RESUMEN
Although FOXP3+ regulatory T cells (Treg) depend on IL-2 produced by other cells for their survival and function, the levels of IL-2 in inflamed tissue are low, making it unclear how Treg access this critical resource. Here, we show that Treg use heparanase (HPSE) to access IL-2 sequestered by heparan sulfate (HS) within the extracellular matrix (ECM) of inflamed central nervous system tissue. HPSE expression distinguishes human and murine Treg from conventional T cells and is regulated by the availability of IL-2. HPSE-/- Treg have impaired stability and function in vivo, including in the experimental autoimmune encephalomyelitis (EAE) mouse model of multiple sclerosis. Conversely, endowing monoclonal antibody-directed chimeric antigen receptor (mAbCAR) Treg with HPSE enhances their ability to access HS-sequestered IL-2 and their ability to suppress neuroinflammation in vivo. Together, these data identify a role for HPSE and the ECM in immune tolerance, providing new avenues for improving Treg-based therapy of autoimmunity.
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Encefalomielitis Autoinmune Experimental , Linfocitos T Reguladores , Ratones , Animales , Humanos , Interleucina-2/metabolismo , Glucuronidasa/genética , Glucuronidasa/metabolismo , Matriz Extracelular/metabolismo , Heparitina Sulfato/metabolismoRESUMEN
BACKGROUND & AIMS: Inflammatory bowel disease (IBD) causes a marked increase in the number of T cells in the intestinal mucosa. Debate exists about whether these excess cells arise from local clonal proliferation or recruitment from the periphery. METHODS: CD8+ T cells were sorted from colon biopsy specimens and blood for T-cell receptor (TCR) ß-chain sequencing. Biopsy specimens from inflamed or uninflamed colon from ulcerative colitis or Crohn's disease cohorts were compared with colon biopsy specimens from people without IBD, as well as with autologous blood α4ß7+, α4ß7- effector/memory, terminal effector/memory CD45RA+ T cell, and mucosal-associated invariant T-cell CD8 subpopulations. RESULTS: CD8 TCR diversity in mucosa and blood did not correlate with inflammation. Repertoire overlap between any 2 distinct locations of a given person's colon was consistently high, although often lower between inflamed and uninflamed sites. CD8 TCR repertoires overlapped between the colon and each peripheral blood subpopulation studied, with the highest overlap seen for integrin α4ß7+ T cells. Inflamed tissue consistently overlapped more than uninflamed tissue with each blood subpopulation. CONCLUSIONS: CD8 T-cell clones are spread homogenously throughout the length of the colon. Although TCR repertoire overlap is greater within than between inflamed and uninflamed colon segments, a similar TCR diversity in both argues against local clonal expansion being the main source of excess cytotoxic T cells in inflamed mucosa. Rather, the increased TCR overlap observed between blood and inflamed mucosa supports the significance of T-cell trafficking in IBD pathogenesis, particularly concerning α4ß7+ T-cell populations.
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Colitis Ulcerosa , Enfermedad de Crohn , Enfermedades Inflamatorias del Intestino , Humanos , Enfermedades Inflamatorias del Intestino/patología , Enfermedad de Crohn/patología , Receptores de Antígenos de Linfocitos T alfa-beta/genéticaRESUMEN
OBJECTIVES: Mucosal-Associated Invariant T (MAIT) cells are T cells with a semi-invariant T cell receptor (TCR), recognizing riboflavin precursors presented by a non-polymorphic MR1 molecule. As these precursors are produced by the gut microbiome, we characterized the frequency, phenotype and clonality of MAIT cells in human colons with and without Crohn's disease (CD). METHODS: The transcriptome of MAIT cells sorted from blood and intestinal lamina propria cells from colectomy recipients were compared with other CD8+ T cells. Colon biopsies from an additional ten CD patients and ten healthy controls (HC) were analyzed by flow cytometry. TCR genes were sequenced from individual MAIT cells from these biopsies and compared with those of MAIT cells from autologous blood. RESULTS: MAIT cells in the blood and colon showed a transcriptome distinct from other CD8 T cells, with more expression of the IL-23 receptor. MAIT cells were enriched in the colons of CD patients, with less NKG2D in inflamed versus uninflamed segments. Regardless of disease, most MAIT cells expressed integrin α4ß7 in the colon but not in the blood, where they were enriched for α4ß7 expression. TCR sequencing revealed heterogeneity in the colon and blood, with few public sequences associated with cohorts. CONCLUSION: MAIT cells are enriched in the colons of CD patients and disproportionately express molecules (IL-23R, integrin α4ß7) targeted by CD therapeutics, to suggest a pathogenic role for them in CD. Public TCR sequences were neither common nor sufficiently restricted to a cohort to suggest protective or pathogenic antigen-specificities.
