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1.
Nature ; 615(7950): 151-157, 2023 03.
Artigo em Inglês | MEDLINE | ID: mdl-36509106

RESUMO

In the past decade, single-cell transcriptomics has helped to uncover new cell types and states and led to the construction of a cellular compendium of health and disease. Despite this progress, some difficult-to-sequence cells remain absent from tissue atlases. Eosinophils-elusive granulocytes that are implicated in a plethora of human pathologies1-5-are among these uncharted cell types. The heterogeneity of eosinophils and the gene programs that underpin their pleiotropic functions remain poorly understood. Here we provide a comprehensive single-cell transcriptomic profiling of mouse eosinophils. We identify an active and a basal population of intestinal eosinophils, which differ in their transcriptome, surface proteome and spatial localization. By means of a genome-wide CRISPR inhibition screen and functional assays, we reveal a mechanism by which interleukin-33 (IL-33) and interferon-γ (IFNγ) induce the accumulation of active eosinophils in the inflamed colon. Active eosinophils are endowed with bactericidal and T cell regulatory activity, and express the co-stimulatory molecules CD80 and PD-L1. Notably, active eosinophils are enriched in the lamina propria of a small cohort of patients with inflammatory bowel disease, and are closely associated with CD4+ T cells. Our findings provide insights into the biology of eosinophils and highlight the crucial contribution of this cell type to intestinal homeostasis, immune regulation and host defence. Furthermore, we lay a framework for the characterization of eosinophils in human gastrointestinal diseases.


Assuntos
Colite , Eosinófilos , Imunidade , Intestinos , Animais , Humanos , Camundongos , Colite/imunologia , Colite/patologia , Eosinófilos/classificação , Eosinófilos/citologia , Eosinófilos/imunologia , Eosinófilos/metabolismo , Doenças Inflamatórias Intestinais/imunologia , Análise da Expressão Gênica de Célula Única , Transcriptoma , Proteoma , Interleucina-33 , Interferon gama , Linfócitos T , Antígeno B7-1/metabolismo , Intestinos/imunologia , Intestinos/patologia
2.
Proc Natl Acad Sci U S A ; 116(48): 24231-24241, 2019 11 26.
Artigo em Inglês | MEDLINE | ID: mdl-31699819

RESUMO

Trisomy 21 (T21) causes Down syndrome (DS), a condition characterized by high prevalence of autoimmune disorders. However, the molecular and cellular mechanisms driving this phenotype remain unclear. Building upon our previous finding that T cells from people with DS show increased expression of interferon (IFN)-stimulated genes, we have completed a comprehensive characterization of the peripheral T cell compartment in adults with DS with and without autoimmune conditions. CD8+ T cells from adults with DS are depleted of naïve subsets and enriched for differentiated subsets, express higher levels of markers of activation and senescence (e.g., IFN-γ, Granzyme B, PD-1, KLRG1), and overproduce cytokines tied to autoimmunity (e.g., TNF-α). Conventional CD4+ T cells display increased differentiation, polarization toward the Th1 and Th1/17 states, and overproduction of the autoimmunity-related cytokines IL-17A and IL-22. Plasma cytokine analysis confirms elevation of multiple autoimmunity-related cytokines (e.g., TNF-α, IL17A-D, IL-22) in people with DS, independent of diagnosis of autoimmunity. Although Tregs are more abundant in DS, functional assays show that CD8+ and CD4+ effector T cells with T21 are resistant to Treg-mediated suppression, regardless of Treg karyotype. Transcriptome analysis of white blood cells and T cells reveals strong signatures of T cell differentiation and activation that correlate positively with IFN hyperactivity. Finally, mass cytometry analysis of 8 IFN-inducible phosphoepitopes demonstrates that T cell subsets with T21 show elevated levels of basal IFN signaling and hypersensitivity to IFN-α stimulation. Therefore, these results point to T cell dysregulation associated with IFN hyperactivity as a contributor to autoimmunity in DS.


