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OBJECTIVES: Compared to low-grade irAEs, high-grade irAEs are more often dose-limiting and can alter the long-term treatment options for a patient. Predicting the incidence of high-grade irAEs would help with treatment selection and therapeutic drug monitoring. MATERIALS AND METHODS: We performed a retrospective study of 430 stage III and IV patients with non-small cell lung cancer (NSCLC) who received an immune checkpoint inhibitor (ICI), either with or without chemotherapy, at a single comprehensive cancer center from 2015 to 2022. The study team retrieved sequencing data and complete clinical information, including detailed irAEs medical records. Fisher's exact test was used to determine the association between mutations and the presence or absence of high-grade irAEs. Patients were analyzed separately based on tumor subtypes and sequencing platforms. RESULTS: High-grade and low-grade irAEs occurred in 15.2% and 46.2% of patients, respectively. Respiratory and gastrointestinal irAEs were the 2 most common irAEs. The distribution of patients with or without irAEs was similar between ICI and ICI+chemotherapy-treated patients. By analyzing the mutation data, we identified 5 genes (MYC, TEK, FANCA, FAM123B, and MET) with mutations that were correlated with an increased risk of high-grade irAEs. For the adenocarcinoma subtype, mutations in TEK, MYC, FGF19, RET, and MET were associated with high-grade irAEs; while for the squamous subtype, ERBB2 mutations were associated with high-grade irAEs. CONCLUSION: This study is the first to demonstrate that specific tumor mutations correlate with the incidence of high-grade irAEs in patients with NSCLC treated with an ICI, providing molecular guidance for treatment selection and drug monitoring.
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Studies over the last 100 years have suggested a link between inflammation, infectious disease, and Alzheimer's Disease (AD). Understanding how the immune system changes during the development of AD may facilitate new treatments. Here, we studied an aging cohort who had been assessed for AD pathology with amyloid positron emission tomography and cognitive testing, and conducted high dimensional flow cytometry on peripheral blood mononuclear and cerebrospinal fluid cells. Participants were assigned a classification of being amyloid negative cognitively normal, amyloid positive cognitively normal (APCN), or amyloid positive mild cognitive impairment (APMCI), an early stage of AD. We observed major alterations in the peripheral innate immune system including increased myeloid and plasmacytoid dendritic cells in the blood of APMCI participants. When the adaptive immune system was examined, amyloid positive participants, regardless of cognitive status, had increased CD3+ T cells. Further analyses of CD4+ and CD8+ T cells revealed that APMCI participants had an increase in more differentiated phenotype T cells, such as effector memory and effector memory CD45RA expressing (TEMRA), compared to those with normal cognition. When T cell function was measured, we observed that T cells from APCN participants had increased IFNγ+GzB- producing cells compared to the other participants. In contrast, we demonstrate that APMCI participants had a major increase in T cells that lacked cytokine production following restimulation and expressed increased levels of PD-1 and Tox, suggesting these are exhausted cells. Rejuvenation of these cells may provide a potential treatment for AD.
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Doença de Alzheimer , Exaustão das Células T , Humanos , Linfócitos T CD8-Positivos , Leucócitos Mononucleares , Tomografia Computadorizada por Raios X , Proteínas AmiloidogênicasRESUMO
Cells are the basic building blocks of human organisms, and the identification of their types and states in transcriptomic data is an important and challenging task. Many of the existing approaches to cell-type prediction are based on clustering methods that optimize only one criterion. In this paper, a multi-objective Genetic Algorithm for cluster analysis is proposed, implemented, and systematically validated on 48 experimental and 60 synthetic datasets. The results demonstrate that the performance and the accuracy of the proposed algorithm are reproducible, stable, and better than those of single-objective clustering methods. Computational run times of multi-objective clustering of large datasets were studied and used in supervised machine learning to accurately predict the execution times of clustering of new single-cell transcriptomes.
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The Three Prime Repair EXonuclease I (TREX1) is critical for degrading post-apoptosis DNA. Mice expressing catalytically inactive TREX1 (TREX1 D18N) develop lupus-like autoimmunity due to chronic sensing of undegraded TREX1 DNA substrates, production of the inflammatory cytokines, and the inappropriate activation of innate and adaptive immunity. This study aimed to investigate Thelper (Th) dysregulation in the TREX1 D18N model system as a potential mechanism for lupus-like autoimmunity. Comparison of immune cells in secondary lymphoid organs, spleen and peripheral lymph nodes (LNs) between TREX1 D18N mice and the TREX1 null mice revealed that the TREX1 D18N mice exhibit a Th1 bias. Additionally, the T-follicular helper cells (Tfh) and the germinal celter (GC) B cells were also elevated in the TREX1 D18N mice. Targeting Bcl6, a lineage-defining transcription factor for Tfh and GC B cells, with a commercially available Bcl6 inhibitor, FX1, attenuated Tfh, GC, and Th1 responses, and rescued TREX1 D18N mice from autoimmunity. The study presents Tfh and GC B-cell responses as potential targets in autoimmunity and that Bcl6 inhibitors may offer therapeutic approach in TREX1-associated or other lupus-like diseases.
