RESUMEN
Scurfy mice have a deletion in the Foxp3 gene, resulting in a failure to generate Foxp3(+) regulatory T cells, and they subsequently develop severe CD4(+) T-cell-mediated autoimmune inflammation. Multiple organs are involved, but the skin is one of the main organs affected. During the course of disease, Scurfy mice develop autoantibodies; however, the targeted antigens are unknown. In this study, we show that Scurfy mice develop autoantibodies directed against skin antigens. Using western blot analysis, we found that Scurfy serum reacted with proteins in total skin lysate, as well as in a keratinocyte lysate. Most of the Scurfy sera tested identified a major band at 50 kDa. Transfer of Scurfy CD4(+) T cells into nu/nu mice yielded autoantibodies with similar reactivity. Further analysis using 2D western blots, followed by peptide mass fingerprinting, identified several keratins as targets. To confirm this observation, we chose one of the identified targets, keratin 14, and prepared recombinant proteins encompassing the N-terminal, middle, and C-terminal portions of the keratin 14 protein. Scurfy serum predominantly recognized the C-terminal fragment. Sera from patients with immunodysregulation, polyendocrinopathy, enteropathy, X-linked (IPEX) syndrome, the human disease resulting from FOXP3 mutations, also recognized skin antigens, including keratin 14. Thus, the results of our study indicate that autoantibodies in Scurfy mice and patients with IPEX target keratins.
Asunto(s)
Enfermedades Autoinmunes/inmunología , Dermatitis/inmunología , Factores de Transcripción Forkhead/inmunología , Queratina-14/inmunología , Animales , Especificidad de Anticuerpos , Autoanticuerpos/sangre , Autoanticuerpos/inmunología , Epítopos , Femenino , Factores de Transcripción Forkhead/genética , Humanos , Queratina-14/química , Queratinocitos/inmunología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Mutantes , Ratones Desnudos , Mapeo Peptídico , Poliendocrinopatías Autoinmunes/inmunología , Estructura Terciaria de Proteína , Linfocitos T Reguladores/inmunologíaRESUMEN
TLRs are a class of conserved pattern recognition receptors that are used by cells of the innate immune system. Recent studies have demonstrated the expression of TLRs on both human and mouse T cells raising the possibility that TLRs play a direct role in adaptive immunity. TLR2 is activated primarily by bacterial wall components including peptidoglycan and lipoproteins. Several studies have shown that mouse regulatory T (Treg) cells express TLR2 and claimed that engagement of TLR2 by synthetic ligands reversed their suppressive function. In contrary, enhancement of Treg function was observed following engagement of TLR2 on human Treg. We have reexamined the expression and function of TLR2 on mouse Treg purified from Foxp3-GFP knock-in mice. TLR2 ligation by TLR2 agonist, the synthetic bacterial lipoprotein Pam3CSK4, enhanced the proliferative responses of both conventional T cells and Treg in response to TLR stimulation in the absence of APC. Treatment of Foxp3+ Treg with Pam3CSK4 did not alter their suppressive function in vitro or in vivo and did not reduce their level of Foxp3 expression. An additional effect of TLR2 stimulation of Treg was induction of Bcl-x(L) resulting in enhanced survival in vitro. Treatment of mice with the TLR2 agonist enhanced the Ag-driven proliferation of Treg in vivo, but did not abolish their ability to suppress the development of experimental autoimmune encephalomyelitis. Development of methods to selectively stimulate TLR2 on Treg may lead to a novel approaches for the treatment of autoimmune diseases.
