RESUMEN
OBJECTIVE: Monogenic defects in the interleukin-10 (IL-10) pathway are extremely rare and cause infantile-onset inflammatory bowel disease (IBD)-like pathology. Understanding how immune responses are dysregulated in monogenic IBD-like diseases can provide valuable insight in "classical" IBD pathogenesis. Here, we studied long-term immune cell development and function in an adolescent IL-10 receptor (IL10RA)-deficient patient who presented in infancy with severe colitis and fistulizing perianal disease and is currently treated with immune suppressants. METHODS: Biomaterial was collected from the IL10RA-deficient patient, pediatric patients with IBD, and healthy controls. The frequency and phenotype of immune cells were determined in peripheral blood and intestinal biopsies by flow cytometry and immunohistochemistry. Functional changes in monocyte-derived dendritic cells and T cells were assessed by in vitro activation assays. RESULTS: The IL10RA-deficient immune system developed normally with respect to numbers and phenotype of circulating immune cells. Despite normal co-stimulatory molecule expression, bacterial lipopolysaccharide-stimulated monocyte-derived dendritic cells from the IL10RA-deficient patient released increased amounts of tumor necrosis factor α compared to healthy controls. Upon T-cell receptor ligation, IL10RA-deficient peripheral blood mononuclear cells released increased amounts of T-cell cytokines interferon γ and IL-17 agreeing with high numbers of T-bet and IL-17 cells in intestinal biopsies taken at disease onset. In vitro, the immunosuppressive drug thalidomide used to treat the patient's decreased peripheral blood mononuclear cell-derived tumor necrosis factor production. CONCLUSIONS: With time and during immunosuppressive treatment the IL10RA-deficient immune system develops relatively normally. Upon activation, IL-10 is crucial for controlling excessive inflammatory cytokine release by dendritic cells and preventing interferon γ and IL-17-mediated T-cell responses.
Asunto(s)
Inmunidad Adaptativa/fisiología , Células Dendríticas/metabolismo , Inmunidad Innata/fisiología , Enfermedades Inflamatorias del Intestino/inmunología , Subunidad alfa del Receptor de Interleucina-10/deficiencia , Subgrupos de Linfocitos T/metabolismo , Adolescente , Adulto , Biomarcadores/metabolismo , Estudios de Casos y Controles , Niño , Preescolar , Codón sin Sentido , Femenino , Mutación del Sistema de Lectura , Marcadores Genéticos , Humanos , Lactante , Enfermedades Inflamatorias del Intestino/genética , Subunidad alfa del Receptor de Interleucina-10/genética , Masculino , Persona de Mediana EdadRESUMEN
OBJECTIVES: The aim of this study was to identify new markers of mucosal T cells to monitor ongoing intestinal immune responses in peripheral blood. METHODS: Expression of cell-surface markers was studied in mice on ovalbumin (OVA)-specific T cells in the gut-draining mesenteric lymph nodes (MLN) after OVA feed. The effect of the local mucosal mediators retinoic acid (RA) and transforming growth factor-ß (TGF-ß) on the induction of a mucosal phenotype was determined in in vitro T-cell differentiation assays with murine and human T cells. Tetramer stainings were performed to study gluten-specific T cells in the circulation of patients with celiac disease, a chronic small-intestinal inflammation. RESULTS: In mice, proliferating T cells in MLN were CD62L(neg)CD38(+) during both tolerance induction and abrogation of intestinal homeostasis. This mucosal CD62L(neg)CD38(+) T-cell phenotype was efficiently induced by RA and TGF-ß in mice, whereas for human CD4(+) T cells RA alone was sufficient. The CD4(+)CD62L(neg)CD38(+) T-cell phenotype could be used to identify T cells with mucosal origin in human peripheral blood, as expression of the gut-homing chemokine receptor CCR9 and ß(7) integrin were highly enriched in this subset whereas expression of cutaneous leukocyte-associated antigen was almost absent. Tetramer staining revealed that gluten-specific T cells appearing in blood of treated celiac disease patients after oral gluten challenge were predominantly CD4(+)CD62L(neg)CD38(+). The total percentage of circulating CD62L(neg)CD38(+) of CD4 T cells was not an indicator of intestinal inflammation as percentages did not differ between pediatric celiac disease patients, inflammatory bowel disease patients and respective controls. However, the phenotypic selection of mucosal T cells allowed cytokine profiling as upon restimulation of CD62L(neg)CD38(+) cells interleukin-10 (IL-10) and interferon-γ (IFN-γ) transcripts were readily detected in circulating mucosal T cells. CONCLUSIONS: By selecting for CD62L(neg)CD38(+) expression that comprises 5-10% of the cells within the total CD4(+) T-cell pool we are able to highly enrich for effector T cells with specificity for mucosal antigens. This is of pivotal importance for functional studies as this purification enhances the sensitivity of cytokine detection and cellular activation.
