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1.
Immunity ; 51(5): 856-870.e5, 2019 11 19.
Artigo em Inglês | MEDLINE | ID: mdl-31747582

RESUMO

Naive CD8+ T cells differentiating into effector T cells increase glucose uptake and shift from quiescent to anabolic metabolism. Although much is known about the metabolism of cultured T cells, how T cells use nutrients during immune responses in vivo is less well defined. Here, we combined bioenergetic profiling and 13C-glucose infusion techniques to investigate the metabolism of CD8+ T cells responding to Listeria infection. In contrast to in vitro-activated T cells, which display hallmarks of Warburg metabolism, physiologically activated CD8+ T cells displayed greater rates of oxidative metabolism, higher bioenergetic capacity, differential use of pyruvate, and prominent flow of 13C-glucose carbon to anabolic pathways, including nucleotide and serine biosynthesis. Glucose-dependent serine biosynthesis mediated by the enzyme Phgdh was essential for CD8+ T cell expansion in vivo. Our data highlight fundamental differences in glucose use by pathogen-specific T cells in vivo, illustrating the impact of environment on T cell metabolic phenotypes.


Assuntos
Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/metabolismo , Metabolismo Energético , Glucose/metabolismo , Ativação Linfocitária/imunologia , Metaboloma , Metabolômica , Animais , Proliferação de Células , Cromatografia Gasosa-Espectrometria de Massas , Glicólise , Interações Hospedeiro-Patógeno/genética , Interações Hospedeiro-Patógeno/imunologia , Ativação Linfocitária/genética , Metabolômica/métodos , Camundongos , Estresse Oxidativo , Viroses/genética , Viroses/imunologia , Viroses/metabolismo , Viroses/virologia
2.
Nat Commun ; 10(1): 4768, 2019 10 18.
Artigo em Inglês | MEDLINE | ID: mdl-31628339

RESUMO

B-1a cells are long-lived, self-renewing innate-like B cells that predominantly inhabit the peritoneal and pleural cavities. In contrast to conventional B-2 cells, B-1a cells have a receptor repertoire that is biased towards bacterial and self-antigens, promoting a rapid response to infection and clearing of apoptotic cells. Although B-1a cells are known to primarily originate from fetal tissues, the mechanisms by which they arise has been a topic of debate for many years. Here we show that in the fetal liver versus bone marrow environment, reduced IL-7R/STAT5 levels promote immunoglobulin kappa gene recombination at the early pro-B cell stage. As a result, differentiating B cells can directly generate a mature B cell receptor (BCR) and bypass the requirement for a pre-BCR and pairing with surrogate light chain. This 'alternate pathway' of development enables the production of B cells with self-reactive, skewed specificity receptors that are peculiar to the B-1a compartment. Together our findings connect seemingly opposing lineage and selection models of B-1a cell development and explain how these cells acquire their unique properties.


Assuntos
Subpopulações de Linfócitos B/imunologia , Linfócitos B/imunologia , Diferenciação Celular/imunologia , Receptores de Células Precursoras de Linfócitos B/imunologia , Receptores de Antígenos de Linfócitos B/imunologia , Animais , Subpopulações de Linfócitos B/metabolismo , Linfócitos B/metabolismo , Medula Óssea/imunologia , Medula Óssea/metabolismo , Diferenciação Celular/genética , Cadeias Leves Substitutas da Imunoglobulina/genética , Cadeias Leves Substitutas da Imunoglobulina/imunologia , Cadeias Leves Substitutas da Imunoglobulina/metabolismo , Fígado/embriologia , Fígado/imunologia , Fígado/metabolismo , Ativação Linfocitária/genética , Ativação Linfocitária/imunologia , Camundongos Endogâmicos C57BL , Camundongos Knockout , Receptores de Células Precursoras de Linfócitos B/genética , Receptores de Células Precursoras de Linfócitos B/metabolismo , Receptores de Antígenos de Linfócitos B/genética , Receptores de Antígenos de Linfócitos B/metabolismo , Receptores de Interleucina-7/genética , Receptores de Interleucina-7/imunologia , Receptores de Interleucina-7/metabolismo , Fator de Transcrição STAT5/genética , Fator de Transcrição STAT5/imunologia , Fator de Transcrição STAT5/metabolismo
3.
Nat Commun ; 10(1): 4706, 2019 10 17.
Artigo em Inglês | MEDLINE | ID: mdl-31624246

RESUMO

Human T cells coordinate adaptive immunity in diverse anatomic compartments through production of cytokines and effector molecules, but it is unclear how tissue site influences T cell persistence and function. Here, we use single cell RNA-sequencing (scRNA-seq) to define the heterogeneity of human T cells isolated from lungs, lymph nodes, bone marrow and blood, and their functional responses following stimulation. Through analysis of >50,000 resting and activated T cells, we reveal tissue T cell signatures in mucosal and lymphoid sites, and lineage-specific activation states across all sites including distinct effector states for CD8+ T cells and an interferon-response state for CD4+ T cells. Comparing scRNA-seq profiles of tumor-associated T cells to our dataset reveals predominant activated CD8+ compared to CD4+ T cell states within multiple tumor types. Our results therefore establish a high dimensional reference map of human T cell activation in health for analyzing T cells in disease.


