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1.
Nat Immunol ; 20(10): 1299-1310, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31534238

RESUMO

Resisting and tolerating microbes are alternative strategies to survive infection, but little is known about the evolutionary mechanisms controlling this balance. Here genomic analyses of anatomically modern humans, extinct Denisovan hominins and mice revealed a TNFAIP3 allelic series with alterations in the encoded immune response inhibitor A20. Each TNFAIP3 allele encoded substitutions at non-catalytic residues of the ubiquitin protease OTU domain that diminished IκB kinase-dependent phosphorylation and activation of A20. Two TNFAIP3 alleles encoding A20 proteins with partial phosphorylation deficits seemed to be beneficial by increasing immunity without causing spontaneous inflammatory disease: A20 T108A;I207L, originating in Denisovans and introgressed in modern humans throughout Oceania, and A20 I325N, from an N-ethyl-N-nitrosourea (ENU)-mutagenized mouse strain. By contrast, a rare human TNFAIP3 allele encoding an A20 protein with 95% loss of phosphorylation, C243Y, caused spontaneous inflammatory disease in humans and mice. Analysis of the partial-phosphorylation A20 I325N allele in mice revealed diminished tolerance of bacterial lipopolysaccharide and poxvirus inoculation as tradeoffs for enhanced immunity.

2.
Nat Commun ; 10(1): 2201, 2019 05 17.
Artigo em Inglês | MEDLINE | ID: mdl-31101814

RESUMO

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 Completo do Exoma , Quinases da Família src/metabolismo
4.
Blood Adv ; 3(3): 219-229, 2019 02 12.
Artigo em Inglês | MEDLINE | ID: mdl-30674456

RESUMO

The asymmetric distribution of phospholipids in the plasma/organellar membranes is generated and maintained through phospholipid flippases in resting cells, but becomes disrupted in apoptotic cells and activated platelets, resulting in phosphatidylserine (PS) exposure on the cell surface. Stable PS exposure during apoptosis requires inactivation of flippases to prevent PS from being reinternalized. Here we show that flippase ATP8A1 is highly expressed in both murine and human platelets, but is not present in the plasma membrane. ATP8A1 is cleaved by the cysteine protease calpain during apoptosis, and the cleavage is prevented indirectly by caspase inhibition, involving blockage of calcium influx into platelets and subsequent calpain activation. In contrast, in platelets activated with thrombin and collagen and exposing PS, ATP8A1 remains intact. These data reveal a novel mechanism of flippase cleavage and suggest that flippase activity in intracellular membranes differs between platelets undergoing apoptosis and activation.

5.
Clin Transl Immunology ; 6(10): e159, 2017 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-29114388

RESUMO

Common variable immunodeficiency disorders (CVID) are a group of primary immunodeficiencies where monogenetic causes account for only a fraction of cases. On this evidence, CVID is potentially polygenic and epistatic although there are, as yet, no examples to support this hypothesis. We have identified a non-consanguineous family, who carry the C104R (c.310T>C) mutation of the Transmembrane Activator Calcium-modulator and cyclophilin ligand Interactor (TACI, TNFRSF13B) gene. Variants in TNFRSF13B/TACI are identified in up to 10% of CVID patients, and are associated with, but not solely causative of CVID. The proband is heterozygous for the TNFRSF13B/TACI C104R mutation and meets the Ameratunga et al. diagnostic criteria for CVID and the American College of Rheumatology criteria for systemic lupus erythematosus (SLE). Her son has type 1 diabetes, arthritis, reduced IgG levels and IgA deficiency, but has not inherited the TNFRSF13B/TACI mutation. Her brother, homozygous for the TNFRSF13B/TACI mutation, is in good health despite profound hypogammaglobulinemia and mild cytopenias. We hypothesised that a second unidentified mutation contributed to the symptomatic phenotype of the proband and her son. Whole-exome sequencing of the family revealed a de novo nonsense mutation (T168fsX191) in the Transcription Factor 3 (TCF3) gene encoding the E2A transcription factors, present only in the proband and her son. We demonstrate mutations of TNFRSF13B/TACI impair immunoglobulin isotype switching and antibody production predominantly via T-cell-independent signalling, while mutations of TCF3 impair both T-cell-dependent and -independent pathways of B-cell activation and differentiation. We conclude that epistatic interactions between mutations of the TNFRSF13B/TACI and TCF3 signalling networks lead to the severe CVID-like disorder and SLE in the proband.

