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1.
Cell ; 2020 Feb 05.
Artigo em Inglês | MEDLINE | ID: mdl-32059783

RESUMO

Pathogenic autoantibodies arise in many autoimmune diseases, but it is not understood how the cells making them evade immune checkpoints. Here, single-cell multi-omics analysis demonstrates a shared mechanism with lymphoid malignancy in the formation of public rheumatoid factor autoantibodies responsible for mixed cryoglobulinemic vasculitis. By combining single-cell DNA and RNA sequencing with serum antibody peptide sequencing and antibody synthesis, rare circulating B lymphocytes making pathogenic autoantibodies were found to comprise clonal trees accumulating mutations. Lymphoma driver mutations in genes regulating B cell proliferation and V(D)J mutation (CARD11, TNFAIP3, CCND3, ID3, BTG2, and KLHL6) were present in rogue B cells producing the pathogenic autoantibody. Antibody V(D)J mutations conferred pathogenicity by causing the antigen-bound autoantibodies to undergo phase transition to insoluble aggregates at lower temperatures. These results reveal a pre-neoplastic stage in human lymphomagenesis and a cascade of somatic mutations leading to an iconic pathogenic autoantibody.

2.
J Exp Med ; 217(2)2020 Feb 03.
Artigo em Inglês | MEDLINE | ID: mdl-31841125

RESUMO

Antibody-mediated autoimmune diseases are a major health burden. However, our understanding of how self-reactive B cells escape self-tolerance checkpoints to secrete pathogenic autoantibodies remains incomplete. Here, we demonstrate that patients with monogenic immune dysregulation caused by gain-of-function mutations in PIK3CD, encoding the p110δ catalytic subunit of phosphoinositide 3-kinase (PI3K), have highly penetrant secretion of autoreactive IgM antibodies. In mice with the corresponding heterozygous Pik3cd activating mutation, self-reactive B cells exhibit a cell-autonomous subversion of their response to self-antigen: instead of becoming tolerized and repressed from secreting autoantibody, Pik3cd gain-of-function B cells are activated by self-antigen to form plasmablasts that secrete high titers of germline-encoded IgM autoantibody and hypermutating germinal center B cells. However, within the germinal center, peripheral tolerance was still enforced, and there was selection against B cells with high affinity for self-antigen. These data show that the strength of PI3K signaling is a key regulator of pregerminal center B cell self-tolerance and thus represents a druggable pathway to treat antibody-mediated autoimmunity.

3.
Immunol Rev ; 292(1): 61-75, 2019 11.
Artigo em Inglês | MEDLINE | ID: mdl-31556969

RESUMO

The adaptive immune system is tasked with producing antibodies that recognize a wide scope of potential pathogens, including those never before encountered, and concurrently avoiding formation of antibodies binding host tissues. The diverse repertoire of antibodies produced by V(D)J recombination inevitably includes autoantibodies that bind to self-antigens, estimated to be as much as 70% of nascent antibodies on immature B cells. Early theoretical models of tolerance hypothesized that such self-reactive clones could not possibly be allowed to survive and mature. However from the first direct view of the fate of nascent B cells carrying a self-binding antibody it was clear that many "forbidden clones" circulate to secondary lymphoid tissues, where they adopt an IgMlow IgD+ cell surface phenotype and are prevented from secreting autoantibodies by a series of tolerance checkpoints referred to as "clonal anergy." Since anergic B cells can be reactivated to secrete pathogenic autoantibodies in certain settings, the advantage of controlling self-reactive antibodies by clonal anergy has until recently remained enigmatic. Here we review this topic and recent advances showing that anergic B cells are recruited into the germinal center to mutate away from self-reactivity, undergoing "clonal redemption" into cells making antibodies with exquisite specificity for foreign immunogens.

