RESUMEN
Allogeneic hematopoietic cell transplantation (allo-HCT) offers a curative option for patients with certain non-malignant hematological diseases. High-dose post-transplant cyclophosphamide (PT-Cy) (200 mg/kg) and sirolimus (3 mg/kg), (HiC) synergistically induce stable mixed chimerism. Further, sirolimus and cytotoxic T lymphocyte-associated antigen-4 immunoglobulin (CTLA4-Ig), also known as Abatacept (Aba), promote immune tolerance and allograft survival. Here, in a major histocompatibility complex (MHC)-mismatched allo-HCT murine model, we combined Aba and/or T-cell depleting anti-Thy1.2 (Thy) with a lower dose of PT-Cy (50 mg/kg) and Sirolimus (3 mg/kg), (LoC). While mice in the LoC group showed graft rejection, the addition of Thy to LoC induced similar donor chimerism levels when compared to the HiC group. However, the addition of Aba to LoC led to graft acceptance only in younger mice. When Thy was added to the LoC+Aba setting, graft acceptance was restored in both age groups. Engrafted groups displayed significantly reduced frequencies of recipient-specific interferon-γ-producing T cells as well as an increased frequency in regulatory T cells (Tregs) except in the LoC+Aba group. Splenocytes from engrafted mice showed no proliferation upon restimulation with Balb/c stimulators. Collectively, in combination with Aba or Thy, LoC may be considered to reduce graft rejection in patients who undergo allo-HCT.
Asunto(s)
Abatacept , Ciclofosfamida , Depleción Linfocítica , Sirolimus , Animales , Ciclofosfamida/farmacología , Sirolimus/farmacología , Ratones , Abatacept/farmacología , Abatacept/uso terapéutico , Trasplante de Células Madre Hematopoyéticas/métodos , Ratones Endogámicos BALB C , Quimera por Trasplante , Trasplante Homólogo/métodos , AloinjertosRESUMEN
Foxp3 is regarded as the major transcription factor for T regulatory (Treg) cells and expression of Foxp3 is used to identify and quantitate Treg cells in mouse models. However, several studies have demonstrated that human CD4+ T conventional (Tconv) cells activated in vitro by T cell receptor (TCR) stimulation can express Foxp3. This observation has raised doubt as to the suitability of Foxp3 as a Treg marker in man. Helios, a member of the Ikaros gene family, has been shown to be expressed by 80-90% of human Foxp3+ Treg cells and can potentially serve as a marker of human Treg. Here, we confirm that Foxp3 expression is readily upregulated by Tconv upon TCR stimulation in vitro, while Helios expression is not altered. More importantly, we show that Foxp3 expression is not elevated by stimulation of hTconv in a humanized mouse model of graft versus host disease (GVHD) and in patients with a wide variety of acute and chronic inflammatory diseases including sickle cell disease, acute and chronic GVHD, systemic lupus erythematosus, as well as critical COVID-19. In all patients studied, an excellent correlation was observed between the percentage of CD4+ T cells expressing Foxp3 and the percentage expressing Helios. Taken together, these studies demonstrate that Foxp3 is not induced upon Tconv cell activation in vivo and that Foxp3 expression alone can be used to quantitate Treg cells in humans. Nevertheless, the combined use of Foxp3 and Helios expression provides a more reliable approach for the characterization of Treg in humans.
Asunto(s)
COVID-19 , Enfermedad Injerto contra Huésped , Ratones , Animales , Humanos , Linfocitos T Reguladores/metabolismo , COVID-19/metabolismo , Factores de Transcripción/metabolismo , Receptores de Antígenos de Linfocitos T/metabolismo , Enfermedad Injerto contra Huésped/metabolismo , Factores de Transcripción Forkhead/genética , Factores de Transcripción Forkhead/metabolismoRESUMEN
Sickle cell disease (SCD) is an inherited red blood cell disorder that leads to significant morbidity and early mortality. The most widely available curative approach remains allogeneic hematopoietic stem cell transplantation (HSCT). HLA-haploidentical (haplo) HSCT expands the donor pool considerably and is a practical alternative for these patients, but traditionally with an increased risk of allograft rejection. Biomarkers in patient plasma could potentially help predict HSCT outcome and allow treatment at an early stage to reverse or prevent graft rejection. Reliable, noninvasive methods to predict engraftment or rejection early after HSCT are needed. We sought to detect variations in the plasma proteomes of patients who engrafted compared with those who rejected their grafts. We used a mass spectrometry-based proteomics approach to identify candidate biomarkers associated with engraftment and rejection by comparing plasma samples obtained from 9 engrafted patients and 10 patients who experienced graft rejection. A total of 1378 proteins were identified, 45 of which were differentially expressed in the engrafted group compared with the rejected group. Based on