RESUMEN
DNA mismatch repair-deficient (MMR-d) cancers present an abundance of neoantigens that is thought to explain their exceptional responsiveness to immune checkpoint blockade (ICB)1,2. Here, in contrast to other cancer types3-5, we observed that 20 out of 21 (95%) MMR-d cancers with genomic inactivation of ß2-microglobulin (encoded by B2M) retained responsiveness to ICB, suggesting the involvement of immune effector cells other than CD8+ T cells in this context. We next identified a strong association between B2M inactivation and increased infiltration by γδ T cells in MMR-d cancers. These γδ T cells mainly comprised the Vδ1 and Vδ3 subsets, and expressed high levels of PD-1, other activation markers, including cytotoxic molecules, and a broad repertoire of killer-cell immunoglobulin-like receptors. In vitro, PD-1+ γδ T cells that were isolated from MMR-d colon cancers exhibited enhanced reactivity to human leukocyte antigen (HLA)-class-I-negative MMR-d colon cancer cell lines and B2M-knockout patient-derived tumour organoids compared with antigen-presentation-proficient cells. By comparing paired tumour samples from patients with MMR-d colon cancer that were obtained before and after dual PD-1 and CTLA-4 blockade, we found that immune checkpoint blockade substantially increased the frequency of γδ T cells in B2M-deficient cancers. Taken together, these data indicate that γδ T cells contribute to the response to immune checkpoint blockade in patients with HLA-class-I-negative MMR-d colon cancers, and underline the potential of γδ T cells in cancer immunotherapy.
Asunto(s)
Neoplasias del Colon , Genes MHC Clase I , Antígenos de Histocompatibilidad Clase I , Inhibidores de Puntos de Control Inmunológico , Inmunoterapia , Receptores de Antígenos de Linfocitos T gamma-delta , Linfocitos T , Humanos , Neoplasias del Colon/tratamiento farmacológico , Neoplasias del Colon/genética , Neoplasias del Colon/inmunología , Neoplasias del Colon/terapia , Antígenos de Histocompatibilidad Clase I/genética , Antígenos de Histocompatibilidad Clase I/inmunología , Inhibidores de Puntos de Control Inmunológico/farmacología , Inhibidores de Puntos de Control Inmunológico/uso terapéutico , Receptores de Antígenos de Linfocitos T gamma-delta/inmunología , Linfocitos T/inmunología , Microglobulina beta-2/deficiencia , Microglobulina beta-2/genética , Reparación de la Incompatibilidad de ADN/genética , Receptores KIR , Línea Celular Tumoral , Organoides , Presentación de Antígeno , Genes MHC Clase I/genéticaRESUMEN
The objective of this study was to examine the protective effect of phytic acid (PA) in reducing oxidative stress in an animal model for human hereditary hemochromatosis (HH) fed high-fat diets. Sixty-four ß2 microglobulin knockout (ß2m KO) mice were randomly assigned to three treatments by feeding: control (basal), atherogenic (AT), and polyunsaturated fatty acid (PUFA) diets. One-half of the mice in each treatment group were fed 2% (wt/wt) PA. The ß2m+/+ mice (wild type (WT)) were fed a basal diet. All seven groups were fed for 10 weeks with a 50-ppm iron-containing diet (AIN-93G). Free iron and lipids were measured in serum samples. Nonheme iron, thiobarbituric acid-reactive substances (TBARS), superoxide dismutase (SOD), and catalase concentrations were measured in the liver tissue. Nonheme iron concentration in ß2m KO mice (on the basal diet) was 20× higher (p < 0.0001) than in the WT mice. Compared to the WT mice, ß2m KO mice had a significantly higher concentration of free iron in the serum (p < 0.0001), six-fold higher hepatic TBARs (p < 0.0001), and 18% lower hepatic SOD level. When PA was added to the ß2m KO basal diet, a reduction (26 to 50%) of iron concentration was seen in the liver and heart. The addition of PA also significantly reduced TBARs in all three dietary groups of the iron-overloaded group, but most effectively in the control group. An increase in SOD concentration was seen only in the PUFA group, but serum triacylglycerol (TG) concentration was reduced in both dietary fat groups. In conclusion, our results suggest that PA protects against oxidative stress-induced by genetic iron overload alone or when fed high fat.
Asunto(s)
Dieta Alta en Grasa , Sobrecarga de Hierro/patología , Estrés Oxidativo/efectos de los fármacos , Ácido Fítico/farmacología , Sustancias Protectoras/farmacología , Microglobulina beta-2/deficiencia , Animales , Biomarcadores/metabolismo , Hierro/metabolismo , Sobrecarga de Hierro/sangre , Lípidos/sangre , Masculino , Ratones Endogámicos C57BL , Ratones Noqueados , Aumento de Peso/efectos de los fármacosRESUMEN
Hermaphroditic invertebrates and plants have a self-recognition system on the cell surface of sperm and eggs, which prevents their self-fusion and enhances non-self-fusion, thereby contributing to genetic variation. However, the system of sperm-egg recognition in mammals is under debate. To address this issue, we explored the role of major histocompatibility complex class I (MHC class I, also known as histocompatibility 2-Kb or H2-Kb and H2-Db in mice) antigens by analyzing H2-Kb-/-H2-Db-/-ß2-microglobulin (ß2M)-/- triple-knockout (T-KO) male mice with full fertility. T-KO sperm exhibited an increased sperm number in the perivitelline space of wild-type (WT) eggs in vitro. Moreover, T-KO sperm showed multiple fusion with zona pellucida (ZP)-free WT eggs, implying that the ability of polyspermy block for sperm from T-KO males was weakened in WT eggs. When T-KO male mice were intercrossed with WT female mice, the percentage of females in progeny increased. We speculate that WT eggs prefer fusion with T-KO sperm, more specifically X-chromosome-bearing sperm (X sperm), suggesting the presence of preferential (non-random) fertilization in mammals, including humans.
