CD24⁺ Cell Depletion Permits Effective Enrichment of Thymic iNKT Cells While Preserving Their Subset Composition

Invariant NKT (iNKT) cells are a small subset of thymus-generated T cells that produce cytokines to control both innate and adaptive immunity. Because of their very low frequency in the thymus, in-depth characterization of iNKT cells can be facilitated by their enrichment from total thymocytes. Magnetic-activated cell sorting (MACS) of glycolipid antigen-loaded CD1d-tetramer-binding cells is a commonly used method to enrich iNKT cells. Surprisingly, we found that this procedure also dramatically altered the subset composition of enriched iNKT cells. As such, NKT2 lineage cells that express large amounts of the transcription factor promyelocytic leukemia zinc finger were markedly over-represented, while NKT1 lineage cells expressing the transcription factor T-bet were significantly reduced. To overcome this limitation, here, we tested magnetic-activated depletion of CD24⁺ immature thymocytes as an alternative method to enrich iNKT cells. We found that the overall recovery in iNKT cell numbers did not differ between these 2 methods. However, enrichment by CD24⁺ cell depletion preserved the subset composition of iNKT cells in the thymus, and thus permitted accurate and reproducible analysis of thymic iNKT cells in further detail.

Mecanismos de activación de las células T asesinas naturales invariantes (iNKT) / Mecanismos de ativação das células T assassinas naturais invariantes (iNKT) / Activation mechanisms of invariant natural killer T cells (iNKTs)

Aunque se ha logrado un conocimiento amplio acerca de las células T asesinas naturales (iNKT), aún no existe consenso sobre sus mecanismos de activación. Dichas células reconocen diferentes antígenos glicolipídicos presentados por medio de la molécula CD1d, los cuales pueden ser endógenos, exógenos derivados de organismos como bacterias y sintéticos desarrollados para aplicaciones clínicas. Existe mucho interés en entender cómo estas distintas variantes glicolipídicas inducen diferentes tipos de polarización, pero ha sido muy difícil llegar a un consenso, debido a que la respuesta depende de varios factores como la naturaleza, la internalización y el procesamiento de los glicolípidos. Además, la activación de las células iNKT la determinan el tipo y estado de activación de la célula presentadora de antígeno, las moléculas coestimuladoras, los mecanismos de transactivación y la localización de los complejos CD1d-glicolípido en distintas microrregiones de la membrana plasmática, como las balsas lipídicas. Esta revisión explora la evidencia sobre los factores que afectan la activación de las células iNKT con el fin de entender su potencial inmunomodulador.

A great amount of knowledge on natural killer T cells (iNKTs) is now available, but a consensus about their activation mechanisms has not been reached. These cells recognize different glycolipid antigens through the CD1d molecule. Such antigens may be endogenous, derived from bacteria (foreign) and synthetic, the latter have been developed for clinical applications. There exists much interest in understanding how these different glycolipid compounds induce different types of polarization, but it has been difficult to reach a consensus due to the fact that responses depend on different factors such as: the nature of the molecule, the internalization process and the presentation of the glycolipids. Moreover, activation of iNKT cells is determined by the type and state of the antigen presenting cell, the co-stimulatory molecules, the transactivation mechanisms and the location of the glycolipid-CD1d complexes on the plasma membrane, such as the lipid rafts. This review explores the evidence about the factors that affect activation of iNKT cells in order to understand their immune-modulatory potential.

Ainda que se conseguiu um conhecimento amplo a respeito das células T assassinas naturais (iNKT), ainda não existe consenso sobre seus mecanismos de ativação. Ditas células reconhecem diferentes antígenos glicolipídicos apresentados por meio da molécula CD1d, os quais pode ser: endógenos, exógenos derivados de organismos como bactérias e sintéticos desenvolvidos para aplicações clínicas. Existe muito interesse em entender como estas diferentes variantes glicolipídicas induzem diferentes tipos de polarização, mas foi muito difícil chegar a um consenso, devido a que a resposta depende de vários fatores como a natureza, a internalização e o processamento dos glicolípidos. Ademais, a ativação das células iNKT a determinam o tipo e estado de ativação da célula apresentadora de antígeno, as moléculas co-estimuladoras, os mecanismos de transativação e a localização dos complexos CD1d-glicolípido em diferentes microrregiões da membrana plasmática, como as balsas lipídicas. Esta revisão explora a evidência sobre os fatores que afetam a ativação das células iNKT com o fim de entender seu potencial imunomodulador.

