RESUMEN
AIMS: Immunomodulation with autoantigens potentially constitutes a specific and safe treatment for type 1 diabetes (T1D). Studies with GAD-alum administrated subcutaneously have shown to be safe, but its efficacy has been inconclusive. Administration of GAD-alum into the lymph nodes, aimed to optimise antigen presentation, has shown promising results in an open-label clinical trial. Herein, we compared the immune response of the individuals included in the trial with a group who received GAD-alum subcutaneously in a previous study. MATERIALS AND METHODS: Samples from T1D individuals collected 15 months after administration of either three doses 1 month apart of 4 µg GAD-alum into lymph nodes (LN, n = 12) or two doses 1 month apart of 20 µg subcutaneously (SC, n = 12) were studied. GADA, GADA subclasses, GAD65 -induced cytokines, peripheral blood mononuclear cell proliferation, and T cells markers were analysed. RESULTS: Low doses of GAD-alum into the lymph nodes induced higher GADA levels than higher doses administrated subcutaneously. Immune response in the LN group was characterised by changes in GADA subclasses, with a relative reduction of IgG1 and enhanced IgG2, IgG3, and IgG4 proportion, higher GAD65 -induced secretion of IL-5, IL-10, and TNF-α, and reduction of cell proliferation and CD8+ T cells. These changes were not observed after subcutaneous (SC) injections of GAD-alum. CONCLUSIONS: GAD-specific immune responses 15 months after lymph node injections of GAD-alum differed from the ones induced by SC administration of the same autoantigen.
Asunto(s)
Diabetes Mellitus Tipo 1 , Compuestos de Alumbre , Linfocitos T CD8-positivos , Glutamato Descarboxilasa , Humanos , Inmunidad , Leucocitos MononuclearesRESUMEN
Administration of Glutamic Acid Decarboxylase (GAD)65 formulated in aluminium hydroxide preserved insulin secretion in a phase II trial in recent onset Type 1 Diabetes. A subsequent European phase III trial was closed at 15months after failing to reach primary endpoint, but the majority of the Swedish patients completed the 21months follow-up. We studied the frequencies and phenotype of T cells, suppressive capacity of Tregs, GAD65-induced proliferation, and frequencies of T cells with a GAD65-specific TCR in Swedes participating in the trial. Stimulation with GAD65 induced activated T cells and also cells with a suppressive phenotype. Activated GAD65-specific effector T cells were detected by tetramer staining while the frequency of GAD65-specific Treg was not affected by the treatment. Additional doses of GAD-alum increased frequencies of CD25+CD127+, but had no effect on CD25hiCD127lo. Our findings indicate that GAD-alum treatment primarily induced activated T cells. GAD65-specific cells were mainly of activated phenotype.
Asunto(s)
Diabetes Mellitus Tipo 1/tratamiento farmacológico , Diabetes Mellitus Tipo 1/inmunología , Glutamato Descarboxilasa/administración & dosificación , Linfocitos T Reguladores/efectos de los fármacos , Linfocitos T Reguladores/inmunología , Adolescente , Adulto , Niño , Método Doble Ciego , Femenino , Estudios de Seguimiento , Humanos , Subunidad alfa del Receptor de Interleucina-2/inmunología , Subunidad alfa del Receptor de Interleucina-7/inmunología , Activación de Linfocitos/efectos de los fármacos , Activación de Linfocitos/inmunología , Masculino , Suecia , Adulto JovenRESUMEN
Regulatory T cells are an important component of the immune system that plays a key role in maintaining homeostasis. Identification of distinct regulatory T cell subsets is essential to understand their function. Mass cytometry or CyTOF is a technology that enables the simultaneous measurement of up to 50 markers in single cells by using antibodies tagged with heavy metals, which are then detected with time-of-flight mass spectrometry. This chapter describes a mass cytometry approach for phenotypic characterization of regulatory T cells and determination of their master transcription factor Foxp3.