Asunto(s)
Fertilidad/genética , Antígenos de Histocompatibilidad Clase I/genética , Óvulo/metabolismo , Razón de Masculinidad , Interacciones Espermatozoide-Óvulo/genética , Espermatozoides/metabolismo , Animales , Proteínas de Unión al ADN/deficiencia , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/inmunología , Femenino , Fertilización In Vitro , Regulación de la Expresión Génica , Antígenos de Histocompatibilidad Clase I/inmunología , Humanos , Masculino , Ratones , Ratones Noqueados , Óvulo/citología , Recuento de Espermatozoides , Espermatozoides/citología , Microglobulina beta-2/deficiencia , Microglobulina beta-2/genética , Microglobulina beta-2/inmunologíaRESUMEN
Macrophages can change their physiology in response to microenvironmental signals. This differentiation into classically activated M1 or alternatively activated M2 macrophages is known as polarization. In this study, we isolated bone marrow-derived macrophages from ß2m-deficient (deficient in both MHC class Ia and Ib) and Kb Db -deficient (deficient only in MHC class Ia) mice and found that ß2m-deficient macrophages showed a significantly lower M2b polarization efficiency. In addition, the absence of constitutive MHC class Ib expression decreased the stability of the Notch-1 intracellular domain. Finally, we found that ß2m-deficient mice exposed to irradiation showed reduced bacterial translocation and sepsis severity. Overall, our study demonstrates that MHC class Ib molecules are essential for M2b macrophage polarization and suggests that MHC class Ib molecules play an important role during infection-induced innate immunity.
Asunto(s)
Polaridad Celular , Rayos gamma , Antígenos de Histocompatibilidad Clase I/metabolismo , Macrófagos/patología , Macrófagos/efectos de la radiación , Sepsis/inmunología , Animales , Traslocación Bacteriana/efectos de la radiación , Polaridad Celular/efectos de la radiación , Enterococcus faecalis/fisiología , Femenino , Ratones Endogámicos C57BL , Sepsis/microbiología , Transducción de Señal/efectos de la radiación , Microglobulina beta-2/deficiencia , Microglobulina beta-2/metabolismoRESUMEN
The ex vivo production of platelets depleted of human leukocyte antigen class I (HLA-I) could serve as a universal measure to overcome platelet transfusion refractoriness caused by HLA-I incompatibility. Here, we developed human induced pluripotent cell-derived HLA-I-deficient platelets (HLA-KO iPLATs) in a clinically applicable imMKCL system by genetic manipulation and assessed their immunogenic properties including natural killer (NK) cells, which reject HLA-I downregulated cells. HLA-KO iPLATs were deficient for all HLA-I but did not elicit a cytotoxic response by NK cells in vitro and showed circulation equal to wild-type iPLATs upon transfusion in our newly established Hu-NK-MSTRG mice reconstituted with human NK cells. Additionally, HLA-KO iPLATs successfully circulated in an alloimmune platelet transfusion refractoriness model of Hu-NK-MISTRG mice. Mechanistically, the lack of NK cell-activating ligands on platelets may be responsible for evading the NK cell response. This study revealed the unique non-immunogenic property of platelets and provides a proof of concept for the clinical application of HLA-KO iPLATs.
Asunto(s)
Plaquetas/citología , Plaquetas/metabolismo , Diferenciación Celular , Antígenos de Histocompatibilidad Clase I/inmunología , Células Madre Pluripotentes Inducidas/citología , Células Asesinas Naturales/inmunología , Células Asesinas Naturales/metabolismo , Animales , Citotoxicidad Inmunológica/genética , Citotoxicidad Inmunológica/inmunología , Técnicas de Inactivación de Genes , Antígenos de Histocompatibilidad Clase I/genética , Humanos , Activación de Linfocitos/genética , Activación de Linfocitos/inmunología , Ratones , Ratones Noqueados , Microglobulina beta-2/deficiencia , Microglobulina beta-2/genéticaRESUMEN
Traumatic brain injury (TBI) leaves many survivors with long-term disabilities. A prolonged immune response in the brain may cause neurodegeneration, resulting in chronic neurological disturbances. In this study, using a TBI mouse model, we correlate changes in the local immune response with neurodegeneration/neurological dysfunction over an 8-month period. Flow cytometric analysis reveals a protracted increase in effector/memory CD8+ T cells (expressing granzyme B) in the injured brain. This precedes interleukin-17+CD4+ T cell infiltration and is associated with progressive neurological/motor impairment, increased circulating brain-specific autoantibodies, and myelin-related pathology. Genetic deficiency or pharmacological depletion of CD8+ T cells, but not depletion of CD4+ T cells, improves neurological outcomes and produces a neuroprotective Th2/Th17 immunological shift, indicating a persistent detrimental role for cytotoxic T cells post-TBI. B cell deficiency results in severe neurological dysfunction and a heightened immune reaction. Targeting these adaptive immune cells offers a promising approach to improve recovery following TBI.
