RESUMEN
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.
Asunto(s)
Autoinmunidad , Centro Germinal , Animales , Diferenciación Celular , ADN , Modelos Animales de Enfermedad , Exodesoxirribonucleasas , Ratones , Ratones Noqueados , Fosfoproteínas , Proteínas Proto-Oncogénicas c-bcl-6/genética , Proteínas Proto-Oncogénicas c-bcl-6/metabolismo , Células T Auxiliares Foliculares , Linfocitos T Colaboradores-InductoresRESUMEN
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.
Asunto(s)
Exodesoxirribonucleasas/genética , Lupus Eritematoso Sistémico/genética , Fosfoproteínas/genética , Linfocitos T/inmunología , Animales , Autoantígenos/inmunología , Autoinmunidad , Células Cultivadas , ADN/inmunología , Modelos Animales de Enfermedad , Humanos , Inmunidad Innata , Interferón-alfa/metabolismo , Proteínas de la Membrana/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Nucleotidiltransferasas/metabolismoRESUMEN
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.
Asunto(s)
Subfamilia B de Transportador de Casetes de Unión a ATP/inmunología , Linfocitos T CD8-positivos/metabolismo , Enfermedad Injerto contra Huésped/prevención & control , Depleción Linfocítica/métodos , Subfamilia B de Transportador de Casetes de Unión a ATP/efectos de los fármacos , Subfamilia B de Transportador de Casetes de Unión a ATP/metabolismo , Animales , Linfocitos T CD8-positivos/efectos de los fármacos , Linfocitos T CD8-positivos/inmunología , Metabolismo Energético , Enfermedad Injerto contra Huésped/inmunología , Humanos , Coriomeningitis Linfocítica/inmunología , Virus de la Coriomeningitis Linfocítica/fisiología , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C3H , Ratones Endogámicos C57BL , Fármacos Fotosensibilizantes/aislamiento & purificación , Fármacos Fotosensibilizantes/farmacología , Trasplante HomólogoRESUMEN
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.
Asunto(s)
Eritema Pernio/enzimología , Eritema Pernio/genética , Daño del ADN , ADN/metabolismo , Exodesoxirribonucleasas/genética , Inflamación/patología , Lupus Eritematoso Cutáneo/enzimología , Lupus Eritematoso Cutáneo/genética , Fosfoproteínas/genética , Alelos , Animales , Anticuerpos/inmunología , Autoinmunidad/inmunología , Secuencia de Bases , Eritema Pernio/patología , ADN/química , ADN/genética , Exodesoxirribonucleasas/química , Humanos , Lupus Eritematoso Cutáneo/patología , Ratones , Datos de Secuencia Molecular , Mutación/genética , Conformación de Ácido Nucleico , Fenotipo , Fosfoproteínas/química , Biosíntesis de ProteínasRESUMEN
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.
Asunto(s)
Linfocitos T CD4-Positivos/efectos de los fármacos , Linfocitos T CD8-positivos/efectos de los fármacos , Compuestos Orgánicos de Oro/farmacología , Acridinas/química , Animales , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD4-Positivos/metabolismo , Linfocitos T CD8-positivos/inmunología , Linfocitos T CD8-positivos/metabolismo , Calcio/metabolismo , Células Cultivadas , Citocinas/inmunología , Citocinas/metabolismo , Relación Dosis-Respuesta a Droga , Femenino , Citometría de Flujo , Humanos , Activación de Linfocitos/efectos de los fármacos , Activación de Linfocitos/inmunología , Coriomeningitis Linfocítica/tratamiento farmacológico , Coriomeningitis Linfocítica/inmunología , Coriomeningitis Linfocítica/virología , Virus de la Coriomeningitis Linfocítica/efectos de los fármacos , Virus de la Coriomeningitis Linfocítica/inmunología , Virus de la Coriomeningitis Linfocítica/fisiología , Ratones Endogámicos C57BL , Compuestos Orgánicos de Oro/química , Oxidación-Reducción/efectos de los fármacos , Platino (Metal)/química , Urea/análogos & derivados , Urea/química , Carga Viral/efectos de los fármacos , Carga Viral/inmunologíaRESUMEN
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.