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Autoimunidade , Centro Germinativo , Animais , Diferenciação Celular , DNA , Modelos Animais de Doenças , Exodesoxirribonucleases , Camundongos , Camundongos Knockout , Fosfoproteínas , Proteínas Proto-Oncogênicas c-bcl-6/genética , Proteínas Proto-Oncogênicas c-bcl-6/metabolismo , Células T Auxiliares Foliculares , Linfócitos T Auxiliares-IndutoresRESUMO
Mutations in the TREX1 3' â 5' exonuclease are associated with a spectrum of autoimmune disease phenotypes in humans and mice. Failure to degrade DNA activates the cGAS-STING DNA-sensing pathway signaling a type-I interferon (IFN) response that ultimately drives immune system activation. TREX1 and the cGAS-STING DNA-sensing pathway have also been implicated in the tumor microenvironment, where TREX1 is proposed to degrade tumor-derived DNA that would otherwise activate cGAS-STING. If tumor-derived DNA were not degraded, the cGAS-STING pathway would be activated to promote IFN-dependent antitumor immunity. Thus, we hypothesize TREX1 exonuclease inhibition as a novel immunotherapeutic strategy. We present data demonstrating antitumor immunity in the TREX1 D18N mouse model and discuss theory surrounding the best strategy for TREX1 inhibition. Potential complications of TREX1 inhibition as a therapeutic strategy are also discussed.
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Doenças Autoimunes/imunologia , DNA/imunologia , Exodesoxirribonucleases/imunologia , Proteínas de Membrana/imunologia , Nucleotidiltransferases/imunologia , Fosfoproteínas/imunologia , Animais , Antineoplásicos/imunologia , Antineoplásicos/uso terapêutico , Doenças Autoimunes/genética , Doenças Autoimunes/metabolismo , Células Cultivadas , DNA/genética , DNA/metabolismo , Modelos Animais de Doenças , Exodesoxirribonucleases/genética , Exodesoxirribonucleases/metabolismo , Humanos , Imunoterapia/métodos , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Camundongos da Linhagem 129 , Camundongos Knockout , Nucleotidiltransferases/genética , Nucleotidiltransferases/metabolismo , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Transdução de Sinais/imunologiaRESUMO
Acute graft-versus-host-disease (GVHD), limits the use of hematopoietic cell transplant (HCT) to treat a variety of malignancies. Any new therapeutic approach must satisfy three requirements: 1) Prevent GVHD, 2) Maintain anti-pathogen immunity, and 3) Maintain anti-tumor immunity. In prior studies we have shown that the selective photosensitizer 2-Se-Cl eliminates highly alloreactive lymphocytes from the graft prior to HCT preventing GVHD and that antiviral immune responses were preserved following incubation with 2-Se-Cl. In this report, we investigated whether 2-Se-Cl treatment preserves antitumor immunity, and then used high dimensional flow cytometry to identify the determinants of successful immune reconstitution. Donor C57BL/6 splenocytes were cocultured for 4 days with irradiated BALB/c splenocytes and then exposed to 2-Se-Cl. Photodepletion (PD)-treated splenocytes were then infused into lethally irradiated BALB/c mice inoculated with A20 leukemia/lymphoma cells. Recipient mice that received PD-treated splenocytes survived > 100 days without evidence of GVHD or leukemia. In contrast, mice that did not receive PD-treated cells at time of HCT died of leukemia progression. Multiparameter flow cytometry of cytokines and surface markers on peripheral blood samples 15 days after HCT demonstrated unique patterns of immune reconstitution. We found that before clinical disease onset GVHD was marked by functionally exhausted T cells, while tumor clearance and long-term survival were associated with an expansion of polyfunctional T cells, monocytes, and DCs early after transplantation. Taken together these results demonstrate that 2-Se-Cl photodepletion is a new treatment that can facilitate HCT by preventing GVHD while preserving antiviral and anti-tumor immunity.
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Doença Enxerto-Hospedeiro/prevenção & controle , Fármacos Fotossensibilizantes/farmacologia , Compostos de Selênio/farmacologia , Animais , Antígeno CTLA-4/metabolismo , Células Dendríticas/efeitos dos fármacos , Células Dendríticas/imunologia , Células Dendríticas/efeitos da radiação , Feminino , Leucemia/imunologia , Leucemia/terapia , Camundongos , Receptor de Morte Celular Programada 1/metabolismo , Linfócitos T/efeitos dos fármacos , Linfócitos T/imunologia , Linfócitos T/efeitos da radiação , Regulação para Cima/efeitos dos fármacos , Regulação para Cima/imunologiaRESUMO
Autoimmunity can result when cells fail to properly dispose of DNA. Mutations in the three-prime repair exonuclease 1 (TREX1) cause a spectrum of human autoimmune diseases resembling systemic lupus erythematosus. The cytosolic dsDNA sensor, cyclic GMP-AMP synthase (cGAS), and the stimulator of IFN genes (STING) are required for pathogenesis, but specific cells in which DNA sensing and subsequent type I IFN (IFN-I) production occur remain elusive. In this study, we demonstrate that TREX1 D18N catalytic deficiency causes dysregulated IFN-I signaling and autoimmunity in mice. Moreover, we show that bone marrow-derived cells drive this process. We identify both innate immune and, surprisingly, activated T cells as sources of pathological IFN-α production. These findings demonstrate that TREX1 enzymatic activity is crucial to prevent inappropriate DNA sensing and IFN-I production in immune cells, including normally low-level IFN-α-producing cells. These results expand our understanding of DNA sensing and innate immunity in T cells and may have relevance to the pathogenesis of human disease caused by TREX1 mutation.