6.
Proc Natl Acad Sci U S A ; 114(26): E5216-E5225, 2017 06 27.
Artigo em Inglês | MEDLINE | ID: mdl-28607084

RESUMO

T-cell immunity requires extremely rapid clonal proliferation of rare, antigen-specific T lymphocytes to form effector cells. Here we identify a critical role for ETAA1 in this process by surveying random germ line mutations in mice using exome sequencing and bioinformatic annotation to prioritize mutations in genes of unknown function with potential effects on the immune system, followed by breeding to homozygosity and testing for immune system phenotypes. Effector CD8+ and CD4+ T-cell formation following immunization, lymphocytic choriomeningitis virus (LCMV) infection, or herpes simplex virus 1 (HSV1) infection was profoundly decreased despite normal immune cell development in adult mice homozygous for two different Etaa1 mutations: an exon 2 skipping allele that deletes Gly78-Leu119, and a Cys166Stop truncating allele that eliminates most of the 877-aa protein. ETAA1 deficiency decreased clonal expansion cell autonomously within the responding T cells, causing no decrease in their division rate but increasing TP53-induced mRNAs and phosphorylation of H2AX, a marker of DNA replication stress induced by the ATM and ATR kinases. Homozygous ETAA1-deficient adult mice were otherwise normal, healthy, and fertile, although slightly smaller, and homozygotes were born at lower frequency than expected, consistent with partial lethality after embryonic day 12. Taken together with recently reported evidence in human cancer cell lines that ETAA1 activates ATR kinase through an exon 2-encoded domain, these findings reveal a surprisingly specific requirement for this ATR activator in adult mice restricted to rapidly dividing effector T cells. This specific requirement may provide new ways to suppress pathological T-cell responses in transplantation or autoimmunity.


Assuntos
Antígenos de Superfície/imunologia , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/imunologia , Divisão Celular/imunologia , Imunidade Celular , Mutação , Animais , Antígenos de Superfície/genética , Proteínas Mutadas de Ataxia Telangiectasia/genética , Proteínas Mutadas de Ataxia Telangiectasia/imunologia , Linfócitos T CD4-Positivos/patologia , Linfócitos T CD8-Positivos/patologia , Divisão Celular/genética , Herpes Simples/genética , Herpes Simples/imunologia , Herpes Simples/patologia , Herpesvirus Humano 1/genética , Herpesvirus Humano 1/imunologia , Coriomeningite Linfocítica/genética , Coriomeningite Linfocítica/imunologia , Coriomeningite Linfocítica/patologia , Vírus da Coriomeningite Linfocítica/genética , Vírus da Coriomeningite Linfocítica/imunologia , Camundongos , Camundongos Mutantes , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/imunologia
7.
Front Immunol ; 8: 549, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28553292

RESUMO

SLC1A5 (solute carrier family 1, member 5) is a small neutral amino acid exchanger that is upregulated in rapidly proliferating lymphocytes but also in many primary human cancers. Furthermore, cancer cell lines have been shown to require SLC1A5 for their survival in vitro. One of SLC1A5's primary substrates is the immunomodulatory amino acid glutamine, which plays an important role in multiple key processes, such as energy supply, macromolecular synthesis, nucleotide biosynthesis, redox homeostasis, and resistance against oxidative stress. These processes are also essential to immune cells, including neutrophils, macrophages, B and T lymphocytes. We show here that mice with a stop codon in Slc1a5 have reduced glutamine uptake in activated lymphocytes and primary fibroblasts. B and T cell populations and maturation in resting mice were not affected by absence of SLC1A5. Antibody production in resting and immunized mice and the germinal center response to immunization were also found to be normal. SLC1A5 has been recently described as a novel target for the treatment of a variety of cancers, and our results indicate that inhibition of SLC1A5 in cancer therapy may be tolerated well by the immune system of cancer patients.