Asunto(s)
Compuestos de Organoselenio/farmacología , Fotoféresis , Fármacos Fotosensibilizantes/farmacología , Rodaminas/farmacología , Linfocitos T/efectos de los fármacos , Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/antagonistas & inhibidores , Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/metabolismo , Línea Celular Tumoral , Humanos , Luz , Linfoma , Mitocondrias/metabolismo , Compuestos de Organoselenio/síntesis química , Fármacos Fotosensibilizantes/síntesis química , Rodaminas/síntesis química , Linfocitos T/metabolismo , Verapamilo/farmacologíaRESUMEN
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.
Asunto(s)
Linfocitos B/inmunología , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD8-positivos/inmunología , Homeostasis/inmunología , Coriomeningitis Linfocítica/inmunología , Virus de la Coriomeningitis Linfocítica/inmunología , Enfermedad Aguda , Animales , Linfocitos B/metabolismo , Linfocitos T CD4-Positivos/metabolismo , Linfocitos T CD8-positivos/metabolismo , Proliferación Celular , Citometría de Flujo , Factores de Transcripción Forkhead/inmunología , Factores de Transcripción Forkhead/metabolismo , Interacciones Huésped-Patógeno/inmunología , Interferón gamma/inmunología , Interferón gamma/metabolismo , Subunidad alfa del Receptor de Interleucina-2/inmunología , Subunidad alfa del Receptor de Interleucina-2/metabolismo , Ganglios Linfáticos/inmunología , Ganglios Linfáticos/metabolismo , Recuento de Linfocitos , Depleción Linfocítica , Coriomeningitis Linfocítica/virología , Virus de la Coriomeningitis Linfocítica/fisiología , Ratones , Ratones Endogámicos C57BL , Bazo/inmunología , Bazo/metabolismo , Linfocitos T Reguladores/inmunología , Linfocitos T Reguladores/metabolismo , Factor de Necrosis Tumoral alfa/inmunología , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
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.
Asunto(s)
Linfocitos T CD8-positivos/inmunología , Diferenciación Celular , Infecciones por Herpesviridae/inmunología , Interferón Tipo I/inmunología , Rhadinovirus/inmunología , Transducción de Señal , Infecciones Tumorales por Virus/inmunología , Animales , Linfocitos T CD8-positivos/fisiología , Citocinas/metabolismo , Ratones , Ratones Noqueados , Receptor de Muerte Celular Programada 1/metabolismoRESUMEN
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.
Asunto(s)
Infecciones por Arenaviridae/inmunología , Linfocitos T CD8-positivos/inmunología , Memoria Inmunológica , Virus de la Coriomeningitis Linfocítica/inmunología , Rhadinovirus/inmunología , Rhadinovirus/fisiología , Latencia del Virus , Animales , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD4-Positivos/fisiología , Linfocitos T CD8-positivos/química , Linfocitos T CD8-positivos/fisiología , Diferenciación Celular , Proliferación Celular , Células Dendríticas/inmunología , Células Dendríticas/fisiología , Modelos Animales de Enfermedad , Lectinas Tipo C , Ratones Endogámicos C57BL , Receptores Inmunológicos/análisis , Miembro 7 de la Superfamilia de Receptores de Factores de Necrosis Tumoral/análisisRESUMEN
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.
Asunto(s)
Encefalomielitis Autoinmune Experimental/enzimología , Activación de Linfocitos/fisiología , Linfopoyesis/fisiología , Proteínas Serina-Treonina Quinasas/fisiología , Subgrupos de Linfocitos T/inmunología , Traslado Adoptivo , Compuestos de Anilina/farmacología , Animales , Células Cultivadas/efectos de los fármacos , Células Cultivadas/inmunología , Encefalomielitis Autoinmune Experimental/inmunología , Factor 4E Eucariótico de Iniciación/metabolismo , Femenino , Interferón gamma/biosíntesis , Interferón gamma/genética , Interleucina-17/biosíntesis , Interleucina-17/genética , Listeriosis/inmunología , Activación de Linfocitos/efectos de los fármacos , Coriomeningitis Linfocítica/inmunología , Linfopoyesis/efectos de los fármacos , Sistema de Señalización de MAP Quinasas , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Glicoproteína Mielina-Oligodendrócito/inmunología , Células T Asesinas Naturales/inmunología , Ovalbúmina/inmunología , Fragmentos de Péptidos/inmunología , Fosforilación/efectos de los fármacos , Procesamiento Proteico-Postraduccional/efectos de los fármacos , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Proteínas Serina-Treonina Quinasas/deficiencia , Proteínas Serina-Treonina Quinasas/genética , Purinas/farmacología , Caperuzas de ARNRESUMEN
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.