Assuntos
Exodesoxirribonucleases/genética , Lúpus Eritematoso Sistêmico/genética , Fosfoproteínas/genética , Linfócitos T/imunologia , Animais , Autoantígenos/imunologia , Autoimunidade , Células Cultivadas , DNA/imunologia , Modelos Animais de Doenças , Humanos , Imunidade Inata , Interferon-alfa/metabolismo , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Nucleotidiltransferases/metabolismoRESUMO
Anaemia is commonly observed in chronic inflammatory conditions, including systemic lupus erythematosus (SLE), where â¼50% of patients display clinical signs of anaemia. Mutation at the aspartate residue 18 of the three prime repair exonuclease 1 (TREX1) gene causes a monogenic form of cutaneous lupus in humans and the genetically precise TREX1 D18N mice recapitulate a lupus-like disease. TREX1 degrades single- and double-stranded DNA (dsDNA), and the link between failed DNA degradation by nucleases, including nucleoside-diphosphate kinases (NM23H1/H2) and Deoxyribonuclease II (DNase II), and anaemia prompted our studies to investigate whether TREX1 dysfunction contributes to anaemia. Utilizing the TREX1 D18N mice we demonstrate that (1) TREX1 mutant mice develop normocytic normochromic anaemia and (2) TREX1 exonuclease participates in the degradation of DNA originating from erythroblast nuclei during definitive erythropoiesis. Gene expression, hematocrit, hemoglobin, immunohistochemistry (IHC) and flow cytometry were used to quantify dysfunctional erythropoiesis. An altered response to induced anaemia in the TREX1 D18N mice was determined through IHC, flow cytometry, and interferon-stimulated gene (ISG) expression analysis of the liver, spleen and erythroblastic islands (EBIs). IHC, flow cytometry, and ISG expression studies were performed in vitro to determine the role of TREX1 in the degradation of erythroblast DNA within EBIs. The TREX1 D18N mice exhibit altered erythropoiesis including a 20% reduction in hematocrit, 10-20 fold increased erythropoietic gene expression levels in the spleen and phenotypic signs of normocytic normochromic anaemia. Anaemia in TREX1 D18N mice is accompanied by increased erythropoietin (Epo), normal hepcidin levels and the TREX1 D18N mice display an inappropriate response to anaemic challenge. Enhanced ISG expression results from failed processing and subsequent sensing of undegraded erythroblast DNA in EBIs. TREX1 participates in the degradation of erythroblast DNA in the EBI and TREX1 D18N mice exhibit a normocytic normochromic anaemia.
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CD8+ T lymphocytes are critical for the control of gammaherpesvirus latency. To determine how memory CD8+ T cells generated during latency differ from those primed during acute or chronic viral infection, we adoptively transferred naive P14 CD8+ T cells into uninfected recipients, and examined surface proteins, cytokines and transcription factors following infection with the Armstrong (acute) or Clone 13 (chronic) strains of lymphocytic choriomeningitis virus (LCMV), or murine gammaherpesvirus 68 (MHV68) expressing the LCMV epitope DbGP33-41. By performing k-means clustering and generating self organizing maps (SOM), we observed increased short-lived effector-like, CD27lo CD62Llo and Bcl-6lo CD8+ T cells following latent infection. In addition, we found that memory CD8+ T cells from latent primed mice underwent less expansion following adoptive transfer and antigen rechallenge. Data from cluster models were combined and visualized by principal component analysis (PCA) demonstrating memory CD8+ T cells from latent infection occupy an intermediate differentiation space.
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Linfócitos T CD8-Positivos/imunologia , Memória Imunológica , Vírus da Coriomeningite Linfocítica/imunologia , Rhadinovirus/imunologia , Subpopulações de Linfócitos T/imunologia , Viroses/imunologia , Viroses/patologia , Animais , Linfócitos T CD8-Positivos/química , Linfócitos T CD8-Positivos/classificação , Doença Crônica , Aprendizado de Máquina , Camundongos , Subpopulações de Linfócitos T/química , Subpopulações de Linfócitos T/classificação , Latência ViralRESUMO
Using a modular library format in conjunction with cell viability (MTS) and flow cytometry assays, 90 cationic complexes [AuPL] n+ (P = phosphine ligand; L = thiourea derivative or chloride) were studied for their antiproliferative activity in CD8+ T lymphocyte cells. The activity of the compounds correlates with the steric bulk of the phosphine ligands. Thiourea serves as a leaving group that is readily replaced by cysteine thiol (NMR, ESI-MS). Taking advantage of selective thiourea ligand exchange, the fragments [Au(PEt3)]+ and [Au(JohnPhos)]+ (JohnPhos = 1,1'-biphenyl-2-yl)di-tert-butylphosphine) in compounds 1 and 2 were transferred to recombinant human serum albumin (rHSA). PEt3 promoted efficient modification of Cys34 in HSA (HSA-1), whereas use of bulky JohnPhos as a carrier ligand led to serum protein nonspecifically modified with multiple gold adducts (HSA-2) (Ellman's test, ESI-TOF MS). HSA-1, but not HSA-2, strongly inhibits T cell proliferation at nanomolar doses. The potential role of HSA as a delivery vehicle in gold-based autoimmune disease treatment is discussed.