Asunto(s)
Regulación hacia Abajo/inmunología , Linfocitos T Reguladores/inmunología , Linfocitos T Reguladores/metabolismo , Receptor Toll-Like 2/metabolismo , Regulación hacia Arriba/inmunología , Animales , Apoptosis/inmunología , Supervivencia Celular/genética , Supervivencia Celular/inmunología , Células Cultivadas , Técnicas de Cocultivo , Regulación hacia Abajo/genética , Encefalomielitis Autoinmune Experimental/genética , Encefalomielitis Autoinmune Experimental/inmunología , Encefalomielitis Autoinmune Experimental/patología , Femenino , Factores de Transcripción Forkhead/genética , Técnicas de Sustitución del Gen , Proteínas Fluorescentes Verdes/genética , Lipopéptidos/administración & dosificación , Activación de Linfocitos/genética , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Mutantes , Ratones Transgénicos , Linfocitos T Reguladores/citología , Receptor Toll-Like 2/deficiencia , Receptor Toll-Like 2/genética , Regulación hacia Arriba/genética , Proteína bcl-X/biosíntesis , Proteína bcl-X/fisiologíaAsunto(s)
Fascitis/patología , Adulto , Fascitis/cirugía , Femenino , Antebrazo , Humanos , Recurrencia , Remisión EspontáneaRESUMEN
Naïve antigen-specific CD4(+) T cells (TxA23) induce autoimmune gastritis when transferred into BALB/c nu/nu mice. Transfer of in vitro pre-differentiated Th1 or Th17 TxA23 effector T cells into BALB/c nu/nu recipients induces distinct histological patterns of disease. We have previously shown that co-transfer of polyclonal naturally occurring Treg (nTreg) suppressed development of Th1-, but not Th17-mediated disease. Therefore, we analysed the suppressive capacity of different types of Treg to suppress Th1- and Th17-mediated autoimmune gastritis. We compared nTreg with polyclonal TGFbeta-induced WT Treg (iTreg) or TGFbeta-induced antigen-specific TxA23 iTreg in co-transfer experiments with Th1 or Th17 TxA23 effector T cells. 6 weeks after transfer in vitro pre-differentiated TxA23 Th1 and Th17 effector cells induced destructive gastritis. Th1-mediated disease was prevented by co-transfer of nTreg and also antigen-specific iTreg, whereas WT iTreg did not show an effect. However, Th17-mediated disease was only suppressed by antigen-specific iTreg. Pre-activation of nTreg in vitro prior to transfer did not increase their suppressive activity in Th17-mediated gastritis. Thus, antigen-specific iTreg are potent suppressors of autoimmune gastritis induced by both, fully differentiated Th1 and Th17 effector cells.
Asunto(s)
Enfermedades Autoinmunes/inmunología , Gastritis/inmunología , Subgrupos de Linfocitos T/metabolismo , Linfocitos T Reguladores/metabolismo , Células TH1/metabolismo , Traslado Adoptivo , Animales , Antígenos/inmunología , Enfermedades Autoinmunes/patología , Diferenciación Celular , Gastritis/patología , Tolerancia Inmunológica , Interleucina-17/metabolismo , Activación de Linfocitos , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Estómago/patología , Especificidad del Receptor de Antígeno de Linfocitos T , Subgrupos de Linfocitos T/inmunología , Subgrupos de Linfocitos T/patología , Linfocitos T Reguladores/inmunología , Linfocitos T Reguladores/patología , Células TH1/inmunología , Células TH1/patología , Timo/patología , Factor de Crecimiento Transformador beta/metabolismoRESUMEN
CD4(+) T cells from the TCR transgenic TxA23 mouse recognize a peptide from the H/K-ATPase alpha-chain. When TxA23 CD4(+) thymocytes are differentiated into Th1, Th2, and Th17 lines, all three subpopulations induced autoimmune gastritis (AIG) upon transfer into nu/nu recipients. The induction of AIG by naive T cells or by Th1 or Th2 cell lines could be prevented by the cotransfer of polyclonal Foxp3(+) T regulatory cells (nTreg), whereas Th17-induced AIG was resistant to suppression. We compared the capacity of different types of Treg to suppress Th17-mediated AIG. Cotransfer of either nTreg or polyclonal TGFbeta-induced Treg (iTreg) did not prevent AIG, while cotransfer of TGFbeta-induced Ag-specific TxA23 iTreg completely prevented the development of disease. Ag-specific iTreg were able to suppress Th17-mediated disease when injected 6 days after the Th17 effectors. The implications of these results for the use of Treg for the cellular biotherapy of autoimmune disease are discussed.
Asunto(s)
Enfermedades Autoinmunes/prevención & control , Epítopos de Linfocito T/inmunología , Gastritis/inmunología , Gastritis/prevención & control , Interleucina-17/fisiología , Linfocitos T Reguladores/inmunología , Factor de Crecimiento Transformador beta/fisiología , Traslado Adoptivo , Animales , Enfermedades Autoinmunes/inmunología , Células Cultivadas , Femenino , Interleucina-17/biosíntesis , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Ratones Transgénicos , Linfocitos T Reguladores/metabolismo , Linfocitos T Reguladores/trasplanteRESUMEN
INTRODUCTION: A number of studies have suggested that transforming growth factor beta (TGF-beta) plays a critical role in immune suppression mediated by Foxp3(+) regulatory T cells. TGF-beta in concert with interleukin 2 is a potent induction factor for the differentiation of Foxp3(+) Treg from naive precursors. Polyclonal TGF-beta-induced Treg (iTreg) are capable of preventing the autoimmune syndrome that develops in scurfy mice that lack Foxp3(+) Treg. Antigen-specific iTreg can be used to both prevent and treat autoimmune gastritis that is induced by transfer of naive or primed autoantigen-specific T cells. TGF-beta complexed with latency-associated peptide is expressed on the surface of activated thymus-derived Treg. Coculture of activated Treg with naive responder T cells results in the de novo generation of fully functional Foxp3(+) T cells in a contact-dependent and TGF-beta-dependent manner. CONCLUSIONS AND SPECULATIONS: Generation of functional Foxp3(+) T cells via this pathway may represent a mechanism by which Treg maintain tolerance and expand their repertoire.