Asunto(s)
Enfermedad de Crohn , Células T Invariantes Asociadas a Mucosa , Humanos , Receptores de Antígenos de Linfocitos T alfa-beta/genética , Receptores de Antígenos de Linfocitos T alfa-beta/metabolismo , Enfermedad de Crohn/genética , Enfermedad de Crohn/metabolismo , Antígenos de Histocompatibilidad Clase I/genética , Receptores de Antígenos de Linfocitos T/metabolismo , Colon/metabolismoRESUMEN
BACKGROUND: Vedolizumab, an antibody blocking integrin α4ß7, is a safe and effective therapy for Crohn's disease and ulcerative colitis. Blocking α4ß7 from binding its cognate addressin MAdCAM-1 on intestinal blood vessel endothelial cells prevents T cells from migrating to the gut mucosa in animal models. However, data supporting this mechanism of action in humans is limited. METHODS: We conducted a cross-sectional case-control study to evaluate the effect of vedolizumab on intestinal immune cell populations while avoiding the confounding effect of resolving inflammation on the cellularity of the colonic mucosa in treatment-responsive patients. Colon biopsies from 65 case subjects receiving vedolizumab were matched with biopsies from 65 control individuals, similar in disease type, medications, anatomic location, and inflammation. Biopsies were analyzed by flow cytometry and full messenger RNA transcriptome sequencing of sorted T cells. RESULTS: No difference was seen between vedolizumab recipients and control individuals in the quantity of any antigen-experienced T lymphocyte subset or in the quality of the transcriptome in any experienced T cell subset. Fewer naïve colonic B and T cells were seen in vedolizumab recipients than control individuals, regardless of response. However, the most striking finding was a marked reduction in CD1c+ (BDCA1+) dendritic cells exclusively in vedolizumab-responsive patients. In blood, these dendritic cells ubiquitously express high levels of α4ß7, which is rapidly downregulated upon vedolizumab exposure. CONCLUSIONS: The clinical effects of vedolizumab reveal integrin α4ß7-dependent dendritic cell migration to the intestinal mucosa to be central to inflammatory bowel disease pathogenesis.
Vedolizumab had no effect on the number or gene expression of memory T lymphocytes in the colons of recipients relative to control individuals. However, the colons of vedolizumab-responsive patients had distinctly fewer dendritic cells, which in blood express the most integrin α4ß7.