Assuntos
Pulmão/metabolismo , Linfonodos/metabolismo , Neoplasias/genética , Análise de Célula Única/métodos , Linfócitos T/metabolismo , Transcriptoma/genética , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/metabolismo , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/metabolismo , Células Cultivadas , Humanos , Linfonodos/imunologia , Ativação Linfocitária/genética , Ativação Linfocitária/imunologia , Membrana Mucosa/imunologia , Membrana Mucosa/metabolismo , Neoplasias/patologia , Linfócitos T/imunologia
4.
Nat Med ; 25(9): 1428-1441, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31501614

RESUMO

Psychological distress has long been suspected to influence cancer incidence and mortality. It remains largely unknown whether and how stress affects the efficacy of anticancer therapies. We observed that social defeat caused anxiety-like behaviors in mice and dampened therapeutic responses against carcinogen-induced neoplasias and transplantable tumors. Stress elevated plasma corticosterone and upregulated the expression of glucocorticoid-inducible factor Tsc22d3, which blocked type I interferon (IFN) responses in dendritic cell (DC) and IFN-γ+ T cell activation. Similarly, close correlations were discovered among plasma cortisol levels, TSC22D3 expression in circulating leukocytes and negative mood in patients with cancer. In murine models, exogenous glucocorticoid injection, or enforced expression of Tsc22d3 in DC was sufficient to abolish therapeutic control of tumors. Administration of a glucocorticoid receptor antagonist or DC-specific Tsc22d3 deletion reversed the negative impact of stress or glucocorticoid supplementation on therapeutic outcomes. Altogether, these results indicate that stress-induced glucocorticoid surge and Tsc22d3 upregulation can subvert therapy-induced anticancer immunosurveillance.


Assuntos
Imunidade Celular , Neoplasias/imunologia , Estresse Psicológico/imunologia , Fatores de Transcrição/genética , Animais , Ansiedade/sangue , Ansiedade/induzido quimicamente , Ansiedade/imunologia , Ansiedade/psicologia , Comportamento Animal/fisiologia , Carcinógenos/toxicidade , Neoplasias Colorretais/sangue , Neoplasias Colorretais/genética , Neoplasias Colorretais/imunologia , Neoplasias Colorretais/psicologia , Corticosterona/sangue , Células Dendríticas/transplante , Regulação Neoplásica da Expressão Gênica , Glucocorticoides/farmacologia , Humanos , Hidrocortisona/sangue , Neoplasias Pulmonares/sangue , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/imunologia , Neoplasias Pulmonares/psicologia , Ativação Linfocitária/genética , Camundongos , Monitorização Imunológica/métodos , Neoplasias/induzido quimicamente , Neoplasias/genética , Neoplasias/psicologia , Receptores de Glucocorticoides/antagonistas & inibidores , Transdução de Sinais/efeitos dos fármacos , Neoplasias Gástricas/sangue , Neoplasias Gástricas/genética , Neoplasias Gástricas/imunologia , Neoplasias Gástricas/psicologia , Estresse Psicológico/induzido quimicamente , Estresse Psicológico/genética , Estresse Psicológico/terapia , Fatores de Transcrição/imunologia
5.
J Microbiol Biotechnol ; 29(9): 1361-1368, 2019 Sep 28.
Artigo em Inglês | MEDLINE | ID: mdl-31434170

RESUMO

Codium fragile is an edible seaweed in Asian countries that has been used as a thrombolytic, anticoagulant, antioxidant, anti-inflammatory, and immune-stimulatory agent. Ginseng has also been known to maintain immune homeostasis and to regulate the immune system via enhancing resistance to diseases and microorganisms. In this study, anionic macromolecules extracted from C. fragile (CFAM) were orally administered with red ginseng extract (100 mg/kg body weight) to cyclophosphamide-induced immunosuppressed male BALB/c mice to investigate the immune-enhancing cooperative effect of Codium fragile and red ginseng. Our results showed that supplementing CFAM with red ginseng extract significantly increased spleen index, T- and B-cell proliferation, NK cell activity, and splenic lymphocyte immuneassociated gene expression compared to those with red ginseng alone, even though a high concentration of CFAM with red ginseng decreased immune biomarkers. These results suggest that CFAM can be used as a co-stimulant to enhance health and immunity in immunosuppressed conditions.