4.
Front Immunol ; 10: 1544, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31396201

RESUMO

One of the primary targets of immune checkpoint inhibition is the negative immune regulatory molecule CTLA-4. Immune-related adverse events are commonly observed following CTLA-4 inhibition in melanoma treatment, and a spectrum of these conditions are also observed in individuals with germline haploinsufficiency of CTLA4. Here we describe a heterozygous de novo missense variant of CTLA4 in a young girl with childhood-onset autoimmune hepatitis and polyarthritis, the latter responding to treatment with CTLA-4-Ig fusion protein. This variant lay within the highly conserved MYPPPY motif of CTLA-4: a critical structural determinant of ligand binding, which is also bound by the anti-CTLA-4 monoclonal antibody ipilimumab. Within the spectrum of CTLA4 variants reported, missense variants in the MYPPPY motif were overrepresented when compared to variants within a control population, highlighting the physiological importance of this motif in both the genetic and pharmacological regulation of autoimmunity and anti-tumor immunity.

5.
Nat Commun ; 10(1): 3120, 2019 07 16.
Artigo em Inglês | MEDLINE | ID: mdl-31311926

RESUMO

High-throughput single-cell RNA sequencing is a powerful technique but only generates short reads from one end of a cDNA template, limiting the reconstruction of highly diverse sequences such as antigen receptors. To overcome this limitation, we combined targeted capture and long-read sequencing of T-cell-receptor (TCR) and B-cell-receptor (BCR) mRNA transcripts with short-read transcriptome profiling of barcoded single-cell libraries generated by droplet-based partitioning. We show that Repertoire and Gene Expression by Sequencing (RAGE-Seq) can generate accurate full-length antigen receptor sequences at nucleotide resolution, infer B-cell clonal evolution and identify alternatively spliced BCR transcripts. We apply RAGE-Seq to 7138 cells sampled from the primary tumor and draining lymph node of a breast cancer patient to track transcriptome profiles of expanded lymphocyte clones across tissues. Our results demonstrate that RAGE-Seq is a powerful method for tracking the clonal evolution from large numbers of lymphocytes applicable to the study of immunity, autoimmunity and cancer.


Assuntos
Evolução Clonal/genética , Linfócitos/metabolismo , Análise de Célula Única/métodos , Evolução Clonal/imunologia , Perfilação da Expressão Gênica , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Humanos , Cultura Primária de Células , Receptores de Antígenos de Linfócitos B/genética , Receptores de Antígenos de Linfócitos B/metabolismo , Receptores de Antígenos de Linfócitos T/genética , Receptores de Antígenos de Linfócitos T/metabolismo , Análise de Sequência de RNA/métodos
6.
Immunol Cell Biol ; 97(8): 740-752, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31087793

RESUMO

FOXP3+ regulatory T (Treg) cells are essential for immunological tolerance and immune homeostasis. Despite a great deal of interest in modulating their number and function for the treatment of autoimmune disease or cancer, the precise mechanisms that control the homeostasis of Treg cells remain unclear. We report a new ENU-induced mutant mouse, lack of costimulation (loco), with atopic dermatitis and Treg cell deficiency typical of Card11 loss-of-function mutants. Three distinct single nucleotide variants were found in the Card11 introns 2, 10 and 20 that cause the loss of CARD11 expression in these mutant mice. These mutations caused the loss of thymic-derived, Neuropilin-1+ (NRP1+ ) Treg cells in neonatal and adult loco mice; however, residual peripherally induced NRP1- Treg cells remained. These peripherally generated Treg cells could be expanded in vivo by the administration of IL-2:anti-IL-2 complexes, indicating that this key homeostatic signaling axis remained intact in CARD11-deficient Treg cells. Furthermore, these expanded Treg cells could mediate near-normal suppression of activated, conventional CD4+ T cells, suggesting that CARD11 is dispensable for Treg cell function. In addition to shedding light on the requirements for CARD11 in Treg cell homeostasis and function, these data reveal novel noncoding Card11 loss-of-function mutations that impair the expression of this critical immune-regulatory protein.