Asunto(s)
Lesiones Traumáticas del Encéfalo/inmunología , Encéfalo/patología , Linfocitos T CD8-positivos/inmunología , Activación de Linfocitos/inmunología , Inmunidad Adaptativa , Animales , Autoanticuerpos/sangre , Linfocitos B/inmunología , Conducta Animal , Lesiones Traumáticas del Encéfalo/sangre , Lesiones Traumáticas del Encéfalo/fisiopatología , Linfocitos T CD4-Positivos/inmunología , ADN/inmunología , Marcha , Memoria Inmunológica , Depleción Linfocítica , Masculino , Ratones Endogámicos C57BL , Vaina de Mielina/inmunología , Médula Espinal/patología , Células Th17/inmunología , Factores de Tiempo , Microglobulina beta-2/deficiencia , Microglobulina beta-2/metabolismoRESUMEN
ß2m-/Thy1+ bone marrow-derived hepatocyte stem cells (BDHSCs) have a potential to be applied for cellular treatment in liver cirrhosis. However, the resultant tissue regeneration is restricted by transplanted cells' death. The accumulation of transforming growth factor beta 1 (TGF-ß1) in liver fibrosis local microenvironment may play an essential role in the rapid cell death of implanted ß2m-/Thy1+ BDHSCs. The main mechanism of poor survival of the target stem cells is still unknown. Delphinidin, an anthocyanidin, has potent antioxidant and anti-inflammatory activities. However, whether this bio-active ingredient can substantially contribute to ß2m-/Thy1+ BDHSCs' protection from TGF-ß1 induced apoptosis in vitro remains to be elucidated. In the present research, we determined whether delphinidin pretreatment can improve the survival of ß2m-/Thy1+ BDHSCs during exposure to TGF-ß1 and elucidated its underlying mechanisms. By using TGF-ß1, we induced the apoptosis of ß2m-/Thy1+ BDHSCs and assessed the apoptotic rates up to 24 h by flow cytometry. ß2m-/Thy1+ BDHSC proliferation was gauged using 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl- 2H-tetrazolium bromide (MTT) assay. The expression grades of Bcl-2, Akt, caspase-3, and Bax were observed through Western blot analysis. We found that delphinidin can significantly impede TGF-ß1-induced apoptosis dose-dependently, scavenge reactive oxygen species (ROS), and inhibit the discharge of caspase-3 in ß2m-/Thy1+ BDHSCs. We also demonstrated that delphinidin can activate the phosphatidylinositol-3-kinase (PI3K)/Akt signaling pathway. The suppression of ROS and succeeding apoptosis was achieved by pretreatment with LY294002, a PI3K/Akt pathway inhibitor. In summary, our findings revealed that delphinidin can protect ß2m-/Thy1+ BDHSCs from apoptosis and ROS-dependent oxidative stress induced by the TGF-ß1 via PI3K/Akt signaling pathway. On the basis of these data, delphinidin can be regarded as a promising anti-apoptotic agent for enhancing ß2m-/Thy1+ BDHSC survival during cell transplantation in liver cirrhosis patients.
