RESUMEN
The adoptive transfer of regulatory T-cells (Tregs) is a promising therapeutic approach in transplantation and autoimmunity. However, because large cell numbers are needed to achieve a therapeutic effect, in vitro expansion is required. By comparing their function, phenotype and transcriptomic profile against ex vivo Tregs, we demonstrate that expanded human Tregs switch their metabolism to aerobic glycolysis and show enhanced suppressive function through hypoxia-inducible factor 1-alpha (HIF1A) driven acquisition of CD73 expression. In conjunction with CD39, CD73 expression enables expanded Tregs to convert ATP to immunosuppressive adenosine. We conclude that for maximum therapeutic benefit, Treg expansion protocols should be optimised for CD39/CD73 co-expression.
Asunto(s)
5'-Nucleotidasa/genética , Regulación de la Expresión Génica , Subunidad alfa del Factor 1 Inducible por Hipoxia/genética , Linfocitos T Reguladores/metabolismo , 5'-Nucleotidasa/metabolismo , Femenino , Proteínas Ligadas a GPI/genética , Proteínas Ligadas a GPI/metabolismo , Humanos , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , MasculinoRESUMEN
Susceptibility to multiple autoimmune diseases is associated with common gene polymorphisms influencing IL-2 signaling and Treg function, making Treg-specific expansion by IL-2 a compelling therapeutic approach to treatment. As an in vivo IL-2 half-life enhancer we used a non-targeted, effector-function-silent human IgG1 as a fusion protein. An IL-2 mutein (N88D) with reduced binding to the intermediate affinity IL-2Rßγ receptor was engineered with a stoichiometry of two IL-2N88D molecules per IgG, i.e. IgG-(IL-2N88D)2. The reduced affinity of IgG-(IL-2N88D)2 for the IL-2Rßγ receptor resulted in a Treg-selective molecule in human whole blood pSTAT5 assays. Treatment of cynomolgus monkeys with single low doses of IgG-(IL-2N88D)2 induced sustained preferential activation of Tregs accompanied by a corresponding 10-14-fold increase in CD4+ and CD8+ CD25+FOXP3+ Tregs; conditions that had no effect on CD4+ or CD8+ memory effector T cells. The expanded cynomolgus Tregs had demethylated FOXP3 and CTLA4 epigenetic signatures characteristic of functionally suppressive cells. Humanized mice had similar selective in vivo responses; IgG-(IL-2N88D)2 increased Tregs while wild-type IgG-IL-2 increased NK cells in addition to Tregs. The expanded human Tregs had demethylated FOXP3 and CTLA4 signatures and were immunosuppressive. These results describe a next-generation immunotherapy using a long-lived and Treg-selective IL-2 that activates and expands functional Tregsin vivo. Patients should benefit from restored immune homeostasis in a personalized fashion to the extent that their autoimmune disease condition dictates opening up the possibility for remissions and cures.
Asunto(s)
Enfermedades Autoinmunes/terapia , Inmunoglobulina G/inmunología , Inmunoterapia/métodos , Interleucina-2/inmunología , Linfotoxina-alfa/inmunología , Proteínas Recombinantes de Fusión/inmunología , Linfocitos T Reguladores/efectos de los fármacos , Animales , Enfermedades Autoinmunes/genética , Enfermedades Autoinmunes/inmunología , Enfermedades Autoinmunes/patología , Sitios de Unión , Linfocitos T CD8-positivos/efectos de los fármacos , Linfocitos T CD8-positivos/inmunología , Linfocitos T CD8-positivos/patología , Antígeno CTLA-4/genética , Antígeno CTLA-4/inmunología , Proliferación Celular , Metilación de ADN/efectos de los fármacos , Modelos Animales de Enfermedad , Epigénesis Genética , Factores de Transcripción Forkhead/genética , Factores de Transcripción Forkhead/inmunología , Humanos , Inmunoglobulina G/administración & dosificación , Inmunoglobulina G/química , Inmunoglobulina G/genética , Interleucina-2/administración & dosificación , Interleucina-2/química , Interleucina-2/genética , Subunidad beta del Receptor de Interleucina-2/genética , Subunidad beta del Receptor de Interleucina-2/inmunología , Activación de Linfocitos/efectos de los fármacos , Linfotoxina-alfa/administración & dosificación , Linfotoxina-alfa/química , Linfotoxina-alfa/genética , Macaca fascicularis , Masculino , Ratones , Ratones Transgénicos , Modelos Moleculares , Unión Proteica , Isoformas de Proteínas/genética , Isoformas de Proteínas/inmunología , Estructura Secundaria de Proteína , Proteínas Recombinantes de Fusión/administración & dosificación , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/genética , Factor de Transcripción STAT5/genética , Factor de Transcripción STAT5/inmunología , Transducción de Señal , Linfocitos T Reguladores/inmunología , Linfocitos T Reguladores/patologíaRESUMEN
Although the first mouse embryonic stem (ES) cell lines were derived 25 years ago using feeder-layer-based blastocyst cultures, subsequent efforts to extend the approach to other mammals, including both laboratory and domestic species, have been relatively unsuccessful. The most notable exceptions were the derivation of non-human primate ES cell lines followed shortly thereafter by their derivation of human ES cells. Despite the apparent common origin and the similar pluripotency of mouse and human embryonic stem cells, recent studies have revealed that they use different signalling pathways to maintain their pluripotent status. Mouse ES cells depend on leukaemia inhibitory factor and bone morphogenetic protein, whereas their human counterparts rely on activin (INHBA)/nodal (NODAL) and fibroblast growth factor (FGF). Here we show that pluripotent stem cells can be derived from the late epiblast layer of post-implantation mouse and rat embryos using chemically defined, activin-containing culture medium that is sufficient for long-term maintenance of human embryonic stem cells. Our results demonstrate that activin/Nodal signalling has an evolutionarily conserved role in the derivation and the maintenance of pluripotency in these novel stem cells. Epiblast stem cells provide a valuable experimental system for determining whether distinctions between mouse and human embryonic stem cells reflect species differences or diverse temporal origins.