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T lymphocytes play a central role in many human immunologic disorders, including autoimmune and alloimmune diseases. In hematopoietic stem cell transplantation, acute graft-versus-host-disease (GVHD) is caused by an attack on the recipient's tissues from donor allogeneic T cells. Selectively depleting GVHD-causing cells prior to transplant may prevent GVHD. In this study, we evaluated 24 chalcogenorhodamine photosensitizers for their ability to selectively deplete reactive T lymphocytes and identified the photosensitizer 2-Se-Cl, which accumulates in stimulated T cells in proportion to oxidative phosphorylation. The photosensitizer is also a potent stimulator of P-glycoprotein (P-gp). Enhanced P-gp activity promotes the efficient removal of photosensitizer not sequestered in mitochondria and protects resting lymphocytes that are essential for antipathogen and antitumor responses. To evaluate the selective depletion of alloimmune responses, donor C57BL/6 splenocytes were cocultured for 5 d with irradiated BALB/c splenocytes and then photodepleted (PD). PD-treated splenocytes were infused into lethally irradiated BALB/c (same-party) or C3H/HeJ (third-party) mice. Same-party mice that received PD-treated splenocytes at the time of transplant lived 100 d without evidence of GVHD. In contrast, all mice that received untreated primed splenocytes and third-party mice that received PD-treated splenocytes died of lethal GVHD. To evaluate the preservation of antiviral immune responses, acute lymphocytic choriomeningitis virus infection was used. After photodepletion, expansion of Ag-specific naive CD8(+) T cells and viral clearance remained fully intact. The high selectivity of this novel photosensitizer may have broad applications and provide alternative treatment options for patients with T lymphocyte-mediated diseases.
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Subfamília B de Transportador de Cassetes de Ligação de ATP/imunologia , Linfócitos T CD8-Positivos/metabolismo , Doença Enxerto-Hospedeiro/prevenção & controle , Depleção Linfocítica/métodos , Subfamília B de Transportador de Cassetes de Ligação de ATP/efeitos dos fármacos , Subfamília B de Transportador de Cassetes de Ligação de ATP/metabolismo , Animais , Linfócitos T CD8-Positivos/efeitos dos fármacos , Linfócitos T CD8-Positivos/imunologia , Metabolismo Energético , Doença Enxerto-Hospedeiro/imunologia , Humanos , Coriomeningite Linfocítica/imunologia , Vírus da Coriomeningite Linfocítica/fisiologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C3H , Camundongos Endogâmicos C57BL , Fármacos Fotossensibilizantes/isolamento & purificação , Fármacos Fotossensibilizantes/farmacologia , Transplante HomólogoRESUMO
Extracorporeal photopheresis (ECP) has been used successfully in the treatment of erythrodermic cutaneous T cell lymphoma (CTCL), and other T cell-mediated disorders. Not all patients obtain a significant or durable response from ECP. The design of a selective photosensitizer that spares desirable lymphocytes while targeting malignant T cells may promote cytotoxic T cell responses and improve outcomes after ECP. A series of selenorhodamines built with variations of the Texas red core targeted the mitochondria of malignant T cells, were phototoxic to malignant T cells presumably via their ability to generate singlet oxygen, and were transported by P-glycoprotein (P-gp). To determine the selectivity of the photosensitizers in the ECP milieu, staphylococcal enterotoxin B (SEB)-stimulated and non-stimulated human lymphocytes were combined with HUT-78 cells (a CTCL) to simulate ECP. The amide-containing analogues of the selenorhodamines were transported more rapidly than the thioamide analogues in monolayers of MDCKII-MDR1 cells and, consequently, were extruded more rapidly from P-gp-expressing T cells than the corresponding thioamide analogues. Selenorhodamine 6 with the Texas red core and a piperidylamide functionality was phototoxic to >90% of malignant T cells while sparing >60% of both stimulated and non-stimulated T cells. In the resting T cells, (63±7)% of the CD4+ T cell compartment, and (78±2.5)% of the CD8+ cytotoxic T cell population were preserved, resulting in an enrichment of healthy and cytotoxic T cells after photodepletion.