Asunto(s)
Antocianinas/farmacología , Apoptosis/efectos de los fármacos , Hepatocitos/efectos de los fármacos , Estrés Oxidativo/efectos de los fármacos , Transducción de Señal/efectos de los fármacos , Células Madre/efectos de los fármacos , Factor de Crecimiento Transformador beta1/farmacología , Animales , Células de la Médula Ósea/citología , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Hepatocitos/citología , Hepatocitos/metabolismo , Masculino , Estructura Molecular , Fosfatidilinositol 3-Quinasas/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Ratas , Ratas Wistar , Especies Reactivas de Oxígeno/metabolismo , Células Madre/citología , Células Madre/metabolismo , Relación Estructura-Actividad , Antígenos Thy-1/metabolismo , Microglobulina beta-2/deficiencia , Microglobulina beta-2/metabolismoAsunto(s)
Linfocitos T CD8-positivos/enzimología , Dermatitis Alérgica por Contacto/enzimología , Granzimas/metabolismo , Antígenos de Histocompatibilidad Clase I/metabolismo , Tolerancia a Radiación , Animales , Linfocitos T CD8-positivos/inmunología , Linfocitos T CD8-positivos/efectos de la radiación , Dermatitis Alérgica por Contacto/genética , Dermatitis Alérgica por Contacto/inmunología , Dinitrofluorobenceno , Modelos Animales de Enfermedad , Granzimas/inmunología , Antígenos de Histocompatibilidad Clase I/inmunología , Interferón gamma/inmunología , Interferón gamma/metabolismo , Ratones Noqueados , Microglobulina beta-2/deficiencia , Microglobulina beta-2/genéticaRESUMEN
T cells directed against mutant neo-epitopes drive cancer immunity. However, spontaneous immune recognition of mutations is inefficient. We recently introduced the concept of individualized mutanome vaccines and implemented an RNA-based poly-neo-epitope approach to mobilize immunity against a spectrum of cancer mutations. Here we report the first-in-human application of this concept in melanoma. We set up a process comprising comprehensive identification of individual mutations, computational prediction of neo-epitopes, and design and manufacturing of a vaccine unique for each patient. All patients developed T cell responses against multiple vaccine neo-epitopes at up to high single-digit percentages. Vaccine-induced T cell infiltration and neo-epitope-specific killing of autologous tumour cells were shown in post-vaccination resected metastases from two patients. The cumulative rate of metastatic events was highly significantly reduced after the start of vaccination, resulting in a sustained progression-free survival. Two of the five patients with metastatic disease experienced vaccine-related objective responses. One of these patients had a late relapse owing to outgrowth of ß2-microglobulin-deficient melanoma cells as an acquired resistance mechanism. A third patient developed a complete response to vaccination in combination with PD-1 blockade therapy. Our study demonstrates that individual mutations can be exploited, thereby opening a path to personalized immunotherapy for patients with cancer.
Asunto(s)
Vacunas contra el Cáncer/genética , Vacunas contra el Cáncer/inmunología , Melanoma/inmunología , Melanoma/terapia , Mutación/genética , Medicina de Precisión/métodos , ARN/genética , Anticuerpos Monoclonales/farmacología , Anticuerpos Monoclonales/uso terapéutico , Antígeno B7-H1/inmunología , Antígenos CD8/inmunología , Vacunas contra el Cáncer/uso terapéutico , Epítopos/genética , Epítopos/inmunología , Humanos , Inmunoterapia/métodos , Melanoma/genética , Metástasis de la Neoplasia , Recurrencia Local de Neoplasia/prevención & control , Nivolumab , Receptor de Muerte Celular Programada 1/antagonistas & inhibidores , Linfocitos T/inmunología , Vacunación , Microglobulina beta-2/deficienciaRESUMEN
Donor major histocompatibility complex class I (MHC I) molecules are the main targets of the host immune response after organ allotransplantation. Whether and how MHC I-deficiency of pig donor tissues affects rejection after xenotransplantation has not been assessed. Beta2-microglobulin (B2M) is indispensable for the assembly of MHC I receptors and therefore provides an effective target to disrupt cell surface MHC I expression. Here, we report the one-step generation of mutant pigs with targeted disruptions in B2m by injection of porcine zygotes with B2m exon 2-specific TALENs. After germline transmission of mutant B2m alleles, we obtained F1 pigs with biallelic B2m frameshift mutations. F1 pigs lacked detectable B2M expression in tissues derived from the three germ layers, and their lymphocytes were devoid of MHC I surface receptors. Skin grafts from B2M deficient pigs exhibited remarkably prolonged survival on xenogeneic wounds compared to tissues of non-mutant littermates. Mutant founder pigs with bi-allelic disruption in B2m and B2M deficient F1 offspring did not display visible abnormalities, suggesting that pigs are tolerant to B2M deficiency. In summary, we show the efficient generation of pigs with germline mutations in B2m, and demonstrate a beneficial effect of donor MHC I-deficiency on xenotransplantation.
Asunto(s)
Animales Modificados Genéticamente/genética , Eliminación de Gen , Marcación de Gen/métodos , Porcinos/genética , Nucleasas de los Efectores Tipo Activadores de la Transcripción , Cigoto , Microglobulina beta-2/deficiencia , AnimalesRESUMEN
OBJECTIVE: This review focuses on the current knowledge on the implication and significance of beta 2 microglobulin (ß2M), a conservative immune molecule in vertebrate. DATA SOURCES: The data used in this review were obtained from PubMed up to October 2015. Terms of ß2M, immune response, and infection were used in the search. STUDY SELECTIONS: Articles related to ß2M were retrieved and reviewed. Articles focusing on the characteristic and function of ß2M were selected. The exclusion criteria of articles were that the studies on ß2M-related molecules. RESULTS: ß2M is critical for the immune surveillance and modulation in vertebrate animals. The dysregulation of ß2M is associated with multiple diseases, including endogenous and infectious diseases. ß2M could directly participate in the development of cancer cells, and the level of ß2M is deemed as a prognostic marker for several malignancies. It also involves in forming major histocompatibility complex (MHC class I or MHC I) or like heterodimers, covering from antigen presentation to immune homeostasis. CONCLUSIONS: Based on the characteristic of ß2M, it or its signaling pathway has been targeted as biomedical or therapeutic tools. Moreover, ß2M is highly conserved among different species, and overall structures are virtually identical, implying the versatility of ß2M on applications.