Assuntos
Adjuvantes Imunológicos/farmacologia , Clorófitas/química , Substâncias Macromoleculares/farmacologia , Panax/química , Extratos Vegetais/farmacologia , Adjuvantes Imunológicos/química , Animais , Ânions/isolamento & purificação , Ânions/farmacologia , Ciclofosfamida/toxicidade , Quimioterapia Combinada , Imunossupressão , Ativação Linfocitária/efeitos dos fármacos , Ativação Linfocitária/genética , Linfócitos/citologia , Linfócitos/imunologia , Substâncias Macromoleculares/isolamento & purificação , Masculino , Camundongos Endogâmicos BALB C , Extratos Vegetais/administração & dosagem , Extratos Vegetais/química , Baço/imunologia
6.
Nat Immunol ; 20(7): 879-889, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-31182807

RESUMO

CD8+ T cells and natural killer (NK) cells are central cellular components of immune responses against pathogens and cancer, which rely on interleukin (IL)-15 for homeostasis. Here we show that IL-15 also mediates homeostatic priming of CD8+ T cells for antigen-stimulated activation, which is controlled by a deubiquitinase, Otub1. IL-15 mediates membrane recruitment of Otub1, which inhibits ubiquitin-dependent activation of AKT, a kinase that is pivotal for T cell activation and metabolism. Otub1 deficiency in mice causes aberrant responses of CD8+ T cells to IL-15, rendering naive CD8+ T cells hypersensitive to antigen stimulation characterized by enhanced metabolic reprograming and effector functions. Otub1 also controls the maturation and activation of NK cells. Deletion of Otub1 profoundly enhances anticancer immunity by unleashing the activity of CD8+ T cells and NK cells. These findings suggest that Otub1 controls the activation of CD8+ T cells and NK cells by functioning as a checkpoint of IL-15-mediated priming.


Assuntos
Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/metabolismo , Cisteína Endopeptidases/metabolismo , Interleucina-15/metabolismo , Células Matadoras Naturais/imunologia , Células Matadoras Naturais/metabolismo , Ativação Linfocitária/genética , Ativação Linfocitária/imunologia , Animais , Diferenciação Celular/genética , Diferenciação Celular/imunologia , Cisteína Endopeptidases/deficiência , Enzimas Desubiquitinantes/metabolismo , Modelos Animais de Doenças , Metabolismo Energético , Interações Hospedeiro-Patógeno/genética , Interações Hospedeiro-Patógeno/imunologia , Humanos , Interleucina-15/genética , Melanoma Experimental , Camundongos , Camundongos Transgênicos , Ligação Proteica , Proteínas Proto-Oncogênicas c-akt/metabolismo , Receptores de Interleucina-15/metabolismo , Tolerância a Antígenos Próprios/genética , Tolerância a Antígenos Próprios/imunologia , Transdução de Sinais , Especificidade do Receptor de Antígeno de Linfócitos T , Ubiquitinação
7.
Nat Immunol ; 20(7): 793-801, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-31213715

RESUMO

Unlike other cells in the body, immune cells have to be able to enter and adapt to life within diverse tissues. Immune cells develop within dedicated immune system organs, such as the bone marrow, thymus and lymphoid tissues, but also inhabit other tissues, wherein they not only provide defense against infection and malignancies but also contribute to homeostatic tissue function. Because different tissues have widely divergent metabolic rates and fuel requirements, this raises interesting questions about the adaptation of immune cells in specific tissues. When immune cells take up residence in different tissues, they develop a transcriptional signature that reflects adaptation to life and function within that tissue. Genes encoding metabolic-pathway proteins are strongly represented within these signatures, reflective of the importance of metabolic adaptation to tissue residence. In this Review, we discuss the available data on the metabolic adaptation of immune cells to life in different tissue sites, within the broader framework of how functional adaptation versus maladaptation in the niche can affect tissue homeostasis.


Assuntos
Adaptação Biológica , Metabolismo Energético , Sistema Imunitário/citologia , Sistema Imunitário/fisiologia , Especificidade de Órgãos/imunologia , Animais , Biomarcadores , Homeostase , Interações Hospedeiro-Patógeno/imunologia , Humanos , Ativação Linfocitária/genética , Ativação Linfocitária/imunologia , Ativação de Macrófagos/genética , Ativação de Macrófagos/imunologia , Macrófagos/imunologia , Macrófagos/metabolismo , Transdução de Sinais
8.
Nature ; 571(7765): 403-407, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-31217581

RESUMO

Activated CD4 T cells proliferate rapidly and remodel epigenetically before exiting the cell cycle and engaging acquired effector functions. Metabolic reprogramming from the naive state is required throughout these phases of activation1. In CD4 T cells, T-cell-receptor ligation-along with co-stimulatory and cytokine signals-induces a glycolytic anabolic program that is required for biomass generation, rapid proliferation and effector function2. CD4 T cell differentiation (proliferation and epigenetic remodelling) and function are orchestrated coordinately by signal transduction and transcriptional remodelling. However, it remains unclear whether these processes are regulated independently of one another by cellular biochemical composition. Here we demonstrate that distinct modes of mitochondrial metabolism support differentiation and effector functions of mouse T helper 1 (TH1) cells by biochemically uncoupling these two processes. We find that the tricarboxylic acid cycle is required for the terminal effector function of TH1 cells through succinate dehydrogenase (complex II), but that the activity of succinate dehydrogenase suppresses TH1 cell proliferation and histone acetylation. By contrast, we show that complex I of the electron transport chain, the malate-aspartate shuttle and mitochondrial citrate export are required to maintain synthesis of aspartate, which is necessary for the proliferation of T helper cells. Furthermore, we find that mitochondrial citrate export and the malate-aspartate shuttle promote histone acetylation, and specifically regulate the expression of genes involved in T cell activation. Combining genetic, pharmacological and metabolomics approaches, we demonstrate that the differentiation and terminal effector functions of T helper cells are biochemically uncoupled. These findings support a model in which the malate-aspartate shuttle, mitochondrial citrate export and complex I supply the substrates needed for proliferation and epigenetic remodelling early during T cell activation, whereas complex II consumes the substrates of these pathways, which antagonizes differentiation and enforces terminal effector function. Our data suggest that transcriptional programming acts together with a parallel biochemical network to enforce cell state.