7.
Sci Signal ; 12(582)2019 May 21.
Artigo em Inglês | MEDLINE | ID: mdl-31113851

RESUMO

Gasdermin-D (GSDMD) is cleaved by caspase-1, caspase-4, and caspase-11 in response to canonical and noncanonical inflammasome activation. Upon cleavage, GSDMD oligomerizes and forms plasma membrane pores, resulting in interleukin-1ß (IL-1ß) secretion, pyroptotic cell death, and inflammatory pathologies, including periodic fever syndromes and septic shock-a plague on modern medicine. Here, we showed that IRF2, a member of the interferon regulatory factor (IRF) family of transcription factors, was essential for the transcriptional activation of GSDMD. A forward genetic screen with N-ethyl-N-nitrosourea (ENU)-mutagenized mice linked IRF2 to inflammasome signaling. GSDMD expression was substantially attenuated in IRF2-deficient macrophages, endothelial cells, and multiple tissues, which corresponded with reduced IL-1ß secretion and inhibited pyroptosis. Mechanistically, IRF2 bound to a previously uncharacterized but unique site within the GSDMD promoter to directly drive GSDMD transcription for the execution of pyroptosis. Disruption of this single IRF2-binding site abolished signaling by both the canonical and noncanonical inflammasomes. Together, our data illuminate a key transcriptional mechanism for expression of the gene encoding GSDMD, a critical mediator of inflammatory pathologies.

8.
Cell Death Differ ; 26(12): 2727-2739, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31019259

RESUMO

The selection of αß T cells in the thymus is punctuated by checkpoints at which thymocytes differentiate or undergo apoptosis. Wave 1 deletion is defined as apoptosis within nascent αß T-cell antigen receptor (TCR)-signalled thymocytes that lack CCR7 expression. The antigen-presenting cell (APC) types that mediate wave 1 deletion are unclear. To measure wave 1 deletion, we compared the frequencies of TCRß + CD5 + Helios + CCR7- cells in nascent thymocyte cohorts in mice with normal or defective apoptosis. This thymocyte population is small in mice lacking major histocompatibility complex (MHC) expression. The scale of wave 1 deletion was increased by transgenic expression of the self-reactive Yae62 TCRß chain, was almost halved when haemopoietic APCs lacked MHC expression and, surprisingly, was unchanged when epithelial cells lacked MHC expression. These findings demonstrate efficiency, and some redundancy, in the APC types that mediate wave 1 deletion in the normal mouse thymus.

9.
Cancer Cell ; 35(2): 297-314.e8, 2019 02 11.
Artigo em Inglês | MEDLINE | ID: mdl-30753827

RESUMO

Promoter CpG islands are typically unmethylated in normal cells, but in cancer a proportion are subject to hypermethylation. Using methylome sequencing we identified CpG islands that display partial methylation encroachment across the 5' or 3' CpG island borders. CpG island methylation encroachment is widespread in prostate and breast cancer and commonly associates with gene suppression. We show that the pattern of H3K4me1 at CpG island borders in normal cells predicts the different modes of cancer CpG island hypermethylation. Notably, genetic manipulation of Kmt2d results in concordant alterations in H3K4me1 levels and CpG island border DNA methylation encroachment. Our findings suggest a role for H3K4me1 in the demarcation of CpG island methylation borders in normal cells, which become eroded in cancer.


Assuntos
Ilhas de CpG , Metilação de DNA , DNA de Neoplasias/metabolismo , Histonas/metabolismo , Neoplasias/metabolismo , 5-Metilcitosina/análogos & derivados , 5-Metilcitosina/metabolismo , Animais , Linhagem Celular Tumoral , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Feminino , Regulação Neoplásica da Expressão Gênica , Histona-Lisina N-Metiltransferase/genética , Histona-Lisina N-Metiltransferase/metabolismo , Histonas/genética , Humanos , Masculino , Metilação , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteína de Leucina Linfoide-Mieloide/genética , Proteína de Leucina Linfoide-Mieloide/metabolismo , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Neoplasias/genética , Regiões Promotoras Genéticas
11.
Methods Mol Biol ; 1827: 287-309, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30196503

RESUMO

Here we describe methods for screening human blood to isolate peripheral blood mononuclear cells (PBMCs) capable of binding fluorescently labeled antigen, as well as methods for the amplification and sequencing of B cell receptor (BCR) heavy and light chain genes. Detailed protocols are provided for transient mammalian expression in a hexahistidine-tagged Fab format, purification by immobilized metal affinity chromatography (IMAC), and affinity determination by BioLayer interferometry (BLI).