Asunto(s)
Metales Pesados , Linfocitos T Reguladores , Citometría de Flujo/métodos , Factores de Transcripción Forkhead/genética , Inmunofenotipificación , Subgrupos de Linfocitos TRESUMEN
GAD-alum given into lymph nodes to Type 1 diabetes (T1D) patients participating in a multicenter, randomized, placebo-controlled double-blind study seemed to have a positive effect for patients with DR3DQ2 haplotype, who showed better preservation of C-peptide than the placebo group. Here we compared the immunomodulatory effect of GAD-alum administered into lymph nodes of patients with T1D versus placebo with focus on patients with DR3DQ2 haplotype. Methods: GAD autoantibodies, GADA subclasses, GAD65-induced cytokine secretion (Luminex panel) and proliferation of peripheral mononuclear cells were analyzed in T1D patients (n=109) who received either three intra-lymphatic injections (one month apart) with 4 µg GAD-alum and oral vitamin D supplementation (2000 IE daily for 120 days), or placebo. Results: Higher GADA, GADA subclasses, GAD65-induced proliferation and cytokine secretion was observed in actively treated patients after the second injection of GAD-alum compared to the placebo group. Following the second injection of GAD-alum, actively treated subjects with DR3DQ2 haplotype had higher GAD65-induced secretion of several cytokine (IL4, IL5, IL7, IL10, IL13, IFNγ, GM-CSF and MIP1ß) and proliferation compared to treated individuals without DR3DQ2. Stratification of samples from GAD-alum treated patients according to C-peptide preservation at 15 months revealed that "good responder" individuals with better preservation of C-peptide secretion, independently of the HLA haplotype, had increased GAD65-induced proliferation and IL13 secretion at 3 months, and a 2,5-fold increase of IL5 and IL10 as compared to "poor responders". The second dose of GAD-alum also induced a more pronounced cytokine secretion in "good responders" with DR3DQ2, compared to few "good responders" without DR3DQ2 haplotype. Conclusion: Patients with DR3DQ2 haplotype had a distinct early cellular immune response to GAD-alum injections into the lymph node, and predominant GAD65-induced IL13 secretion and proliferation that seems to be associated with a better clinical outcome. If confirmed in the ongoing larger randomized double-blind placebo-controlled clinical trial (DIAGNODE-3), including only patients carrying DR3DQ2 haplotype, these results might be used as early surrogate markers for clinical efficacy.
Asunto(s)
Diabetes Mellitus Tipo 1 , Humanos , Péptido C , Citocinas/uso terapéutico , Glutamato Descarboxilasa , Haplotipos , Inmunidad Celular , Interleucina-10 , Interleucina-13 , Interleucina-5 , Antígenos HLA/inmunologíaRESUMEN
The frequency of apoptotic cells in a given phenotypically defined population is usually calculated the apoptotic index (AI), i.e., the percentage of apoptotic cells displaying a specific linage antigen (LAg) within a population of cells that remain unfragmented and retain the expression of the LAg. However, this approach has two major limitations. Firstly, apoptotic cells fragment into apoptotic bodies that later disintegrate. Secondly, apoptotic cells frequently lose, partially or even completely, the cell surface expression of the LAg used for the identification of specific cell subsets. The present chapter will describe a flow cytometry method to calculate the apoptotic rate (AR) that takes into account both cell fragmentation and loss of lineage antigen expression on measurement of apoptosis using flow cytometry ratiometric cell enumeration that emerges as a more accurate method of measurement of the occurrence of apoptosis in normal and tumoral cell cultures.