8.
Nat Commun ; 7: 13381, 2016 11 10.
Artigo em Inglês | MEDLINE | ID: mdl-27830696

RESUMO

Self-tolerance by clonal anergy of B cells is marked by an increase in IgD and decrease in IgM antigen receptor surface expression, yet the function of IgD on anergic cells is obscure. Here we define the RNA landscape of the in vivo anergy response, comprising 220 induced sequences including a core set of 97. Failure to co-express IgD with IgM decreases overall expression of receptors for self-antigen, but paradoxically increases the core anergy response, exemplified by increased Sdc1 encoding the cell surface marker syndecan-1. IgD expressed on its own is nevertheless competent to induce calcium signalling and the core anergy mRNA response. Syndecan-1 induction correlates with reduction of surface IgM and is exaggerated without surface IgD in many transitional and mature B cells. These results show that IgD attenuates the response to self-antigen in anergic cells and promotes their accumulation. In this way, IgD minimizes tolerance-induced holes in the pre-immune antibody repertoire.


Assuntos
Linfócitos B/imunologia , Anergia Clonal/imunologia , Imunoglobulina D/imunologia , Imunoglobulina M/imunologia , Animais , Linfócitos B/citologia , Linfócitos B/metabolismo , Sinalização do Cálcio/genética , Sinalização do Cálcio/imunologia , Anergia Clonal/genética , Perfilação da Expressão Gênica/métodos , Imunoglobulina D/genética , Imunoglobulina M/genética , Ativação Linfocitária/genética , Ativação Linfocitária/imunologia , Masculino , Camundongos Endogâmicos C57BL , Mutação , Receptores de Antígenos de Linfócitos B/genética , Receptores de Antígenos de Linfócitos B/imunologia , Receptores de Antígenos de Linfócitos B/metabolismo , Tolerância a Antígenos Próprios/genética , Tolerância a Antígenos Próprios/imunologia , Sindecana-1/genética , Sindecana-1/imunologia , Sindecana-1/metabolismo
9.
Nat Immunol ; 17(11): 1300-1311, 2016 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-27668799

RESUMO

Mucosal-associated invariant T cells (MAIT cells) detect microbial vitamin B2 derivatives presented by the antigen-presenting molecule MR1. Here we defined three developmental stages and checkpoints for the MAIT cell lineage in humans and mice. Stage 1 and stage 2 MAIT cells predominated in thymus, while stage 3 cells progressively increased in abundance extrathymically. Transition through each checkpoint was regulated by MR1, whereas the final checkpoint that generated mature functional MAIT cells was controlled by multiple factors, including the transcription factor PLZF and microbial colonization. Furthermore, stage 3 MAIT cell populations were expanded in mice deficient in the antigen-presenting molecule CD1d, suggestive of a niche shared by MAIT cells and natural killer T cells (NKT cells). Accordingly, this study maps the developmental pathway and checkpoints that control the generation of functional MAIT cells.