Assuntos
Antígenos/sangue , Linfócitos B/imunologia , Cromatografia de Afinidade/métodos , Epitopos , Análise de Sequência de Proteína/métodos , Eletroforese em Gel de Poliacrilamida , Corantes Fluorescentes/metabolismo , Humanos , Fragmentos Fab das Imunoglobulinas/metabolismo , Interferometria , Receptores de Antígenos de Linfócitos B/química , Receptores de Antígenos de Linfócitos B/metabolismo
12.
Cell Rep ; 24(3): 577-584, 2018 07 17.
Artigo em Inglês | MEDLINE | ID: mdl-30021156

RESUMO

Escape from peripheral tolerance checkpoints that control cytotoxic CD8+ T cells is important for cancer immunotherapy and autoimmunity, but pathways enforcing these checkpoints are mostly uncharted. We reveal that the HECT-type ubiquitin ligase activator, NDFIP1, enforces a cell-intrinsic CD8+ T cell checkpoint that desensitizes TCR signaling during in vivo exposure to high antigen levels. Ndfip1-deficient OT-I CD8+ T cells responding to high exogenous tolerogenic antigen doses that normally induce anergy aberrantly expanded and differentiated into effector cells that could precipitate autoimmune diabetes in RIP-OVAhi mice. In contrast, NDFIP1 was dispensable for peripheral deletion to low-dose exogenous or pancreatic islet-derived antigen and had little impact upon effector responses to Listeria or acute LCMV infection. These data provide evidence that NDFIP1 mediates a CD8+ T cell tolerance checkpoint, with a different mechanism to CD4+ T cells, and indicates that CD8+ T cell deletion and anergy are molecularly separable checkpoints.


Assuntos
Antígenos/metabolismo , Linfócitos T CD8-Positivos/imunologia , Proteínas de Transporte/metabolismo , Tolerância Imunológica , Proteínas de Membrana/metabolismo , Animais , Autoantígenos/metabolismo , Diferenciação Celular , Proliferação de Células , Anergia Clonal , Relação Dose-Resposta Imunológica , Peptídeos e Proteínas de Sinalização Intercelular , Proteínas de Membrana/deficiência , Camundongos Endogâmicos C57BL , Mutação/genética , Pâncreas/imunologia , Peptídeos/metabolismo , Receptores de Antígenos de Linfócitos T/metabolismo , Transdução de Sinais
13.
Bioessays ; 40(8): e1800050, 2018 08.
Artigo em Inglês | MEDLINE | ID: mdl-29869436

RESUMO

Cancer cells seem to exploit mechanisms that evolve as part of physiological tolerance, which is a complementary and often beneficial form of defense. The study of physiological systems of tolerance can therefore provide insights into the development of a state of host tolerance of cancer, and how to break it. Analysis of these models has the potential to improve our understanding of existing immunological therapeutic targets, and help to identify future targets and rational therapeutic combinations. The treatment of cancer with immune checkpoint inhibitors aims to reverse the progression to tolerance of cancer, and achieve an immunogenic, rather than tolerogenic, homeostasis. Broadening the efficacy and durability of checkpoint inhibitors focuses on reversing tolerance and stimulating immunogenicity in the cancer, host, and environment. Two examples of important physiological states of tolerance that may inform tolerance of cancer are microbial infection and placental reproduction. These states of tolerance result from bilateral shaping of host and non-self, akin to immunoediting in cancer, and offer reliable models to study the immune tolerance paradigm.


Assuntos
Interações Hospedeiro-Patógeno/imunologia , Tolerância Imunológica/fisiologia , Neoplasias/imunologia , Placenta/fisiologia , Aloenxertos/imunologia , Animais , Feminino , Humanos , Microbiota , Myxoma virus/patogenicidade , Infecções por Poxviridae/mortalidade , Gravidez , Microambiente Tumoral/imunologia
14.
Immunol Cell Biol ; 96(6): 553-561, 2018 07.
Artigo em Inglês | MEDLINE | ID: mdl-29726044