Asunto(s)
Apoptosis , Neoplasias , Membrana Celular , Citometría de Flujo/métodosRESUMEN
AIM: To evaluate the long-term effect of intra-lymphatic administration of GAD-alum and a booster dose 2.5 years after the first intervention (DIAGNODE Extension study) in patients with recent-onset type 1 diabetes. METHODS: DIAGNODE-1: Samples were collected from 12 patients after 30 months who had received 3 injections of 4 µg GAD-alum into a lymph node with one-month interval. DIAGNODE Extension study: First in human, a fourth booster dose of autoantigen (GAD-alum) was given to 3 patients at 31.5 months, who were followed for another 12 months. C-peptide was measured during mixed meal tolerance tests (MMTTs). GADA, IA-2A, GADA subclasses, GAD65-induced cytokines, PBMCs proliferation and T cells markers were analyzed. RESULTS: After 30-month treatment, efficacy was still seen in 8/12 patients (good responders, GR). Partial remission (IDAA1c < 9) had decreased compared to 15 months, but did not differ from baseline, and HbA1c remained stable. GAD65-specific immune responses induced by the treatment started to wane after 30 months, and most changes observed at 15 months were undetectable. GADA subclasses IgG2, IgG3 and IgG4 were predominant in the GR along with IgG1. A fourth intra-lymphatic GAD-alum dose to three patients after 31.5 months gave no adverse events. In all three patients, C-peptide seemed to increase the first 6 months, and thereafter, C-peptide, HbA1c, insulin requirement and IDAA1c remained stable. CONCLUSION: The effect of intra-lymphatic injections of GAD-alum had decreased after 30 months. Good responders showed a specific immune response. Administration of a fourth booster dose after 31.5 months was safe, and there was no decline in C-peptide observed during the 12-month follow-up.
Asunto(s)
Diabetes Mellitus Tipo 1 , Compuestos de Alumbre , Autoanticuerpos , Péptido C , Diabetes Mellitus Tipo 1/terapia , Estudios de Seguimiento , Glutamato Descarboxilasa , Hemoglobina Glucada , Humanos , Inmunoglobulina GRESUMEN
Human lymphocytes lose the expression of lineage antigens (LAgs) along apoptosis. Our aim was to extent our previous studies of LAg loss to rodent species, quantifying LAg expression on apoptotic murine lymphocytes using flow cytometry to measure alterations in cell permeability, phosphatidylserine exposure and caspase activation of CD3, CD5, CD4, CD8, CD19 and CD28 LAgs in highly purified lymphocyte populations. We found loss of expression by apoptotic cells of all LAgs studied in the three species analyzed except for CD3 antigen in mouse. We also found an early, rapid and dramatic reduction in the expression of CD28 by early apoptotic cells. We found several homologies across the three species in the kinetic of loss of several LAgs such as CD5, CD4 and CD28. These data suggest that the loss of expression of LAgs by apoptotic lymphocytes is a common and conserved feature of lymphocytes undergoing apoptosis in several mammalian species.
Asunto(s)
Antígenos de Superficie/inmunología , Apoptosis/inmunología , Linfocitos/inmunología , Animales , Antígenos CD19/inmunología , Antígenos CD19/metabolismo , Antígenos de Superficie/metabolismo , Antígenos CD28/inmunología , Antígenos CD28/metabolismo , Complejo CD3/inmunología , Complejo CD3/metabolismo , Antígenos CD4/inmunología , Antígenos CD4/metabolismo , Antígenos CD5/inmunología , Antígenos CD5/metabolismo , Antígenos CD8/inmunología , Antígenos CD8/metabolismo , Caspasa 3/inmunología , Caspasa 3/metabolismo , Caspasa 8/inmunología , Caspasa 8/metabolismo , Caspasa 9/inmunología , Caspasa 9/metabolismo , Caspasas/inmunología , Caspasas/metabolismo , Células Cultivadas , Citometría de Flujo , Linfocitos/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Ratas , Ratas WistarRESUMEN
Antigen-specific immunotherapy is an appealing strategy to preserve beta-cell function in type 1 diabetes, although the approach has yet to meet its therapeutic endpoint. Direct administration of autoantigen into lymph nodes has emerged as an alternative administration route that can improve the efficacy of the treatment. In the first open-label clinical trial in humans, injection of aluminum-formulated glutamic acid decarboxylase (GAD-alum) into an inguinal lymph node led to the promising preservation of C-peptide in patients with recent-onset type 1 diabetes. The treatment induced a distinct immunomodulatory effect, but the response at the cell level has not been fully characterized. Here we used mass cytometry to profile the immune landscape in peripheral blood mononuclear cells from 12 participants of the study before and after 15 months of treatment. The immunomodulatory effect of the therapy included reduction of naïve and unswitched memory B cells, increase in follicular helper T cells and expansion of PD-1+ CD69+ cells in both CD8+ and double negative T cells. In vitro stimulation with GAD65 only affected effector CD8+ T cells in samples collected before the treatment. However, the recall response to antigen after 15 months included induction of CXCR3+ and CD11c+Tbet+ B cells, PD-1+ follicular helper T cells and exhausted-like CD8+ T cells. This study provides a deeper insight into the immunological changes associated with GAD-alum administration directly into the lymph nodes.
