Immunosuppressive Mechanisms of Regulatory B Cells.

Regulatory B cells (Bregs) is a term that encompasses all B cells that act to suppress immune responses. Bregs contribute to the maintenance of tolerance, limiting ongoing immune responses and reestablishing immune homeostasis. The important role of Bregs in restraining the pathology associated with exacerbated inflammatory responses in autoimmunity and graft rejection has been consistently demonstrated, while more recent studies have suggested a role for this population in other immune-related conditions, such as infections, allergy, cancer, and chronic metabolic diseases. Initial studies identified IL-10 as the hallmark of Breg function; nevertheless, the past decade has seen the discovery of other molecules utilized by human and murine B cells to regulate immune responses. This new arsenal includes other anti-inflammatory cytokines such IL-35 and TGF-ß, as well as cell surface proteins like CD1d and PD-L1. In this review, we examine the main suppressive mechanisms employed by these novel Breg populations. We also discuss recent evidence that helps to unravel previously unknown aspects of the phenotype, development, activation, and function of IL-10-producing Bregs, incorporating an overview on those questions that remain obscure.

New Insights into Asthma Inflammation: Focus on iNKT, MAIT, and γδT Cells.

Asthma is a chronic immunological disease affecting all age groups, but often starting in childhood. Although it has long been ascribed to a single pathology, recent studies have highlighted its heterogeneity due to the potential involvement of various pathogenic mechanisms. Here, we present our current understanding of the role of innate-like T (ILT) cells in asthma pathogenesis. These cells constitute a specific family mainly comprising γδT, invariant natural killer (iNKT) and mucosal-associated invariant (MAIT) T cells. They all share the ability to massively secrete a wide range of cytokines in a T-cell receptor (TCR)-dependent or -independent manner. ILT cells are prevalent in mucosal tissues, including airways, where their innate and adaptive immune functions consist primarily in protecting tissue integrity. However, ILT cells may also have detrimental effects leading to asthma symptoms. The immune mechanisms through which this pathogenic effect occurs will be discussed in this overview.

Decreased invariant natural killer T-cell-mediated antitumor immune response in patients with gastric cancer.

Gastric cancer (GC) is the third most common cause of cancer-related death worldwide. Invariant natural killer T (iNKT) cells are innate-like cytotoxic T lymphocytes involved in tumor immune surveillance. They can be activated either through CD1d-presented glycolipid antigens recognized by their invariant T-cell receptor, cytokines or by sensing tumor-associated stress-induced ligands through the natural killer group 2, member D (NKG2D) receptor. Although the number and functionality of iNKT cells may be decreased in several types of cancer, here we show that GC patients presented a mild increase in iNKT cell frequencies and numbers in the blood compared with healthy donors. In GC patients, iNKT cells, expanded in vitro with α-galactosyl ceramide and stimulated with phorbol 12-myristate 13-acetate and ionomycin, produced higher levels of interleukin-2 and transforming growth factor-beta, while their capacity to degranulate remained preserved. Because tumor-derived epithelial cell adhesion molecule-positive epithelial cells did not display surface CD1d, and NKG2D ligands (NKG2DLs) were detected in the gastric tumor milieu, we envisioned a role for NKG2D in iNKT cell functions. Peripheral iNKT cells from GC patients and controls presented similar levels of NKG2D; nevertheless, the percentages of interferon-γ-producing and CD107a-positive iNKT cells from patients were reduced upon challenge with CD1d-negative, NKG2DL-positive K562 cells, suggesting a compromised response by iNKT cells in GC patients, which may not result from impaired NKG2D/NKG2DL signaling. The decreased response of iNKT cells may explain the fact that higher frequencies of circulating iNKT cells did not confer a survival benefit for GC patients. Therefore, functional impairment of iNKT cells in GC may contribute to tumor immune escape and favor disease progression.

Development of a novel mechanism-based glycolipid adjuvant for vaccination.

The inability to elicit strong and durable cellular responses is a major obstacle in the development of successful vaccines, in particular those against malaria. In this regard, the generation of novel adjuvants that will potently boost cell-mediated immunity induced by candidate vaccines is helpful. We and others have found a glycolipid, called α-galactosylceramide (α-GalCer), which could be presented on CD1d expressed by antigen-presenting cells (APCs) and stimulate natural killer T (NKT) cells. This triggers the activation/maturation of APCs, particularly dendritic cells (DCs). By activating NKT cells and subsequently DCs, α-GalCer has been shown to enhance adaptive immune responses, particularly of CD8 + T cells, induced by the vaccines. More recently, we identified an analogue of α-GalCer, which can display a potent adjuvant activity in conjunction with malaria vaccines in mice and non-human primates. It is anticipated that CD1d-binding, NKT cell-stimulating glycolipids will be tested as adjuvants in humans in the near future.

NKT cells in leishmaniasis.

The role of NKT cells in the resistance or susceptibility towards Leishmania infections remains to be defined, since controversial data persist. The response of these cells seems to depend on many variables such as the infection site, the number of infecting parasites, the virulence of the strain and the Leishmania species. We here revise the activation pathways leading to NKT cell activation. NKT cells can be activated by the direct pathway, in which Leishmania glycolipids are presented by CD1d molecules on antigen presenting cells, such as dendritic cells (DC), leading to the secretion of diverse cytokines by NKT. NKT cells can also be activated by the indirect pathway, in which Leishmania glycolipids, such as LPG, stimulate TLR2 in DC, inducing their IL-12 production, which in turn activates NKT cells. The review further analyzes the role of NKT cells in disease development, both in humans as in mouse models. Finally we propose the activation of NKT cells for controlling Leishmania infections.

The tumor antigen N-glycolyl-GM3 is a human CD1d ligand capable of mediating B cell and natural killer T cell interaction.

The expression of N-glycolyl-monosialodihexosyl-ganglioside (NGcGM3) in humans is restricted to cancer cells; therefore, it is a tumor antigen. There are measurable quantities of circulating anti-NGcGM3 antibodies (aNGcGM3 Abs) in human serum. Interestingly, some people have circulating Ag-specific immunoglobulins G (IgGs) that are capable of complement mediated cytotoxicity against NGcGM3 positive cells, which is relevant for tumor surveillance. In light of the chemical nature of Ag, we postulated it as a candidate ligand for CD1d. Furthermore, we hypothesize that the immune mechanism involved in the generation of these Abs entails cross talk between B lymphocytes (Bc) and invariant natural killer T cells (iNKT). Combining cellular techniques, such as flow cytometry and biochemical assays, we demonstrated that CD1d binds to NGcGM3 and that human Bc present NGcGM3 in a CD1d context according to two alternative strategies. We also showed that paraformaldehyde treatment of cells expressing CD1d affects the presentation. Finally, by co-culturing primary human Bc with iNKT and measuring Ki-67 expression, we detected a reproducible increment in the proliferation of the iNKT population when Ag was on the medium. Our findings identify a novel, endogenous, human CD1d ligand, which is sufficiently competent to stimulate iNKT. We postulate that CD1d-restricted Bc presentation of NGcGM3 drives effective iNKT activation, an immunological mechanism that has not been previously described for humans, which may contribute to understanding aNGcGM3 occurrence.

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.