RESUMEN
BACKGROUND: Janus kinase (JAK) inhibitors (JAKinibs) are effective small molecule therapies for treating Crohn's Disease (CD) and Ulcerative Colitis (UC), collectively known as inflammatory bowel disease (IBD). By preventing JAKs from phosphorylating signal transducer and activator of transcription proteins, JAKinibs disrupt cytokine signaling pathways that promote inflammation. Despite considerable overlap in the JAKs they target, first and second generation JAKinibs display different clinical efficacies in CD and UC. METHODS: We conducted a comparative phosflow study of four JAKinibs (filgotinib, upadacitinib, tofacitinib, and deucravacitinib) to observe subtle mechanistic differences that may dictate their clinical behavior. Resected mesenteric lymph node (MLN) cells from 19 patients (9 CD, 10 UC) were analyzed by flow cytometry in the presence or absence of different cytokine stimuli and titrated JAKinibs. RESULTS: We found a higher potency of the JAK 1/3-preferential inhibitor, tofacitinib, for JAK 3-dependent cytokine signaling pathways in comparison to filgotinib, but a higher potency of the JAK 1-preferential inhibitors, filgotinib and upadacitinib, for JAK 3-independent cytokine signaling pathways. Deucravacitinib, a TYK2-preferential inhibitor, demonstrated a much narrower selectivity by inhibiting only IL-10 and IFN-ßpathways, albeit more potently than the other JAKinibs . Additionally, we found some differences in the sensitivity of immune cells from CD versus UC and patients with versus without a CD-associated NOD2 polymorphism to phosphorylate signal transducer and activator of transcriptions in response to specific cytokine stimulation. CONCLUSIONS: Despite their similarities, differences exist in the relative potencies of different JAKinibs against distinct cytokine families to explain their clinical efficacy.
RESUMEN
FOXP3+ regulatory T cells (Treg) depend on exogenous IL-2 for their survival and function, but circulating levels of IL-2 are low, making it unclear how Treg access this critical resource in vivo. Here, we show that Treg use heparanase (HPSE) to access IL-2 sequestered by heparan sulfate (HS) within the extracellular matrix (ECM) of inflamed central nervous system tissue. HPSE expression distinguishes human and murine Treg from conventional T cells and is regulated by the availability of IL-2. HPSE-/- Treg have impaired stability and function in vivo, including the experimental autoimmune encephalomyelitis (EAE) mouse model of multiple sclerosis. Conversely, endowing Treg with HPSE enhances their ability to access HS-sequestered IL-2 and their tolerogenic function in vivo. Together, these data identify novel roles for HPSE and the ECM in immune tolerance, providing new avenues for improving Treg-based therapy of autoimmunity.
RESUMEN
Successful treatment of inflammatory bowel disease (IBD) with the anti-integrin α4ß7 mAb vedolizumab suggests that interaction of this integrin with addressin mucosal addressin cell adhesion molecule-1 (MAdCAM-1) is central to IBD pathogenesis. Although this was presumed to be due to an inhibition of lymphocyte trafficking to the gut, as has been observed in animal models, we report no depletion of CD4 T cells from the colonic mucosa as a consequence of vedolizumab treatment in humans, regardless of efficacy. Likewise, no upregulation of alternative trafficking mechanisms was observed as a consequence of therapy to suggest that this homeostasis is maintained in patients by a mechanistic escape from inhibition. Instead, we explore a role for MAdCAM-integrin interaction as a gut-specific costimulatory signal, demonstrating that it can replace CD28 ligation to activate human T cells in vitro. This activation through integrin α4ß7 is mediated through the gut-restricted molecule MAdCAM-1, and it cannot be replicated by matrix molecules or proteins that bind other integrins. A detailed analysis of mRNA expression by human T cell subsets following suboptimal TCR stimulation in the presence or absence of CD28 versus MAdCAM-1 costimulation reveals marked similarity in the effect that these two signals have upon T cells, with temporal or quantitative differences detected in the expression of cytokines associated with Th17 cells or pyogenic inflammation. Thus, we describe an alternative costimulatory pathway for T cells in the intestine, through ligation of integrin α4ß7 by MAdCAM-1, which may explain the therapeutic efficacy of vedolizumab and have implications concerning the treatment of IBD.