Asunto(s)
Linfocitos B/inmunología , Linfocitos T CD8-positivos/inmunología , Linfocitos T CD8-positivos/metabolismo , Diabetes Mellitus Tipo 1/etiología , Glutamato Descarboxilasa/administración & dosificación , Inmunomodulación/efectos de los fármacos , Receptor de Muerte Celular Programada 1/metabolismo , Células T Auxiliares Foliculares/metabolismo , Linfocitos B/metabolismo , Biomarcadores , Diabetes Mellitus Tipo 1/metabolismo , Susceptibilidad a Enfermedades , Humanos , Inmunofenotipificación , Inyecciones Intralinfáticas , Células T de Memoria/inmunología , Células T de Memoria/metabolismo , Células T Asesinas Naturales/inmunología , Células T Asesinas Naturales/metabolismo , Células T Auxiliares Foliculares/inmunologíaRESUMEN
In spite of intensive treatment Type 1 diabetes leads to serious complications. Preservation of residual beta cell function makes the disease milder, facilitates treatment, prevents complications and increase survival. So far immune interventions have had limited effect, and some serious adverse events and risks. In an open pilot trial we aimed to improve efficacy of GAD-alum treatment using lymph-node administration in combination with oral vitamin D. Here we report the clinical effect and focus on biomarkers for response to treatment. Patients (n = 12) aged 12 to 24 years with recent onset of Type 1 diabetes received 4 µg GAD-alum into lymph-node at day 30, 60, and 90, and oral Vitamin D 2000 U/d, days 1 to 120. Beta cell function was estimated by Mixed Meal Tolerance Tests. GADA, GADA subclasses, GAD65-induced cytokines and proliferation, and T cells markers were analyzed. The treatment was tolerable with no adverse events. Fasting C-peptide and insulin requirement remained stable at 15 months, while HbA1c was lower than baseline. Stimulated C-peptide showed no change at 6 months but declined after 15 months (81% of baseline). Eleven patients remained in partial remission (IDAAC < 9). Patients (n = 9) with better clinical outcome had reduced proportion of IgG1 and increased IgG2, IgG3, and IgG4, increased IL-10 secretion, and reduction of proliferation and CD8+ T cells activation. Patients with poorer clinical response had higher baseline levels of GAD65-induced cytokines and T-cell activation, and an increased ratio of effector/central memory T cells. Intra-lymphatic GAD treatment combined with Vitamin D might preserve beta cell function and improve clinical course in T1D. Patients with less benefit have a different quality of immune response both before and after treatment. Clinical Trial Registration: clinicaltrials.gov, identifier NCT02352974.