Assuntos
Diferenciação Celular , Mitocôndrias/metabolismo , Células Th1/citologia , Células Th1/imunologia , Acetilação , Animais , Ácido Aspártico/metabolismo , Diferenciação Celular/genética , Linhagem Celular , Proliferação de Células/genética , Ácido Cítrico/metabolismo , Ciclo do Ácido Cítrico , Transporte de Elétrons , Feminino , Histonas/metabolismo , Humanos , Ativação Linfocitária/genética , Malatos/metabolismo , Masculino , Camundongos , Succinato Desidrogenase/metabolismo , Células Th1/metabolismo , Transcrição Genética
9.
Nat Immunol ; 20(6): 747-755, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-31061531

RESUMO

Despite gathering evidence that ubiquitylation can direct non-degradative outcomes, most investigations of ubiquitylation in T cells have focused on degradation. Here, we integrated proteomic and transcriptomic datasets from primary mouse CD4+ T cells to establish a framework for predicting degradative or non-degradative outcomes of ubiquitylation. Di-glycine remnant profiling was used to reveal ubiquitylated proteins, which in combination with whole-cell proteomic and transcriptomic data allowed prediction of protein degradation. Analysis of ubiquitylated proteins identified by di-glycine remnant profiling indicated that activation of CD4+ T cells led to an increase in non-degradative ubiquitylation. This correlated with an increase in non-proteasome-targeted K29, K33 and K63 polyubiquitin chains. This study revealed over 1,200 proteins that were ubiquitylated in primary mouse CD4+ T cells and highlighted the relevance of non-proteasomally targeted ubiquitin chains in T cell signaling.


Assuntos
Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/metabolismo , Ativação Linfocitária/imunologia , Proteoma , Proteômica , Animais , Perfilação da Expressão Gênica , Ativação Linfocitária/genética , Espectrometria de Massas , Camundongos , Poliubiquitina/metabolismo , Proteômica/métodos , Receptores de Antígenos de Linfócitos T/metabolismo , Transdução de Sinais , Subpopulações de Linfócitos T/imunologia , Subpopulações de Linfócitos T/metabolismo , Transcriptoma , Ubiquitinação
10.
Biomed Pharmacother ; 115: 108921, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31078045

RESUMO

Colorectal cancer is the third most common cancer worldwide and shows resistance to immune checkpoint inhibitors which have been demonstrated to be effective in many other types of cancers. Pre-existing T-cell response in tumor microenvironment often determines the therapeutic benefit of immune checkpoint blockade. Tumor-infiltrating CD8+ T-cells are considered as the major effector immune cells in antitumor immunity. In this study, we aimed to identify the intrinsic oncogenic pathway that contributes to a reduction of CD8+ T-cell infiltration in colorectal cancer. To achieve this, human colon adenocarcinoma samples derived from The Cancer Genome Altas (TCGA) were stratified into low T-cell-inflamed and high T-cell-inflamed groups based on the expression of T-cell signature genes. Gene set enrichment analysis of revealed a close correlation between activation of the Wnt/ß-catenin signaling pathway and absence of T-cell infiltration. By immunohistochemical analysis of 155 colorectal cancer tissues, we found that tumors with high ß-catenin expression showed a significant reduction of CD8+ T-cell infiltration. Mechanistically, ß-catenin can regulate CCL4 expression to recruit CD103+ dendritic cells to enable CD8+ T cell activation. Collectively, our data indicate that oncogenic ß-catenin signal may mediate colorectal cancer resistance to immunotherapies, pointing to the combined PD-1-immunotherapy with targeting ß-catenin in colorectal cancer.