RESUMO

The thymus plays a crucial role in immune tolerance by exposing developing T cells (thymocytes) to a myriad of self-antigens. Strong T-cell receptor (TCR) engagement induces tolerance in self-reactive thymocytes by stimulating apoptosis or selection into specialized T-cell lineages, including intestinal TCRαß+ CD8αα+ intraepithelial lymphocytes (IEL). TCR-intrinsic amino acid motifs that can be used to predict whether a TCR will be strongly self-reactive remain elusive. Here, a novel TCR sequence alignment approach revealed that T-cell lineages in C57BL/6 mice had divergent usage of cysteine within two positions of the amino acid at the apex of the complementarity-determining region 3 (CDR3) of the TCRα or TCRß chain. Compared to pre-selection thymocytes, central CDR3 cysteine usage was increased in IEL and Type A IEL precursors (IELp) and markedly decreased in Foxp3+ regulatory T cells (T-reg) and naïve T cells. These findings reveal a TCR-intrinsic motif that distinguishes Type A IELp and IEL from T-reg and naïve T cells.


Assuntos
Linfócitos T CD8-Positivos/citologia , Regiões Determinantes de Complementaridade/química , Linfócitos Intraepiteliais/citologia , Receptores de Antígenos de Linfócitos T alfa-beta/química , Timócitos/citologia , Animais , Linhagem da Célula , Cisteína/química , Camundongos , Camundongos Endogâmicos C57BL
15.
Science ; 360(6385): 223-226, 2018 04 13.
Artigo em Inglês | MEDLINE | ID: mdl-29650674

RESUMO

Antibodies have the specificity to differentiate foreign antigens that mimic self antigens, but it remains unclear how such specificity is acquired. In a mouse model, we generated B cells displaying an antibody that cross-reacts with two related protein antigens expressed on self versus foreign cells. B cell anergy was imposed by self antigen but reversed upon challenge with high-density foreign antigen, leading to germinal center recruitment and antibody gene hypermutation. Single-cell analysis detected rapid selection for mutations that decrease self affinity and slower selection for epistatic mutations that specifically increase foreign affinity. Crystal structures revealed that these mutations exploited subtle topological differences to achieve 5000-fold preferential binding to foreign over self epitopes. Resolution of antigenic mimicry drove the optimal affinity maturation trajectory, highlighting the value of retaining self-reactive clones as substrates for protective antibody responses.


Assuntos
Anticorpos/genética , Formação de Anticorpos/genética , Autoantígenos/imunologia , Centro Germinativo/imunologia , Mimetismo Molecular/genética , Tolerância a Antígenos Próprios , Animais , Anticorpos/química , Anticorpos/imunologia , Afinidade de Anticorpos/genética , Linfócitos B/imunologia , Anergia Clonal , Reações Cruzadas , Cristalografia por Raios X , Camundongos , Camundongos Mutantes , Mutação , Nucleoproteínas/genética , Nucleoproteínas/imunologia , Seleção Genética , Análise de Célula Única
16.
Bioinformatics ; 34(16): 2846-2847, 2018 08 15.
Artigo em Inglês | MEDLINE | ID: mdl-29659703

RESUMO

Motivation: The B-cell receptor (BCR) performs essential functions for the adaptive immune system including recognition of pathogen-derived antigens. The vast repertoire and adaptive variation of BCR sequences due to V(D)J recombination and somatic hypermutation necessitates single-cell characterization of BCR sequences. Single-cell RNA sequencing presents the opportunity for simultaneous capture of paired BCR heavy and light chains and the transcriptomic signature. Results: We developed VDJPuzzle, a novel bioinformatic tool that reconstructs productive, full-length B-cell receptor sequences of both heavy and light chains and extract somatic mutations on the VDJ region. VDJPuzzle successfully reconstructed BCRs from 100% (n=117) human and 96.5% (n=200) murine B cells. The reconstructed BCRs were successfully validated with single-cell Sanger sequencing. Availability and implementation: VDJPuzzle is available at https://bitbucket.org/kirbyvisp/vdjpuzzle2. Supplementary information: Supplementary data are available at Bioinformatics online.