Asunto(s)
Hidróxido de Aluminio/administración & dosificación , Diabetes Mellitus Tipo 1/tratamiento farmacológico , Diabetes Mellitus Tipo 1/inmunología , Glutamato Descarboxilasa/administración & dosificación , Inmunidad/efectos de los fármacos , Células Secretoras de Insulina/efectos de los fármacos , Ganglios Linfáticos/inmunología , Vitamina D/administración & dosificación , Vitaminas/administración & dosificación , Administración Oral , Adolescente , Péptido C/metabolismo , Linfocitos T CD8-positivos/inmunología , Niño , Femenino , Humanos , Inmunoglobulina G/metabolismo , Inyecciones Intralinfáticas , Interleucina-10/metabolismo , Activación de Linfocitos/efectos de los fármacos , Masculino , Transducción de Señal/efectos de los fármacos , Resultado del Tratamiento , Adulto JovenRESUMEN
Although there is evidence that autoimmune diseases share similar immunogenetic mechanisms, studies comparing peripheral CD45+ cells from patients with autoimmune endocrine diseases in parallel are limited. In this study, we applied high-dimensional single-cell mass cytometry to phenotypically characterize PBMC from patients with new-onset (N-T1D) and long-standing type 1 diabetes, Hashimoto's thyroiditis (HT), Graves' disease and autoimmune Addison's disease (AD), as well as healthy controls. The frequency of CD20loCD27hiCD38hiHLA-DRint plasmablasts, CD86+CD14loCD16+ non-classical monocytes and two subsets of CD56dimHLA-DR+IFN-γ+ NK cells were increased in patients with HT. Subsets of CD56dimCD69+HLA-DR- NK cells and CD8+ TEMRA cells, both expressing IFN-γ, were expanded and reduced, respectively, in the N-T1D group. In addition, patients with AD were characterized by an increased percentage of central memory CD8+ T cells that expressed CCR4, GATA3, and IL-2. We demonstrate that patients with N-T1D, HT, and AD had altered frequencies of distinct subsets within antigen-presenting and cytotoxic cell lineages. Previously unreported alterations of specific cell subsets were identified in samples from patients with HT and AD. Our study might contribute to a better understanding of shared and diverging immunological features between autoimmune endocrine diseases.
Asunto(s)
Enfermedades Autoinmunes/metabolismo , Células Dendríticas/inmunología , Enfermedades del Sistema Endocrino/metabolismo , Células Asesinas Naturales/inmunología , Espectrometría de Masas/métodos , Linfocitos T/inmunología , Adulto , Anticuerpos/química , Anticuerpos/metabolismo , Enfermedades Autoinmunes/patología , Diferenciación Celular , Linaje de la Célula , Enfermedades del Sistema Endocrino/patología , Femenino , Citometría de Flujo , Humanos , Elementos de la Serie de los Lantanoides/química , MasculinoRESUMEN
Abnormal apoptosis has been reported in circulating T lymphocytes in patients with multiple sclerosis. The effects of 12 months of IFNbeta treatment in T and B lymphocyte spontaneous ex vivo apoptosis were studied in patients with MS. Peripheral blood mononuclear cells were obtained from 48 patients before and at 1, 6 and 12 months after treatment with IFNbeta. Spontaneous ex vivo apoptosis was quantified by four-color flow cytometry. A significant reduction and normalization of the percentage of apoptotic cells was found in all T lymphocyte subsets. B cell apoptosis values were unaffected by therapy; Relapses of the clinical activity of the disease were associated to transitory upturns of lymphocyte apoptosis. In conclusion, IFNbeta therapy progressively normalizes the increased ex vivo T lymphocyte apoptosis observed in MS. However, it is not clear if this reduction in spontaneous T lymphocyte apoptosis is due to direct effect of IFNbeta or secondary to decreased clinical and sub-clinical activity.
