RESUMEN
Regulatory T cells (Tregs) are critical homeostatic components in preventing the development of autoimmunity, and are a major focus for their therapeutic potential for autoimmune diseases. To enhance the efficacy of Tregs in adoptive therapy, we developed a strategy for generating engineered Tregs that have the capacity to target autoimmune T cells in an Ag-specific manner. Using a retroviral expression system encoding Foxp3 and HLA-DR1 covalently linked to the immunodominant peptide of the autoantigen type II collagen (DR1-CII), naive T cells were engineered to become Tregs that express DR1-CII complexes on their surface. When these cells were tested for their ability to prevent the development of collagen induced arthritis, both the engineered DR1-CII-Foxp3 and Foxp3 only Tregs significantly reduced the severity and incidence of disease. However, the mechanism by which these two populations of Tregs inhibited disease differed significantly. Disease inhibition by the DR1-CII-Foxp3 Tregs was accompanied by significantly lower numbers of autoimmune CII-specific T cells in vivo and lower levels of autoantibodies in comparison with engineered Tregs expressing Foxp3 alone. In addition, the numbers of IFN-γ- and IL-17-expressing T cells in mice treated with DR1-CII-Foxp3 Tregs were also significantly reduced in comparison with mice treated with Foxp3 engineered Tregs or vector control cells. These data indicate that the coexpression of class II autoantigen-peptide complexes on Tregs provides these cells with a distinct capacity to regulate autoimmune T cell responses that differs from that used by conventional Tregs.
Asunto(s)
Expresión Génica , Antígeno HLA-DR1/genética , Antígeno HLA-DR1/inmunología , Linfocitos T Reguladores/inmunología , Linfocitos T Reguladores/metabolismo , Animales , Artritis/genética , Artritis/inmunología , Artritis/prevención & control , Artritis Experimental , Enfermedades Autoinmunes/genética , Enfermedades Autoinmunes/inmunología , Enfermedades Autoinmunes/prevención & control , Autoinmunidad , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD4-Positivos/metabolismo , Línea Celular , Supervivencia Celular , Colágeno Tipo II/genética , Colágeno Tipo II/inmunología , Colágeno Tipo II/metabolismo , Citocinas/inmunología , Citocinas/metabolismo , Orden Génico , Vectores Genéticos/genética , Humanos , Ratones , Ratones Transgénicos , Péptidos/inmunología , Fenotipo , Retroviridae/genética , Transducción GenéticaRESUMEN
Although it is clear that CD4(+) T cells play a major role in mediating the pathogenesis of autoimmunity, they often represent only a minor population at the site of inflammation in autoimmune diseases. To investigate the migration and specificity of autoimmune T cells to the inflammatory site, we used the collagen-induced arthritis model to determine the frequency, clonotype, and specificity of T cells that infiltrate arthritic joints. We demonstrate that despite the fact that CD4(+) T cells are a minor population of the synovial infiltrate, the CD4(+) T cells present are a highly selective subset of the TCR repertoire and, based on CDR3 length polymorphisms, have a limited clonality. Although a similar repertoire of type II collagen (CII)-specific TCR-BV8 and BV14-expressing T cells was found in peripheral lymphoid organs, the clonality of the TCR-BV8 and BV14 T cells that migrate to the arthritic joint generally made up a single CDR3 length. T cell hybridomas produced from these joint-derived cells revealed that many of these infiltrating T cells are CII specific, and the majority recognize mouse CII. These data suggest that despite being a minor population at the site of inflammation, autoantigen-specific T cells are selectively recruited and/or retained in the arthritic joint and may be playing a significant role in the pathogenesis of the autoimmune arthritis. In addition, this model may be very useful for studying the function in situ and the mechanism by which autoimmune T cells are recruited to the site of inflammation.