bioinformatics analysis results, information from the literature, and immunoassay availability, 7 proteins-thrombospondin-1 (Tsp-1), platelet factor 4 (Pf-4), talin-1, moesin, cell division control protein 42 homolog (CDC42), galectin-1 (Gal-1), and CD9-were selected for further analysis. We compared these protein concentrations among 35 plasma samples (engrafted, n = 9; rejected, n = 10; healthy volunteers, n = 8; nontransplanted SCD, n = 8). ELISA analysis confirmed the significant up-regulation of Tsp-1, Pf-4, and Gal-1 in plasma samples from engrafted patients compared with rejected patients, healthy African American volunteers, and the nontransplanted SCD group (P < .01). By receiver operating characteristic analysis, these 3 proteins distinguished engrafted patients from the other groups (area under the curve, >0.8; P < .05). We then evaluated the concentration of these 3 proteins in samples collected pre-HSCT and at days +30, +60, +100, and +180 post-HSCT. The results demonstrate that Tsp-1 and Pf-4 stratified engrafted patients as early as day 60 post-HSCT (P < .01), and that Gal-1 was significantly higher in engrafted patients as early as day 30 post-HSCT (P < .01). We also divided the rejected group into those who experienced primary (n = 5) and secondary graft rejection (n = 5) and found that engrafted patients had significantly higher Tsp-1 levels compared with patients who developed primary graft rejection at days +60 and +100 (P < .05), as well as higher Pf-4 levels compared with patients who developed primary graft rejection at post-transplantation (PT) day 100. Furthermore, Tsp-1 levels were significantly higher at PT days 60 and 100 and Pf-4 levels were higher at PT day 100 in engrafted patients compared with those who experienced secondary graft rejection. Increased concentrations of plasma Gal-1, Tsp-1, and Pf-4 could reflect increased T regulatory cells, IL-10, and TGF-ß, which are essential players in the initiation of immunologic tolerance. These biomarkers may provide opportunities for preemptive intervention to minimize the incidence of graft rejection.
Asunto(s)
Anemia de Células Falciformes , Trasplante de Células Madre Hematopoyéticas , Anemia de Células Falciformes/terapia , Factores de Transcripción Básicos con Cremalleras de Leucinas y Motivos Hélice-Asa-Hélice , Biomarcadores , Galectina 1 , Trasplante de Células Madre Hematopoyéticas/efectos adversos , Humanos , Factores Inmunológicos , Factor Plaquetario 4 , Trombospondina 1 , Acondicionamiento Pretrasplante/métodosRESUMEN
Haploidentical hematopoietic stem cell transplantation (haplo-HSCT) is a widely available curative option for patients with sickle cell disease (SCD). Our original non-myeloablative haplo-HSCT trial employing post-transplant (PT) cyclophosphamide had a low incidence of GVHD but had high rejection rates. Here, we aimed to evaluate immune reconstitution following haplo-HSCT and identify cytokines and cells associated with graft rejection/engraftment. 50 cytokines and 10 immune cell subsets were screened using multiplex-ELISA and flow cytometry, respectively, at baseline and PT-Days 30, 60, 100, and 180. We observed the most significant differences in cytokine levels between the engrafted and rejected groups at PT-Day 60, corresponding with clinical findings of secondary graft rejection. Of the 44 cytokines evaluated, plasma concentrations of 19 cytokines were different between the two groups at PT-Day 60. Factor analysis suggested two independent factors. The first factor (IL-17A, IL-10, IL-7, G-CSF, IL-2, MIP-1a, VEGF, and TGFb1 contributed significantly) was strongly associated with engraftment with OR = 2.7 (95%CI of 1.4 to 5.4), whereas the second factor (GROa and IL-18 contributed significantly) was not significantly associated with engraftment. Sufficient donor myeloid chimerism (DMC) is critical for the success of HSCT; here, we evaluated immune cells among high (H) DMC (DMC≥20%) and low (L) DMC (DMC<20%) groups along with engrafted and rejected groups. We found that early myeloid-derived suppressor cell (eMDSC) frequencies were elevated in engrafted patients and patients with HDMC at PT-Day 30 (P< 0.04 & P< 0.003, respectively). 9 of 20 patients were evaluated for the source of eMDSCs. The HDMC group had high mixed chimeric eMDSCs as compared to the LDMC group (P< 0.00001). We found a positive correlation between the frequencies of eMDSCs and Tregs at PT-Day 100 (r=0.72, P <0.0007); eMDSCs at BSL and Tregs at PT-Day 100 (r=0.63, P <0.004). Of 10 immune regulatory cells and 50 cytokines, we observed mixed chimeric eMDSCs and IL-17A, IL-10, IL-7, G-CSF, IL-2, MIP-1a, VEGF, TGFb1 as potential hits which could serve as prognostic markers in predicting allograft outcome towards engraftment following haploidentical HSCT employing post-transplant cyclophosphamide. The current findings need to be replicated and further explored in a larger cohort.