Asunto(s)
Apoptosis/efectos de los fármacos , Interferón beta/uso terapéutico , Esclerosis Múltiple/tratamiento farmacológico , Linfocitos T/efectos de los fármacos , Adulto , Complejo CD3/inmunología , Antígenos CD5/inmunología , Antígenos CD8/inmunología , Femenino , Citometría de Flujo/métodos , Humanos , Factores Inmunológicos/administración & dosificación , Factores Inmunológicos/uso terapéutico , Interferón beta/administración & dosificación , Antígenos Comunes de Leucocito/inmunología , Masculino , Persona de Mediana Edad , Esclerosis Múltiple/sangre , Esclerosis Múltiple/inmunología , Método Simple Ciego , Linfocitos T/inmunología , Linfocitos T/patología , Factores de Tiempo , Resultado del TratamientoRESUMEN
INTRODUCTION: Given the pivotal role of T lymphocytes in the immune system, patients with septic shock may show T cell abnormalities. We have characterised the T cell compartment in septic shock and assess its clinical implications. METHODS: T lymphocytes from the peripheral blood of 52 patients with septic shock and 36 healthy control subjects were analysed on admission to the intensive care unit, baseline, and 3, 7, 14 and 28 days later. T cell phenotypes (CD3+CD4+/CD3+CD8+, CD45RA+/CD45RO+, CD62L+/CD28+) were assessed by quantitative flow cytometry. RESULTS: CD3+, CD3+CD4+ and CD3+CD8+ lymphocyte counts were significantly lower in patients with septic shock than control subjects. In surviving patients, CD3+CD4+ lymphocytes had normalised after 14 days, yet CD3+CD8+ numbers were still low. Non effector CD45RA+CD45RO- subsets of CD3+CD4+ and CD3+CD8+ were persistently low during patient follow up. CD3+CD8+CD28+ and CD3+CD8+CD62L+ were reduced in patients versus controls and survivors versus nonsurvivors in the first three days. A prediction receptor operative curve revealed that for the CD3+CD8+CD28+ subset, a cutoff of 136 cells/ml showed 70% sensitivity and 100% specificity for predicting death and the area under the curve was 0.84 at admission. Corresponding values for CD3+CD8+CD62L+ were 141 cells/ml, 60% sensitivity, 100% specificity and an area under the curve of 0.75. CONCLUSIONS: A severe redistribution of T lymphocyte subsets is found in septic shock patients. A different kinetic pattern of T cell subset involvement is observed in surviving and nonsurviving patients, with lower numbers of circulating CD3+CD8+CD28+ and CD3+CD8+CD62L+ associated with a better disease outcome.
Asunto(s)
Choque Séptico/sangre , Choque Séptico/patología , Subgrupos de Linfocitos T/patología , Anciano , Femenino , Estudios de Seguimiento , Humanos , Leucocitos Mononucleares/patología , Recuento de Linfocitos/métodos , Recuento de Linfocitos/tendencias , Masculino , Persona de Mediana EdadRESUMEN
Type 1 diabetes (T1D) is characterized by autoimmune destruction of insulin producing ß-cells. The time from onset of islet autoimmunity to manifest clinical disease can vary widely in length, and it is fairly uncharacterized both clinically and immunologically. In the current study, peripheral blood mononuclear cells from autoantibody-positive children with high risk for T1D, and from age-matched healthy individuals, were analyzed by mass cytometry using a panel of 32 antibodies. Surface markers were chosen to identify multiple cell types including T, B, NK, monocytes, and DC, and antibodies specific for identification of differentiation, activation and functional markers were also included in the panel. By applying dimensional reduction and computational unsupervised clustering approaches, we delineated in an unbiased fashion 132 phenotypically distinct subsets within the major immune cell populations. We were able to identify an effector memory Treg subset expressing HLA-DR, CCR4, CCR6, CXCR3, and GATA3 that was increased in the high-risk group. In addition, two subsets of NK cells defined by CD16+ CD8+ CXCR3+ and CD16+ CD8+ CXCR3+ CD11c+ were also higher in the same subjects. High-risk individuals did not show impaired glucose tolerance at the time of sampling, suggesting that the changes observed were not the result of metabolic imbalance, and might be potential biomarkers predictive of T1D.
Asunto(s)
Diabetes Mellitus Tipo 1/inmunología , Células Asesinas Naturales/inmunología , Subgrupos de Linfocitos T/inmunología , Linfocitos T Reguladores/inmunología , Adolescente , Niño , Técnicas Citológicas , Femenino , Humanos , Masculino , Factores de RiesgoRESUMEN
Most authors currently quantify the frequency of apoptotic cells in a given phenotypically defined population after calculating the apoptotic index (AI), that is, the percentage of apoptotic cells displaying a specific lineage antigen (LAg) within a population of cells that remain unfragmented and retain the expression of the LAg. However, this approach has two major limitations. First, apoptotic cells fragment into apoptotic bodies that later disintegrate. Second, apoptotic cells frequently lose, partially or even completely, the cell surface expression of the LAg used for the identification of specific cell subsets. This chapter will describe a flow cytometry method to calculate the apoptotic rate (AR) that takes into account both cell fragmentation and loss of LAg expression on measurement of apoptosis using flow cytometry ratiometric cell enumeration that emerges as a more accurate method of measurement of the occurrence of apoptosis in normal and tumoral cell cultures.