Assuntos
Autoimunidade/genética , Síndrome de Down/imunologia , Subpopulações de Linfócitos T/imunologia , Adulto , Autoimunidade/imunologia , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/metabolismo , Estudos de Casos e Controles , Diferenciação Celular/fisiologia , Linhagem da Célula , Senescência Celular , Feminino , Perfilação da Expressão Gênica , Humanos , Interferon-alfa/farmacologia , Interferon gama/imunologia , Ativação Linfocitária/genética , Masculino , Subpopulações de Linfócitos T/citologia , Subpopulações de Linfócitos T/efeitos dos fármacos , Linfócitos T Reguladores/imunologia , Adulto Jovem
3.
Breast Cancer Res ; 23(1): 40, 2021 03 25.
Artigo em Inglês | MEDLINE | ID: mdl-33766090

RESUMO

BACKGROUND: Characterization of breast cancer (BC) through the determination of conventional markers such as ER, PR, HER2, and Ki67 has been useful as a predictive and therapeutic tool. Also, assessment of tumor-infiltrating lymphocytes has been proposed as an important prognostic aspect to be considered in certain BC subtypes. However, there is still a need to identify additional biomarkers that could add precision in distinguishing therapeutic response of individual patients. To this end, we focused in the expression of interferon regulatory factor 8 (IRF8) in BC cells. IRF8 is a transcription factor which plays a well-determined role in myeloid cells and that seems to have multiple antitumoral roles: it has tumor suppressor functions; it acts downstream IFN/STAT1, required for the success of some therapeutic regimes, and its expression in neoplastic cells seems to depend on a cross talk between the immune contexture and the tumor cells. The goal of the present study was to examine the relationship between IRF8 with the therapeutic response and the immune contexture in BC, since its clinical significance in this type of cancer has not been thoroughly addressed. METHODS: We identified the relationship between IRF8 expression and the clinical outcome of BC patients and validated IRF8 as predictive biomarker by using public databases and then performed in silico analysis. To correlate the expression of IRF8 with the immune infiltrate in BC samples, we performed quantitative multiplex immunohistochemistry. RESULTS: IRF8 expression can precisely predict the complete pathological response to monoclonal antibody therapy or to select combinations of chemotherapy such as FAC (fluorouracil, adriamycin, and cytoxan) in ER-negative BC subtypes. Analysis of immune cell infiltration indicates there is a strong correlation between activated and effector CD8+ T cell infiltration and tumoral IRF8 expression. CONCLUSIONS: We propose IRF8 expression as a potent biomarker not only for prognosis, but also for predicting therapy response in ER-negative BC phenotypes. Its expression in neoplastic cells also correlates with CD8+ T cell activation and infiltration. Therefore, our results justify new efforts towards understanding mechanisms regulating IRF8 expression and how they can be therapeutically manipulated.


Assuntos
Neoplasias da Mama/metabolismo , Linfócitos T CD8-Positivos/patologia , Fatores Reguladores de Interferon/metabolismo , Linfócitos do Interstício Tumoral/patologia , Receptores de Estrogênio/deficiência , Biomarcadores Tumorais/metabolismo , Neoplasias da Mama/patologia , Neoplasias da Mama/terapia , Linhagem Celular Tumoral , Progressão da Doença , Feminino , Humanos , Prognóstico , Resultado do Tratamento
4.
Acta Neuropathol ; 141(6): 901-915, 2021 06.
Artigo em Inglês | MEDLINE | ID: mdl-33774709