Asunto(s)
Anemia de Células Falciformes/terapia , Trasplante de Células Madre Hematopoyéticas , Reconstitución Inmune/inmunología , Quimera por Trasplante , Adulto , Anemia de Células Falciformes/inmunología , Quimerismo , Ciclofosfamida/uso terapéutico , Citocinas/inmunología , Rechazo de Injerto/inmunología , Supervivencia de Injerto/inmunología , Humanos , Inmunosupresores/uso terapéutico , Células Supresoras de Origen Mieloide , Pronóstico , Acondicionamiento Pretrasplante , Trasplante Haploidéntico , Resultado del TratamientoRESUMEN
Coxsackievirus B3 (CVB3), is commonly implicated in myocarditis, which can lead to dilated cardiomyopathy, in addition to causing acute pancreatitis and meningitis. Yet, no vaccines are currently available to prevent this infection. Here, we describe the derivation of a live attenuated vaccine virus, termed mutant (Mt) 10, encoding a single amino acid substitution H790A within the viral protein 1, that prevents CVB3 infection in mice and protects from both myocarditis and pancreatitis in challenge studies. We noted that animals vaccinated with Mt 10 developed virus-neutralizing antibodies, predominantly containing IgG2a and IgG2b, and to a lesser extent IgG3 and IgG1. Furthermore, by using major histocompatibility complex class II dextramers and tetramers, we demonstrated that Mt 10 induces antigen-specific T cell responses that preferentially produce interferon-γ. Finally, neither vaccine recipients nor those challenged with the wild-type virus revealed evidence of autoimmunity or cardiac injury as determined by T cell response to cardiac myosin and measurement of circulating cardiac troponin I levels, respectively. Together, our data suggest that Mt 10 is a vaccine candidate that prevents CVB3 infection through the induction of neutralizing antibodies and antigen-specific T cell responses, the two critical components needed for complete protection against virus infections in vaccine studies.
Asunto(s)
Infecciones por Coxsackievirus/prevención & control , Enterovirus Humano B/inmunología , Miocarditis/prevención & control , Pancreatitis/prevención & control , Vacunas Virales/inmunología , Animales , Anticuerpos Neutralizantes/sangre , Anticuerpos Neutralizantes/inmunología , Antígenos Virales/inmunología , Sitios de Unión/genética , Proteína de la Membrana Similar al Receptor de Coxsackie y Adenovirus/metabolismo , Infecciones por Coxsackievirus/virología , Modelos Animales de Enfermedad , Enterovirus Humano B/genética , Femenino , Humanos , Inmunogenicidad Vacunal/genética , Masculino , Ratones , Mutación , Miocarditis/virología , Pancreatitis/virología , Linfocitos T/inmunología , Vacunas Atenuadas/administración & dosificación , Vacunas Atenuadas/genética , Vacunas Atenuadas/inmunología , Vacunas Virales/administración & dosificación , Vacunas Virales/genéticaRESUMEN
BACKGROUND: Despite multi-model therapy of maximal surgical resection, radiation, chemotherapy, and tumor-treating fields, the median survival of glioblastoma (GBM) patients is less than 15 months. Protein arginine methyltransferase 5 (PRMT5) catalyzes the symmetric dimethylation of arginine residues and is overexpressed in GBM. Inhibition of PRMT5 causes senescence in stem-like GBM tumor cells. LB100, a first-in-class small molecular inhibitor of protein phosphatase 2A (PP2A), can sensitize therapy-resistant tumor cells. Here, we tested the anti-GBM effect of concurrent PRMT5 and PP2A inhibition. METHODS: Patient-derived primary GBM neurospheres (GBMNS), transfected with PRMT5 target-specific siRNA, were treated with LB100 and subjected to in vitro assays including PP2A activity and western blot. The intracranial mouse xenograft model was used to test the in vivo antitumor efficacy of combination treatment. RESULTS: We found that PRMT5 depletion increased PP2A activity in GBMNS. LB100 treatment significantly reduced the viability of PRMT5-depleted GBMNS compared to PRMT5-intact GBMNS. LB100 enhanced G1 cell cycle arrest induced by PRMT5 depletion. Combination therapy also increased the expression of phospho-MLKL. Necrostatin-1 rescued PRMT5-depleted cells from the cytotoxic effects of LB100, indicating that necroptosis caused the enhanced cytotoxicity of combination therapy. In the in vivo mouse tumor xenograft model, LB100 treatment combined with transient depletion of PRMT5 significantly decreased tumor size and prolonged survival, while LB100 treatment alone had no survival benefit. CONCLUSION: Overall, combined PRMT5 and PP2A inhibition had significantly greater antitumor effects than PRMT5 inhibition alone.
Asunto(s)
Glioblastoma , Animales , Línea Celular Tumoral , Glioblastoma/tratamiento farmacológico , Humanos , Ratones , Piperazinas , Proteína Fosfatasa 2 , Proteína-Arginina N-Metiltransferasas/genética , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Group B coxsackieviruses (CVBs) belonging to the genus, Enterovirus and contain six serotypes that induce various diseases, whose occurrence may involve the mediation of more than one serotype. We recently identified immunogenic epitopes within coxsackieviruses B3 (CVB3) viral protein 1 that induce anti-viral T cell responses in mouse models of CVB infections. In our investigations to determine the protective responses of the viral epitopes, we unexpectedly noted that animals immunized with complete Freund's adjuvant (CFA) alone and later challenged with CVB3 were completely protected against myocarditis. Similarly, the pancreatitis-inducing ability of CVB3 was remarkably reduced to only 10% in the CFA group as opposed to 73.3% in the control group that received no CFA. Additionally, no mortalities were noted in the CFA group, whereas 40% of control animals died during the course of 21 days post-infection with CVB3. Taken together, our data suggest that the adjuvant effects of CFA may be sufficient for protection against CVB infections. These observations may provide new insights into our understanding of the occurrence of viral infections.