Assuntos
Adenocarcinoma/imunologia , Linfócitos T CD8-Positivos/imunologia , Neoplasias Colorretais/imunologia , Linfócitos do Interstício Tumoral/imunologia , Via de Sinalização Wnt/imunologia , beta Catenina/metabolismo , Adenocarcinoma/genética , Adenocarcinoma/metabolismo , Quimiocina CCL4/genética , Neoplasias Colorretais/genética , Neoplasias Colorretais/metabolismo , Regulação Neoplásica da Expressão Gênica , Células HCT116 , Humanos , Imunoterapia , Ativação Linfocitária/genética , Transdução de Sinais/genética , Microambiente Tumoral/genética , Microambiente Tumoral/imunologia , Via de Sinalização Wnt/genética , beta Catenina/genética
11.
Life Sci Alliance ; 2(3)2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-31097471

RESUMO

A fast antibody response can be critical to contain rapidly dividing pathogens. This can be achieved by the expansion of antigen-specific B cells in response to T-cell help followed by differentiation into plasmablasts. MicroRNA-155 (miR-155) is required for optimal T-cell-dependent extrafollicular responses via regulation of PU.1, although the cellular processes underlying this defect are largely unknown. Here, we show that miR-155 regulates the early expansion of B-blasts and later on the survival and proliferation of plasmablasts in a B-cell-intrinsic manner, by tracking antigen-specific B cells in vivo since the onset of antigen stimulation. In agreement, comparative analysis of the transcriptome of miR-155-sufficient and miR-155-deficient plasmablasts at the peak of the response showed that the main processes regulated by miR-155 were DNA metabolic process, DNA replication, and cell cycle. Thus, miR-155 controls the extent of the extrafollicular response by regulating the survival and proliferation of B-blasts, plasmablasts and, consequently, antibody production.


Assuntos
Subpopulações de Linfócitos B/metabolismo , MicroRNAs/genética , Plasmócitos/metabolismo , Animais , Formação de Anticorpos/genética , Formação de Anticorpos/imunologia , Subpopulações de Linfócitos B/imunologia , Biomarcadores , Proliferação de Células , Sobrevivência Celular/genética , Imunofenotipagem , Ativação Linfocitária/genética , Ativação Linfocitária/imunologia , Camundongos , Camundongos Knockout , Plasmócitos/imunologia
12.
Immun Inflamm Dis ; 7(3): 95-104, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-30997749

RESUMO

INTRODUCTION: Zoonotic cutaneous leishmaniasis (ZCL), due to infection by Leishmania (L). major, is characterized by polymorphic clinical manifestations which could be attributed to the host's immune response. In this study we investigated the involvement of cytotoxic cells on the outcome of the disease. METHODS: Expression of granzyme B (GrB), granulysine (Grly), and interferon (IFN)-γ was evaluated within ZCL lesion specimens using the technique of real-time quantitative polymerase chain reaction (RT-qPCR). Immunohistochemical staining was performed using anti-CD3, CD4, CD8, CD56, GrB, and IFN-γ antibodies to identify the phenotype of GrB and IFN-γ-producing cells. RESULTS: GrB and Grly mRNA was detected within 75% and 80% of ZCL lesions, respectively. Statistical analysis demonstrated a significant correlation between levels of GrB and Grly. Interestingly, expression of these molecules correlates negatively with the lesion's age. The highest levels were measured in early lesions (E-ZCL) (lesion age ≤1 month) comparing to late lesions (L-ZCL) (lesion age >1 month). Otherwise, IFN-γ mRNA was detected only within 56% and a positive correlation was found between levels of this cytokine and those of GrB. Immunohistochemical analysis showed that GrB is produced essentially by CD8+ T cells whereas IFN-γ is produced by both CD4+ and CD8+ T cells. CONCLUSION: Together our results demonstrate the presence of cytotoxic cells producing GrB and Grly within leishmaniasis cutaneous lesions.


Assuntos
Leishmania major/imunologia , Leishmaniose Cutânea/imunologia , Linfócitos T Citotóxicos/imunologia , Zoonoses/imunologia , Adulto , Animais , Antígenos de Diferenciação de Linfócitos T/genética , Antígenos de Diferenciação de Linfócitos T/imunologia , Antígenos de Diferenciação de Linfócitos T/metabolismo , Criança , Pré-Escolar , Citocinas/imunologia , Citocinas/metabolismo , Feminino , Expressão Gênica/imunologia , Granzimas/genética , Granzimas/imunologia , Granzimas/metabolismo , Humanos , Lactente , Recém-Nascido , Interferon gama/genética , Interferon gama/metabolismo , Leishmania major/parasitologia , Leishmaniose Cutânea/metabolismo , Leishmaniose Cutânea/parasitologia , Ativação Linfocitária/genética , Ativação Linfocitária/imunologia , Masculino , Pessoa de Meia-Idade , Pele/imunologia , Pele/parasitologia , Linfócitos T Citotóxicos/metabolismo , Zoonoses/parasitologia
13.
Nat Commun ; 10(1): 1842, 2019 04 23.
Artigo em Inglês | MEDLINE | ID: mdl-31015529

RESUMO

The CRISPR-Cas9 system has raised hopes for developing personalized gene therapies for complex diseases. Its application for genetic and epigenetic therapies in humans raises concerns over immunogenicity of the bacterially derived Cas9 protein. Here we detect antibodies to Streptococcus pyogenes Cas9 (SpCas9) in at least 5% of 143 healthy individuals. We also report pre-existing human CD8+T cell immunity in the majority of healthy individuals screened. We identify two immunodominant SpCas9 T cell epitopes for HLA-A*02:01 using an enhanced prediction algorithm that incorporates T cell receptor contact residue hydrophobicity and HLA binding and evaluated them by T cell assays using healthy donor PBMCs. In a proof-of-principle study, we demonstrate that Cas9 protein can be modified to eliminate immunodominant epitopes through targeted mutation while preserving its function and specificity. Our study highlights the problem of pre-existing immunity against CRISPR-associated nucleases and offers a potential solution to mitigate the T cell immune response.