RESUMO

Myasthenia gravis (MG) is an autoimmune disease characterized by impaired neuromuscular signaling due to autoantibodies targeting the acetylcholine receptor. Although its auto-antigens and effector mechanisms are well defined, the cellular and molecular drivers underpinning MG remain elusive. Here, we employed high-dimensional single-cell mass and spectral cytometry of blood and thymus samples from MG patients in combination with supervised and unsupervised machine-learning tools to gain insight into the immune dysregulation underlying MG. By creating a comprehensive immune map, we identified two dysregulated subsets of inflammatory circulating memory T helper (Th) cells. These signature ThCD103 and ThGM cells populated the diseased thymus, were reduced in the blood of MG patients, and were inversely correlated with disease severity. Both signature Th subsets rebounded in the blood of MG patients after surgical thymus removal, indicative of their role as cellular markers of disease activity. Together, this in-depth analysis of the immune landscape of MG provides valuable insight into disease pathogenesis, suggests novel biomarkers and identifies new potential therapeutic targets for treatment.


Assuntos
Imunofenotipagem/métodos , Miastenia Gravis/imunologia , Miastenia Gravis/patologia , Análise de Célula Única , Linfócitos T/patologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Autoanticorpos , Autoimunidade , Linfócitos B/imunologia , Biomarcadores , Feminino , Humanos , Aprendizado de Máquina , Masculino , Pessoa de Meia-Idade , Miastenia Gravis/sangue , Receptores Colinérgicos/imunologia , Linfócitos T/imunologia , Timectomia , Timo
7.
J Immunol ; 190(3): 948-60, 2013 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-23284054

RESUMO

ssRNA can interact with dendritic cells (DCs) through binding to TLR7, inducing secretion of proinflammatory cytokines and type I IFN. Triggering TLR7 enhances cross-priming of CD8(+) T cells, which requires cross-presentation of exogenous Ag to DCs. However, how TLR triggering can affect Ag cross-presentation is still not clear. Using OVA as an Ag model, we observed that stimulation of TLR7 in DCs by polyuridylic acid (polyU), a synthetic ssRNA analog, generates a strong specific cytotoxic response in C57BL/6 mice. PolyU stimulate CD8α(+) DCs to cross-prime naive CD8(+) T cells in a type I IFN-dependent fashion. This enhanced cross-priming is accompanied by a higher density of OVA(256-264)/H-2K(b) complexes on CD8α(+) DCs treated with polyU, as well as by upregulation of costimulatory molecules and increased secretion of proinflammatory cytokines by DCs. Cross-priming of CD8(+) T cells by DCs treated with polyU requires proteasome and Ag translocation to cytosol through the Sec61 channel in DCs. The observed enhancement in OVA cross-presentation with polyU in DCs could be mediated by a limited Ag degradation in endophagosomal compartments and a higher permanence of OVA peptide/MHC class I complexes on DCs. These observations clearly reveal that key steps of Ag processing for cross-presentation can be modulated by TLR ligands, opening new avenues for understanding their mechanisms as adjuvants of the immune response.


Assuntos
Adjuvantes Imunológicos/farmacologia , Apresentação de Antígeno/efeitos dos fármacos , Linfócitos T CD8-Positivos/efeitos dos fármacos , Células Dendríticas/efeitos dos fármacos , Antígenos H-2/imunologia , Glicoproteínas de Membrana/efeitos dos fármacos , Ovalbumina/imunologia , Fragmentos de Peptídeos/imunologia , Poli U/farmacologia , Receptor 7 Toll-Like/efeitos dos fármacos , Animais , Apresentação de Antígeno/imunologia , Antígenos/imunologia , Linfócitos T CD8-Positivos/imunologia , Compartimento Celular , Células Cultivadas/imunologia , Citotoxicidade Imunológica , Células Dendríticas/imunologia , Endossomos/imunologia , Feminino , Glicoproteínas de Membrana/imunologia , Proteínas de Membrana/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fagossomos/imunologia , Complexo de Endopeptidases do Proteassoma/metabolismo , Transporte Proteico , Receptores de Antígenos de Linfócitos T alfa-beta/imunologia , Canais de Translocação SEC , Baço/imunologia , Receptor 7 Toll-Like/imunologia
8.
Oncoimmunology ; 12(1): 2261326, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-37808403