Asunto(s)
Anticuerpos Antivirales/sangre , Antioxidantes/farmacología , Infecciones por Arenaviridae/tratamiento farmacológico , Infecciones por Arenaviridae/inmunología , Inmunidad Humoral/efectos de los fármacos , Virus de la Coriomeningitis Linfocítica/inmunología , Metaloporfirinas/farmacología , Enfermedad Aguda , Animales , Anticuerpos Antivirales/inmunología , Células Productoras de Anticuerpos/efectos de los fármacos , Células Productoras de Anticuerpos/inmunología , Apoptosis , Proteínas Reguladoras de la Apoptosis/genética , Proteínas Reguladoras de la Apoptosis/metabolismo , Infecciones por Arenaviridae/metabolismo , Proteína 11 Similar a Bcl2 , Células de la Médula Ósea/efectos de los fármacos , Linfocitos T CD4-Positivos/efectos de los fármacos , Linfocitos T CD8-positivos/inmunología , Células Cultivadas , Citoprotección , Femenino , Depuradores de Radicales Libres/farmacología , Memoria Inmunológica , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas/metabolismo , Especies Reactivas de Oxígeno/metabolismoRESUMEN
By the peak of the CD8(+) T cell response, the effector cell pool consists of a heterogeneous population of cells that includes both those with an increased propensity to become long-lived memory cells (memory precursor effector cells; MPEC) and those that are terminally differentiated cells (short-lived effector cells; SLEC). Numerous studies have established the critical role that functional avidity plays in determining the in vivo efficacy of CD8(+) effector cells. Currently, how functional avidity differs in MPEC versus SLEC and the evolution of this property within these two populations during the expansion and contraction of the response are unknown. The data presented in this study show that at the peak of the effector response generated after poxvirus infection, SLEC were of higher functional avidity than their MPEC counterpart. Over time, however, SLEC exhibited a decrease in peptide sensitivity. This is in contrast to MPEC, which showed a modest increase in peptide sensitivity as the response reached equilibrium. The decrease in functional avidity in SLEC was independent of CD8 modulation or the amount of Ag receptor expressed by the T cell. Instead, the loss in sensitivity was correlated with decreased expression and activation of ZAP70 and Lck, critical components of TCR membrane proximal signaling. These results highlight the potential contribution of avidity in the differentiation and evolution of the T cell effector response after viral infection.
Asunto(s)
Antígenos CD8/biosíntesis , Linfocitos T CD8-positivos/inmunología , Linfocitos T CD8-positivos/virología , Diferenciación Celular/inmunología , Senescencia Celular/inmunología , Memoria Inmunológica , Virus de la Coriomeningitis Linfocítica/inmunología , Virus Vaccinia/inmunología , Animales , Células Presentadoras de Antígenos/inmunología , Células Presentadoras de Antígenos/metabolismo , Células Presentadoras de Antígenos/virología , Antígenos CD8/fisiología , Linfocitos T CD8-positivos/metabolismo , Adhesión Celular/inmunología , Ratones , Ratones Endogámicos C57BL , Receptores de Antígenos de Linfocitos T/metabolismo , Transducción de Señal/inmunología , Factores de TiempoRESUMEN
The manipulation of signals downstream of the TCR can have profound consequences for T cell development, function, and homeostasis. Diacylglycerol (DAG) produced after TCR stimulation functions as a secondary messenger and mediates the signaling to Ras-MEK-Erk and NF-κB pathways in T cells. DAG kinases (DGKs) convert DAG into phosphatidic acid, resulting in termination of DAG signaling. In this study, we demonstrate that DAG metabolism by DGKs can serve a crucial function in viral clearance upon lymphocytic choriomeningitis virus infection. Ag-specific CD8(+) T cells from DGKα(-/-) and DGKζ(-/-) mice show enhanced expansion and increased cytokine production after lymphocytic choriomeningitis virus infection, yet DGK-deficient memory CD8(+) T cells exhibit impaired expansion after rechallenge. Thus, DGK activity plays opposing roles in the expansion of CD8(+) T cells during the primary and memory phases of the immune response, whereas consistently inhibiting antiviral cytokine production.