Asunto(s)
Microglobulina beta-2/fisiología , Antígenos CD1/fisiología , Proteína de la Hemocromatosis/análisis , Antígenos de Histocompatibilidad Clase I/fisiología , Humanos , Receptores Fc/fisiología , Microglobulina beta-2/sangre , Microglobulina beta-2/química , Microglobulina beta-2/deficienciaRESUMEN
BACKGROUND: Most patients with MHC class I (MHC-I) deficiency carry genetic defects in transporter associated with antigen processing 1 (TAP1) or TAP2. The clinical presentation can vary, and about half of the patients have severe skin disease. Previously, one report described ß2-microglobulin (ß2m) deficiency as another monogenetic cause of MHC-I deficiency, but no further immunologic evaluation was performed. OBJECTIVE: We sought to describe the molecular and immunologic features of ß2m deficiency in 2 Turkish siblings with new diagnoses. METHODS: Based on clinical and serologic findings, the genetic defect was detected by means of candidate gene analysis. The immunologic characterization comprises flow cytometry, ELISA, functional assays, and immunohistochemistry. RESULTS: Here we provide the first extensive clinical and immunologic description of ß2m deficiency in 2 siblings. The sister had recurrent respiratory tract infections and severe skin disease, whereas the brother was fairly asymptomatic but had bronchiectasis. Not only polymorphic MHC-I but also the related CD1a, CD1b, CD1c, and neonatal Fc receptor molecules were absent from the surfaces of ß2m-deficient cells. Absent neonatal Fc receptor surface expression led to low serum IgG and albumin levels in both siblings, whereas the heterozygous parents had normal results for all tested parameters except ß2m mRNA (B2M) expression. Similar to TAP deficiency in the absence of a regular CD8 T-cell compartment, CD8(+) γδ T cells were strongly expanded. Natural killer cells were normal in number but not "licensed to kill." CONCLUSION: The clinical presentation of patients with ß2m deficiency resembles that of patients with other forms of MHC-I deficiency, but because of the missing stabilizing effect of ß2m on other members of the MHC-I family, the immunologic defect is more extensive than in patients with TAP deficiency.
Asunto(s)
Bronquiectasia/inmunología , Síndromes de Inmunodeficiencia/inmunología , Infecciones del Sistema Respiratorio/inmunología , Úlcera Cutánea/inmunología , Microglobulina beta-2/inmunología , Inmunidad Adaptativa , Adolescente , Adulto , Antígenos CD1/genética , Antígenos CD1/inmunología , Bronquiectasia/complicaciones , Bronquiectasia/genética , Bronquiectasia/patología , Linfocitos T CD8-positivos/inmunología , Linfocitos T CD8-positivos/patología , Consanguinidad , Femenino , Eliminación de Gen , Antígenos de Histocompatibilidad Clase I/genética , Antígenos de Histocompatibilidad Clase I/inmunología , Humanos , Inmunidad Innata , Inmunoglobulina G/sangre , Inmunoglobulina G/genética , Síndromes de Inmunodeficiencia/complicaciones , Síndromes de Inmunodeficiencia/genética , Síndromes de Inmunodeficiencia/patología , Células Asesinas Naturales/inmunología , Células Asesinas Naturales/patología , Masculino , Linaje , Isoformas de Proteínas/deficiencia , Isoformas de Proteínas/genética , Isoformas de Proteínas/inmunología , Receptores Fc/deficiencia , Receptores Fc/genética , Receptores Fc/inmunología , Recurrencia , Infecciones del Sistema Respiratorio/complicaciones , Infecciones del Sistema Respiratorio/genética , Infecciones del Sistema Respiratorio/patología , Hermanos , Úlcera Cutánea/complicaciones , Úlcera Cutánea/genética , Úlcera Cutánea/patología , Microglobulina beta-2/deficiencia , Microglobulina beta-2/genéticaRESUMEN
Turnover of blood serum proteins is a vital function in mammals, but technical challenges have thus far prevented comprehensive measurements of serum protein half-lives. Here, we injected native serum from heavy stable isotope labeled (SIL) mice into nonlabeled recipients to quantify turnover of more than 200 proteins using mass spectrometry with high reproducibility and accuracy. We found a median of 19.4 h and a total range of 6-70 h for calculated half-lives. Moreover, we observed similar half-lives for proteins with equal function. To demonstrate the value and effectiveness of SILflood, we investigated the impaired serum clearance in ß2-microglobulin (B2M-/-) deficient mice. Notably, we found that serum albumin and IgG half-lives were clearly reduced in B2M deficient animals compared to control animals. Taken together, our results demonstrate that SILflood is a versatile tool to investigate serum half-lives under regular and pathological conditions in living animals.