RESUMO

IL-17 immune responses in cancer are controversial, with both tumor-promoting and tumor-repressing effects observed. To clarify the role of IL-17 signaling in cancer progression, we used syngeneic tumor models from different tissue origins. We found that deficiencies in host IL-17RA or IL-17A/F expression had varying effects on the in vivo growth of different solid tumors including melanoma, sarcoma, lymphoma, and leukemia. In each tumor type, the absence of IL-17 led to changes in the expression of mediators associated with inflammation and metastasis in the tumor microenvironment. Furthermore, IL-17 signaling deficiencies in the hosts resulted in decreased anti-tumor CD8+ T cell immunity and caused tumor-specific changes in several lymphoid cell populations. Our findings were associated with distinct patterns of IL-17A/F cytokine and receptor subunit expression in the injected tumor cell lines. These patterns affected tumor cell responsiveness to IL-17 and downstream intracellular signaling, leading to divergent effects on cancer progression. Additionally, we identified IL-17RC as a critical determinant of the IL-17-mediated response in tumor cells and a potential biomarker for IL-17 signaling effects in tumor progression. Our study offers insight into the molecular mechanisms underlying IL-17 activities in cancer and lays the groundwork for developing personalized immunotherapies.


Assuntos
Neoplasias , Receptores de Interleucina-17 , Humanos , Receptores de Interleucina-17/genética , Receptores de Interleucina-17/metabolismo , Interleucina-17 , Transdução de Sinais , Linfócitos T CD8-Positivos , Inflamação , Neoplasias/genética
9.
Front Immunol ; 12: 713132, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34386013

RESUMO

Senescent T cells have been described during aging, chronic infections, and cancer; however, a comprehensive study of the phenotype, function, and transcriptional program of this T cell population in breast cancer (BC) patients is missing. Compared to healthy donors (HDs), BC patients exhibit an accumulation of KLRG-1+CD57+ CD4+ and CD8+ T cells in peripheral blood. These T cells infiltrate tumors and tumor-draining lymph nodes. KLRG-1+CD57+ CD4+ and CD8+ T cells from BC patients and HDs exhibit features of senescence, and despite their inhibitory receptor expression, they produce more effector cytokines and exhibit higher expression of Perforin, Granzyme B, and CD107a than non-senescent subsets. When compared to blood counterparts, tumor-infiltrating senescent CD4+ T cells show similar surface phenotype but reduced cytokine production. Transcriptional profiling of senescent CD4+ T cells from the peripheral blood of BC patients reveals enrichment in genes associated with NK or CD8+-mediated cytotoxicity, TCR-mediated stimulation, and cell exhaustion compared to non-senescent T cells. Comparison of the transcriptional profile of senescent CD4+ T cells from peripheral blood of BC patients with those of HDs highlighted marked similarities but also relevant differences. Senescent CD4+ T cells from BC patients show enrichment in T-cell signaling, processes involved in DNA replication, p53 pathways, oncogene-induced senescence, among others compared to their counterparts in HDs. High gene expression of CD4, KLRG-1, and B3GAT1 (CD57), which correlates with increased overall survival for BC patients, underscores the usefulness of the evaluation of the frequency of senescent CD4+ T cells as a biomarker in the follow-up of patients.


Assuntos
Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/metabolismo , Senescência Celular , Linfócitos do Interstício Tumoral/imunologia , Linfócitos do Interstício Tumoral/metabolismo , Neoplasias da Mama/etiologia , Antígenos CD57/metabolismo , Estudos de Casos e Controles , Senescência Celular/genética , Senescência Celular/imunologia , Citotoxicidade Imunológica , Feminino , Perfilação da Expressão Gênica , Humanos , Imunofenotipagem , Lectinas Tipo C/metabolismo , Contagem de Linfócitos , Linfócitos do Interstício Tumoral/patologia , Receptores Imunológicos/metabolismo , Subpopulações de Linfócitos T/imunologia , Subpopulações de Linfócitos T/metabolismo , Subpopulações de Linfócitos T/patologia
10.
Front Immunol ; 9: 2347, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30364284