Perfil das células T Natural Killer (NKT)de indivíduos asmáticos desafiadas com antígenos solúvel do ovo de schistosoma mansoni / Natural Killer T cell (NKT) profile of asthmatic individuals challenged with soluble schistosoma mansoni egg antigens

INTRODUÇÃO: A asma é uma doença inflamatória crônica, caracterizada por hiper-reatividade das vias aéreas inferiores e por limitação variável e reversível ao fluxo aéreo. Apresenta manifestações clínicas na forma de sibilância, dispneia, sensação de aperto no peito e tosse, podendo ser considerada como atópica ou não atópica, de acordo com seus aspectos imunopatogênicos. Células do sistema imune, como neutrófilos, macrófagos, células dendríticas e as células T Natural Killer (NKT), apresentam importante papel no desenvolvimento ou regulação da resposta inflamatória da asma. Desta forma é possível que antígenos com propriedades regulatórias, como no caso dos antígenos de ovo do Schistosoma mansoni (SEA), sejam capazes de alterar o perfil destas células e regular a resposta imune da asma. OBJETIVOS: Avaliar a frequência de NKT e expressão de moléculas de ativação e coestimulação, além de citocinas nestas células, em indivíduos com asma. METODOLOGIA: Trata-se de um estudo de corte transversal realizado com 24 voluntários, sendo 14 indivíduos asmáticos e 10 voluntários não asmáticos. Células mononucleares de sangue periférico (PNMC)...

INTRODUCTION: Asthma is a chronic inflammatory disease characterized by hyperreactivity of lower airways and variable limitation and reversible airflow. The main clinical manifestations are wheezing, breathlessness, chest pain that feel like tightness and coughing, being considered as atopic or non-atopic, according to its immunopathogenic aspect. Immune cells, such as neutrophils, macrophages, dendritic cells and Natural Killer T cells (NKT) play an important role in the regulation or development of inflammatory response of asthma. Thus, it is possible that antigens with regulatory properties, such as Schistosoma mansoni soluble egg antigen (SEA), are able to alter the profile of these cells and regulate the immune response of asthma. AIM: To evaluate the frequency of NKT cells, expression of activation and costimulatory markers, as well as cytokine expression in NKT cells from individuals with asthma. METHODOLOGY: This is a cross-sectional study of 24 volunteers, of which 14 were asthmatic and 10 nonasthmatic volunteers. Peripheral blood mononuclear cells (PBMC)...

Murine invariant natural killer T cells recognize glycolipids derived from extracts of the lichen Stereocaulon ramulosum / Células T asesinas naturales invariantes murinas reconocen glicolipidos derivados de extractos del liquen Stereocaulon ramulosum

Background: Invariant natural killer T cells (iNKT) can be activated by certain types of glycolipids that have the potential to generate adjuvant effects which could be used to develop effective and safe immunotherapies. Many of these glycolipids have been isolated from natural organisms, but there is a great amount of these organisms completely unexplored as a source of these types of compounds. Some of these organisms are lichens which are complex symbiotic organisms that have been showed to contain glycolipids. Objectives: We decide to test if glycolipids isolated from lichens would be able to activate iNKT cells in vitro and in vivo. Methods: We have used extracted glycolipids from 43 different species of lichens from Colombia. We have used iNKT hybridoma cells, C57BL/6 mice, IL-2 ELISA and the B16 melanoma to test for the adjuvant capabilities of glycolipids isolated from lichens. Results: In this study we have found two glycolipids with the capacity to activate iNKT cells in vivo. One of the glycolipids was able to activate iNKT cells in vivo, and was competent to induce protection against the B16 melanoma in the mouse model. Conclusions: We propose a possible chemical structure for a novel glycolipid called ß-GalCer-lich (1) derived from the lichen Stereocaulon ramulosum.