Asunto(s)
Marcaje Isotópico , Suero/química , Animales , Isótopos de Carbono , Células Cultivadas , Semivida , Ensayos Analíticos de Alto Rendimiento , Inmunoglobulina G/sangre , Masculino , Espectrometría de Masas , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Albúmina Sérica/análisis , Microglobulina beta-2/deficienciaRESUMEN
PURPOSE: CD8(+) T lymphocytes can kill autologous melanoma cells, but their activity is impaired when poorly immunogenic tumor phenotypes evolve in the course of disease progression. Here, we analyzed three consecutive melanoma lesions obtained within one year of developing stage IV disease for their recognition by autologous T cells. EXPERIMENTAL DESIGN: One skin (Ma-Mel-48a) and two lymph node (Ma-Mel-48b, Ma-Mel-48c) metastases were analyzed for T-cell infiltration. Melanoma cell lines established from the respective lesions were characterized, determining the T-cell-stimulatory capacity, expression of surface molecules involved in T-cell activation, and specific genetic alterations affecting the tumor-T-cell interaction. RESULTS: Metastases Ma-Mel-48a and Ma-Mel-48b, in contrast with Ma-Mel-48c, were infiltrated by T cells. The T-cell-stimulatory capacity was found to be strong for Ma-Mel-48a, lower for Ma-Mel-48b, and completely abrogated for Ma-Mel-48c cells. The latter proved to be HLA class I-negative due to an inactivating mutation in one allele of the beta-2-microglobulin (B2M) gene and concomitant loss of the other allele by a deletion on chromosome 15q. The same deletion was already present in Ma-Mel-48a and Ma-Mel-48b cells, pointing to an early acquired genetic event predisposing to development of ß2m deficiency. Notably, the same chronology of genetic alterations was also observed in a second ß2m-deficient melanoma model. CONCLUSION: Our study reveals a progressive loss in melanoma immunogenicity during the course of metastatic disease. The genetic evolvement of T-cell resistance suggests screening tumors for genetic alterations affecting immunogenicity could be clinically relevant in terms of predicting patient responses to T-cell-based immunotherapy.
Asunto(s)
Evolución Molecular , Melanoma/genética , Melanoma/inmunología , Subgrupos de Linfocitos T/inmunología , Subgrupos de Linfocitos T/metabolismo , Alelos , Antígenos B7/genética , Antígenos B7/inmunología , Butorfanol , Línea Celular Tumoral , Análisis por Conglomerados , Citocinas/metabolismo , Progresión de la Enfermedad , Expresión Génica , Predisposición Genética a la Enfermedad , Antígenos de Histocompatibilidad Clase I/genética , Antígenos de Histocompatibilidad Clase I/inmunología , Humanos , Linfocitos Infiltrantes de Tumor/inmunología , Linfocitos Infiltrantes de Tumor/patología , Melanoma/patología , Melanoma/terapia , Mutación , Metástasis de la Neoplasia , Estadificación de Neoplasias , Fenotipo , Polimorfismo de Nucleótido Simple , Microglobulina beta-2/deficiencia , Microglobulina beta-2/genéticaRESUMEN
Expression of MHC class I molecules, which provide immune surveillance against intracellular pathogens, is higher on lymphoid cells than on any other cell types. In T cells, this is a result of activation of class I transcription by the T cell enhanceosome consisting of Runx1, CBFß, and LEF1. We now report that MHC class I transcription in T cells also is enhanced by Foxp3, resulting in higher levels of class I in CD4(+)CD25(+) T regulatory cells than in conventional CD4(+)CD25(-) T cells. Interestingly, the effect of Foxp3 regulation of MHC class I transcription is cell type specific: Foxp3 increases MHC class I expression in T cells but represses it in epithelial tumor cells. In both cell types, Foxp3 targets the upstream IFN response element and downstream core promoter of the class I gene. Importantly, expression of MHC class I contributes to the function of CD4(+)CD25(+) T regulatory cells by enhancing immune suppression, both in in vitro and in vivo. These findings identify MHC class I genes as direct targets of Foxp3 whose expression augments regulatory T cell function.
Asunto(s)
Factores de Transcripción Forkhead/inmunología , Regulación de la Expresión Génica/inmunología , Antígenos de Histocompatibilidad Clase I/inmunología , Linfocitos T Reguladores/inmunología , Animales , Secuencia de Bases , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD4-Positivos/metabolismo , Células Cultivadas , Citometría de Flujo , Factores de Transcripción Forkhead/genética , Factores de Transcripción Forkhead/metabolismo , Células HeLa , Antígenos de Histocompatibilidad Clase I/genética , Antígenos de Histocompatibilidad Clase I/metabolismo , Humanos , Células Jurkat , Células MCF-7 , Ratones , Ratones Endogámicos BALB C , Ratones Noqueados , Ratones Transgénicos , Datos de Secuencia Molecular , Regiones Promotoras Genéticas/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Homología de Secuencia de Ácido Nucleico , Linfocitos T Reguladores/metabolismo , Timocitos/inmunología , Timocitos/metabolismo , Microglobulina beta-2/deficiencia , Microglobulina beta-2/genética , Microglobulina beta-2/inmunologíaRESUMEN
BACKGROUND & AIMS: CD4(+) T cells specific for dietary gluten and interleukin 15 (IL15) contribute to the pathogenesis of celiac disease. We investigated whether and how they interact to damage the intestine using mice that overexpress human IL15 in the intestinal epithelium and have CD4(+) T cells specific for ovalbumin, a dietary antigen. METHODS: We crossed mice with CD4(+) T cells specific for ovalbumin (OTII) with mice that overexpress human IL15 under an intestine-specific promoter (B6 × IL15Tge). The offspring (OTII × IL15Tge mice) received control or ovalbumin-containing diets until 3 months of age. Enteropathy was monitored by weight, ratio of villous:crypt length, and the number of intestinal lymphocytes. Phenotype, cytokine production, and degranulation of mucosal and spleen lymphocytes were analyzed by multicolor flow cytometry or enzyme-linked immunosorbent assay. Regulatory T-cell function and CD8(+) T-cell activation were analyzed in co-culture assays. RESULTS: Exposure to ovalbumin reduced growth and led to enteropathy in OTII × IL15Tge mice but not in control OTII × B6 littermates. Enteropathy was associated with expansion of mucosal granzyme B(+) CD8(+) T cells, and developed despite increased frequency of functional ovalbumin-specific regulatory T cells. Ovalbumin-activated CD4(+) T cells secreted IL2, which along with IL15 stimulated expansion of noncognate intestinal cytotoxic CD8(+) T cells, which did not respond to regulatory T cells and induced epithelial damage. CONCLUSIONS: We observed that in mice given food antigen, cooperation between IL15 and CD4(+) T cells is necessary and sufficient to activate CD8(+) T cells and damage the small intestine. We propose that this process is involved in the development of celiac disease.