Asunto(s)
Apoptosis , Citometría de Flujo/métodos , Células Tumorales Cultivadas/citología , Apoptosis/fisiología , Recuento de Células/métodos , Humanos , Fracciones Subcelulares/fisiologíaRESUMEN
GAD-alum given into lymph nodes to type 1 diabetes patients participating in an open-label pilot trial resulted in preservation of C-peptide similar to promising results from other trials. Here, we compared the immunomodulatory effect of giving GAD-alum directly into lymph nodes versus that induced by subcutaneous administration. Samples from T1D patients (n = 6) who received 4 µg GAD-alum into lymph nodes (LNs), followed by two booster injections one month apart, and from patients (n = 6) who received two subcutaneous injections (SC) (20 µg) given one month apart were compared. GADA, IA-2A, GADA subclasses, IgE, GAD65-induced cytokines, PBMC proliferation, and T cell markers were analyzed. Lower doses of GAD-alum into LN induced higher GADA levels than SC injections and reduced proliferation and IgG1 GADA subclass, while enhancing IgG2, IgG3, and IgG4. The cytokine profile was dominated by the Th2-associated cytokine IL-13, and GAD65 stimulation induced activated CD4 T cells. Patients responding clinically best account for most of the immunological changes. In contrast, SC treatment resulted in predominant IgG1, predominant IFN-γ, higher proliferation, and activated CD4 and CD8 cells. Patients from the LN group with best metabolic outcome seemed to have common immune correlates related to the treatment. This trial is registered with DIAGNODE (NCT02352974, clinicaltrials.gov) and DIABGAD (NCT01785108, clinicaltrials.gov).
Asunto(s)
Diabetes Mellitus Tipo 1/inmunología , Diabetes Mellitus Tipo 1/terapia , Glutamato Descarboxilasa/farmacología , Células Th2/inmunología , Adolescente , Compuestos de Alumbre/farmacología , Proliferación Celular , Niño , Citocinas/inmunología , Femenino , Humanos , Inmunoglobulina G/inmunología , Inmunomodulación , Inmunofenotipificación , Leucocitos Mononucleares/inmunología , Ganglios Linfáticos/inmunología , Activación de Linfocitos , Masculino , Proyectos Piloto , Resultado del Tratamiento , Adulto JovenRESUMEN
Most authors currently quantify the frequency of apoptotic cells in a given phenotypically defined population after calculating the apoptotic index (AI), i.e., the percentage of apoptotic cells displaying a specific linage antigen (LAg) within a population of cells that remain unfragmented and retain the expression of the LAg. However, this approach has two major limitations. Firstly, apoptotic cells fragment into apoptotic bodies that later disintegrate. Secondly, apoptotic cells frequently lose, partially or even completely, the cell surface expression of the LAg used for the identification of specific cell subsets. This chapter describes a flow cytometry method to calculate the apoptotic rate (AR) that takes into account both cell fragmentation and loss of lineage antigen expression on measurement of apoptosis using flow cytometry ratiometric cell enumeration that emerges as a more accurate method of measurement of the occurrence of apoptosis in normal and tumoral cell cultures.