RESUMEN
NK cells recognize MHC class I (MHC-I) Ags via stochastically expressed MHC-I-specific inhibitory receptors that prevent NK cell activation via cytoplasmic ITIM. We have identified a pan anti-MHC-I mAb that blocks NK cell inhibitory receptor binding at a site distinct from the TCR binding site. Treatment of unmanipulated mice with this mAb disrupted immune homeostasis, markedly activated NK and memory phenotype T cells, enhanced immune responses against transplanted tumors, and augmented responses to acute and chronic viral infection. mAbs of this type represent novel checkpoint inhibitors in tumor immunity, potent tools for the eradication of chronic infection, and may function as adjuvants for the augmentation of the immune response to weak vaccines.
Asunto(s)
Antígenos de Histocompatibilidad Clase I/inmunología , Memoria Inmunológica , Células Asesinas Naturales/inmunología , Activación de Linfocitos , Neoplasias Experimentales/inmunología , Receptores de Células Asesinas Naturales/inmunología , Virosis/inmunología , Animales , Femenino , Células Asesinas Naturales/patología , Ratones , Ratones Endogámicos BALB C , Neoplasias Experimentales/patología , Ratas , Virosis/patologíaRESUMEN
Autoreactive T cells may contribute to post-viral myocarditis induced with Coxsackievirus B3 (CVB3), but the underlying mechanisms of their generation are unclear. Here, we have comprehensively analyzed the generation of antigen-specific, autoreactive T cells in the mouse model of CVB3 infection for antigens implicated in patients with myocarditis/dilated cardiomyopathy. First, comparative analysis of CVB3 proteome with five autoantigens led us to identify three mimicry epitopes, one each from adenine nucleotide translocator 1 (ANT), sarcoplasmic/endoplasmic reticulum Ca2+ ATPase 2a (SERCA2a) and cardiac troponin I. None of these induced cross-reactive T cell responses. Next, we generated major histocompatibility complex (MHC) class II dextramers to enumerate the frequencies of antigen-specific T cells to determine whether T cells with multiple antigen specificities are generated by CVB3 infection. These analyses revealed appearance of CD4 T cells positive for SERCA2a 971-990, and cardiac myosin heavy chain-α (Myhc) 334-352 dextramers, both in the periphery and also in the hearts of CVB3-infected animals. While ANT 21-40 dextramer+ T cells were inconsistently detected, the ß1-adrenergic receptor 181-200/211-230 or branched chain α-ketoacid dehydrogenase kinase 111-130 dextramer+ cells were absent. Interestingly, SERCA2a 971-990, Myhc 334-352 and ANT 21-40 dextramer+ cells were also detected in the liver indicating that they may have a pathogenic role. Finally, we demonstrate that the SERCA2a 971-990-reactive T cells generated in CVB3 infection could transfer disease to naïve mice. The data suggest that CVB3 infection can lead to the generation of autoreactive T cells for multiple antigens indicating a possibility that the autoreactive T cells localized in the liver can potentially circulate and contribute to the development of viral myocarditis.
Asunto(s)
Autoantígenos/inmunología , Linfocitos T CD4-Positivos/inmunología , Infecciones por Coxsackievirus/inmunología , Miocarditis/inmunología , Miocarditis/virología , Animales , Autoinmunidad/inmunología , Reacciones Cruzadas , Modelos Animales de Enfermedad , Enterovirus Humano B , Femenino , Masculino , RatonesRESUMEN
Coxsackievirus group B (CVB) contains six serotypes that can affect various organs. Some of these organ-specific diseases such as myocarditis and pancreatitis can be caused by more than one serotype. Thus, development of immunological tools common to multiple serotypes is desired. This is especially critical for analyzing antigen-specific T cell responses at a single cell level. To this end, we made efforts to identify the immunogenic epitopes of CVB3 leading us to localize three T cell epitopes within the viral protein 1 (VP1) namely, VP1 681-700, VP1 721-740 and VP1 771-790. First, we confirmed their immunogenicity in the immunization settings. Second, we sought to verify the ability of VP1 epitopes to bind major histocompatibility complex (MHC) class II (IAk) molecules. Third, we created MHC class II (IAk) dextramers and tetramers and ascertained the T cell responses to be antigen-specific. Fourth, we analyzed the T cell responses in animals infected with CVB3 and noted the magnitude of antigen-specific T cell responses occurring in the order of VP1 721-740 and VP1 681-700 followed by VP1 771-790 as verified by proliferation assay and IAk tetramer staining. All epitopes induced interferon (IFN)-γ as a major cytokine. Finally, we investigated whether the VP1 tools generated for CVB3 can also be used to verify T cell responses in infections caused by other serotypes. To this end, we established the CVB4 infection model in A/J mice and found that the CVB4 infection led to the induction of IFN-γ-producing T cell responses primarily for VP1 721-740 and VP1 681-700. Thus, the VP1-specific tools, particularly IAk tetramers can be used to monitor anti-viral T cell responses in multiple CVB serotypes.