Assuntos
Diferenciação Celular/imunologia , Células T Invariáveis Associadas à Mucosa/citologia , Células T Invariáveis Associadas à Mucosa/fisiologia , Timo/imunologia , Timo/metabolismo , Animais , Antígenos CD1d/genética , Biomarcadores , Diferenciação Celular/genética , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Humanos , Imunofenotipagem , Células Progenitoras Linfoides/imunologia , Células Progenitoras Linfoides/metabolismo , Masculino , Camundongos , Camundongos Knockout , MicroRNAs/genética
10.
Eur J Immunol ; 46(2): 265-8, 2016 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-26840197

RESUMO

The German Society for Immunology (DGfI) and the Australasian Society for Immunology (ASI) hosted the first DGfI-ASI joint workshop from December 3-4, 2015 in Canberra, Australia. A delegation of 15 distinguished German immunologists discussed the workshop topic "immune regulation in infections and immune mediated diseases" with the aim to establish new German-Australasian collaborations, discuss new concepts in the field of immune regulation and build a scientific network to create more utilizable resources for excellent (trans-border) immunological research. The workshop was associated with the 45(th) Annual Scientific Meeting of the ASI held from Nov 29-Dec 3, 2015, opening up even more opportunities for finding new collaboration partners. A return meeting will be linked to the annual DGfI meeting that will take place in 2017 in Erlangen.


Assuntos
Alergia e Imunologia , Doenças Transmissíveis/imunologia , Doenças do Sistema Imunitário/imunologia , Austrália , Comportamento Cooperativo , Alemanha , Processos Grupais , Humanos , Sociedades Científicas
11.
PLoS One ; 11(1): e0146774, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-26799398

RESUMO

Organization of the plasma membrane into specialized substructures in different blood lineages facilitates important biological functions including proper localization of receptors at the plasma membrane as well as the initiation of crucial intracellular signaling cascades. The eukaryotic plasma membrane is a lipid bilayer that consists of asymmetrically distributed phospholipids. This asymmetry is actively maintained by membrane-embedded lipid transporters, but there is only limited data available about the molecular identity of the predominantly active transporters and their substrate specificity in different leukocyte subsets. We demonstrate here that the P4-type ATPase ATP11C mediates significant flippase activity in all murine leukocyte subsets. Loss of ATP11C resulted in a defective internalization of phosphatidylserine (PS) and phosphatidylethanolamine (PE) in comparison to control cells. The diminished flippase activity caused increased PS exposure on 7-aminoactinomycin D- (7-AAD-) viable pro-B cells freshly isolated from the bone marrow of ATP11C-deficient mice, which was corrected upon a 2-hour resting period in vitro. Despite the impaired flippase activity in all immune cell subsets, the only other blood cell type with an accumulation of PS on the surface were viable 7-AAD- developing T cells but this did not result in any discernable effect on their development in the thymus. These findings show that all leukocyte lineages exhibit flippase activity, and identify ATP11C as an aminophospholipid translocase in immune cells.


Assuntos
Adenosina Trifosfatases/genética , Linfócitos B/metabolismo , Transporte Biológico Ativo/fisiologia , Fosfatidiletanolaminas/metabolismo , Fosfatidilserinas/metabolismo , Proteínas de Transferência de Fosfolipídeos/metabolismo , Linfócitos T/metabolismo , Adenosina Trifosfatases/metabolismo , Animais , Células Cultivadas , Dactinomicina/análogos & derivados , Dactinomicina/metabolismo , Leucócitos/metabolismo , Camundongos , Camundongos Endogâmicos C57BL
13.
Proc Natl Acad Sci U S A ; 112(37): E5189-98, 2015 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-26269570