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Compostos Organosselênicos/farmacologia , Fotoferese , Fármacos Fotossensibilizantes/farmacologia , Rodaminas/farmacologia , Linfócitos T/efeitos dos fármacos , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/antagonistas & inibidores , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/metabolismo , Linhagem Celular Tumoral , Humanos , Luz , Linfoma , Mitocôndrias/metabolismo , Compostos Organosselênicos/síntese química , Fármacos Fotossensibilizantes/síntese química , Rodaminas/síntese química , Linfócitos T/metabolismo , Verapamil/farmacologiaRESUMO
Although T cells play a critical role in protection from viruses, bacteria, and tumors, they also cause autoimmune diseases such as systemic lupus erythematosus, rheumatoid arthritis, and multiple sclerosis. Unwanted T cell responses during organ transplant, graft-versus-host disease, and allergies are also major clinical problems. Although drugs are available to suppress unwanted immune responses, they have limited efficacy with serious side effects. Thus, new therapeutics limiting T cell activation, proliferation, and function can make an immediate clinical impact. To identify new suppressors of lymphocyte activation, proliferation, and function, we examined the immunosuppressive activity of gold(I) analogs of platinum-acridine antitumor agents. We found that the gold complex Au-ACRAMTU-PEt3 is a potent suppressor of murine and human T cell activation. Preincubation with Au-ACRAMTU-PEt3 suppresses the proliferation of CD4(+) and CD8(+) T cells at a similar concentration as pharmaceutical grade cyclosporine A. Au-ACRAMTU-PEt3 pretreatment decreases the production of IFN-γ, TNF-α, IL-2, and IL-17 by human and murine CD4(+) and CD8(+) T cells. When mice were treated with Au-ACRAMTU-PEt3 during viral infection, the expansion of virus-specific CD8(+) T cells was decreased 10-fold and viral load was elevated. Taken together, these results demonstrate that Au-ACRAMTU-PEt3 has potent immunosuppressive activity that could be used to suppress immune responses during transplantation and autoimmunity.
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Linfócitos T CD4-Positivos/efeitos dos fármacos , Linfócitos T CD8-Positivos/efeitos dos fármacos , Compostos Organoáuricos/farmacologia , Acridinas/química , Animais , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/metabolismo , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/metabolismo , Cálcio/metabolismo , Células Cultivadas , Citocinas/imunologia , Citocinas/metabolismo , Relação Dose-Resposta a Droga , Feminino , Citometria de Fluxo , Humanos , Ativação Linfocitária/efeitos dos fármacos , Ativação Linfocitária/imunologia , Coriomeningite Linfocítica/tratamento farmacológico , Coriomeningite Linfocítica/imunologia , Coriomeningite Linfocítica/virologia , Vírus da Coriomeningite Linfocítica/efeitos dos fármacos , Vírus da Coriomeningite Linfocítica/imunologia , Vírus da Coriomeningite Linfocítica/fisiologia , Camundongos Endogâmicos C57BL , Compostos Organoáuricos/química , Oxirredução/efeitos dos fármacos , Platina/química , Ureia/análogos & derivados , Ureia/química , Carga Viral/efeitos dos fármacos , Carga Viral/imunologiaRESUMO
The TREX1 gene encodes a potent DNA exonuclease, and mutations in TREX1 cause a spectrum of lupus-like autoimmune diseases. Most lupus patients develop autoantibodies to double-stranded DNA (dsDNA), but the source of DNA antigen is unknown. The TREX1 D18N mutation causes a monogenic, cutaneous form of lupus called familial chilblain lupus, and the TREX1 D18N enzyme exhibits dysfunctional dsDNA-degrading activity, providing a link between dsDNA degradation and nucleic acid-mediated autoimmune disease. We determined the structure of the TREX1 D18N protein in complex with dsDNA, revealing how this exonuclease uses a novel DNA-unwinding mechanism to separate the polynucleotide strands for single-stranded DNA (ssDNA) loading into the active site. The TREX1 D18N dsDNA interactions coupled with catalytic deficiency explain how this mutant nuclease prevents dsDNA degradation. We tested the effects of TREX1 D18N in vivo by replacing the TREX1 WT gene in mice with the TREX1 D18N allele. The TREX1 D18N mice exhibit systemic inflammation, lymphoid hyperplasia, vasculitis, and kidney disease. The observed lupus-like inflammatory disease is associated with immune activation, production of autoantibodies to dsDNA, and deposition of immune complexes in the kidney. Thus, dysfunctional dsDNA degradation by TREX1 D18N induces disease in mice that recapitulates many characteristics of human lupus. Failure to clear DNA has long been linked to lupus in humans, and these data point to dsDNA as a key substrate for TREX1 and a major antigen source in mice with dysfunctional TREX1 enzyme.