Asunto(s)
Antígenos Virales/inmunología , Enterovirus Humano B/clasificación , Enterovirus Humano B/inmunología , Infecciones por Enterovirus/inmunología , Infecciones por Enterovirus/virología , Epítopos de Linfocito T/inmunología , Linfocitos T/inmunología , Secuencia de Aminoácidos , Animales , Antígenos Virales/química , Citocinas/metabolismo , Infecciones por Enterovirus/complicaciones , Epítopos de Linfocito T/química , Células HeLa , Antígenos de Histocompatibilidad/química , Antígenos de Histocompatibilidad/inmunología , Antígenos de Histocompatibilidad/metabolismo , Interacciones Huésped-Patógeno/inmunología , Humanos , Inmunofenotipificación , Activación de Linfocitos , Miocarditis/etiología , Miocarditis/metabolismo , Miocarditis/patología , Unión Proteica , Serogrupo , Linfocitos T/metabolismoRESUMEN
Host conditioning has emerged as an important component of effective adoptive cell transfer-based immunotherapy for cancer. High levels of IL-1ß are induced by host conditioning, but its impact on the antitumor function of T cells remains unclear. We found that the administration of IL-1ß increased the population size and functionality of adoptively transferred T cells within the tumor. Most importantly, IL-1ß enhanced the ability of tumor-specific T cells to trigger the regression of large, established B16 melanoma tumors in mice. Mechanistically, we showed that the increase in T cell numbers was associated with superior tissue homing and survival abilities and was largely mediated by IL-1ß-stimulated host cells. In addition, IL-1ß enhanced T cell functionality indirectly via its actions on radio-resistant host cells in an IL-2- and IL-15-dependent manner. Our findings not only underscore the potential of provoking inflammation to enhance antitumor immunity but also uncover novel host regulations of T cell responses.
Asunto(s)
Inmunoterapia Adoptiva/métodos , Interleucina-1beta/inmunología , Activación de Linfocitos/inmunología , Melanoma Experimental/terapia , Linfocitos T/inmunología , Traslado Adoptivo , Animales , Línea Celular Tumoral , Citocinas/sangre , Interleucina-1beta/metabolismo , Interleucina-6/sangre , Melanoma Experimental/inmunología , Melanoma Experimental/metabolismo , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Linfocitos T/metabolismo , Linfocitos T/trasplante , Factor de Necrosis Tumoral alfa/sangreRESUMEN
Eos (lkzf4) is a member of the Ikaros family of transcription factors and is preferentially expressed in T-regulatory (Treg) cells. However, the role of Eos in Treg function is controversial. One study using siRNA knock down of Eos demonstrated that it was critical for Treg suppressor function. In contrast, Treg from mice with a global deficiency of Eos had normal Treg function in vitro and in vivo. To further dissect the function of Eos in Tregs, we generated mice with a conditional knock out of Eos in Treg cells (lkzf4fl/fl X Foxp3YFP-cre, Eos cKO). Deletion of Eos in Treg resulted in activation of CD4+Foxp3- and CD8+ T cells at the age of 3 months, cellular infiltration in non-lymphoid tissues, hyperglobulinemia, and anti-nuclear antibodies. While Tregs from Eos cKO mice displayed normal suppressive function in vitro, Eos cKO mice developed severe Experimental Autoimmune Encephalomyletis (EAE) following immunization with myelin oligodendrocyte glycoprotein (MOG) and Eos cKO Treg were unable to suppress Inflammatory Bowel Disease (IBD). Eos cKO mice had decreased growth of the transplantable murine adenocarcinoma MC38 tumor accompanied by enhanced IFN-γ/TNF-α production by CD8+ T cells in tumor draining lymph nodes. Mice with a global deficiency of Eos or a deficiency of Eos only in T cells developed autoimmunity at a much older age (12 months or 7-8 months, respectively). Taken together, Eos appears to play an essential role in multiple aspects of Treg suppressor function, but also plays an as yet unknown role in the function of CD4+Foxp3- and CD8+ T cells and potentially in non-T cells.
Asunto(s)
Autoinmunidad/inmunología , Proteínas de Unión al ADN/inmunología , Proteínas del Tejido Nervioso/inmunología , Linfocitos T Reguladores/inmunología , Animales , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD8-positivos/inmunología , Diferenciación Celular/inmunología , Línea Celular Tumoral , Encefalomielitis Autoinmune Experimental/inmunología , Femenino , Factores de Transcripción Forkhead/inmunología , Enfermedades Inflamatorias del Intestino/inmunología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Glicoproteína Mielina-Oligodendrócito/inmunologíaRESUMEN
CD4+ T cell differentiation into multiple T helper (Th) cell lineages is critical for optimal adaptive immune responses. This report identifies an intrinsic mechanism by which programmed death-1 receptor (PD-1) signaling imparted regulatory phenotype to Foxp3+ Th1 cells (denoted as Tbet+iTregPDL1 cells) and inducible regulatory T (iTreg) cells. Tbet+iTregPDL1 cells prevented inflammation in murine models of experimental colitis and experimental graft versus host disease (GvHD). Programmed death ligand-1 (PDL-1) binding to PD-1 imparted regulatory function to Tbet+iTregPDL1 cells and iTreg cells by specifically downregulating endo-lysosomal protease asparaginyl endopeptidase (AEP). AEP regulated Foxp3 stability and blocking AEP imparted regulatory function in Tbet+iTreg cells. Also, Aep-/- iTreg cells significantly inhibited GvHD and maintained Foxp3 expression. PD-1-mediated Foxp3 maintenance in Tbet+ Th1 cells occurred both in tumor infiltrating lymphocytes (TILs) and during chronic viral infection. Collectively, this report has identified an intrinsic function for PD-1 in maintaining Foxp3 through proteolytic pathway.