RESUMO

The IL-17 family contributes to host defense against many intracellular pathogens by mechanisms that are not fully understood. CD8+ T lymphocytes are key elements against intracellular microbes, and their survival and ability to mount cytotoxic responses are orchestrated by several cytokines. Here, we demonstrated that IL-17RA-signaling cytokines sustain pathogen-specific CD8+ T cell immunity. The absence of IL-17RA and IL-17A/F during Trypanosoma cruzi infection resulted in increased tissue parasitism and reduced frequency of parasite-specific CD8+ T cells. Impaired IL-17RA-signaling in vivo increased apoptosis of parasite-specific CD8+ T cells, while in vitro recombinant IL-17 down-regulated the pro-apoptotic protein BAD and promoted the survival of activated CD8+ T cells. Phenotypic, functional, and transcriptomic profiling showed that T. cruzi-specific CD8+ T cells derived from IL-17RA-deficient mice presented features of cell dysfunction. PD-L1 blockade partially restored the magnitude of CD8+ T cell responses and parasite control in these mice. Adoptive transfer experiments established that IL-17RA-signaling is intrinsically required for the proper maintenance of functional effector CD8+ T cells. Altogether, our results identify IL-17RA and IL-17A as critical factors for sustaining CD8+ T cell immunity to T. cruzi.


Assuntos
Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/metabolismo , Doença de Chagas/imunologia , Doença de Chagas/metabolismo , Receptores de Interleucina-17/metabolismo , Transdução de Sinais , Trypanosoma cruzi/imunologia , Transferência Adotiva , Animais , Apoptose , Sobrevivência Celular , Doença de Chagas/microbiologia , Citocinas/metabolismo , Feminino , Interações Hospedeiro-Patógeno/genética , Interações Hospedeiro-Patógeno/imunologia , Imunomodulação/genética , Interleucina-17/metabolismo , Ativação Linfocitária/genética , Ativação Linfocitária/imunologia , Contagem de Linfócitos , Masculino , Camundongos , Camundongos Knockout , Receptores de Interleucina-17/deficiência , Transcrição Gênica
11.
Sci Transl Med ; 10(469)2018 11 28.
Artigo em Inglês | MEDLINE | ID: mdl-30487251

RESUMO

Allogeneic hematopoietic cell transplantation (allo-HCT) not only is an effective treatment for several hematologic malignancies but can also result in potentially life-threatening graft-versus-host disease (GvHD). GvHD is caused by T cells within the allograft attacking nonmalignant host tissues; however, these same T cells mediate the therapeutic graft-versus-leukemia (GvL) response. Thus, there is an urgent need to understand how to mechanistically uncouple GvL from GvHD. Using preclinical models of full and partial MHC-mismatched HCT, we here show that the granulocyte-macrophage colony-stimulating factor (GM-CSF) produced by allogeneic T cells distinguishes between the two processes. GM-CSF drives GvHD pathology by licensing donor-derived phagocytes to produce inflammatory mediators such as interleukin-1ß and reactive oxygen species. In contrast, GM-CSF did not affect allogeneic T cells or their capacity to eliminate leukemic cells, retaining undiminished GvL responses. Last, tissue biopsies and peripheral blood mononuclear cells from patients with grade IV GvHD showed an elevation of GM-CSF-producing T cells, suggesting that GM-CSF neutralization has translational potential in allo-HCT.