Asunto(s)
Antígenos , Linfocitos T CD4-Positivos/inmunología , Enfermedad Celíaca/inmunología , Dieta , Interleucina-15/metabolismo , Intestino Delgado/inmunología , Ovalbúmina/inmunología , Traslado Adoptivo , Animales , Linfocitos T CD4-Positivos/metabolismo , Linfocitos T CD4-Positivos/trasplante , Linfocitos T CD8-positivos/inmunología , Linfocitos T CD8-positivos/metabolismo , Enfermedad Celíaca/metabolismo , Enfermedad Celíaca/patología , Degranulación de la Célula , Proliferación Celular , Células Cultivadas , Técnicas de Cocultivo , Citocinas/metabolismo , Proteínas de Unión al ADN/deficiencia , Proteínas de Unión al ADN/genética , Modelos Animales de Enfermedad , Granzimas/metabolismo , Antígenos de Histocompatibilidad Clase II/genética , Antígenos de Histocompatibilidad Clase II/metabolismo , Humanos , Inmunidad Mucosa , Interleucina-15/genética , Interleucina-2/metabolismo , Mucosa Intestinal/inmunología , Mucosa Intestinal/metabolismo , Intestino Delgado/metabolismo , Intestino Delgado/patología , Activación de Linfocitos , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ovalbúmina/administración & dosificación , Fenotipo , Transducción de Señal , Bazo/inmunología , Bazo/metabolismo , Linfocitos T Reguladores/inmunología , Linfocitos T Reguladores/metabolismo , Microglobulina beta-2/deficiencia , Microglobulina beta-2/genéticaRESUMEN
MHC class I molecules play an important role in synaptic plasticity of the mammalian nervous system. Proteolytic complexes (proteasomes) produce oligopeptides that are presented on cell surfaces in complexes with MHC class I molecules and regulate many cellular processes beside this. The goal of the present work was to study peculiarities in functioning of proteasomes and associated signaling pathways along with evaluation of NeuN and gFAP expression in different sections of the brain in mice with knockout of ß2-microglobulin, a constituent of MHC class I molecules. It was found that the frontal cortex and the brainstem, structures with different ratio of NeuN and gFAP expression, are characterized by opposite changes in the proteasome pool under constant total proteasome levels in B2m-knockout mice in comparison with those in control animals. ChTL-activity as well as expression of LMP7 immune subunit and PA28 regulator of proteasomes was elevated in the cortex of B2m-knockout mice, while these indicators were decreased in the brainstem. The concentrations of the signaling molecules nNOS and HSP70 in B2m-knockout mice were increased in the cortex, while being decreased in the brainstem, and this indicates the possibility of control of expression of the LMP7 subunit and the regulator PA28 by these molecules. Changes in the proteasome pool observed in striatum of B2m-knockout mice are similar to those observed in the brainstem. At the same time, the cerebellum is characterized by a specific pattern of proteasome functioning in comparison with that in all other brain structures. In cerebellum the expression of immune subunits LMP7 and LMP2 and the regulator PA28 was increased, while expression of regulator PA700 was decreased. Deficiency of NeuN and gFAP was revealed in most brain compartments of B2m-knockout mice. Thus, increased expression of the above-mentioned immune subunits and the proteasome regulator PA28 in the cortex and cerebellum may compensate disturbances revealed in the brain structures and the absence of MHC class I molecules. Apparently, this promotes production of peptides necessary for cell-to-cell interactions and maintains nervous system plasticity in B2m-knockout mice.