Asunto(s)
Linfocitos T CD8-positivos/enzimología , Linfocitos T CD8-positivos/inmunología , Diacilglicerol Quinasa/fisiología , Memoria Inmunológica , Coriomeningitis Linfocítica/inmunología , Coriomeningitis Linfocítica/patología , Animales , Antivirales/antagonistas & inhibidores , Antivirales/metabolismo , Linfocitos T CD8-positivos/virología , Diferenciación Celular/genética , Diferenciación Celular/inmunología , Línea Celular , Células Clonales , Cricetinae , Citocinas/antagonistas & inhibidores , Citocinas/biosíntesis , Diacilglicerol Quinasa/deficiencia , Diacilglicerol Quinasa/genética , Modelos Animales de Enfermedad , Regulación hacia Abajo/genética , Regulación hacia Abajo/inmunología , Epítopos de Linfocito T/inmunología , Memoria Inmunológica/genética , Coriomeningitis Linfocítica/genética , Virus de la Coriomeningitis Linfocítica/inmunología , Ratones , Ratones Noqueados , Ratones TransgénicosRESUMEN
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.
RESUMEN
B-cell receptor (BCR) ligation generates reactive oxygen intermediates (ROIs) that play a role in cellular responses. Although ROIs can oxidize all macromolecules, it was unclear which modifications control B-cell responses. In this study, we demonstrate the importance of the first oxidation product of cysteine, sulfenic acid, and its reversible formation in B-cell activation. Upon BCR crosslinking, B cells increase ROI levels with maximal production occurring within 15 min. Increased ROIs preceded elevated cysteine sulfenic acid, which localized to the cytoplasm and nucleus. Analysis of individual proteins revealed that the protein tyrosine phosphatases (PTPs) SHP-1, SHP-2, and PTEN, as well as actin, were modified to sulfenic acid following BCR ligation. Additionally, we used 5,5-dimethyl-1,3-cyclohexanedione (dimedone), a compound that covalently reacts with sulfenic acid to prevent its further oxidation or reduction, to determine the role of reversible cysteine sulfenic acid formation in regulating B-cell responses. Dimedone incubation resulted in a concentration-dependent block in anti-IgM-induced cell division, accompanied by a failure to induce capacitative calcium entry (CCE), and maintain tyrosine phosphorylation. These studies illustrate that reversible cysteine sulfenic acid formation is a mechanism by which B cells modulate pathways critical for activation and proliferation.
Asunto(s)
Linfocitos B/inmunología , Cisteína/análogos & derivados , Cisteína/metabolismo , Activación de Linfocitos , Especies Reactivas de Oxígeno/inmunología , Actinas/metabolismo , Animales , Linfocitos B/metabolismo , Transporte Biológico/efectos de los fármacos , División Celular/efectos de los fármacos , Proliferación Celular , Ciclohexanonas/farmacología , Ratones , Fosfohidrolasa PTEN/metabolismo , Fosforilación/efectos de los fármacos , Proteína Tirosina Fosfatasa no Receptora Tipo 11/metabolismo , Proteína Tirosina Fosfatasa no Receptora Tipo 6/metabolismo , Receptores de Antígenos de Linfocitos BRESUMEN
Reactive oxygen intermediates (ROI) generated in response to receptor stimulation play an important role in cellular responses. However, the effect of increased H(2)O(2) on an antigen-specific CD8(+) T cell response was unknown. Following T cell receptor (TCR) stimulation, the expression and oxidation of peroxiredoxin II (PrdxII), a critical antioxidant enzyme, increased in CD8(+) T cells. Deletion of PrdxII increased ROI, S phase entry, division, and death during in vitro division. During primary acute viral and bacterial infection, the number of effector CD8(+) T cells in PrdxII-deficient mice was increased, while the number of memory cells were similar to those of the wild-type cells. Adoptive transfer of P14 TCR transgenic cells demonstrated that the increased expansion of effector cells was T cell autonomous. After rechallenge, effector CD8(+) T cells in mutant animals were more skewed to memory phenotype than cells from wild-type mice, resulting in a larger secondary memory CD8(+) T cell pool. During chronic viral infection, increased antigen-specific CD8(+) T cells accumulated in the spleens of PrdxII mutant mice, causing mortality. These results demonstrate that PrdxII controls effector CD8(+) T cell expansion, secondary memory generation, and immunopathology.