Asunto(s)
Cisteína Endopeptidasas/metabolismo , Factores de Transcripción Forkhead/metabolismo , Receptor de Muerte Celular Programada 1/metabolismo , Linfocitos T Reguladores/inmunología , Células TH1/inmunología , Animales , Diferenciación Celular/inmunología , Células Cultivadas , Colitis/inmunología , Colitis/patología , Femenino , Enfermedad Injerto contra Huésped/inmunología , Enfermedad Injerto contra Huésped/patología , Coriomeningitis Linfocítica/inmunología , Coriomeningitis Linfocítica/patología , Virus de la Coriomeningitis Linfocítica/inmunología , Melanoma Experimental/inmunología , Melanoma Experimental/patología , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Noqueados , Linfocitos T Reguladores/citología , Células TH1/citologíaRESUMEN
Regulatory T cells (Tregs) play a cardinal role in the immune system by suppressing detrimental autoimmune responses, but their role in acute, chronic infectious diseases and tumor microenvironment remains unclear. We recently demonstrated that IFN-α/ß receptor (IFNAR) signaling promotes Treg function in autoimmunity. Here we dissected the functional role of IFNAR-signaling in Tregs using Treg-specific IFNAR deficient (IFNARfl/flxFoxp3YFP-Cre) mice in acute LCMV Armstrong, chronic Clone-13 viral infection, and in tumor models. In both viral infection and tumor models, IFNARfl/flxFoxp3YFP-Cre mice Tregs expressed enhanced Treg associated activation antigens. LCMV-specific CD8+ T cells and tumor infiltrating lymphocytes from IFNARfl/flxFoxp3YFP-Cre mice produced less antiviral and antitumor IFN-γ and TNF-α. In chronic viral model, the numbers of antiviral effector and memory CD8+ T cells were decreased in IFNARfl/flxFoxp3YFP-Cre mice and the effector CD4+ and CD8+ T cells exhibited a phenotype compatible with enhanced exhaustion. IFNARfl/flxFoxp3YFP-Cre mice cleared Armstrong infection normally, but had higher viral titers in sera, kidneys and lungs during chronic infection, and higher tumor burden than the WT controls. The enhanced activated phenotype was evident through transcriptome analysis of IFNARfl/flxFoxp3YFP-Cre mice Tregs during infection demonstrated differential expression of a unique gene signature characterized by elevated levels of genes involved in suppression and decreased levels of genes mediating apoptosis. Thus, IFN signaling in Tregs is beneficial to host resulting in a more effective antiviral response and augmented antitumor immunity.
Asunto(s)
Infecciones por Arenaviridae/inmunología , Neoplasias del Colon/inmunología , Interferón Tipo I/farmacología , Coriomeningitis Linfocítica/inmunología , Melanoma Experimental/inmunología , Linfocitos T Reguladores/inmunología , Microambiente Tumoral/inmunología , Animales , Antivirales/farmacología , Infecciones por Arenaviridae/tratamiento farmacológico , Infecciones por Arenaviridae/metabolismo , Infecciones por Arenaviridae/virología , Linfocitos T CD8-positivos/efectos de los fármacos , Linfocitos T CD8-positivos/inmunología , Linfocitos T CD8-positivos/metabolismo , Células Cultivadas , Neoplasias del Colon/tratamiento farmacológico , Neoplasias del Colon/metabolismo , Neoplasias del Colon/virología , Inmunidad Innata/efectos de los fármacos , Inmunidad Innata/inmunología , Interferón gamma/metabolismo , Coriomeningitis Linfocítica/tratamiento farmacológico , Coriomeningitis Linfocítica/metabolismo , Coriomeningitis Linfocítica/virología , Virus de la Coriomeningitis Linfocítica/efectos de los fármacos , Melanoma Experimental/tratamiento farmacológico , Melanoma Experimental/metabolismo , Melanoma Experimental/virología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Receptor de Interferón alfa y beta/fisiología , Transducción de Señal/efectos de los fármacos , Linfocitos T Reguladores/efectos de los fármacos , Linfocitos T Reguladores/metabolismo , Linfocitos T Reguladores/virología , Microambiente Tumoral/efectos de los fármacosRESUMEN
CD47 is a ubiquitous cell surface receptor that directly regulates T cell immunity by interacting with its inhibitory ligand thrombospondin-1 and limits clearance of cells by phagocytes that express its counter-receptor signal-regulatory protein-α. Murine natural killer (NK) cells express higher levels of CD47 than other lymphocytes, but the role of CD47 in regulating NK cell homeostasis and immune function remains unclear. Cd47-/- mice exhibited depletion of NK precursors in bone marrow, consistent with the antiphagocytic function of CD47. In contrast, antisense CD47 knockdown or gene disruption resulted in a dose dependent accumulation of immature and mature NK cells in spleen. Mature Cd47-/- NK cells exhibited increased expression of NK effector and interferon gene signatures and an increased proliferative response to interleukin-15 in vitro. Cd47-/- mice showed no defect in their early response to acute Armstrong lymphocytic choriomeningitis virus (LCMV) infection but were moderately impaired in controlling chronic Clone-13 LCMV infection, which was associated with depletion of splenic NK cells and loss of effector cytokine and interferon response gene expression in Cd47-/- NK cells. Broad CD47-dependent differences in NK activation, survival, and exhaustion pathways were observed in NK cell transcriptional signatures in LCMV infected mice. These data identify CD47 as a cell-intrinsic and systemic regulator of NK cell homeostasis and NK cell function in responding to a viral infection.