RESUMO

Each person's genome sequence has thousands of missense variants. Practical interpretation of their functional significance must rely on computational inferences in the absence of exhaustive experimental measurements. Here we analyzed the efficacy of these inferences in 33 de novo missense mutations revealed by sequencing in first-generation progeny of N-ethyl-N-nitrosourea-treated mice, involving 23 essential immune system genes. PolyPhen2, SIFT, MutationAssessor, Panther, CADD, and Condel were used to predict each mutation's functional importance, whereas the actual effect was measured by breeding and testing homozygotes for the expected in vivo loss-of-function phenotype. Only 20% of mutations predicted to be deleterious by PolyPhen2 (and 15% by CADD) showed a discernible phenotype in individual homozygotes. Half of all possible missense mutations in the same 23 immune genes were predicted to be deleterious, and most of these appear to become subject to purifying selection because few persist between separate mouse substrains, rodents, or primates. Because defects in immune genes could be phenotypically masked in vivo by compensation and environment, we compared inferences by the same tools with the in vitro phenotype of all 2,314 possible missense variants in TP53; 42% of mutations predicted by PolyPhen2 to be deleterious (and 45% by CADD) had little measurable consequence for TP53-promoted transcription. We conclude that for de novo or low-frequency missense mutations found by genome sequencing, half those inferred as deleterious correspond to nearly neutral mutations that have little impact on the clinical phenotype of individual cases but will nevertheless become subject to purifying selection.


Assuntos
Mutação de Sentido Incorreto , Animais , Códon , Biologia Computacional , Simulação por Computador , Exoma , Variação Genética , Genoma , Genoma Humano , Genótipo , Humanos , Sistema Imunitário , Síndromes de Imunodeficiência/genética , Camundongos , Camundongos Endogâmicos C57BL , Modelos Genéticos , Neoplasias/genética , Fenótipo , Software , Proteína Supressora de Tumor p53/genética
14.
Blood ; 126(14): 1658-69, 2015 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-26289640

RESUMO

Omenn syndrome (OS) is a severe immunodeficiency associated with erythroderma, lymphoproliferation, elevated IgE, and hyperactive oligoclonal T cells. A restricted T-cell repertoire caused by defective thymic T-cell development and selection, lymphopenia with homeostatic proliferation, and lack of regulatory T cells are considered key factors in OS pathogenesis. We report 2 siblings presenting with cytomegalovirus (CMV) and Pneumocystis jirovecii infections and recurrent sepsis; one developed all clinical features of OS. Both carried homozygous germline mutations in CARD11 (p.Cys150*), impairing NF-κB signaling and IL-2 production. A somatic second-site mutation reverting the stop codon to a missense mutation (p.Cys150Leu) was detected in tissue-infiltrating T cells of the OS patient. Expression of p.Cys150Leu in CARD11-deficient T cells largely reconstituted NF-κB signaling. The reversion likely occurred in a prethymic T-cell precursor, leading to a chimeric T-cell repertoire. We speculate that in our patient the functional advantage of the revertant T cells in the context of persistent CMV infection, combined with lack of regulatory T cells, may have been sufficient to favor OS. This first observation of OS in a patient with a T-cell activation defect suggests that severely defective T-cell development or homeostatic proliferation in a lymphopenic environment are not required for this severe immunopathology.


Assuntos
Proteínas Adaptadoras de Sinalização CARD/genética , Guanilato Ciclase/genética , Ativação Linfocitária/genética , Mutação , Imunodeficiência Combinada Severa/genética , Linfócitos T Reguladores/imunologia , Animais , Proteínas Adaptadoras de Sinalização CARD/deficiência , Proteínas Adaptadoras de Sinalização CARD/imunologia , Feminino , Citometria de Fluxo , Guanilato Ciclase/deficiência , Guanilato Ciclase/imunologia , Humanos , Immunoblotting , Imuno-Histoquímica , Imunofenotipagem , Lactente , Ativação Linfocitária/imunologia , Masculino , Camundongos , Reação em Cadeia da Polimerase em Tempo Real , Imunodeficiência Combinada Severa/imunologia , Irmãos
15.
J Exp Med ; 212(7): 1095-108, 2015 Jun 29.
Artigo em Inglês | MEDLINE | ID: mdl-26101265