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Pérnio/enzimologia , Pérnio/genética , Dano ao DNA , DNA/metabolismo , Exodesoxirribonucleases/genética , Inflamação/patologia , Lúpus Eritematoso Cutâneo/enzimologia , Lúpus Eritematoso Cutâneo/genética , Fosfoproteínas/genética , Alelos , Animais , Anticorpos/imunologia , Autoimunidade/imunologia , Sequência de Bases , Pérnio/patologia , DNA/química , DNA/genética , Exodesoxirribonucleases/química , Humanos , Lúpus Eritematoso Cutâneo/patologia , Camundongos , Dados de Sequência Molecular , Mutação/genética , Conformação de Ácido Nucleico , Fenótipo , Fosfoproteínas/química , Biossíntese de ProteínasRESUMO
UNLABELLED: CD8(+) T cell responses are critical to the control of replication and reactivation associated with gammaherpesvirus infection. Type I interferons (IFNs) have been shown to have direct and indirect roles in supporting CD8(+) T cell development and function during viral infection; however, the role of type I interferons during latent viral infection has not been examined. Mice deficient in type I IFN signaling (IFNAR1(-/-) mice) have high levels of reactivation during infection with murine gammaherpesvirus 68 (MHV68), a murine gammaherpesvirus model for Epstein-Barr virus. We hypothesized that type I IFNs function to enhance the anti-gammaherpesvirus CD8(+) T cell response. To test this, IFNAR1(-/-) mice were infected with MHV68 and the CD8(+) T cell response was analyzed. In the absence of type I IFN signaling, there was a marked increase in short-lived effector CD8(+) T cells, and MHV68-specific CD8(+) T cells had upregulated expression of PD-1 and reduced tumor necrosis factor alpha (TNF-α), gamma IFN (IFN-γ), and interleukin-2 (IL-2) production. Suppressing MHV68 replication early in infection using the antiviral cidofovir rescued CD8(+) T cell cytokine production and reduced PD-1 expression. However, suppressing high levels of reactivation in IFNAR1(-/-) mice failed to improve CD8(+) T cell cytokine production during latency. T cell-specific abrogation of type I IFN signaling showed that the effects of type I IFNs on the CD8(+) T cell response during MHV68 infection are independent of direct type I IFN signaling on T cells. Our findings support a model in which type I IFNs likely suppress MHV68 replication, thus limiting viral antigen and facilitating an effective gammaherpesvirus-directed CD8(+) T cell response. IMPORTANCE: The murine gammaherpesvirus MHV68 has both genetic and biologic homology to the human gammaherpesvirus Epstein-Barr virus (EBV), which infects over 90% of humans. Latent EBV infection and reactivation are associated with various life-threatening diseases and malignancies. Host suppression of gammaherpesvirus latency and reactivation requires both CD8(+) T cells as well as type I interferon signaling. Type I IFNs have been shown to critically support the antiviral CD8(+) T cell response in other virus models. Here, we identify an indirect role for type I IFN signaling in enhancing gammaherpesvirus-specific CD8(+) T cell cytokine production. Further, this function of type I IFN signaling can be partially rescued by suppressing viral replication during early MHV68 infection. Our data suggest that type I IFN signaling on non-T cells can enhance CD8(+) T cell function during gammaherpesvirus infection, potentially through suppression of MHV68 replication.
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Linfócitos T CD8-Positivos/imunologia , Diferenciação Celular , Infecções por Herpesviridae/imunologia , Interferon Tipo I/imunologia , Rhadinovirus/imunologia , Transdução de Sinais , Infecções Tumorais por Vírus/imunologia , Animais , Linfócitos T CD8-Positivos/fisiologia , Citocinas/metabolismo , Camundongos , Camundongos Knockout , Receptor de Morte Celular Programada 1/metabolismoRESUMO
UNLABELLED: Unlike laboratory animals, humans are infected with multiple pathogens, including the highly prevalent herpesviruses. The purpose of these studies was to determine the effect of gammaherpesvirus latency on T cell number and differentiation during subsequent heterologous viral infections. Mice were first infected with murine gammaherpesvirus 68 (MHV68), a model of Epstein-Barr virus (EBV) infection, and then after latency was established, they were challenged with the Armstrong strain of lymphocytic choriomeningitis virus (LCMV). The initial replication of LCMV was lower in latently infected mice, and the maturation of dendritic cells was abated. Although the number of LCMV-specific effector CD8(+) T cells was not altered, they were skewed to a memory phenotype. In contrast, LCMV-specific effector CD4(+) T cells were increased in latently infected mice compared to those in mice infected solely with LCMV. When the memory phase was reached, latently infected mice had an LCMV-specific memory T cell pool that was increased relative to that found in singly infected mice. Importantly, LCMV-specific memory CD8(+) T cells had decreased CD27 and increased killer cell lectin-like receptor G1 (KLRG1) expression. Upon secondary challenge, LCMV-specific secondary effector CD8(+) T cells expanded and cleared the infection. However, the LCMV-specific secondary memory CD8(+) T cell pool was decreased in latently infected animals, abrogating the boosting effect normally observed following rechallenge. Taken together, these results demonstrate that ongoing gammaherpesvirus latency affects the number and phenotype of primary versus secondary memory CD8(+) T cells during acute infection. IMPORTANCE: CD8(+) T cells are critical for the clearance of intracellular pathogens, including viruses, certain bacteria, and tumors. However, current models for memory CD8(+) T cell differentiation are derived from pathogen-free laboratory mice challenged with a single pathogen or vaccine vector. Unlike laboratory animals, all humans are infected with multiple acute and chronic pathogens, including the highly prevalent herpesviruses Epstein-Barr virus (EBV), cytomegalovirus (CMV), herpes simplex viruses (HSV), and varicella-zoster virus (VZV). The purpose of these studies was to determine the effect of gammaherpesvirus latency on T cell number and differentiation during subsequent heterologous viral infections. We observed that ongoing gammaherpesvirus latency affects the number and phenotype of primary versus secondary memory CD8(+) T cells during acute infection. These results suggest that unlike pathogen-free laboratory mice, infection or immunization of latently infected humans may result in the generation of T cells with limited potential for long-term protection.