Assuntos
Linfócitos T CD8-Positivos/imunologia , Proteína 9 Associada à CRISPR/imunologia , Epitopos de Linfócito T/genética , Mutagênese/imunologia , Streptococcus pyogenes/imunologia , Anticorpos Antibacterianos/sangue , Anticorpos Antibacterianos/imunologia , Células Apresentadoras de Antígenos/imunologia , Proteína 9 Associada à CRISPR/genética , Engenharia Celular/métodos , Mapeamento de Epitopos/métodos , Epitopos de Linfócito T/imunologia , Terapia Genética/efeitos adversos , Terapia Genética/métodos , Células HEK293 , Antígenos HLA-A/imunologia , Voluntários Saudáveis , Humanos , Ativação Linfocitária/genética , Ativação Linfocitária/imunologia , Medicina de Precisão/efeitos adversos , Medicina de Precisão/métodos , Streptococcus pyogenes/genética
14.
Immunity ; 50(5): 1202-1217.e7, 2019 05 21.
Artigo em Inglês | MEDLINE | ID: mdl-31027997

RESUMO

Stable changes in chromatin states and gene expression in cells of the immune system form the basis for memory of infections and other challenges. Here, we used naturally occurring cis-regulatory variation in wild-derived inbred mouse strains to explore the mechanisms underlying long-lasting versus transient gene regulation in CD8 T cells responding to acute viral infection. Stably responsive DNA elements were characterized by dramatic and congruent chromatin remodeling events affecting multiple neighboring sites and required distinct transcription factor (TF) binding motifs for their accessibility. Specifically, we found that cooperative recruitment of T-box and Runx family transcription factors to shared targets mediated stable chromatin remodeling upon T cell activation. Our observations provide insights into the molecular mechanisms driving virus-specific CD8 T cell responses and suggest a general mechanism for the formation of transcriptional and epigenetic memory applicable to other immune and non-immune cells.


Assuntos
Linfócitos T CD8-Positivos/imunologia , Montagem e Desmontagem da Cromatina/genética , Subunidade alfa 2 de Fator de Ligação ao Core/genética , Regulação da Expressão Gênica/genética , Coriomeningite Linfocítica/imunologia , Vírus da Coriomeningite Linfocítica/imunologia , Proteínas com Domínio T/genética , Animais , Linfócitos T CD8-Positivos/virologia , Linhagem Celular , Cromatina/genética , Epigênese Genética/genética , Feminino , Expressão Gênica/genética , Regulação da Expressão Gênica/imunologia , Variação Genética , Memória Imunológica/genética , Memória Imunológica/imunologia , Ativação Linfocitária/genética , Ativação Linfocitária/imunologia , Coriomeningite Linfocítica/virologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Transcrição Genética/genética
15.
Ann Rheum Dis ; 78(6): 773-780, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-30936065

RESUMO

OBJECTIVE: The strong genetic contribution of the major histocompatibility complex (MHC) region to rheumatoid arthritis (RA) has been generally attributed to human leukocyte antigen (HLA)-DRB1. However, due to the high polymorphisms and linkage disequilibrium within MHC, it is difficult to define novel and/or independent genetic risks using conventional HLA genotyping or chip-based microarray technology. This study aimed to identify novel RA risk variants by performing deep sequencing for MHC. METHODS: We first conducted target sequencing for the entire MHC region in 357 anticitrullinated protein antibodies (ACPA)-positive patients with RA and 1001 healthy controls, and then performed HLA typing in an independent case-control cohort consisting of 1415 samples for validation. All study subjects were Han Chinese. Genetic associations for RA susceptibility and severity were analysed. Comparative modelling was constructed to predict potential functions for the newly discovered RA association variants. RESULTS: HLA-DQα1:160D conferred the strongest and independent susceptibility to ACPA-positive RA (p=6.16×10-36, OR=2.29). DRß1:37N had an independent protective effect (p=5.81×10-16, OR=0.49). As predicted by comparative modelling, the negatively charged DQα1:160D stabilises the dimer of dimers, thus may lead to an increased T cell activation. The negatively charged DRß1:37N encoding alleles preferentially bind with epitope P9 arginine, thus may result in a decreased RA susceptibility. CONCLUSIONS: We provide the first evidence that HLA-DQα1:160D, instead of HLA-DRB1*0405, is the strongest and independent genetic risk for ACPA-positive RA in Han Chinese. Our study also illustrates the value of deep sequencing for fine-mapping disease risk variants in the MHC region.