Asunto(s)
Encéfalo/metabolismo , Complejo de la Endopetidasa Proteasomal/metabolismo , Microglobulina beta-2/deficiencia , Animales , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Complejo de la Endopetidasa Proteasomal/química , Transducción de Señal , Microglobulina beta-2/genéticaRESUMEN
Immune rejection hinders the application of human embryonic stem cells (hESCs) in transplantation therapy. Human leukocyte antigens (HLAs) on the cell surface are the major cause of graft rejection. In this study, we generated HLA class I-deficient hESCs via disruption of beta 2-microglobulin (ß2m), the light chain of HLA Class I. We found that HLA class I proteins were not present on the cell surface of ß2m-null hESCs. These cells showed the same pluripotency as wildtype hESCs and demonstrated hypoimmunogenicity. Thus, HLA class I-deficient hESCs might serve as an unlimited cell source for the generation of universally compatible "off-the-shelf" cell grafts, tissues or organs in the future.
Asunto(s)
Células Madre Embrionarias/inmunología , Microglobulina beta-2/metabolismo , Secuencia de Bases , Células Madre Embrionarias/citología , Antígenos de Histocompatibilidad Clase I/metabolismo , Humanos , Datos de Secuencia Molecular , Células Madre Pluripotentes/citología , Células Madre Pluripotentes/metabolismo , Factores de Transcripción/metabolismo , Microglobulina beta-2/deficienciaRESUMEN
We investigated the role of effector CD8 T cells in the pathogenesis of immune glomerular injury. BALB/c mice are not prone to autoimmune disease, but after 12 immunizations with OVA they developed a variety of autoantibodies and glomerulonephritis accompanied by immune complex (IC) deposition. In these mice, IFN-γ-producing effector CD8 T cells were significantly increased concomitantly with glomerulonephritis. In contrast, after 12 immunizations with keyhole limpet hemocyanin, although autoantibodies appeared, IFN-γ-producing effector CD8 T cells did not develop, and glomerular injury was not induced. In ß2-microglobulin-deficient mice lacking CD8 T cells, glomerular injury was not induced after 12 immunizations with OVA, despite massive deposition of IC in the glomeruli. In mice containing a targeted disruption of the exon encoding the membrane-spanning region of the Ig µ-chain (µMT mice), 12 immunizations with OVA induced IFN-γ-producing effector CD8 T cells but not IC deposition or glomerular injury. When CD8 T cells from mice immunized 12 times with OVA were transferred into naive recipients, glomerular injury could be induced, but only when a single injection of OVA was also given simultaneously. Importantly, injection of OVA could be replaced by one injection of the sera from mice that had been fully immunized with OVA. This indicates that deposition of IC is required for effector CD8 T cells to cause immune tissue injury. Thus, in a mouse model of systemic lupus erythematosus, glomerular injury is caused by effector CD8 T cells that recognize Ag presented as IC on the target renal tissue.
Asunto(s)
Presentación de Antígeno/inmunología , Complejo Antígeno-Anticuerpo/inmunología , Linfocitos T CD8-positivos/inmunología , Glomerulonefritis/inmunología , Interferón gamma/biosíntesis , Animales , Presentación de Antígeno/genética , Complejo Antígeno-Anticuerpo/metabolismo , Linfocitos T CD8-positivos/metabolismo , Linfocitos T CD8-positivos/patología , Femenino , Glomerulonefritis/metabolismo , Glomerulonefritis/patología , Cadenas mu de Inmunoglobulina/genética , Interferón gamma/fisiología , Lupus Eritematoso Sistémico/inmunología , Lupus Eritematoso Sistémico/patología , Ratones , Ratones Endogámicos BALB C , Ratones Noqueados , Ovalbúmina/administración & dosificación , Ovalbúmina/inmunología , Linfocitos T Citotóxicos/inmunología , Linfocitos T Citotóxicos/patología , Microglobulina beta-2/deficienciaRESUMEN
Human leukocyte antigen-G (HLA-G) is a nonclassical HLA class-I molecule and plays a role in tissue specific immunoregulation. Many studies have addressed functional aspects of ß2-microglobulin (ß2m)-associated HLA-G1. ß2m-free HLA-G has been found in human placental cytotrophoblasts and pancreatic ß cells although its function remains unclear. In the present study, we investigated the function of ß2m-free HLA-G by transfecting HLA-G1 and -G3 into human ß2m deficient rat pancreatic ß cell carcinoma (BRIN-BD11) cells. RT-PCR and western blots studies confirmed high expression of HLA-G1 and -G3 in -G1 and -G3 transfectants, respectively. HLA-G1 and -G3 were detected mainly in intracellular compartments of BRIN-BD11 transductants by confocal fluorescent microscopy and flow cytometry. Functional analysis revealed that ß2m-free HLA-G promoted xenogeneic cytotoxic lysis of BRIN-BD11 cells by natural killer (NK) cells and increased production of IL-1ß, TNF-α, and IFN-γ. Stimulation of cytotoxic lysis was impaired by blocking the MAPK and DNA-PKcs pathways in NK cells. Importantly, treatment with 33mAb, a KLR2DL4 receptor agonist, induced NK-mediated cytotoxic lysis of BRIN-BD11 cells transfected with a mock vector. Our data suggest that ß2m-free HLA-G activates NK cells via engagement of KLR2DL4 receptors.