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1.
Nature ; 2021 Jan 20.
Artigo em Inglês | MEDLINE | ID: mdl-33472215

RESUMO

Plasma membrane rupture (PMR) is the final cataclysmic event in lytic cell death. PMR releases intracellular molecules known as damage-associated molecular patterns (DAMPs) that propagate the inflammatory response1-3. The underlying mechanism of PMR, however, is unknown. Here we show that the cell-surface NINJ1 protein4-8, which contains two transmembrane regions, has an essential role in the induction of PMR. A forward-genetic screen of randomly mutagenized mice linked NINJ1 to PMR. Ninj1-/- macrophages exhibited impaired PMR in response to diverse inducers of pyroptotic, necrotic and apoptotic cell death, and were unable to release numerous intracellular proteins including HMGB1 (a known DAMP) and LDH (a standard measure of PMR). Ninj1-/- macrophages died, but with a distinctive and persistent ballooned morphology, attributable to defective disintegration of bubble-like herniations. Ninj1-/- mice were more susceptible than wild-type mice to infection with Citrobacter rodentium, which suggests a role for PMR in anti-bacterial host defence. Mechanistically, NINJ1 used an evolutionarily conserved extracellular domain for oligomerization and subsequent PMR. The discovery of NINJ1 as a mediator of PMR overturns the long-held idea that cell death-related PMR is a passive event.

2.
J Exp Med ; 218(2)2021 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-33107914

RESUMO

NF-κB2/p100 (p100) is an inhibitor of κB (IκB) protein that is partially degraded to produce the NF-κB2/p52 (p52) transcription factor. Heterozygous NFKB2 mutations cause a human syndrome of immunodeficiency and autoimmunity, but whether autoimmunity arises from insufficiency of p52 or IκB function of mutated p100 is unclear. Here, we studied mice bearing mutations in the p100 degron, a domain that harbors most of the clinically recognized mutations and is required for signal-dependent p100 degradation. Distinct mutations caused graded increases in p100-degradation resistance. Severe p100-degradation resistance, due to inheritance of one highly degradation-resistant allele or two subclinical alleles, caused thymic medullary hypoplasia and autoimmune disease, whereas the absence of p100 and p52 did not. We inferred a similar mechanism occurs in humans, as the T cell receptor repertoires of affected humans and mice contained a hydrophobic signature of increased self-reactivity. Autoimmunity in autosomal dominant NFKB2 syndrome arises largely from defects in nonhematopoietic cells caused by the IκB function of degradation-resistant p100.

3.
Immunol Cell Biol ; 2020 Dec 17.
Artigo em Inglês | MEDLINE | ID: mdl-33331104

RESUMO

The RNA-binding protein heterogeneous nuclear ribonucleoprotein L-like (hnRNPLL) controls alternative splicing of protein tyrosine phosphatase receptor type C (Ptprc) which encodes CD45. hnRNPLL deficiency leads to a failure in silencing Ptprc exons 4-6 causing aberrant expression of the corresponding CD45 isoforms, namely, CD45RA, RB and RC. While an N-ethyl-N-nitrosourea-induced point mutation in murine Hnrnpll results in loss of peripheral naïve T cells, its role in B-cell biology remains unclear. Here, we demonstrate that B-cell development in the bone marrow of Hnrnpllthu/thu mice is normal and the number of mature B-cell subsets in the spleen and peritoneal cavity is comparable to control littermates. In response to in vivo immunization, Hnrnpllthu/thu mice were deficient in generating germinal center (GC) B cells, and analysis of mixed bone marrow chimeras revealed that the GC B-cell deficiency was a B-cell extrinsic effect of the hnRNPLL mutation. Mature Hnrnpllthu/thu B cells proliferated normally in response to various B-cell receptor- and Toll-like receptor-mediated stimuli. Similarly, in vitro stimulation of mutant B cells led to normal generation of plasmablasts, but mutant plasmablasts failed to downregulate B220 expression because of the inability of cells to undergo proper CD45 pre-messenger RNA alternative splicing. These findings collectively suggest that, like in T and natural killer T cells, the mutation disrupts hnRNPLL-mediated alternative splicing of the Ptprc gene in plasmablasts, but this dysregulation of Ptprc alternative splicing does not affect the development and function of B cells.