RESUMO

Studies on the biology of mucosal-associated invariant T cells (MAIT cells) in mice have been hampered by a lack of specific reagents. Using MR1-antigen (Ag) tetramers that specifically bind to the MR1-restricted MAIT T cell receptors (TCRs), we demonstrate that MAIT cells are detectable in a broad range of tissues in C57BL/6 and BALB/c mice. These cells include CD4(-)CD8(-), CD4(-)CD8(+), and CD4(+)CD8(-) subsets, and their frequency varies in a tissue- and strain-specific manner. Mouse MAIT cells have a CD44(hi)CD62L(lo) memory phenotype and produce high levels of IL-17A, whereas other cytokines, including IFN-γ, IL-4, IL-10, IL-13, and GM-CSF, are produced at low to moderate levels. Consistent with high IL-17A production, most MAIT cells express high levels of retinoic acid-related orphan receptor γt (RORγt), whereas RORγt(lo) MAIT cells predominantly express T-bet and produce IFN-γ. Most MAIT cells express the promyelocytic leukemia zinc finger (PLZF) transcription factor, and their development is largely PLZF dependent. These observations contrast with previous reports that MAIT cells from Vα19 TCR transgenic mice are PLZF(-) and express a naive CD44(lo) phenotype. Accordingly, MAIT cells from normal mice more closely resemble human MAIT cells than previously appreciated, and this provides the foundation for further investigations of these cells in health and disease.


Assuntos
Antígenos de Histocompatibilidade Classe I/metabolismo , Membrana Mucosa/imunologia , Células T Matadoras Naturais/metabolismo , Receptores de Antígenos de Linfócitos T/metabolismo , Animais , Proliferação de Células , Citocinas/metabolismo , Técnicas Histológicas , Fatores de Transcrição Kruppel-Like/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Antígenos de Histocompatibilidade Menor , Membrana Mucosa/citologia , Células T Matadoras Naturais/citologia , Proteína com Dedos de Zinco da Leucemia Promielocítica , Especificidade da Espécie
16.
Bioinformatics ; 31(14): 2377-9, 2015 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-25755272

RESUMO

MOTIVATION: Increasingly, cost-effective high-throughput DNA sequencing technologies are being utilized to sequence human pedigrees to elucidate the genetic cause of a wide variety of human diseases. While numerous tools exist for variant prioritization within a single genome, the ability to concurrently analyze variants within pedigrees remains a challenge, especially should there be no prior indication of the underlying genetic cause of the disease. Here, we present a tool, variant analysis of sequenced pedigrees (VASP), a flexible data integration environment capable of producing a summary of pedigree variation, providing relevant information such as compound heterozygosity, genome phasing and disease inheritance patterns. Designed to aggregate data across a sequenced pedigree, VASP allows both powerful filtering and custom prioritization of both single nucleotide variants (SNVs) and small indels. Hence, clinical and research users with prior knowledge of a disease are able to dramatically reduce the variant search space based on a wide variety of custom prioritization criteria. AVAILABILITY AND IMPLEMENTATION: Source code available for academic non-commercial research purposes at https://github.com/mattmattmattmatt/VASP.


Assuntos
Ligação Genética , Predisposição Genética para Doença , Variação Genética/genética , Software , Feminino , Marcadores Genéticos/genética , Heterozigoto , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Humanos , Mutação INDEL/genética , Masculino , Linhagem , Polimorfismo de Nucleotídeo Único/genética
17.
J Immunol ; 194(6): 2725-34, 2015 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-25672755

RESUMO

Defining the minimal thresholds for effective antiviral T cell immunity is important for clinical decisions in immunodeficient patients. TCR signaling is critical for T cell development, activation, and effector functions. In this article, we analyzed which of these TCR-mediated processes is limiting for antiviral immunity in a mouse strain with reduced expression of SLP-76 (twp mice). Despite severe T cell activation defects in vitro, twp mice generated a normal proportion of antiviral effector T cells postinfection with lymphocytic choriomeningitis virus (LCMV). Twp CD8(+) T cells showed impaired polyfunctional cytokine production, whereas cytotoxicity as the crucial antiviral effector function for LCMV control was normal. The main limiting factor in the antiviral response of twp mice was impaired T cell proliferation and survival, leading to a 5- to 10-fold reduction of antiviral T cells at the peak of the immune response. This was still sufficient to control infection with the LCMV Armstrong strain, but the more rapidly replicating LCMV-WE induced T cell exhaustion and viral persistence. Thus, under conditions of impaired TCR signaling, reduced T cell expansion was the limiting factor in antiviral immunity. These findings have implications for understanding antiviral immunity in patients with T cell deficiencies.