Asunto(s)
Factores de Transcripción Forkhead/biosíntesis , Interleucina-2/metabolismo , Linfocitos T Reguladores/inmunología , Factor de Crecimiento Transformador beta/fisiología , Animales , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD4-Positivos/metabolismo , Diferenciación Celular/inmunología , Tolerancia Inmunológica/inmunología , Interleucina-2/inmunología , Ratones , Ratones Noqueados , Linfocitos T Reguladores/metabolismo , Factor de Crecimiento Transformador beta/genéticaRESUMEN
Th cells can be subdivided into IFN-gamma-secreting Th1, IL-4/IL-5-secreting Th2, and IL-17-secreting Th17 cells. We have evaluated the capacity of fully differentiated Th1, Th2, and Th17 cells derived from a mouse bearing a transgenic TCR specific for the gastric parietal cell antigen, H(+)K(+)-ATPase, to induce autoimmune gastritis after transfer to immunodeficient recipients. We have also determined the susceptibility of the disease induced by each of the effector T cell types to suppression by polyclonal regulatory T cells (Treg) in vivo. Each type of effector cell induced autoimmune gastritis with distinct histological patterns. Th17 cells induced the most destructive disease with cellular infiltrates composed primarily of eosinophils accompanied by high levels of serum IgE. Polyclonal Treg could suppress the capacity of Th1 cells, could moderately suppress Th2 cells, but could suppress Th17-induced disease only at early time points. The major effect of the Treg was to inhibit the expansion of the effector T cells. However, effector cells isolated from protected animals were not anergic and were fully competent to proliferate and produce effector cytokines ex vivo. The strong inhibitory effect of polyclonal Treg on the capacity of some types of differentiated effector cells to induce disease provides an experimental basis for the clinical use of polyclonal Treg in the treatment of autoimmune disease in humans.
Asunto(s)
Enfermedades Autoinmunes/inmunología , Enfermedades Autoinmunes/patología , Gastritis/inmunología , Gastritis/patología , Interleucina-17/inmunología , Linfocitos T Colaboradores-Inductores/inmunología , Linfocitos T Reguladores/inmunología , Animales , Enfermedades Autoinmunes/sangre , Diferenciación Celular/inmunología , Separación Celular , Células Cultivadas , Susceptibilidad a Enfermedades/inmunología , Susceptibilidad a Enfermedades/patología , Femenino , Mucosa Gástrica/inmunología , Mucosa Gástrica/patología , Gastritis/sangre , Inmunoglobulina G/sangre , Ganglios Linfáticos/citología , Ganglios Linfáticos/inmunología , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Linfocitos T Colaboradores-Inductores/citologíaRESUMEN
Scurfy mice have a deletion in the forkhead domain of the forkhead transcription factor p3 (Foxp3), fail to develop thymic-derived, naturally occurring Foxp3+ regulatory T cells (nTreg), and develop a fatal lymphoproliferative syndrome with multi-organ inflammation. Transfer of thymic-derived Foxp3+ nTreg into neonatal Scurfy mice prevents the development of disease. Stimulation of conventional CD4+Foxp3(-) via the TCR in the presence of TGF-beta and IL-2 induces the expression of Foxp3 and an anergic/suppressive phenotype. To determine whether the TGF-beta-induced Treg (iTreg) were capable of suppressing disease in the Scurfy mouse, we reconstituted newborn Scurfy mice with polyclonal iTreg. Scurfy mice treated with iTreg do not show any signs of disease and have drastically reduced cell numbers in peripheral lymph nodes and spleen in comparison to untreated Scurfy controls. The iTreg retained their expression of Foxp3 in vivo for 21 days, migrated into the skin, and prevented the development of inflammation in skin, liver and lung. Thus, TGF-beta-differentiated Foxp3+ Treg appear to possess all of the functional properties of thymic-derived nTreg and represent a potent population for the cellular immunotherapy of autoimmune and inflammatory diseases.