Assuntos
RNA/genética , Receptores de Antígenos de Linfócitos B/genética , Animais , Biologia Computacional , Humanos , Camundongos , Análise de Sequência de RNA , Análise de Célula Única , Transcriptoma
17.
Arthritis Rheumatol ; 70(10): 1617-1625, 2018 10.
Artigo em Inglês | MEDLINE | ID: mdl-29697211

RESUMO

OBJECTIVE: Rheumatoid factors (RFs) are associated with systemic disease in primary Sjögren's syndrome (SS) and may be pathogenic as mixed cryoglobulins. Current detection methods cannot resolve RFs at a molecular level. This study was undertaken to perform the first proteomic and transcriptomic analysis of secreted and membrane-bound IgM-RF in primary SS and identify unique heavy-chain peptide signatures for RF clonotype tracking. METHODS: Purified heavy chains of serum RFs from 15 patients with primary SS were subjected to de novo mass spectrometric sequencing. The circulating B cell Ig repertoire was determined by massively parallel sequencing of IGH RNA from matched peripheral blood mononuclear cells (n = 7). RF-specific heavy-chain third complementarity-determining region (CDR3) peptides were identified by searching RF heavy-chain peptide sequences against the corresponding IGH RNA sequence libraries. Heavy-chain CDR3 peptides were used as biomarkers to track serum RF clonotypes using quantitative multiple reaction monitoring. RESULTS: Serum RFs were clonally restricted and composed of shared sets of IgM heavy-chain variable region (Ig VH ) 1-69, 3-15, 3-7, and 3-74 subfamilies. Cryoprecipitable RFs from patients with mixed cryoglobulinemia (MC) were distinguishable from nonprecipitating RFs by a higher frequency of amino acid substitutions and identification of stereotypic heavy-chain CDR3 transcripts. Potentially pathogenic RF clonotypes were detected in serum by multiple reaction monitoring years before patients presented with MC. Levels of Ig VH 4-34 IgM-RF decreased following immunosuppression and remission of MC. CONCLUSION: Cryoprecipitable RF clonotypes linked to vasculitis in primary SS have different molecular profiles than nonprecipitating RFs, suggesting different underlying mechanisms of production. The combined omics workflow presented herein provides molecular biomarkers for tracking and removal of pathogenic RF clones.


Assuntos
Cadeias Pesadas de Imunoglobulinas/sangue , Leucócitos Mononucleares/fisiologia , Fator Reumatoide/sangue , Síndrome de Sjogren/sangue , Adulto , Linfócitos B/metabolismo , Compostos de Boro/metabolismo , Rastreamento de Células , Feminino , Perfilação da Expressão Gênica , Humanos , Cadeias Pesadas de Imunoglobulinas/imunologia , Imunoglobulina M/imunologia , Masculino , Espectrometria de Massas , Pessoa de Meia-Idade , Proteômica , Fator Reumatoide/imunologia , Síndrome de Sjogren/imunologia
18.
Immunology ; 154(3): 522-532, 2018 07.
Artigo em Inglês | MEDLINE | ID: mdl-29411880

RESUMO

Acquisition of T-cell central tolerance involves distinct pathways of self-antigen presentation to thymocytes. One pathway termed indirect presentation requires a self-antigen transfer step from thymic epithelial cells (TECs) to bone marrow-derived cells before the self-antigen is presented to thymocytes. The role of indirect presentation in central tolerance is context-dependent, potentially due to variation in self-antigen expression, processing and presentation in the thymus. Here, we report experiments in mice in which TECs expressed a membrane-bound transgenic self-antigen, hen egg lysozyme (HEL), from either the insulin (insHEL) or thyroglobulin (thyroHEL) promoter. Intrathymic HEL expression was less abundant and more confined to the medulla in insHEL mice compared with thyroHEL mice. When indirect presentation was impaired by generating mice lacking MHC class II expression in bone marrow-derived antigen-presenting cells, insHEL-mediated thymocyte deletion was abolished, whereas thyroHEL-mediated deletion occurred at a later stage of thymocyte development and Foxp3+ regulatory T-cell differentiation increased. Indirect presentation increased the strength of T-cell receptor signalling that both self-antigens induced in thymocytes, as assessed by Helios expression. Hence, indirect presentation limits the differentiation of naive and regulatory T cells by promoting deletion of self-reactive thymocytes.