Antecedentes: Las células asesinas naturales T (iNKT) pueden ser activadas por ciertos tipos de glicolípidos que tienen el potencial para generar efectos adyuvantes los cuales pueden ser usados para desarrollar inmunoterapias efectivas. Muchos de estos glicolípidos han sido aislados de organismos naturales, pero hay una gran cantidad de organismos completamente inexplorados como fuente de este tipo de compuestos. Algunos de estos organismos son los líquenes, los cuales son organismos simbiontes complejos para los que se ha mostrado que contienen glicolípidos. Objetivos: Nosotros decidimos probar si los glicolípidos aislados de líquenes podrían ser capaces de activar alas celulas iNKT in vitro e in vivo. Metodos: Nosotros hemos extraído glicolípidos de 43 especies de líquenes de Colombia. Nosotros hemos usado células de un hibridoma de iNKTs, ratones C57BL/6, ELISA para IL-2 y el melanoma B16 para probar la capacidad adyuvante de los glicolipidos aislados de los líquenes. Resultados: En este estudio nosotros hemos encontrado dos glicolípidos con la capacidad de activar iNKTs in vitro. Uno de los glicolípidos fue capaz de activar células iNKT in vivo, y fue competente para inducir protección contra el melanoma B16 en el modelo de ratón. Conclusiones: Nosotros proponemos una posible estructura química para el nuevo glicolípido llamado ß-GalCer-lich (1) derivado del liquen Stereocaulon ramulosum.

Subpoblaciones de linfocitos B y su expresión de CD1d en pacientes con lupus eritematoso sistémico / B cell subsets and their expression of CD1d in patients with systemic lupus erythematosus

Resumen Introducción: Los linfocitos B (LB) se consideran el centro de la desregulación inmune en pacientes con lupus eritematoso sistémico (LES), principalmente, por su producción de autoanticuerpos. Recientemente, se demostró la existencia de LB, incluidos en los B transicionales, con capacidad reguladora (Breg) y fenotipo CD19+CD24hiCD38hi. En humanos se demostró la importancia de CD80 y CD86 en su función reguladora. El papel de CD1d aún no ha sido evaluado. Objetivo: Evaluar la frecuencia de LB maduros, memoria y transicionales, en controles y pacientes con LES, además de la expresión de CD1d y correlacionarla con la actividad de la enfermedad medida por SLEDAI (Systemic Lupus Erythematosus Disease Activity Index). Materiales y métodos: Se evaluó por citometría de flujo la frecuencia de subpoblaciones de LB basados en la expresión de CD19, CD24 y CD38, además de CD1d, en controles con otras enfermedades autoinmunes (OEA), individuos sanos y pacientes con LES, y se correlacionó con SLEDAI. Resultados: Se evidenció una disminución significativa en el porcentaje de LB de memoria en pacientes LES y OEA, sin alteraciones en las subpoblaciones de LB maduros y transicionales. La expresión de CD1d no evidenció diferencias significativas en ninguna de las subpoblaciones ni se correlacionó con SLEDAI. Conclusión: La disminución de la subpoblación de memoria fue previamente descrita en LES y se ha asociado a algunos tipos de tratamiento. Aunque CD1d se ha asociado a la función de Breg en murinos, no hubo diferencias significativas en su expresión en las subpoblaciones y queda por clarificar su papel en la función de las Breg humanas.

Abstract Introduction: B lymphocytes are considered the center of immune dysregulation in Systemic Lupus Erythematosus (SLE). It has recently been demonstrated that there is a B cell with regulatory capacities (Breg) included in transitional B lymphocytes with the phenotype CD19+CD24hiCD38hi. The importance of CD80 and CD86 in the regulatory function of the Bregs has been demonstrated in humans, but the role of CD1d has not been evaluated. Objective: To evaluate the frequency of mature, memory and transitional B cells in SLE patients and controls, the expression of CD1d among these cells, and its correlation with the activity of the disease measured using the Systemic Lupus Erythematosus Disease Activity Index (SLEDAI). Materials and methods: The frequency of the B cell subsets was evaluated by flow cytometry based on the expression of CD19, CD24 and CD38, as well as CD1d in these cells in SLE patients and controls, and were correlated with the activity of the disease measured using the SLEDAI. Results: A significant reduction in the percentage of memory B cells was observed in SLE patients and other autoimmune conditions, with no changes in the mature or transitional B cell subsets. Similarly, no significant differences were observed in the expression of CD1d in any of the subsets, nor was there any correlation with the SLEDAI. Conclusion: The reduction of the memory subset has been previously described in SLE, and has been associated with some types of treatment. The expression of CD1d in all the subsets was observed, but its role in the regulatory function of the CD19+CD24hiCD38hi cells is still not clear.