Assuntos
Artrite Reumatoide/genética , Cadeias alfa de HLA-DQ/genética , Adulto , Idoso , Anticorpos Anti-Proteína Citrulinada/imunologia , Artrite Reumatoide/imunologia , Grupo com Ancestrais do Continente Asiático/genética , Estudos de Casos e Controles , Feminino , Predisposição Genética para Doença , Cadeias HLA-DRB1/genética , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Teste de Histocompatibilidade/métodos , Humanos , Ativação Linfocitária/genética , Masculino , Pessoa de Meia-Idade , Linfócitos T/imunologia
16.
Fish Shellfish Immunol ; 89: 281-289, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-30953781

RESUMO

H-Ras is a guanosine triphosphatase (GTPase), which acts as a molecular switch and controls multiple important cellular processes including lymphocyte activation and function. However, regulatory mechanism of adaptive immune response by H-Ras remains unclear in non-mammalian animals. In the present study, we investigated the involvement of H-Ras in lymphocyte activation with a teleost model Oreochromis niloticus. H-Ras from O. niloticus (On-H-Ras) is highly conserved with those from other vertebrates. The mRNA of On-H-Ras showed a wide expression pattern in the lymphoid-tissues and with the highest level in liver. After Aeromonas hydrophila infection, transcription of On-H-Ras was significantly induced on day 8 but came back to basal level on day 16, suggesting that On-H-Ras potentially participated in primary response during the adaptive immunity. Furthermore, On-H-Ras mRNA was obviously up-regulated when leukocytes were activated by T lymphocyte mitogen PHA in vitro. Meanwhile, protein level of H-Ras was also augmented once leukocytes were stimulated with lymphocyte receptor signaling agonist PMA and ionomycin. More importantly, once Ras activity was inhibited by specific inhibitor, the up-regulation of lymphocyte activation marker CD122 was obviously impaired during lymphocyte activation process. Therefore, On-H-Ras regulated lymphocyte activation through both mRNA and protein level. Altogether, our results illustrated the involvement of H-Ras in teleost adaptive immunity via controlling lymphocyte activation, and thus provided a novel perspective to understand evolution of the lymphocyte-mediated adaptive immunity.


Assuntos
Imunidade Adaptativa/genética , Ciclídeos/genética , Doenças dos Peixes/imunologia , Proteínas de Peixes/genética , Genes ras/imunologia , Ativação Linfocitária/genética , Aeromonas hydrophila/fisiologia , Animais , Ciclídeos/imunologia , Proteínas de Peixes/metabolismo , Infecções por Bactérias Gram-Negativas/imunologia , Infecções por Bactérias Gram-Negativas/veterinária
17.
J Immunol ; 202(8): 2220-2228, 2019 04 15.
Artigo em Inglês | MEDLINE | ID: mdl-30850477

RESUMO

Abs can acquire N-linked glycans in their V regions during Ag-specific B cell responses. Among others, these N-linked glycans can affect Ag binding and Ab stability. Elevated N-linked glycosylation has furthermore been associated with several B cell-associated pathologies. Basic knowledge about patterns of V region glycosylation at different stages of B cell development is scarce. The aim of the current study is to establish patterns of N-glycosylation sites in Ab V regions of naive and memory B cell subsets. We analyzed the distribution and acquisition of N-glycosylation sites within Ab V regions of peripheral blood and bone marrow B cells of 12 healthy individuals, eight myasthenia gravis patients, and six systemic lupus erythematosus patients, obtained by next-generation sequencing. N-glycosylation sites are clustered around CDRs and the DE loop for both H and L chains, with similar frequencies for healthy donors and patients. No evidence was found for an overall selection bias against acquiring an N-glycosylation site, except for the CDR3 of the H chain. Interestingly, both IgE and IgG4 subsets have a 2-fold higher propensity to acquire Fab glycans compared with IgG1 or IgA. When expressed as rmAb, 35 out of 38 (92%) nongermline N-glycosylation sites became occupied. These results point toward a differential selection pressure of N-glycosylation site acquisition during affinity maturation of B cells, which depends on the location within the V region and is isotype and subclass dependent. Elevated Fab glycosylation represents an additional hallmark of TH2-like IgG4/IgE responses.


Assuntos
Linfócitos B/imunologia , Imunoglobulina G , Região Variável de Imunoglobulina , Lúpus Eritematoso Sistêmico/genética , Lúpus Eritematoso Sistêmico/imunologia , Ativação Linfocitária/genética , Linfócitos B/patologia , Glicosilação , Humanos , Imunoglobulina G/genética , Imunoglobulina G/imunologia , Região Variável de Imunoglobulina/genética , Região Variável de Imunoglobulina/imunologia , Lúpus Eritematoso Sistêmico/patologia
18.
Nat Biotechnol ; 37(3): 259-266, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30742126

RESUMO

Selective differentiation of naive T cells into multipotent T cells is of great interest clinically for the generation of cell-based cancer immunotherapies. Cellular differentiation depends crucially on division state and time. Here we adapt a dye dilution assay for tracking cell proliferative history through mass cytometry and uncouple division, time and regulatory protein expression in single naive human T cells during their activation and expansion in a complex ex vivo milieu. Using 23 markers, we defined groups of proteins controlled predominantly by division state or time and found that undivided cells account for the majority of phenotypic diversity. We next built a map of cell state changes during naive T-cell expansion. By examining cell signaling on this map, we rationally selected ibrutinib, a BTK and ITK inhibitor, and administered it before T cell activation to direct differentiation toward a T stem cell memory (TSCM)-like phenotype. This method for tracing cell fate across division states and time can be broadly applied for directing cellular differentiation.