Asunto(s)
Enfermedades Inflamatorias del Intestino , Integrinas , Animales , Antígenos CD28/metabolismo , Linfocitos T CD4-Positivos/metabolismo , Expresión Génica , Humanos , Enfermedades Inflamatorias del Intestino/tratamiento farmacológico , Enfermedades Inflamatorias del Intestino/metabolismo , Integrinas/metabolismoRESUMEN
Type 2 inflammation is associated with epithelial cell responses, including goblet cell hyperplasia, that promote worm expulsion during intestinal helminth infection. How these epithelial responses are regulated remains incompletely understood. Here, we show that mice deficient in the prostaglandin D2 (PGD2) receptor CRTH2 and mice with CRTH2 deficiency only in nonhematopoietic cells exhibited enhanced worm clearance and intestinal goblet cell hyperplasia following infection with the helminth Nippostrongylus brasiliensis. Small intestinal stem, goblet, and tuft cells expressed CRTH2. CRTH2-deficient small intestinal organoids showed enhanced budding and terminal differentiation to the goblet cell lineage. During helminth infection or in organoids, PGD2 and CRTH2 down-regulated intestinal epithelial Il13ra1 expression and reversed Type 2 cytokine-mediated suppression of epithelial cell proliferation and promotion of goblet cell accumulation. These data show that the PGD2-CRTH2 pathway negatively regulates the Type 2 cytokine-driven epithelial program, revealing a mechanism that can temper the highly inflammatory effects of the anti-helminth response.
Asunto(s)
Citocinas/metabolismo , Mucosa Intestinal/parasitología , Prostaglandina D2/metabolismo , Receptores Inmunológicos/metabolismo , Receptores de Prostaglandina/metabolismo , Infecciones por Strongylida/parasitología , Animales , Femenino , Gastroenteritis/parasitología , Gastroenteritis/patología , Células Caliciformes/patología , Interacciones Huésped-Parásitos/fisiología , Mucosa Intestinal/patología , Masculino , Ratones Endogámicos C57BL , Nippostrongylus/patogenicidad , Organoides , Receptores Inmunológicos/genética , Receptores de Prostaglandina/genética , Infecciones por Strongylida/patologíaRESUMEN
BACKGROUND & AIMS: Crohn's disease (CD) likely represents decreased immune tolerance to intestinal bacterial antigens. Most CD patients have high titers of antibodies to intestinal commensal proteins, including the outer membrane porin C (OmpC) of Escherichia coli. METHODS: By using major histocompatibility complex II tetramers, we identified an HLA-DRB1∗15:01-restricted peptide epitope of OmpC recognized by CD4+ T cells in peripheral blood mononuclear cells from HLA-DRB1∗15:01+ healthy control (HC) and CD patients. RESULTS: The precursor frequency of these cells in CD correlated with anti-OmpC IgA titers, but did not differ from that of HCs. In both cohorts, they showed a CD161+, integrin α4ß7+ phenotype ex vivo by flow cytometry, distinct from the C-X-C Motif Chemokine Receptor 3 phenotype of autologous influenza hemagglutinin (Flu) peptide-specific T cells. The T-cell receptor α and ß chains of in vitro-expanded OmpC-specific T-cell clones often contained public amino acid sequences that were identical in cells from different patients. Expanded T-cell clones from CD subjects produced significantly less interleukin (IL)10 (P < .0001) than those from HCs, and a trend toward decreased production of the T helper 2 cell-associated IL4, IL5, and IL13 by CD clones also was seen. CONCLUSIONS: Both HCs and CD patients have detectable OmpC-specific T cells in circulation, with similar immunophenotypes and often identical T-cell-receptor sequences. However, expanded clones from patients with CD produce less of the immunoregulatory cytokine IL10, showing a selective defect in the regulatory function of intestinal microbial antigen-specific T cells in patients with CD.