Assuntos
Infecções por Arenaviridae/imunologia , Linfócitos T CD8-Positivos/imunologia , Memória Imunológica , Vírus da Coriomeningite Linfocítica/imunologia , Rhadinovirus/imunologia , Rhadinovirus/fisiologia , Latência Viral , Animais , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/fisiologia , Linfócitos T CD8-Positivos/química , Linfócitos T CD8-Positivos/fisiologia , Diferenciação Celular , Proliferação de Células , Células Dendríticas/imunologia , Células Dendríticas/fisiologia , Modelos Animais de Doenças , Lectinas Tipo C , Camundongos Endogâmicos C57BL , Receptores Imunológicos/análise , Membro 7 da Superfamília de Receptores de Fatores de Necrose Tumoral/análiseRESUMO
B cells provide humoral protection against pathogens and promote cellular immunity through diverse nonclassical effector functions. To assess B cell function in promoting T cell homeostasis, mature B cells were either acutely or chronically depleted in mice using CD20 mAb. Acute B cell depletion in either 2- or 4-mo-old mice significantly reduced spleen and lymph node CD4(+) and CD8(+) T cell numbers, including naive, activated, and Foxp3(+)CD25(+)CD4(+) regulatory T cell subsets. The numbers of IFN-γ- and TNF-α-producing T cells were also significantly reduced. Chronic B cell depletion for 6 mo in aged naive mice resulted in a 40-70% reduction in activated CD4(+) and CD8(+) T cell numbers and 20-50% reductions in IFN-γ-producing T cells. Therefore, B cells were necessary for maintaining naive CD4(+) and CD8(+) T cell homeostasis for subsequent optimal T cell expansion in young and old mice. To determine the significance of this finding, a week of B cell depletion in 4-mo-old mice was followed by acute viral infection with lymphocytic choriomeningitis virus Armstrong. Despite their expansion, activated and cytokine-producing CD4(+) and CD8(+) T cell numbers were still significantly reduced 1 wk later. Moreover, viral peptide-specific CD4(+) and CD8(+) T cell numbers and effector cell development were significantly reduced in mice lacking B cells, whereas lymphocytic choriomeningitis virus titers were dramatically increased. Thus, T cell function is maintained in B cell-depleted mice, but B cells are required for optimal CD4(+) and CD8(+) T cell homeostasis, activation, and effector development in vivo, particularly during responses to acute viral infection.
Assuntos
Linfócitos B/imunologia , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/imunologia , Homeostase/imunologia , Coriomeningite Linfocítica/imunologia , Vírus da Coriomeningite Linfocítica/imunologia , Doença Aguda , Animais , Linfócitos B/metabolismo , Linfócitos T CD4-Positivos/metabolismo , Linfócitos T CD8-Positivos/metabolismo , Proliferação de Células , Citometria de Fluxo , Fatores de Transcrição Forkhead/imunologia , Fatores de Transcrição Forkhead/metabolismo , Interações Hospedeiro-Patógeno/imunologia , Interferon gama/imunologia , Interferon gama/metabolismo , Subunidade alfa de Receptor de Interleucina-2/imunologia , Subunidade alfa de Receptor de Interleucina-2/metabolismo , Linfonodos/imunologia , Linfonodos/metabolismo , Contagem de Linfócitos , Depleção Linfocítica , Coriomeningite Linfocítica/virologia , Vírus da Coriomeningite Linfocítica/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Baço/imunologia , Baço/metabolismo , Linfócitos T Reguladores/imunologia , Linfócitos T Reguladores/metabolismo , Fator de Necrose Tumoral alfa/imunologia , Fator de Necrose Tumoral alfa/metabolismoRESUMO
Recognition of peptide Major Histocompatibility Complexes (MHC) by the T cell receptor causes rapid production of reactive oxygen intermediates (ROI) in naïve CD8(+) T cells. Because ROI such as H2O2 are membrane permeable, mechanisms must exist to prevent overoxidation of surface proteins. In this study we used fluorescently labeled conjugates of maleimide to measure the level of cell surface free thiols (CSFT) during the development, activation and differentiation of CD8(+) T cells. We found that during development CSFT were higher on CD8 SP compared to CD4 SP or CD4CD8 DP T cells. After activation CSFT became elevated prior to division but once proliferation started levels continued to rise. During acute viral infection CSFT levels were elevated on antigen-specific effector cells compared to memory cells. Additionally, the CSFT level was always higher on antigen-specific CD8(+) T cells in lymphoid compared to nonlymphoid organs. During chronic viral infection, CSFT levels were elevated for extended periods on antigen-specific effector CD8(+) T cells. Finally, CSFT levels on effector CD8(+) T cells, regardless of infection, identified cells undergoing TCR stimulation. Taken together these data suggest that CD8(+) T cells upregulate CSFT following receptor ligation and ROI production during infection to prevent overoxidation of surface proteins.