4.
Immunol Cell Biol ; 2020 Oct 28.
Artigo em Inglês | MEDLINE | ID: mdl-33113212

RESUMO

Coronavirus disease 2019 (COVID-19) is a zoonosis like most of the great plagues sculpting human history, from smallpox to pandemic influenza and human immunodeficiency virus. When viruses jump into a new species the outcome of infection ranges from asymptomatic to lethal, historically ascribed to "genetic resistance to viral disease." People have exploited these differences for good and bad, for developing vaccines from cowpox and horsepox virus, controlling rabbit plagues with myxoma virus and introducing smallpox during colonization of America and Australia. Differences in resistance to viral disease are at the core of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) crisis, yet our understanding of the mechanisms in any interspecies leap falls short of the mark. Here I review how the two key parameters of viral disease are countered by fundamentally different genetic mechanisms for resistance: (1) virus transmission, countered primarily by activation of innate and adaptive immune responses; and (2) pathology, countered primarily by tolerance checkpoints to limit innate and adaptive immune responses. I discuss tolerance thresholds and the role of CD8 T cells to limit pathological immune responses, the problems posed by tolerant superspreaders and the signature coronavirus evasion strategy of eliciting only short-lived neutralizing antibody responses. Pinpointing and targeting the mechanisms responsible for varying pathology and short-lived antibody were beyond reach in previous zoonoses, but this time we are armed with genomic technologies and more knowledge of immune checkpoint genes. These known unknowns must now be tackled to solve the current COVID-19 crisis and the inevitable zoonoses to follow.

5.
Proc Natl Acad Sci U S A ; 117(36): 22341-22350, 2020 09 08.
Artigo em Inglês | MEDLINE | ID: mdl-32855302

RESUMO

Conformational diversity and self-cross-reactivity of antigens have been correlated with evasion from neutralizing antibody responses. We utilized single cell B cell sequencing, biolayer interferometry and X-ray crystallography to trace mutation selection pathways where the antibody response must resolve cross-reactivity between foreign and self-proteins bearing near-identical contact surfaces, but differing in conformational flexibility. Recurring antibody mutation trajectories mediate long-range rearrangements of framework (FW) and complementarity determining regions (CDRs) that increase binding site conformational diversity. These antibody mutations decrease affinity for self-antigen 19-fold and increase foreign affinity 67-fold, to yield a more than 1,250-fold increase in binding discrimination. These results demonstrate how conformational diversity in antigen and antibody does not act as a barrier, as previously suggested, but rather facilitates high affinity and high discrimination between foreign and self.


Assuntos
Anticorpos , Diversidade de Anticorpos/genética , Autoantígenos , Rearranjo Gênico do Linfócito B/genética , Mutação/genética , Animais , Anticorpos/química , Anticorpos/genética , Anticorpos/metabolismo , Afinidade de Anticorpos/genética , Autoanticorpos/química , Autoanticorpos/genética , Autoanticorpos/metabolismo , Autoantígenos/química , Autoantígenos/metabolismo , Regiões Determinantes de Complementaridade/genética , Imunidade Humoral/genética , Camundongos , Modelos Moleculares , Conformação Proteica , Hipermutação Somática de Imunoglobulina/genética
6.
Cell ; 180(5): 878-894.e19, 2020 03 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.


Assuntos
Autoanticorpos/genética , Doenças Autoimunes/genética , Linfócitos B/imunologia , Linfoma/genética , Animais , Autoanticorpos/imunologia , Doenças Autoimunes/imunologia , Doenças Autoimunes/patologia , Linfócitos B/patologia , Proteínas Adaptadoras de Sinalização CARD/genética , Proteínas de Transporte/genética , Evolução Clonal/genética , Evolução Clonal/imunologia , Ciclina D3/genética , Guanilato Ciclase/genética , Humanos , Proteínas Imediatamente Precoces/genética , Região Variável de Imunoglobulina/genética , Região Variável de Imunoglobulina/imunologia , Proteínas Inibidoras de Diferenciação/genética , Linfoma/imunologia , Linfoma/patologia , Camundongos , Mutação/genética , Mutação/imunologia , Proteínas de Neoplasias/genética , Análise de Sequência de DNA/métodos , Análise de Sequência de RNA/métodos , Análise de Célula Única/métodos , Proteína 3 Induzida por Fator de Necrose Tumoral alfa/genética , Proteínas Supressoras de Tumor/genética , Recombinação V(D)J/genética
7.
J Exp Med ; 217(2)2020 02 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.