Asunto(s)
Linfocitos T CD4-Positivos/inmunología , Enfermedad de Crohn/inmunología , Proteínas de Escherichia coli/inmunología , Microbioma Gastrointestinal/inmunología , Porinas/inmunología , Adulto , Anciano , Linfocitos T CD4-Positivos/metabolismo , Estudios de Casos y Controles , Enfermedad de Crohn/sangre , Enfermedad de Crohn/microbiología , Epítopos de Linfocito T/inmunología , Escherichia coli/inmunología , Femenino , Cadenas HLA-DRB1/metabolismo , Voluntarios Sanos , Interacciones Microbiota-Huesped/inmunología , Humanos , Interleucina-10/metabolismo , Masculino , Persona de Mediana Edad , Receptores de Antígenos de Linfocitos T/metabolismo , Adulto JovenRESUMEN
Integrin alpha4/beta7 on circulating lymphocytes identifies them as gut-tropic, and can be targeted by the humanized antibody vedolizumab to treat inflammatory bowel disease (IBD). We found lymphocytes expressing alpha4/beta7 were significantly more responsive to the pro-inflammatory cytokines IL-6, IL-7, and IL-21, and less responsive to the regulatory T cell (Treg)-supporting cytokine IL-2. Alpha4/beta7 was expressed by a smaller percent of FOXP3â¯+â¯Helios+ thymically-derived Tregs (tTregs) than FOXP3â¯+â¯Helios- peripherally-derived Tregs (pTregs) or FOXP3- effector T cells. Integrin alpha4/beta7+ CD4 T cells were also rare among cells expressing the Th2 marker CRTh2, but enriched in cells bearing the circulating T follicular helper cell marker CXCR5. Thus the effect of this anti-integrin therapy on the mucosal immune system may be more qualitative than quantitative, and selectively replace pro-inflammatory effector cells with Tregs and Th2 cells to facilitate immune tolerance in the mucosa without globally depleting lymphocytes from the intestinal mucosa.
Asunto(s)
Anticuerpos Monoclonales Humanizados/uso terapéutico , Fármacos Gastrointestinales/uso terapéutico , Enfermedades Inflamatorias del Intestino/inmunología , Integrinas/metabolismo , Intestinos/inmunología , Linfocitos T Reguladores/inmunología , Células TH1/inmunología , Células Th2/inmunología , Adulto , Circulación Sanguínea , Citocinas/metabolismo , Femenino , Factores de Transcripción Forkhead/metabolismo , Humanos , Factor de Transcripción Ikaros/metabolismo , Tolerancia Inmunológica , Mediadores de Inflamación/metabolismo , Enfermedades Inflamatorias del Intestino/tratamiento farmacológico , Integrinas/inmunología , Masculino , Persona de Mediana Edad , Receptores CXCR5/metabolismoRESUMEN
BACKGROUND/AIMS: Vedolizumab is an anti-α4ß7 monoclonal antibody approved for the treatment of inflammatory bowel disease (IBD). This exploratory study aimed to identify biomarkers associated with vedolizumab response. METHODS: Twenty-six IBD patients (15 with Crohn's, 11 with ulcerative or indeterminate colitis) initiating vedolizumab at a single center between 2014 and 2016 underwent sampling of serum and peripheral blood mononuclear cells (PBMCs) before and during vedolizumab therapy. Response was defined as steroid-free improvement in endoscopic score or Harvey-Bradshaw index/simple clinical colitis activity index (reduction greater than 3 or total less than 3). PBMCs were evaluated for immunophenotype and expression of α4ß7 integrin on lymphocytes before and during vedolizumab therapy. Serum vedolizumab levels and α4ß7 saturation were measured serially after induction. RESULTS: Fourteen out of 26 (54%) patients treated with vedolizumab responded to therapy. Pretreatment α4ß7 expression was higher in responders on multiple subsets of T, B, and NK cells, with terminal effector memory (p = .0009 for CD4 and .0043 for CD8) and NK cells (p = .0047) best discriminating between responders and nonresponders. During therapy, log10 serum vedolizumab levels at trough were higher in responders than nonresponders (p = .0007). Conversely, the percentage of effector memory T cells with free α4ß7 at trough was lower in responders than nonresponders (p < .0001). However, loss of α4ß7 saturation with vedolizumab was more sensitive to low serum vedolizumab in nonresponders. CONCLUSIONS: Pretreatment α4ß7 expression and α4ß7 receptor saturation during maintenance therapy were identified as candidate biomarkers for vedolizumab response.