Asunto(s)
Antígeno CD47/metabolismo , Células Asesinas Naturales/inmunología , Coriomeningitis Linfocítica/inmunología , Virus de la Coriomeningitis Linfocítica/inmunología , Animales , Trasplante de Médula Ósea , Antígeno CD47/genética , Antígeno CD47/inmunología , Proliferación Celular , Modelos Animales de Enfermedad , Femenino , Humanos , Células Asesinas Naturales/metabolismo , Coriomeningitis Linfocítica/virología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Bazo/citología , Bazo/inmunología , Quimera por TrasplanteRESUMEN
Sarcoplasmic/endoplasmic reticulum Ca2+ adenosine triphosphatase (SERCA)2a, a critical regulator of calcium homeostasis, is known to be decreased in heart failure. Patients with myocarditis or dilated cardiomyopathy develop autoantibodies to SERCA2a suggesting that they may have pathogenetic significance. In this report, we describe epitope mapping analysis of SERCA2a in A/J mice that leads us to make five observations: 1) SERCA2a contains multiple T cell epitopes that induce varying degrees of myocarditis. One epitope, SERCA2a 971-990, induces widespread atrial inflammation without affecting noncardiac tissues; the cardiac abnormalities could be noninvasively captured by echocardiography, electrocardiography, and magnetic resonance microscopy imaging. 2) SERCA2a 971-990-induced disease was associated with the induction of CD4 T cell responses and the epitope preferentially binds MHC class II/IAk rather than IEk By creating IAk/and IEk/SERCA2a 971-990 dextramers, the T cell responses were determined by flow cytometry to be Ag specific. 3) SERCA2a 971-990-sensitized T cells produce both Th1 and Th17 cytokines. 4) Animals immunized with SERCA2a 971-990 showed Ag-specific Abs with enhanced production of IgG2a and IgG2b isotypes, suggesting that SERCA2a 971-990 can potentially act as a common epitope for both T cells and B cells. 5) Finally, SERCA2a 971-990-sensitized T cells were able to transfer disease to naive recipients. Together, these data indicate that SERCA2a is a critical autoantigen in the mediation of atrial inflammation in mice and that our model may be helpful to study the inflammatory events that underlie the development of conditions such as atrial fibrillation in humans.
Asunto(s)
Mapeo Epitopo , Epítopos/inmunología , Miocarditis/inmunología , ATPasas Transportadoras de Calcio del Retículo Sarcoplásmico/inmunología , Alelos , Animales , Proteínas Bacterianas , Citocinas/inmunología , Citocinas/metabolismo , Modelos Animales de Enfermedad , Mapeo Epitopo/métodos , Epítopos de Linfocito B/inmunología , Técnica del Anticuerpo Fluorescente , Expresión Génica , Atrios Cardíacos/inmunología , Atrios Cardíacos/metabolismo , Atrios Cardíacos/patología , Ventrículos Cardíacos/inmunología , Ventrículos Cardíacos/metabolismo , Ventrículos Cardíacos/patología , Antígenos de Histocompatibilidad Clase II/genética , Antígenos de Histocompatibilidad Clase II/inmunología , Epítopos Inmunodominantes/inmunología , Inmunohistoquímica , Ratones , Ratones Endogámicos , Miocarditis/diagnóstico por imagen , Miocarditis/patología , Miocitos Cardíacos/inmunología , Miocitos Cardíacos/metabolismo , Miocitos Cardíacos/patología , Péptidos/inmunología , ATPasas Transportadoras de Calcio del Retículo Sarcoplásmico/genética , ATPasas Transportadoras de Calcio del Retículo Sarcoplásmico/metabolismo , Subgrupos de Linfocitos T/inmunología , Subgrupos de Linfocitos T/metabolismoRESUMEN
Myocarditis/dilated cardiomyopathy (DCM) patients can develop autoantibodies to various cardiac antigens and one major antigen is ß1-adrenergic receptor (ß1AR). Previous reports indicate that animals immunized with a ß1AR fragment encompassing, 197-222 amino acids for a prolonged period can develop DCM by producing autoantibodies, but existence of T cell epitopes, if any, were unknown. Using A/J mice that are highly susceptible to lymphocytic myocarditis, we have identified ß1AR 171-190, ß1AR 181-200, and ß1AR 211-230 as the major T cell epitopes that bind major histocompatibility complex class II/IAk or IEk alleles, and by creating IAk and IEk dextramers, we demonstrate that the CD4 T cell responses to be antigen-specific. Of note, all the three epitopes were found also to stimulate CD8 T cells suggesting that they can act as common epitopes for both CD4 and CD8 T cells. While, all epitopes induced only mild myocarditis, the disease-incidence