Asunto(s)
Linfocitos T CD8-positivos/inmunología , Memoria Inmunológica , Peroxirredoxinas/fisiología , Animales , Western Blotting , Proliferación Celular , Ratones , Ratones Transgénicos , Peroxirredoxinas/genética , Reacción en Cadena en Tiempo Real de la PolimerasaRESUMEN
Flagellin is a highly effective adjuvant for CD4(+) T cell and humoral immune responses. However, there is conflicting data in the literature regarding the ability of flagellin to promote a CD8(+) T cell response. In this article, we report that immunization of wild-type, TLR5(-/-), and MyD88(-/-) adoptive transfer recipient mice revealed the ability of flagellin fusion proteins to promote OVA-specific CD8(+) T cell proliferation independent of TLR5 or MyD88 expression by the recipient animal. Wild-type and TLR5(-/-) APCs were able to stimulate high levels of OVA-specific CD8(+) T cell proliferation in vitro in response to a flagellin fusion protein containing full-length OVA or the SIINFEKL epitope and 10 flanking amino acids (OVAe), but not to OVA and flagellin added as separate proteins. This effect was independent of the conserved regions of flagellin and occurred in response to OVAe alone. Comparison of IFN-γ production by CD8(+) effector cells revealed higher levels of SIINFEKL peptide-MHC I complexes on the surface of APCs that had been pulsed with OVAe-flagellin fusion proteins than on cells pulsed with OVA. Inhibition of the proteasome significantly reduced Ag-specific proliferation in response to OVAe fusion proteins. In summary, our data are consistent with the conclusion that flagellin-OVA fusion proteins induce an epitope-specific CD8(+) T cell response by facilitating Ag processing and not through stimulatory signaling via TLR5 and MyD88. Our findings raise the possibility that flagellin might be an efficient Ag carrier for Ags that are poorly processed in their native state.
Asunto(s)
Presentación de Antígeno/inmunología , Linfocitos T CD8-positivos/inmunología , Flagelina/inmunología , Proteínas Recombinantes de Fusión/inmunología , Traslado Adoptivo , Secuencia de Aminoácidos , Animales , Células Presentadoras de Antígenos/inmunología , Células Presentadoras de Antígenos/metabolismo , Linfocitos T CD8-positivos/metabolismo , Linfocitos T CD8-positivos/trasplante , Proliferación Celular , Femenino , Flagelina/genética , Flagelina/metabolismo , Citometría de Flujo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Factor 88 de Diferenciación Mieloide/genética , Factor 88 de Diferenciación Mieloide/inmunología , Ovalbúmina/genética , Ovalbúmina/inmunología , Ovalbúmina/metabolismo , Fragmentos de Péptidos/inmunología , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Salmonella enteritidis/genética , Salmonella enteritidis/inmunología , Salmonella enteritidis/metabolismo , Receptor Toll-Like 5/genética , Receptor Toll-Like 5/inmunologíaRESUMEN
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.
RESUMEN
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.
Asunto(s)
Enfermedad de Alzheimer , Agotamiento de Células T , Humanos , Linfocitos T CD8-positivos , Leucocitos Mononucleares , Tomografía Computarizada por Rayos X , Proteínas AmiloidogénicasRESUMEN
The immune system is complex, with multiple layers of regulation that serve to prevent the production of self-antigens. One layer of regulation involves regulatory T cells (Tregs) that play an essential role in maintaining peripheral self-tolerance. Patients with autoimmune diseases such as systemic lupus erythematosus and rheumatoid arthritis have decreased levels of HDL, suggesting that apoA-I concentrations may be important in preventing autoimmunity and the loss of self-tolerance. In published studies, hypercholesterolemic mice lacking HDL apoA-I or LDLr(-/-), apoA-I(-/-) (DKO), exhibit characteristics of autoimmunity in response to an atherogenic diet. This phenotype is characterized by enlarged cholesterol-enriched lymph nodes (LNs), as well as increased T cell activation, proliferation, and the production of autoantibodies in plasma. In this study, we investigated whether treatment of mice with lipid-free apoA-I could attenuate the autoimmune phenotype. To do this, DKO mice were first fed an atherogenic diet containing 0.1% cholesterol, 10% fat for 6 weeks, after which treatment with apoA-I was begun. Subcutaneous injections of 500 µg of lipid-free apoA-I was administered every 48 h during the treatment phase. These and control mice were maintained for an additional 6 weeks on the diet. At the end of the 12-week study, DKO mice showed decreased numbers of LN immune cells, whereas Tregs were proportionately increased. Accompanying this increase in Tregs was a decrease in the percentage of effector/effector memory T cells. Furthermore, lipid accumulation in LN and skin was reduced. These results suggest that treatment with apoA-I reduces inflammation in DKO mice by augmenting the effectiveness of the LN Treg response.