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Anticuerpos Monoclonales Humanizados/uso terapéutico , Colitis Ulcerosa/tratamiento farmacológico , Enfermedad de Crohn/tratamiento farmacológico , Fármacos Gastrointestinales/uso terapéutico , Integrinas/antagonistas & inhibidores , Subgrupos Linfocitarios/efectos de los fármacos , Adulto , Anticuerpos Monoclonales Humanizados/sangre , Biomarcadores/sangre , Separación Celular/métodos , Colitis Ulcerosa/sangre , Colitis Ulcerosa/diagnóstico , Colitis Ulcerosa/inmunología , Enfermedad de Crohn/sangre , Enfermedad de Crohn/diagnóstico , Enfermedad de Crohn/inmunología , Endoscopía Gastrointestinal , Femenino , Citometría de Flujo , Fármacos Gastrointestinales/sangre , Humanos , Inmunofenotipificación/métodos , Integrinas/sangre , Integrinas/inmunología , Subgrupos Linfocitarios/inmunología , Subgrupos Linfocitarios/metabolismo , Masculino , Persona de Mediana Edad , Fenotipo , Inducción de Remisión , Factores de Tiempo , Resultado del Tratamiento , WashingtónRESUMEN
AIM: To identify which blood and mucosal lymphocyte populations are specifically depleted by thiopurine use in vivo. METHODS: The thiopurines azathioprine and 6-mercaptopurine have been a mainstay of inflammatory bowel disease (IBD) therapy for decades, but their mechanism of action in vivo remains obscure. Although thiopurines are lymphotoxic at high doses, and have been reported to cause T cell apoptosis in vitro, their ability to control IBD at lower doses suggests that they may selectively deplete particular lymphocyte populations. Blood cells from 19 IBD patients on a thiopurine, 19 IBD patients not on a thiopurine, and 38 matched healthy control subjects were analyzed by multiple multi-color flow cytometry panels to quantify the immune cell subsets contained therein, both as a percent of cells, and as an absolute cell count. Similar analyses were performed on colon biopsies from 17 IBD patients on a thiopurine, 17 IBD patients not on a thiopurine, and 49 healthy screening colonoscopy recipients. RESULTS: Complete blood counts revealed lower lymphocyte, but not monocyte or granulocyte, counts in IBD patients who were taking thiopurines at the time of sampling. This reduction was restricted to CD3-negative lymphocytes, wherein both natural killer (NK) and B cells were significantly reduced among thiopurine recipients. Among CD19+ B cells, the transitional B cells were particularly depleted, being nearly absent in both blood and colon biopsies of thiopurine recipients. No differences were associated with thiopurine use in CD8+ T cells, mucosa-associated invariant T (MAIT) cells, invariant natural killer T (iNKT) cells, gamma/delta T cells, Th1, Th17, regulatory T cells (Tregs) or naïve CD4+ T cells. However, patients with IBD had significantly more circulating FOXP3+, Helios+ Tregs and fewer iNKT and MAIT cells than healthy controls. CONCLUSION: Thiopurine use is associated with reduced B and NK cell, but not T cell, subpopulations in the blood of IBD patients.