Asunto(s)
ADP-Ribosil Ciclasa 1/inmunología , Linfocitos T CD4-Positivos/inmunología , Enfermedad Celíaca/inmunología , Glútenes/metabolismo , Selectina L/metabolismo , ADP-Ribosil Ciclasa 1/genética , Adulto , Animales , Biomarcadores/análisis , Biopsia con Aguja , Linfocitos T CD4-Positivos/patología , Estudios de Casos y Controles , Enfermedad Celíaca/patología , Niño , Modelos Animales de Enfermedad , Duodeno/inmunología , Duodeno/patología , Femenino , Regulación de la Expresión Génica , Glútenes/inmunología , Humanos , Mucosa Intestinal/inmunología , Mucosa Intestinal/patología , Selectina L/inmunología , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Transgénicos , Reacción en Cadena de la Polimerasa/métodos , Especificidad de la EspecieRESUMEN
BACKGROUND: Celiac disease (CD) is an intestinal inflammation driven by gluten-reactive CD4(+) T cells. Due to lack of selective markers it has not been determined whether defects in inducible regulatory T cell (Treg) differentiation are associated with CD. This is of importance as changes in numbers of induced Treg could be indicative of defects in mucosal tolerance development in CD. Recently, we have shown that, after encounter of retinoic acid during differentiation, circulating gut-imprinted T cells express CD62L(neg)CD38(+). Using this new phenotype, we now determined whether alterations occur in the frequency of natural CD62L(+)Foxp3(+) Treg or mucosally-imprinted CD62L(neg)CD38(+)Foxp3(+) Treg in peripheral blood of CD patients. In particular, we compared pediatric CD, aiming to select for disease at onset, with adult CD. METHODS: Cell surface markers, intracellular Foxp3 and Helios were determined by flow cytometry. Foxp3 expression was also detected by immunohistochemistry in duodenal tissue of CD patients. RESULTS: In children, the percentages of peripheral blood CD4(+)Foxp3(+) Treg were comparable between CD patients and healthy age-matched controls. Differentiation between natural and mucosally-imprinted Treg on the basis of CD62L and CD38 did not uncover differences in Foxp3. In adult patients on gluten-free diet and in refractory CD increased percentages of circulating natural CD62L(+)Foxp3(+) Treg, but normal mucosally-imprinted CD62L(neg)CD38(+)Foxp3(+) Treg frequencies were observed. CONCLUSIONS: Our data exclude that significant numeric deficiency of mucosally-imprinted or natural Foxp3(+) Treg explains exuberant effector responses in CD. Changes in natural Foxp3(+) Treg occur in a subset of adult patients on a gluten-free diet and in refractory CD patients.
Asunto(s)
ADP-Ribosil Ciclasa 1/metabolismo , Enfermedad Celíaca/inmunología , Factores de Transcripción Forkhead/metabolismo , Impresión Genómica , Mucosa Intestinal/inmunología , Selectina L/metabolismo , Linfocitos T Reguladores/inmunología , Adolescente , Adulto , Estudios de Casos y Controles , Enfermedad Celíaca/sangre , Enfermedad Celíaca/patología , Movimiento Celular , Niño , Preescolar , Humanos , Lactante , Interleucina-15/sangre , Mucosa Intestinal/patología , Recuento de LinfocitosRESUMEN
Sézary syndrome (SS) is a cutaneous T-cell lymphoma (CTCL) with malignant CD4+ T cells (SS cells) in skin, lymph nodes, and blood. Signal transducer and activator of transcription 3 (STAT3) is constitutively activated in SS cells, whereas this activation is lost upon in vitro culturing, indicating that STAT3 activation observed in vivo is the result of activating factors in the micromilieu of the malignant cells. We investigated which factors are involved in STAT3 activation in SS, focusing on cytokines of the common γ-chain family because of their crucial role in T-cell activation. Furthermore, downstream effects of STAT3 signaling in SS cells were assayed. In SS cells, STAT3 was strongly activated by IL-21, and increased expression of IL-21 and its receptor chains was observed in peripheral blood SS cells. IL-21 and IL-21R protein expression was detectable on neoplastic cells in SS skin biopsies. Using short-term culturing experiments, we demonstrate that IL-21 itself and the α-chain of the IL-2 receptor are STAT3 target genes in SS cells, thereby rendering cells more sensitive to stimulation with the T-cell proliferation and activating cytokine IL-2. Combined, our data point toward a pivotal role for an autocrine positive feedback loop involving IL-21 and consequent persistent STAT3 activation in the pathogenesis of SS, thereby indicating IL-21 and IL-21R as new therapeutical targets.