was enhanced in animals immunized with all the three peptides together as a cocktail. Although, antigen-sensitized T cells produced mainly interleukin-17A, their transfer into naive animals yielded no disease. But, steering for T helper 1 response led the T cells reacting to one epitope, ß1AR 181-200 to induce severe myocarditis in naive mice. Finally, we demonstrate that all three ß1AR epitopes to be unique for T cells as none of them induced antibody responses. Conversely, animals immunized with a non-T cell activator, ß1AR 201-220, an equivalent of ß1AR 197-222, had antibodies comprising of all IgG isotypes and IgM except, IgA and IgE. Thus, identification of T cell and B cell epitopes of ß1AR may be helpful to determine ß1AR-reactive autoimmune responses in various experimental settings in A/J mice.
RESUMEN
INTRODUCTION: Organ-specific autoimmune diseases are believed to result from immune responses generated against self-antigens specific to each organ. However, when such responses target antigens expressed promiscuously in multiple tissues, then the immune-mediated damage may be wide spread. METHODS: In this report, we describe a mitochondrial protein, branched chain α-ketoacid dehydrogenase kinase (BCKDk ) that can act as a target autoantigen in the development of autoimmune inflammatory reactions in both heart and liver. RESULTS: We demonstrate that BCKDk protein contains at least nine immunodominant epitopes, three of which, BCKDk 71-90, BCKDk 111-130 and BCKDk 141-160, were found to induce varying degrees of myocarditis in immunized mice. One of these, BCKDk 111-130, could also induce hepatitis without affecting lungs, kidneys, skeletal muscles, and brain. In immunogenicity testing, all three peptides induced antigen-specific T cell responses, as verified by proliferation assay and/or major histocompatibility complex class II/IAk dextramer staining. Finally, the disease-inducing abilities of BCKDk peptides were correlated with the production of interferon-γ, and the activated T cells could transfer disease to naive recipients. CONCLUSIONS: The disease induced by BCKDk peptides could serve as a useful model to study the autoimmune events of inflammatory heart and liver diseases.
Asunto(s)
Enfermedades Autoinmunes/inmunología , Epítopos de Linfocito T/inmunología , Hepatitis Autoinmune/inmunología , Miocarditis/inmunología , Proteínas Quinasas/inmunología , Secuencia de Aminoácidos , Animales , Autoantígenos/química , Autoantígenos/inmunología , Enfermedades Autoinmunes/patología , Biopsia , Citocinas/metabolismo , Modelos Animales de Enfermedad , Epítopos de Linfocito T/química , Hepatitis Autoinmune/patología , Antígenos de Histocompatibilidad Clase II/genética , Antígenos de Histocompatibilidad Clase II/inmunología , Inmunización , Ratones , Miocarditis/patología , Fragmentos de Péptidos/química , Fragmentos de Péptidos/inmunología , Proteínas Quinasas/química , Multimerización de Proteína , Linfocitos T/inmunología , Linfocitos T/metabolismoRESUMEN
Heart failure, a leading cause of death in humans, can emanate from myocarditis. Although most individuals with myocarditis recover spontaneously, some develop chronic dilated cardiomyopathy. Myocarditis may result from both infectious and noninfectious causes, including autoimmune responses to cardiac antigens. In support of this notion, intracellular cardiac antigens, like cardiac myosin heavy chain-α, cardiac troponin-I, and adenine nucleotide translocator 1 (ANT1), have been identified as autoantigens in cardiac autoimmunity. Herein, we demonstrate that ANT1 can induce autoimmune myocarditis in A/J mice by generating autoreactive T cells. We show that ANT1 encompasses multiple immunodominant epitopes (namely, ANT1 21-40, ANT1 31-50, ANT1 171-190, and ANT1 181-200). Although all four peptides induce comparable T-cell responses, only ANT1 21-40 was found to be a major myocarditogenic epitope in immunized animals. The myocarditis-inducing ability of ANT1 21-40 was associated with the generation of T cells producing predominantly IL-17A, and the antigen-sensitized T cells could transfer the disease to naïve recipients. These data indicate that cardiac mitochondrial proteins can be target autoantigens in myocarditis, supporting the notion that the antigens released as a result of primary damage may contribute to the persistence of chronic inflammation through autoimmunity.