The requirement of natural killer T-cells in tolerogenic APCs-mediated suppression of collagen-induced arthritis

TGF-beta-induced tolerogenic-antigen presenting cells (Tol-APCs) could induce suppression of autoimmune diseases such as collagen-induced arthritis (CIA) and allergic asthma. In contrast, many studies have shown that NKT cells are involved in the pathogenesis of Th1-mediated autoimmune joint inflammation and Th2-mediated allergic pulmonary inflammation. In this study, we investigated the effect of CD1d-restricted NKT cells in the Tol-APCs-mediated suppression of autoimmune disease using a murine CIA model. When CIA-induced mice were treated with Tol-APCs obtained from CD1d+/- or CD1d-/- mice, unlike CD1d+/- APCs, CD1d-/- Tol-APCs failed to suppress CIA. More specifically, CD1d-/- Tol-APCs failed to suppress the production of inflammatory cytokines and the induction of Th2 responses by antigen-specific CD4 T cells both in vitro and in vivo. Our results demonstrate that the presence of CD1d-restricted NKT cells is critical for the induction of Tol-APCs-mediated suppression of CIA.

Natural killer T cell and pathophysiology of asthma / 소아과

Natural killer T (NKT) cell is a special type of T lymphocytes that has both receptor of natural killer (NK) cell (NK1.1, CD161c) and T cell (TCR) and express a conserved or invariant T cell receptor called Valpha14Jalpha18 in mice or Va24 in humans. Invariant NKT (iNKT) cell recognizes lipid antigen presented by CD1d molecules. Marine-sponge-derived glycolipid, alpha-galactosylceremide (alpha-GalCer), binds CD1d at the cell surface of antigen-presenting cells and is presented to iNKT cells. Within hours, iNKT cells become activated and start to secrete Interleukin-4 and interferon-gamma. NKT cell prevents autoimmune diseases, such as type 1 diabetes, experimental allergic encephalomyelitis, systemic lupus erythematous, inflammatory colitis, and Graves' thyroiditis, by activation with alpha-GalCer. In addition, NKT cell is associated with infectious diseases by mycobacteria, leshmania, and virus. Moreover NKT cell is associated with asthma, especially CD4+ iNKT cells. In this review, I will discuss the characteristics of NKT cell and the association with inflammatory diseases, especially asthma.

The presence of CD8+ invariant NKT cells in mice

Invariant natural killer T (iNKT) cells develop in the thymus upon recognition of CD1d expressed on developing thymocytes. Although CD4 and CD8 coreceptors are not directly involved in the interaction between CD1d and the T cell receptors (TCRs) of iNKT cells, a conspicuous lack of CD8+ iNKT cells in mice raised the question of whether CD8+ iNKT cells are excluded due to negative selection during their thymic development, or if there is no lineage commitment for the development of murine CD8+ iNKT cells. To address this question, we analyzed iNKT cell-specific TCR Valpha14+ transgenic mice, where the Valpha14 transgene forces the generation of iNKT cells. This allows detailed study of the iNKT cell repertoire. We were able to identify CD8+ iNKT cells which respond to the NKT cell-specific glycolipid ligand alpha-galactosylceramide. Unlike conventional iNKT cells, CD8+ iNKT cells produce predominantly IFN-gamma but not IL-4 upon antigen stimulation. We also confirmed the presence of CD8+ iNKT cells in wild type mice. Our results suggest that CD8+ NKT cells do exist in mice, although their population size is quite small. Their Th1-skewed phenotype might explain why the population size of this subtype needs to be controlled tightly.