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Linfocitos B/efectos de los fármacos , Enfermedades Inflamatorias del Intestino/tratamiento farmacológico , Células Asesinas Naturales/efectos de los fármacos , Mercaptopurina/análogos & derivados , Mercaptopurina/uso terapéutico , Membrana Mucosa/efectos de los fármacos , Antígenos CD19/metabolismo , Apoptosis , Azatioprina/uso terapéutico , Linfocitos B/citología , Complejo CD3/metabolismo , Estudios de Casos y Controles , Colon/efectos de los fármacos , Humanos , Enfermedades Inflamatorias del Intestino/inmunología , Células Asesinas Naturales/citología , Células T Asesinas Naturales/citología , Riesgo , Linfocitos T Reguladores/citologíaRESUMEN
Since their discovery two decades ago, CD4(+)CD25(+)Foxp3(+) regulatory T cells (Tregs) have become the subject of intense investigation by immunologists. Unlike other T cells, which promote an immune response, Tregs actively inhibit inflammation when activated by their cognate antigen, thus raising hope that these cells could be engineered into a highly targeted, antigen-specific, immunosuppressant therapy. Although Tregs represent less than 10% of circulating CD4(+)T cells, they have been shown to play an essential role in preventing or limiting inflammation in a variety of animal models and human diseases. In particular, spontaneous intestinal inflammation has been shown to occur in the absence of Tregs, suggesting that there may be a Treg defect central to the pathogenesis of human inflammatory bowel disease (IBD). However, over the past decade, multiple groups have reported no qualitative or quantitative deficits in Tregs from the intestines and blood of IBD patients to explain why these cells fail to regulate inflammation in Crohn's disease and ulcerative colitis. In this review, we will discuss the history of Tregs, what is known about them in IBD, and what progress and obstacles have been seen with efforts to employ them for therapeutic benefit.
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Colitis Ulcerosa/inmunología , Enfermedad de Crohn/inmunología , Intestinos/inmunología , Linfocitos T Reguladores/inmunología , Animales , Colitis Ulcerosa/microbiología , Colitis Ulcerosa/patología , Colitis Ulcerosa/terapia , Enfermedad de Crohn/microbiología , Enfermedad de Crohn/patología , Enfermedad de Crohn/terapia , Factores de Transcripción Forkhead/inmunología , Factores de Transcripción Forkhead/metabolismo , Microbioma Gastrointestinal , Interacciones Huésped-Patógeno , Humanos , Inmunoterapia/métodos , Intestinos/microbiología , Intestinos/patología , Fenotipo , Linfocitos T Reguladores/microbiología , Células Th17/inmunologíaRESUMEN
BACKGROUND: FOXP3+ regulatory T cells (Tregs) are critical for preventing intestinal inflammation. However, FOXP3+ T cells are paradoxically increased in the intestines of patients with the inflammatory bowel disease (IBD) ulcerative colitis (UC) or Crohn's disease (CD). We determined whether these FOXP3+ cells in IBD patients share or lack the phenotype of such cells from patients without IBD. METHODS: We quantified and characterized FOXP3+ Treg populations, as well as FOXP3- CD4+ T cells, in the lamina propria lymphocytes (LPL) of intestine surgically resected from patients with and without IBD, and in the blood of controls or Crohn's patients with or without disease activity. RESULTS: In all samples, a similar fraction of FOXP3+ cells expressed the "natural" Treg (nTreg) marker Helios, suggesting that, in IBD, these cells are not entirely "induced" Tregs (iTregs) derived from activated effector T cells. Helios+ and Helios- FOXP3+ T cells demonstrated similar expression of maturation markers, activation markers, and inhibitory molecules between IBD patients and controls, while FOXP3- cells paradoxically expressed more of the inhibitory receptors CD39, CTLA4, and PD-1 in inflamed mucosa. Greater expression of activation markers was also seen in both Helios+ and Helios- Tregs, relative to FOXP3- cells, in both IBD patients and controls, indicating that Tregs are effectively activated by antigen in IBD. CONCLUSIONS: Extensive immunophenotyping revealed that Helios+ and Helios- mucosal Tregs exist at a similar frequency, and have a similar expression of inhibitory molecules and activation markers in patients with IBD as in healthy controls.