Assuntos
Diferenciação Celular/genética , Proliferação de Células/genética , Ativação Linfocitária/genética , Células-Tronco Multipotentes/citologia , Rastreamento de Células/métodos , Humanos , Análise de Célula Única/métodos , Linfócitos T/citologia
19.
Nat Commun ; 10(1): 794, 2019 02 15.
Artigo em Inglês | MEDLINE | ID: mdl-30770827

RESUMO

Prolonged exposure of CD8+ T cells to antigenic stimulation, as in chronic viral infections, leads to a state of diminished function termed exhaustion. We now demonstrate that even during exhaustion there is a subset of functional CD8+ T cells defined by surface expression of SIRPα, a protein not previously reported on lymphocytes. On SIRPα+ CD8+ T cells, expression of co-inhibitory receptors is counterbalanced by expression of co-stimulatory receptors and it is only SIRPα+ cells that actively proliferate, transcribe IFNγ and show cytolytic activity. Furthermore, target cells that express the ligand for SIRPα, CD47, are more susceptible to CD8+ T cell-killing in vivo. SIRPα+ CD8+ T cells are evident in mice infected with Friend retrovirus, LCMV Clone 13, and in patients with chronic HCV infections. Furthermore, therapeutic blockade of PD-L1 to reinvigorate CD8+ T cells during chronic infection expands the cytotoxic subset of SIRPα+ CD8+ T cells.


Assuntos
Infecções por Arenaviridae/imunologia , Linfócitos T CD8-Positivos/imunologia , Coriomeningite Linfocítica/imunologia , Vírus da Coriomeningite Linfocítica/imunologia , Receptores Imunológicos/imunologia , Animais , Infecções por Arenaviridae/genética , Infecções por Arenaviridae/virologia , Linfócitos T CD8-Positivos/metabolismo , Linfócitos T CD8-Positivos/virologia , Feminino , Expressão Gênica/imunologia , Perfilação da Expressão Gênica , Interações Hospedeiro-Patógeno/imunologia , Humanos , Ativação Linfocitária/genética , Ativação Linfocitária/imunologia , Coriomeningite Linfocítica/genética , Coriomeningite Linfocítica/virologia , Vírus da Coriomeningite Linfocítica/fisiologia , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Receptores Imunológicos/genética , Receptores Imunológicos/metabolismo , Linfócitos T Citotóxicos/imunologia , Linfócitos T Citotóxicos/metabolismo , Linfócitos T Citotóxicos/virologia
20.
PLoS Genet ; 15(2): e1007930, 2019 02.
Artigo em Inglês | MEDLINE | ID: mdl-30779742

RESUMO

DNA cytosine methylation is involved in the regulation of gene expression during development and its deregulation is often associated with disease. Mammalian genomes are predominantly methylated at CpG dinucleotides. Unmethylated CpGs are often associated with active regulatory sequences while methylated CpGs are often linked to transcriptional silencing. Previous studies on CpG methylation led to the notion that transcription initiation is more sensitive to CpG methylation than transcriptional elongation. The immunoglobulin heavy chain (IgH) constant locus comprises multiple inducible constant genes and is expressed exclusively in B lymphocytes. The developmental B cell stage at which methylation patterns of the IgH constant genes are established, and the role of CpG methylation in their expression, are unknown. Here, we find that methylation patterns at most cis-acting elements of the IgH constant genes are established and maintained independently of B cell activation or promoter activity. Moreover, one of the promoters, but not the enhancers, is hypomethylated in sperm and early embryonic cells, and is targeted by different demethylation pathways, including AID, UNG, and ATM pathways. Combined, the data suggest that, rather than being prominently involved in the regulation of the IgH constant locus expression, DNA methylation may primarily contribute to its epigenetic pre-marking.


Assuntos
Metilação de DNA , Genes de Cadeia Pesada de Imunoglobulina , Regiões Constantes de Imunoglobulina/genética , Cadeias Pesadas de Imunoglobulinas/genética , Animais , Linfócitos B/citologia , Linfócitos B/imunologia , Linfócitos B/metabolismo , Linhagem da Célula/genética , Linhagem da Célula/imunologia , Ilhas de CpG/genética , Citosina/metabolismo , Epigênese Genética , Regulação da Expressão Gênica no Desenvolvimento , Ativação Linfocitária/genética , Camundongos , Regiões Promotoras Genéticas
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