Asunto(s)
Artritis Experimental/inmunología , Artritis Experimental/patología , Autoantígenos/inmunología , Movimiento Celular/inmunología , Colágeno Tipo II/inmunología , Antígeno HLA-DR1/inmunología , Subgrupos de Linfocitos T/inmunología , Subgrupos de Linfocitos T/patología , Animales , Artritis Experimental/metabolismo , Artritis Reumatoide/inmunología , Artritis Reumatoide/metabolismo , Artritis Reumatoide/patología , Autoantígenos/genética , Bovinos , Movimiento Celular/genética , Células Clonales , Colágeno Tipo II/genética , Antígenos HLA-A/biosíntesis , Antígenos HLA-A/genética , Antígeno HLA-DR1/biosíntesis , Antígeno HLA-DR1/genética , Cadenas HLA-DRB1 , Humanos , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Receptores de Antígenos de Linfocitos T alfa-beta/genética , Receptores de Antígenos de Linfocitos T alfa-beta/inmunología , Índice de Severidad de la Enfermedad , Subgrupos de Linfocitos T/metabolismoRESUMEN
The collagen-induced arthritis (CIA) mouse model is the most commonly studied autoimmune model of rheumatoid arthritis. Autoimmune arthritis is induced in this model by immunization with an emulsion of complete Freund's adjuvant and type II collagen (CII). This protocol describes the steps necessary for acquisition, handling and preparation of CII, as well as selection of mouse strains, proper immunization technique and evaluation of the arthritis incidence and severity. Typically, the first signs of arthritis appear in this model 21-28 days after immunization, and identification of the arthritic limbs is not difficult. Using the protocol described, the investigator should be able to reproducibly induce a high incidence of CIA in various strains of genetically susceptible mice as well as learn how to critically evaluate the pathology of the disease. The total time for the preparation of reagents and the immunization of ten mice is about 1.5 h.
Asunto(s)
Artritis Experimental/etiología , Colágeno Tipo II/administración & dosificación , Modelos Animales de Enfermedad , Adyuvante de Freund/administración & dosificación , Inmunización/métodos , Animales , Artritis Experimental/inmunología , Artritis Experimental/patología , Colágeno Tipo II/inmunología , Adyuvante de Freund/inmunología , RatonesRESUMEN
Although the pathogenesis of collagen-induced arthritis (CIA), a model of rheumatoid arthritis, is mediated by both collagen-specific CD4(+) T cells and Ab specific for type II collagen (CII), the role of CII-specific T cells in the pathogenesis of CIA remains unclear. Using tetrameric HLA-DR1 with a covalently bound immunodominant CII peptide, CII(259-273), we studied the development of the CII-specific T cell response in the periphery and arthritic joints of DR1 transgenic mice. Although the maximum number of DR1-CII-tetramer(+) cells was detected in draining lymph nodes 10 days postimmunization, these T cells accounted for only 1% or less of the CD4(+) population. After day 10, their numbers gradually decreased, but were still detectable on day 130. Examination of TCR expression and changes in CD62L, CD44(high), and CD69 expression by these T cells indicated that they expressed a limited TCR-BV repertoire and had clearly undergone activation. RT-PCR analysis of cytokine expression by the tetramer(+) T cells compared with tetramer(-) cells indicated the tetramer(+) cells expressed high levels of Th1 and proinflammatory cytokines, including IL-2, IFN-gamma, IL-6, TNF-alpha, and especially IL-17. Additionally, analysis of the synovium from arthritic paws indicated that the same CD4(+)/BV8(+)/BV14(+)/tetramer(+) T cells were present in the arthritic joints. These data demonstrate that although only small numbers of CII-specific T cells are generated during the development of CIA, these cells express very high levels of cytokine mRNA and appear to preferentially migrate to the arthritic joint, indicating a potential direct role of CII-specific T cells in the pathogenesis of CIA.