Assuntos
Formação de Anticorpos/genética , Autoanticorpos/imunologia , Classe I de Fosfatidilinositol 3-Quinases/genética , Mutação com Ganho de Função , Tolerância Imunológica/imunologia , Plasmócitos/imunologia , Animais , Autoanticorpos/sangue , Autoantígenos/imunologia , Autoimunidade/genética , Classe I de Fosfatidilinositol 3-Quinases/sangue , Classe I de Fosfatidilinositol 3-Quinases/metabolismo , Feminino , Centro Germinativo/imunologia , Humanos , Imunoglobulina M/sangue , Imunoglobulina M/imunologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Transdução de Sinais/genética
8.
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.


Assuntos
Linfócitos B/imunologia , Anergia Clonal/imunologia , Tolerância Imunológica/imunologia , Imunidade/imunologia , Animais , Autoanticorpos/imunologia , Autoantígenos/imunologia , Autoantígenos/metabolismo , Linfócitos B/metabolismo , Centro Germinativo/imunologia , Centro Germinativo/metabolismo , Humanos , Receptores de Antígenos de Linfócitos B/imunologia , Receptores de Antígenos de Linfócitos B/metabolismo
9.
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.


Assuntos
Artrite Juvenil/genética , Antígeno CTLA-4/genética , Hepatite Autoimune/genética , Abatacepte/uso terapêutico , Motivos de Aminoácidos , Antirreumáticos/uso terapêutico , Artrite Juvenil/tratamento farmacológico , Pré-Escolar , Feminino , Hepatite Autoimune/tratamento farmacológico , Humanos , Mutação de Sentido Incorreto
10.
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
11.
Sci Signal ; 12(582)2019 05 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.


Assuntos
Fator Regulador 2 de Interferon/genética , Peptídeos e Proteínas de Sinalização Intracelular/genética , Proteínas de Ligação a Fosfato/genética , Piroptose/genética , Transcrição Genética/genética , Animais , Células Endoteliais/citologia , Células Endoteliais/metabolismo , Inflamassomos/genética , Inflamassomos/metabolismo , Fator Regulador 2 de Interferon/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Macrófagos/citologia , Macrófagos/metabolismo , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas de Ligação a Fosfato/metabolismo , Transdução de Sinais/genética , Ativação Transcricional/genética
12.
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.


Assuntos
Proteínas Adaptadoras de Sinalização CARD/deficiência , Dermatite Atópica/imunologia , Homeostase/imunologia , Transdução de Sinais/imunologia , Linfócitos T Reguladores/imunologia , Animais , Proteínas Adaptadoras de Sinalização CARD/genética , Proteínas Adaptadoras de Sinalização CARD/imunologia , Dermatite Atópica/genética , Modelos Animais de Doenças , Etilnitrosoureia/toxicidade , Fatores de Transcrição Forkhead/imunologia , Fatores de Transcrição Forkhead/metabolismo , Homeostase/genética , Humanos , Íntrons/efeitos dos fármacos , Íntrons/genética , Íntrons/imunologia , Mutação com Perda de Função/efeitos dos fármacos , Mutação com Perda de Função/imunologia , Camundongos , Camundongos Transgênicos , Mutagênese/imunologia , Mutagênicos/toxicidade , Neuropilina-1/imunologia , Neuropilina-1/metabolismo , Polimorfismo de Nucleotídeo Único/efeitos dos fármacos , Polimorfismo de Nucleotídeo Único/imunologia , Transdução de Sinais/genética , Linfócitos T Reguladores/metabolismo
13.
Cell Death Differ ; 26(12): 2727-2739, 2019 12.
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.


Assuntos
Receptores CCR7/deficiência , Timo/metabolismo , Animais , Apoptose , Células Epiteliais/citologia , Células Epiteliais/imunologia , Células Epiteliais/metabolismo , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Receptores de Antígenos de Linfócitos T alfa-beta , Receptores CCR7/metabolismo , Timo/citologia , Timo/imunologia , Família de Proteínas da Síndrome de Wiskott-Aldrich
15.
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
16.
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
17.
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
18.
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 , Infecções/imunologia , Neoplasias/imunologia , Placenta/fisiologia , Aloenxertos/imunologia , Animais , Feminino , Humanos , Microbiota , Myxoma virus/patogenicidade , Infecções por Poxviridae/mortalidade , Gravidez , Microambiente Tumoral/imunologia
19.
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
20.
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
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