Assuntos
Coriomeningite Linfocítica/imunologia , Vírus da Coriomeningite Linfocítica/imunologia , Receptores de Antígenos de Linfócitos T/imunologia , Linfócitos T/imunologia , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/imunologia , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Apoptose/genética , Apoptose/imunologia , Western Blotting , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/metabolismo , Proliferação de Células/genética , Sobrevivência Celular/genética , Sobrevivência Celular/imunologia , Citocinas/imunologia , Citocinas/metabolismo , Resistência à Doença/genética , Resistência à Doença/imunologia , Citometria de Fluxo , Interações Hospedeiro-Patógeno/imunologia , Humanos , Síndromes de Imunodeficiência/genética , Síndromes de Imunodeficiência/imunologia , Contagem de Linfócitos , Coriomeningite Linfocítica/virologia , Vírus da Coriomeningite Linfocítica/fisiologia , Camundongos Mutantes , Fosfoproteínas/genética , Fosfoproteínas/imunologia , Fosfoproteínas/metabolismo , Receptores de Antígenos de Linfócitos T/metabolismo , Linfócitos T/metabolismo
18.
J Biol Chem ; 289(28): 19531-7, 2014 Jul 11.
Artigo em Inglês | MEDLINE | ID: mdl-24898253

RESUMO

Transmembrane lipid transporters are believed to establish and maintain phospholipid asymmetry in biological membranes; however, little is known about the in vivo function of the specific transporters involved. Here, we report that developing erythrocytes from mice lacking the putative phosphatidylserine flippase ATP11C showed a lower rate of PS translocation in vitro compared with erythrocytes from wild-type littermates. Furthermore, the mutant mice had an elevated percentage of phosphatidylserine-exposing mature erythrocytes in the periphery. Although erythrocyte development in ATP11C-deficient mice was normal, the mature erythrocytes had an abnormal shape (stomatocytosis), and the life span of mature erythrocytes was shortened relative to that in control littermates, resulting in anemia in the mutant mice. Thus, our findings uncover an essential role for ATP11C in erythrocyte morphology and survival and provide a new candidate for the rare inherited blood disorder stomatocytosis with uncompensated anemia.


Assuntos
Adenosina Trifosfatases/metabolismo , Membrana Eritrocítica/enzimologia , Fosfolipídeos/metabolismo , Desequilíbrio Ácido-Base/genética , Desequilíbrio Ácido-Base/metabolismo , Desequilíbrio Ácido-Base/patologia , Adenosina Trifosfatases/genética , Anemia Hemolítica Congênita/genética , Anemia Hemolítica Congênita/metabolismo , Anemia Hemolítica Congênita/patologia , Animais , Transporte Biológico Ativo , Sobrevivência Celular/fisiologia , Membrana Eritrocítica/genética , Eritrócitos Anormais/metabolismo , Eritrócitos Anormais/patologia , Erros Inatos do Metabolismo/genética , Erros Inatos do Metabolismo/metabolismo , Erros Inatos do Metabolismo/patologia , Camundongos , Camundongos Mutantes , Fosfolipídeos/genética
19.
Proc Natl Acad Sci U S A ; 111(12): 4513-8, 2014 Mar 25.
Artigo em Inglês | MEDLINE | ID: mdl-24616512