Asunto(s)
Artritis/etiología , Autoinmunidad , Colágeno Tipo II/inmunología , Antígeno HLA-DR1/inmunología , Linfocitos T/inmunología , Animales , Artritis/inducido químicamente , Artritis/inmunología , Quimiotaxis de Leucocito/inmunología , Colágeno/efectos adversos , Citocinas/genética , Modelos Animales de Enfermedad , Epítopos de Linfocito T/inmunología , Antígeno HLA-DR1/genética , Humanos , Epítopos Inmunodominantes , Articulaciones/inmunología , Articulaciones/patología , Activación de Linfocitos , Ratones , Ratones Transgénicos , Membrana Sinovial/inmunología , Membrana Sinovial/patología , Factores de TiempoRESUMEN
A number of clinical and experimental observations have been made relating elevated estrogen levels with the amelioration of autoimmune diseases, yet questions remain about the levels required for efficacy as well as the mechanism of disease inhibition. Using the collagen-induced arthritis (CIA) model, we have studied the effects of physiological, sustained levels of 17beta-estradiol in preventing the development of autoimmune arthritis and analyzed the changes in the autoimmune response. Using time-release pellets of 17beta-estradiol, arthritis development was significantly inhibited in three different strains of CIA-susceptible mice compared with the effect of placebo treatment, and serum estradiol levels similar to those of mice in estrus were found to be equally effective as higher estradiol concentrations. Analysis of the autoimmune response in the estradiol-treated mice indicated that T cell production of IFN-gamma was markedly decreased, and significant decreases were also observed in levels of IL-10 and GM-CSF produced by lymph nodes cells from estradiol-treated mice. Although the total IgG anti-CII response was only minimally affected by estrogen treatment, a significant reduction in the levels of IgG2a anti-CII Abs and an increase in the levels of IgG1 anti-CII Abs were observed in estradiol-treated mice. These data indicate that estradiol treatment altered the Th profile of the autoimmune T cell response, which, in turn, altered the production of IgG Abs to an isotype that is poor at fixing complement, an important component in the immunopathogenesis of CIA.
Asunto(s)
Artritis Experimental/prevención & control , Autoanticuerpos/biosíntesis , Enfermedades Autoinmunes/prevención & control , Estradiol/administración & dosificación , Isotipos de Inmunoglobulinas/biosíntesis , Animales , Artritis Experimental/inmunología , Enfermedades Autoinmunes/inmunología , Bovinos , Colágeno Tipo II/inmunología , Citocinas/biosíntesis , Progresión de la Enfermedad , Susceptibilidad a Enfermedades/inmunología , Regulación hacia Abajo/efectos de los fármacos , Regulación hacia Abajo/inmunología , Implantes de Medicamentos , Humanos , Epítopos Inmunodominantes/inmunología , Inmunoglobulina G/biosíntesis , Ratones , Ratones Endogámicos DBA , Ratones Transgénicos , Linfocitos T/efectos de los fármacos , Linfocitos T/inmunología , Linfocitos T/metabolismoRESUMEN
To study the phenotypic and functional changes in naive type II collagen (CII)-specific autoimmune T cells following a tolerogenic signal, a TCR-transgenic (Tg) mouse model of collagen-induced arthritis was developed. These Tg mice express an I-A(q)-restricted CII (260-267)-specific TCR that confers severe accelerated autoimmune arthritis following immunization with CII. Despite the fact that >90% of the alphabeta T cells express the Tg, these mice can be rendered completely tolerant to the induction of arthritis by i.v. administration of 200 microg of CII. As early as 24 h after CII administration, CII-specific T cells demonstrated a decreased ability to proliferate in response to the CII immunodominant peptide and phenotypically altered the expression of L-selectin to CD62L(low) and of phagocytic glycoprotein-1 to CD44(high), expression levels consistent with the phenotype of memory T cells. In addition, they up-regulated the expression of the activation markers CD71 and CD69. Functionally, following tolerogenic stimulation, the CII-specific T cells produced similar levels of IL-2 in comparison to controls when challenged with CII peptide, however, by 48 h after exposure to tolerogen, IL-2 production dropped and was replaced by high levels of IL-10 and IL-4. Based on their production of Th2 cytokines, these data suggest that T regulatory cells expressing activation and memory markers are induced by the tolerogen and may exert their influence via cytokines to protect the animals from the induction of arthritis.