparan sulfate. To date, all chemokines tested bind to immobilized heparin in vitro, as well as cell surface heparan sulfate in vitro and in vivo. These interactions play an important role in modulating the action of chemokines by facilitating the formation of stable chemokine gradients within the vascular endothelium and directing leukocyte migration, by protecting chemokines from proteolysis, by inducing chemokine oligomerization, and by facilitating transcytosis. Despite the importance of eotaxin in eosinophil differentiation and recruitment being well established, little is known about the interaction between eotaxin and GAGs and the functional consequences of such an interaction. Here we report that eotaxin binds selectively to immobilized heparin with high affinity (K(d) = 1.23 x 10(-8) M), but not to heparan sulfate or a range of other GAGs. The interaction of eotaxin with heparin does not promote eotaxin oligomerization but protects eotaxin from proteolysis directly by plasmin and indirectly by cathepsin G and elastase. In vivo, co-administration of eotaxin and heparin is able to significantly enhance eotaxin-mediated eosinophil recruitment in a mouse air-pouch model. Furthermore, when heparin is co-administered with eotaxin at a concentration that does not normally result in eosinophil infiltration, eosinophil recruitment occurs. In contrast, heparin does not enhance eotaxin-mediated eosinophil chemotaxis in vitro, suggesting protease protection or haptotactic gradient formation as the mechanism by which heparin enhances eotaxin action in vivo. These results suggest a role for mast cell-derived heparin in the recruitment of eosinophils, reinforcing Th2 polarization of inflammatory responses.
Assuntos
Quimiocinas CC/metabolismo , Quimiotaxia de Leucócito , Eosinófilos/metabolismo , Heparina/metabolismo , Heparitina Sulfato/metabolismo , Processamento de Proteína Pós-Traducional , Animais , Anticoagulantes/química , Anticoagulantes/metabolismo , Anticoagulantes/farmacologia , Catepsina G , Catepsinas/metabolismo , Quimiocina CCL11 , Quimiocinas CC/química , Quimiocinas CC/farmacologia , Quimiotaxia de Leucócito/efeitos dos fármacos , Endotélio Vascular/metabolismo , Endotélio Vascular/patologia , Eosinófilos/patologia , Fibrinolisina/metabolismo , Heparina/química , Heparina/farmacologia , Heparitina Sulfato/química , Heparitina Sulfato/farmacologia , Inflamação/metabolismo , Inflamação/patologia , Masculino , Mastócitos/metabolismo , Mastócitos/patologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Transgênicos , Modelos Biológicos , Ligação Proteica/efeitos dos fármacos , Processamento de Proteína Pós-Traducional/efeitos dos fármacos , Serina Endopeptidases/metabolismo , Células Th2/metabolismo , Células Th2/patologiaRESUMO
The role of the immune system in the surveillance of transformed cells has seen a resurgence of interest in the last 10 years, with a substantial body of data in mice and humans supporting a role for the immune system in host protection from tumor development and in shaping tumor immunogenicity. A number of earlier studies have demonstrated that eosinophils, when recruited into tumors, can very effectively eradicate transplantable tumors. In this study, we investigated whether eosinophils also play a role in tumor immune surveillance by determining the incidence of methylcholanthrene (MCA)-induced fibrosarcomas in IL-5 transgenic mice that have greatly enhanced levels of circulating eosinophils, CCL11 (eotaxin-1)-deficient mice that lack a key chemokine that recruits eosinophils into tissues, and the eosinophil-deficient mouse strains, IL-5/CCL11(-/-) and DeltadblGATA. It was found that MCA-induced tumor incidence and growth were significantly attenuated in IL-5 transgenic mice of both the BALB/c and C57BL/6 backgrounds. Histological examination revealed that the protective effect of IL-5 was associated with massively enhanced numbers of eosinophils within and surrounding tumors. Conversely, there was a higher tumor incidence in CCL11(-/-) BALB/c mice, which was associated with a reduced eosinophil influx into tumors. This correlation was confirmed in the eosinophil-deficient IL-5/CCL11(-/-) and DeltadblGATA mouse strains, where tumor incidence was greatly increased in the total absence of eosinophils. In addition, subsequent in vitro studies found that eosinophils could directly kill MCA-induced fibrosarcoma cells. Collectively, our data support a potential role for the eosinophil as an effector cell in tumor immune surveillance.