Alteration of Innate Immune T and B Cells in the NC/Nga Mouse

BACKGROUND: Millions of people in the world are suffering from atopic dermatitis (AD), which is a chronic inflammatory skin disease triggered by Th2 immune responses. The NC/Nga mouse is the most extensively studied animal model of AD. Like human AD, NC/Nga mice demonstrate increased levels of IgE, a hallmark of Th2 immune responses. Adaptive immunity cannot be generated without help of innate immunity. Especially natural killer T (NKT) cells and marginal zone B (MZB) cells have been known to play important roles in linking innate immunity to adaptive immunity. METHODS: Through flow cytometric analysis and ELISA assay, we investigated whether these lymphocytes might be altered in number in NC/Nga mice. RESULTS: Our data demonstrated that the number of NKT cells was reduced in NC/Nga mice and IFNgamma production by NKT cells upon alpha-GalCer stimulation decreased to the levels of CD1d KO mice lacking in NKT cells. However, reduction of NKT cells in NC/Nga mice was not due to CD1d expression, which was normal in the thymus. Interestingly, there was a significant increase of CD1d(high)B220+ cells in the spleen of NC/Nga mice. Further, we confirmed that CD1d(high)B220+ cells are B cells, not dendritic cells. These CD1d(high)B220+ B cells show IgM(high)CD21(high)CD23low, a characteristic phenotype of MZB cells. CONCLUSION: We provide the evidence that there are decreased activities of NKT cells and increased number of MZB cells in the NC/Nga mice. Our findings may thus explain why NC/Nga mice are susceptible to AD.

Study of cotransfection of B7-1 gene and CD1_D gene in pancreatic carcinoma cell and its anti-tumor responses in mice / 中国普通外科杂志

Objective To study the cotransfection mB7-1 and mCD1D gene into pancreatic cancer cells of rats and to observe its anti-tmor responses.Methods Recombinant retroviral vectors expressing mB7-1and mCD1D gene were packaged into GP2-293 cell lines and transfected.The expressions of mB7-1 and mCD1D were detected with PCR and Western blot.The positive cells of mB7-1 and mCD1D were used to induce the anti-tumor immunity in vitro.Results Anti-tumor immunity was induced after B7-1 and CD1D positive cells were coinoculated in syngeneic mice.Furthermore,the growth of tumor was inhibited.Conclusions Cotransfection of B7-1 and CD1D could induce anti-tumor effect.This study provide a foundation for the application of B7-1 and CD1D gene therapy in tumor.

Study on the expansion and the cytokine releasing of human NKT cells in vitro / 中国输血杂志

Objective To expand the NKT cells in vitro and to determine the cytokine releasing of NKT cells during proliferation.Methods Several kind of methods were established to expand TCRV ?24 +/TCRV ?11 + NKT cells from PBMC or purified T lymphocyte.The levels of IL 4,IFN ?and TNF ? of NKT cells were determined by flow cytometry.Results After 19 days' expansion,the largest number of NKT cells increased (23.0?16.7)?10 3 times and the largest ratio of NKT cells to total T lymphocyte was (25.5?7.2)%.The ratio of TCRV ?11 + NKT cells that secreted IL 4,IFN ?and TNF ? was higher than the TCRV?11 T lymphocytes.Conclusion TCRV ?24 +/TCRV ?11 + NKT cells can be expanded in vitro with ? Galcer presented by CD 1d molecule.The NKT cells can also be expanded from PBMC because the monocyte lineage cells within PBMC can express CD 1d molecule and present specific glycolipids like ? Galcer to them.

Progresses in development and function of NKT cells / 中国免疫学杂志

Natural killer T (NKT) cells,a subset of lymphocytes that bridge innate and adaptive immune systems,involve in processes of infection immunity,tumor immunity,transplantation immunity and autoimmunity.A significant progress has been made in the mechanisms of origin,selection,differentiation and maturation of NKT cells.However,some viewpoints are still controversial,and need to be further intensively investigated.The potential therapeutic applications of functional NKT cells have been suggested in the prevention and the treatment of various diseases.