Asunto(s)
Apoptosis , Recuento de Células/métodos , Citometría de Flujo/métodos , Animales , Técnicas de Cultivo de Célula , Citometría de Flujo/instrumentación , HumanosRESUMEN
In order to characterize the immunophenotype and the lymphocyte apoptosis in multiple sclerosis (MS) patients, the peripheral blood lymphocytes of 46 MS patients and 12 healthy volunteers were studied by flow cytometry. Immunophenotypic alterations included significant increases in T CD4+ lymphocytes and reductions in the percentages of CD3+ and CD8+ T cells. After 24 h of culture spontaneous apoptosis was increased in almost T lymphocyte subsets from MS patients and it was significantly higher in those patients who had suffered more than two relapses in the two previous years. The incidence of spontaneous apoptosis was not dependent on FasL-Fas interactions and correlated with the percentages of cells positive for active caspases but not with percentages of Fas+ cells. The increased susceptibility to apoptosis of peripheral blood T lymphocytes from MS patients is difficult to reconcile with the previously proposed role of a defective lymphocyte apoptosis in the pathophysiology of MS.
Asunto(s)
Apoptosis/inmunología , Esclerosis Múltiple/inmunología , Linfocitos T/inmunología , Adulto , Anexina A5/metabolismo , Linfocitos B/inmunología , Linfocitos B/patología , Caspasas/metabolismo , Activación Enzimática , Femenino , Humanos , Inmunofenotipificación , Masculino , Persona de Mediana Edad , Esclerosis Múltiple/sangre , Esclerosis Múltiple/enzimología , Recurrencia , Subgrupos de Linfocitos T/inmunología , Linfocitos T/enzimología , Linfocitos T/patología , Receptor fas/inmunologíaRESUMEN
BACKGROUND: The use of ratiometric cell enumeration methods emerges as a more accurate method of measurement of the occurrence of apoptosis in cell cultures. These new flow cytometry methods were used to quantify the impact of cell fragmentation and loss of lineage antigen (LAg) expression on measurement of apoptosis. METHODS: Highly purified human lymphocyte populations were negatively sorted and cultured for 24 h. Apoptotic cells were identified using annexin V, 7-amino-actinomycin D and their LAgs were stained with antibodies. A new indicator, the apoptotic rate, was used to determine apoptosis occurrence and its validity compared with the widely accepted percentage of apoptotic cells (apoptotic index, AI). RESULTS: Loss of LAg expression and cell fragmentation were observed under all conditions assayed and for all cell populations studied. CONCLUSIONS: Current methods for quantifying of apoptosis involving AI systematically underestimate apoptosis occurrence in all populations and conditions, especially among cells undergoing spontaneous apoptosis.
Asunto(s)
Antígenos de Superficie/metabolismo , Apoptosis/fisiología , Recuento de Células/métodos , Citometría de Flujo/métodos , Leucocitos Mononucleares/metabolismo , Antígenos de Superficie/inmunología , Separación Celular , Células Cultivadas , Humanos , Leucocitos Mononucleares/citología , Leucocitos Mononucleares/inmunología , Subgrupos de Linfocitos T/citología , Subgrupos de Linfocitos T/inmunología , Subgrupos de Linfocitos T/metabolismoRESUMEN
BACKGROUND: Late apoptotic cells divide into apoptotic bodies and are missed by current detection methods. This results in an artificially low apoptotic index (AI). METHODS: This study proposes a flow cytometry-based ratiometric method that uses an internal reference standard of microbeads combined with fluorescein-annexin V binding and 7-aminoactinomycin D to enumerate viable, necrotic, and early and late apoptotic cells within specific subsets of a heterogeneous culture. RESULTS: In the absence of cell growth, the number of apoptotic cells that undergo fragmentation into apoptotic bodies in culture can also be determined accurately by this method. This information can then be used to obtain the apoptotic rate (AR), a new indicator of apoptosis that calculates the proportion of cells that have undergone apoptosis with respect to the total number of seeded cells. The main limitation of the method is that the AR is only suitable for the study of apoptosis in noncycling cells. CONCLUSIONS: This study reveals the superiority of the proposed method over the widely used Nicoletti method and current annexin-V binding methods. The AI did not reflect the true incidence of lymphocyte apoptosis, neither in response to lectins or phorbol esters, nor to serum deprivation. AR was more sensitive than AI, detecting apoptosis at lower concentrations of cell death inducers in all the subsets studied.