Assuntos
Doença Enxerto-Hospedeiro/imunologia , Fator Estimulador de Colônias de Granulócitos e Macrófagos/farmacologia , Imunidade/efeitos dos fármacos , Leucemia/imunologia , Células Mieloides/metabolismo , Animais , Doença Enxerto-Hospedeiro/patologia , Transplante de Células-Tronco Hematopoéticas , Antígenos de Histocompatibilidade/metabolismo , Humanos , Interferon gama/metabolismo , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Células Mieloides/efeitos dos fármacos , Linfócitos T/efeitos dos fármacos , Linfócitos T/imunologia , Transplante Homólogo
12.
Neuromuscul Disord ; 17(8): 611-23, 2007 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-17588759

RESUMO

Facioscapulohumeral muscular dystrophy (FSHD) patients carry contractions of the D4Z4-tandem repeat array on chromosome 4q35. Decrease in D4Z4 copy number is thought to alter a chromatin structure and activate expression of neighboring genes. D4Z4 contains a putative double-homeobox gene called DUX4. We identified DUX4 mRNAs in cells transfected with genomic fragments containing the DUX4 gene. Using RT-PCR we also recognized expressed DUX4 mRNAs in primary FSHD myoblasts. Polyclonal antibodies raised against specific DUX4 peptides detected the DUX4 protein in cells transfected with D4Z4 elements. DUX4 localizes in the nucleus of cells transfected with CMV-DUX4 expression vectors. A DUX4-related protein is endogenously expressed in nuclei of adult and fetal human rhabdomyosarcoma cell lines. Overexpression of DUX4 induces cell death, induces caspase 3/7 activity and alters emerin distribution at the nuclear envelope. We propose that DUX4-mediated cell death contributes to the pathogenic pathway in FSHD.


Assuntos
Apoptose/fisiologia , Proteínas de Homeodomínio/genética , Células Musculares/fisiologia , Distrofia Muscular Facioescapuloumeral/genética , Distrofia Muscular Facioescapuloumeral/patologia , Sequência de Aminoácidos , Linhagem Celular Tumoral , Expressão Gênica/fisiologia , Proteínas de Homeodomínio/metabolismo , Humanos , Proteínas de Membrana/metabolismo , Dados de Sequência Molecular , Células Musculares/citologia , Distrofia Muscular Facioescapuloumeral/fisiopatologia , Membrana Nuclear/metabolismo , Proteínas Nucleares/metabolismo , Músculo Quadríceps/citologia , RNA Mensageiro/metabolismo , Rabdomiossarcoma , Transcrição Gênica/fisiologia
13.
PLoS One ; 10(10): e0140672, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-26474053

RESUMO

The aging process is accompanied by altered immune system functioning and an increased risk of infection. Dendritic cells (DCs) are antigen-presenting cells that play a key role in both adaptive and innate immunity, but how aging affects DCs and their influence on immunity has not been thoroughly established. Here we examined the function of conventional DCs (cDCs) in old mice after TLR7 stimulation, focusing on their ability to cross-prime CD8+ T cells. Using polyU, a synthetic ssRNA analog, as TLR7 ligand and OVA as an antigen (Ag) model, we found that cDCs from old mice have a poor ability to stimulate a CD8+ T cell-mediated cytotoxic response. cDCs from old mice exhibit alterations in Ag-processing machinery and TLR7 activation. Remarkably, CD8α+ cDCs from old mice have an impaired ability to activate naïve CD8+ T cells and, moreover, a lower capacity to mature and to process exogenous Ag. Taken together, our results suggest that immunosenescence impacts cDC function, affecting the activation of naïve CD8+ T cells and the generation of effector cytotoxic T cells.


Assuntos
Envelhecimento/imunologia , Apresentação de Antígeno , Linfócitos T CD8-Positivos/enzimologia , Apresentação Cruzada/imunologia , Células Dendríticas/imunologia , Imunidade Celular , Glicoproteínas de Membrana/imunologia , Receptor 7 Toll-Like/imunologia , Animais , Antígenos/imunologia , Antígenos/farmacologia , Apresentação Cruzada/efeitos dos fármacos , Feminino , Camundongos , Poli U/imunologia , Poli U/farmacologia
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