RESUMO

IgD and IgM are produced by alternative splicing of long primary RNA transcripts from the Ig heavy chain (Igh) locus and serve as the receptors for antigen on naïve mature B lymphocytes. IgM is made selectively in immature B cells, whereas IgD is coexpressed with IgM when the cells mature into follicular or marginal zone B cells, but the transacting factors responsible for this regulated change in splicing have remained elusive. Here, we use a genetic screen in mice to identify ZFP318, a nuclear protein with two U1-type zinc fingers found in RNA-binding proteins and no known role in the immune system, as a critical factor for IgD expression. A point mutation in an evolutionarily conserved lysine-rich domain encoded by the alternatively spliced Zfp318 exon 10 abolished IgD expression on marginal zone B cells, decreased IgD on follicular B cells, and increased IgM, but only slightly decreased the percentage of B cells and did not decrease expression of other maturation markers CD21, CD23, or CD62L. A targeted Zfp318 null allele extinguished IgD expression on mature B cells and increased IgM. Zfp318 mRNA is developmentally regulated in parallel with IgD, with little in pro-B cells, moderate amounts in immature B cells, and high levels selectively in mature follicular B cells. These findings identify ZFP318 as a crucial factor regulating the expression of the two major antibody isotypes on the surface of most mature B cells.


Assuntos
Processamento Alternativo , Linfócitos B/metabolismo , Imunoglobulina D/genética , Cadeias Pesadas de Imunoglobulinas/genética , Dedos de Zinco , Sequência de Aminoácidos , Animais , Humanos , Camundongos , Dados de Sequência Molecular , Mutação de Sentido Incorreto , Homologia de Sequência de Aminoácidos
20.
Genome Biol ; 15(1): R26, 2014 Jan 29.
Artigo em Inglês | MEDLINE | ID: mdl-24476532

RESUMO

BACKGROUND: Retention of a subset of introns in spliced polyadenylated mRNA is emerging as a frequent, unexplained finding from RNA deep sequencing in mammalian cells. RESULTS: Here we analyze intron retention in T lymphocytes by deep sequencing polyadenylated RNA. We show a developmentally regulated RNA-binding protein, hnRNPLL, induces retention of specific introns by sequencing RNA from T cells with an inactivating Hnrpll mutation and from B lymphocytes that physiologically downregulate Hnrpll during their differentiation. In Ptprc mRNA encoding the tyrosine phosphatase CD45, hnRNPLL induces selective retention of introns flanking exons 4 to 6; these correspond to the cassette exons containing hnRNPLL binding sites that are skipped in cells with normal, but not mutant or low, hnRNPLL. We identify similar patterns of hnRNPLL-induced differential intron retention flanking alternative exons in 14 other genes, representing novel elements of the hnRNPLL-induced splicing program in T cells. Retroviral expression of a normally spliced cDNA for one of these targets, Senp2, partially corrects the survival defect of Hnrpll-mutant T cells. We find that integrating a number of computational methods to detect genes with differentially retained introns provides a strategy to enrich for alternatively spliced exons in mammalian RNA-seq data, when complemented by RNA-seq analysis of purified cells with experimentally perturbed RNA-binding proteins. CONCLUSIONS: Our findings demonstrate that intron retention in mRNA is induced by specific RNA-binding proteins and suggest a biological significance for this process in marking exons that are poised for alternative splicing.


Assuntos
Processamento Alternativo , Ribonucleoproteínas Nucleares Heterogêneas/metabolismo , Íntrons , Proteínas de Ligação a RNA/metabolismo , Animais , Mapeamento Cromossômico , Biologia Computacional , Cisteína Endopeptidases/genética , Cisteína Endopeptidases/metabolismo , Éxons , Ribonucleoproteínas Nucleares Heterogêneas/genética , Sequenciamento de Nucleotídeos em Larga Escala , Antígenos Comuns de Leucócito/genética , Antígenos Comuns de Leucócito/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/genética , Análise de Sequência de RNA , Linfócitos T/metabolismo
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