Assuntos
Apresentação do Antígeno/imunologia , Diferenciação Celular , Seleção Clonal Mediada por Antígeno/imunologia , Linfócitos T Reguladores/citologia , Linfócitos T Reguladores/imunologia , Timócitos/citologia , Timócitos/imunologia , Animais , Células Apresentadoras de Antígenos/imunologia , Células Apresentadoras de Antígenos/metabolismo , Autoantígenos/imunologia , Biomarcadores , Expressão Gênica , Tolerância Imunológica , Imunofenotipagem , Camundongos , Camundongos Knockout , Fenótipo , Subpopulações de Linfócitos T/imunologia , Subpopulações de Linfócitos T/metabolismo , Linfócitos T Reguladores/metabolismo , Timócitos/metabolismo , Timo/citologia , Timo/imunologia
19.
PLoS Genet ; 13(11): e1007072, 2017 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-29117179

RESUMO

We identified a non-synonymous mutation in Oas2 (I405N), a sensor of viral double-stranded RNA, from an ENU-mutagenesis screen designed to discover new genes involved in mammary development. The mutation caused post-partum failure of lactation in healthy mice with otherwise normally developed mammary glands, characterized by greatly reduced milk protein synthesis coupled with epithelial cell death, inhibition of proliferation and a robust interferon response. Expression of mutant but not wild type Oas2 in cultured HC-11 or T47D mammary cells recapitulated the phenotypic and transcriptional effects observed in the mouse. The mutation activates the OAS2 pathway, demonstrated by a 34-fold increase in RNase L activity, and its effects were dependent on expression of RNase L and IRF7, proximal and distal pathway members. This is the first report of a viral recognition pathway regulating lactation.


Assuntos
2',5'-Oligoadenilato Sintetase/genética , Lactação/genética , 2',5'-Oligoadenilato Sintetase/metabolismo , Nucleotídeos de Adenina/metabolismo , Animais , Técnicas de Cultura de Células , Endorribonucleases/metabolismo , Feminino , Humanos , Glândulas Mamárias Animais/metabolismo , Camundongos , Leite , Mutação/genética , Oligorribonucleotídeos/metabolismo , RNA de Cadeia Dupla/metabolismo , Transdução de Sinais/genética
20.
J Exp Med ; 214(9): 2759-2776, 2017 Sep 04.
Artigo em Inglês | MEDLINE | ID: mdl-28701369

RESUMO

CD79B and MYD88 mutations are frequently and simultaneously detected in B cell malignancies. It is not known if these mutations cooperate or how crosstalk occurs. Here we analyze the consequences of CD79B and MYD88L265P mutations individually and combined in normal activated mouse B lymphocytes. CD79B mutations alone increased surface IgM but did not enhance B cell survival, proliferation, or altered NF-κB responsive markers. Conversely, B cells expressing MYD88L265P decreased surface IgM coupled with accumulation of endoglycosidase H-sensitive IgM intracellularly, resembling the trafficking block in anergic B cells repeatedly stimulated by self-antigen. Mutation or overexpression of CD79B counteracted the effect of MYD88L265P In B cells chronically stimulated by self-antigen, CD79B and MYD88L265P mutations in combination, but not individually, blocked peripheral deletion and triggered differentiation into autoantibody secreting plasmablasts. These results reveal that CD79B and surface IgM constitute a rate-limiting checkpoint against B cell dysregulation by MYD88L265P and provide an explanation for the co-occurrence of MYD88 and CD79B mutations in lymphomas.


Assuntos
Antígenos CD79/genética , Imunoglobulina M/genética , Fator 88 de Diferenciação Mieloide/genética , Receptor Cross-Talk/fisiologia , Animais , Autoanticorpos/imunologia , Autoantígenos/genética , Autoantígenos/fisiologia , Linfócitos B/fisiologia , Antígenos CD79/fisiologia , Imunoglobulina M/fisiologia , Linfoma de Células B/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Mutação/genética , Fator 88 de Diferenciação Mieloide/fisiologia
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