Assuntos
Linfócitos T CD8-Positivos/metabolismo , Membrana Celular/metabolismo , Receptores de Antígenos de Linfócitos T/metabolismo , Compostos de Sulfidrila/metabolismo , Animais , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/virologia , Feminino , Memória Imunológica , Imunofenotipagem , Ativação Linfocitária/imunologia , Coriomeningite Linfocítica/imunologia , Vírus da Coriomeningite Linfocítica/imunologia , Tecido Linfoide/imunologia , Tecido Linfoide/metabolismo , Tecido Linfoide/virologia , Camundongos , Camundongos Transgênicos , FenótipoRESUMO
Generation of reactive oxygen intermediates (ROI) following antigen receptor ligation is critical to promote cellular responses. However, the effect of antioxidant treatment on humoral immunity during a viral infection was unknown. Mice were infected with lymphocytic choriomeningitis virus (LCMV) and treated with Mn(III)tetrakis(4-benzoic acid)porphyrin chloride (MnTBAP), a superoxide dismutase mimetic, from days 0 to 8 postinfection. On day 8, at the peak of the splenic response in vehicle-treated mice, virus-specific IgM and IgG antibody-secreting cells (ASC) were decreased 22- and 457-fold in MnTBAP-treated animals. By day 38, LCMV-specific IgG ASC were decreased 5-fold in the bone marrow of drug-treated mice, and virus-specific antibodies were of lower affinity. Interestingly, antioxidant treatment had no effect on the number of LCMV-specific IgG memory B cells. In addition to decreases in ASC, MnTBAP treatment decreased the number of functional virus-specific CD4(+) T cells. The decreased numbers of ASC observed on day 8 in drug-treated mice were due to a combination of Bim-mediated cell death and decreased proliferation. Together, these data demonstrate that ROI regulate antiviral ASC expansion and have important implications for understanding the effects of antioxidants on humoral immunity during infection and immunization.
Assuntos
Anticorpos Antivirais/sangue , Antioxidantes/farmacologia , Infecções por Arenaviridae/tratamento farmacológico , Infecções por Arenaviridae/imunologia , Imunidade Humoral/efeitos dos fármacos , Vírus da Coriomeningite Linfocítica/imunologia , Metaloporfirinas/farmacologia , Doença Aguda , Animais , Anticorpos Antivirais/imunologia , Células Produtoras de Anticorpos/efeitos dos fármacos , Células Produtoras de Anticorpos/imunologia , Apoptose , Proteínas Reguladoras de Apoptose/genética , Proteínas Reguladoras de Apoptose/metabolismo , Infecções por Arenaviridae/metabolismo , Proteína 11 Semelhante a Bcl-2 , Células da Medula Óssea/efeitos dos fármacos , Linfócitos T CD4-Positivos/efeitos dos fármacos , Linfócitos T CD8-Positivos/imunologia , Células Cultivadas , Citoproteção , Feminino , Sequestradores de Radicais Livres/farmacologia , Memória Imunológica , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/metabolismo , Espécies Reativas de Oxigênio/metabolismoRESUMO
T cell development and activation are usually accompanied by expansion and production of numerous proteins that require active translation. The eukaryotic translation initiation factor 4E (eIF4E) binds to the 5' cap structure of mRNA and is critical for cap-dependent translational initiation. It has been hypothesized that MAPK-interacting kinase 1 and 2 (Mnk1/2) promote cap-dependent translation by phosphorylating eIF4E at serine 209 (S209). Pharmacologic studies using inhibitors have suggested that Mnk1/2 have important roles in T cells. However, genetic evidence supporting such conclusions is lacking. Moreover, the signaling pathways that regulate Mnk1/2 in T cells remain unclear. We demonstrate that TCR engagement activates Mnk1/2 in primary T cells. Such activation is dependent on Ras-Erk1/2 signaling and is inhibited by diacylglycerol kinases α and ζ. Mnk1/2 double deficiency in mice abolishes TCR-induced eIF4E S209 phosphorylation, indicating their absolute requirement for eIF4E S209 phosphorylation. However, Mnk1/2 double deficiency does not affect the development of conventional αß T cells, regulatory T cells, or NKT cells. Furthermore, T cell activation, in vivo primary and memory CD8 T cell responses to microbial infection, and NKT cell cytokine production were not obviously altered by Mnk1/2 deficiency. Although Mnk1/2 deficiency causes decreased IL-17 and IFN-γ production by CD4 T cells following immunization of mice with myelin oligodendrocyte glycoprotein peptide in complete Freund's adjuvant, correlating with milder experimental autoimmune encephalomyelitis scores, it does not affect Th cell differentiation in vitro. Together, these data suggest that Mnk1/2 has a minimal role in T cell development and activation but may regulate non-T cell lineages to control Th1 and Th17 differentiation in vivo.