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Enfermedades Inflamatorias del Intestino/sangre , Enfermedades Inflamatorias del Intestino/inmunología , Mucosa Intestinal/patología , Activación de Linfocitos/inmunología , Receptores Inmunológicos/metabolismo , Linfocitos T Reguladores/inmunología , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Antígenos CD/metabolismo , Biomarcadores/metabolismo , Estudios de Casos y Controles , Colon/patología , Femenino , Humanos , Terapia de Inmunosupresión , Masculino , Persona de Mediana Edad , Adulto JovenRESUMEN
We present a case of an adult patient with new-onset severe, idiopathic, protein-wasting enteropathy, in whom an extensive immunological workup was performed. We found a lack of dendritic cell (DC) subsets in the blood and bowel, as well as elevated circulating TGF-beta levels and decreased numbers of circulating FOXP3+ regulatory T cells with diminished CTLA4 expression. She failed to respond to glucocorticoids and infliximab, and instead developed a constellation of opportunistic infections, including CMV ileitis, Mucormycosis, and Pneumocystis carinii pneumonia, and ultimately passed away. While the cause of her lack of DCs is unknown, this data suggests a key role for these cells in both regulating mucosal immunity and promoting effective cell-mediated immunity against pathogens in humans.
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Células Dendríticas/inmunología , Enteritis/inmunología , Infecciones Oportunistas/inmunología , Colon/inmunología , Citocinas/sangre , Enteritis/complicaciones , Resultado Fatal , Femenino , Humanos , Íleon/inmunología , Inmunidad Celular , Inmunidad Mucosa , Enfermedades Inflamatorias del Intestino/inmunología , Persona de Mediana Edad , Infecciones Oportunistas/complicaciones , Linfocitos T Reguladores/inmunologíaRESUMEN
BACKGROUND: Forkhead box P3 (FOXP3)+ regulatory T cells (Tregs) are critical for controlling inflammation in the gastrointestinal tract. There is a paradoxical increase of mucosal FOXP3+ T cells in patients with inflammatory bowel disease (IBD). These FOXP3+ cells were recently shown to include interleukin (IL)-17A-producing cells in Crohn's disease, resembling Th17 cells implicated in autoimmune diseases. FOXP3 inhibits IL-17A production, but a naturally occurring splice variant of FOXP3 lacking exon 2 (Δexon2) cannot. AIMS: We hypothesized that IBD patients preferentially express the Δexon2 variant of FOXP3 so the paradoxically increased mucosal Tregs in IBD could represent cells expressing only Δexon2. METHODS: We used antibodies and primers that can distinguish between the full-length and Δexon2 splice variant of FOXP3 to evaluate expression of these isoforms in human intestinal tissue by immunohistochemistry and quantitative polymerase chain reaction (PCR), respectively. RESULTS: No difference in the expression pattern of Δexon2 relative to full-length FOXP3 was seen in ulcerative colitis or Crohn's disease versus non-IBD controls. By immunofluorescence microscopy and flow cytometry, we also did not find individual cells which expressed FOXP3 protein exclusively in the Δexon2 isoform in either IBD or control tissue. FOXP3+ mucosal CD4+ T cells from both IBD and control specimens were able to make IL-17A in vitro after phorbol myristate acetate (PMA) and ionomycin stimulation, but these cells did not preferentially express Δexon2. CONCLUSIONS: Our data do not support the hypothesis that selective expression of FOXP3 in the Δexon2 isoform accounts for the inability of copious FOXP3+ T cells to inhibit inflammation or IL-17 expression in IBD.
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Factores de Transcripción Forkhead/metabolismo , Enfermedades Inflamatorias del Intestino/metabolismo , Interleucina-17/metabolismo , Linfocitos T Reguladores/metabolismo , Análisis de Varianza , Biopsia , Exones , Citometría de Flujo , Factores de Transcripción Forkhead/inmunología , Humanos , Inmunohistoquímica , Enfermedades Inflamatorias del Intestino/inmunología , Mucosa Intestinal/inmunología , Mucosa Intestinal/metabolismo , Reacción en Cadena de la Polimerasa , Linfocitos T Reguladores/inmunología , Células Th17/inmunología , Células Th17/metabolismoRESUMEN
We describe a role for ECM as a biosensor for inflammatory microenvironments that plays a critical role in peripheral immune tolerance. We show that hyaluronan (HA) promotes induction of Foxp3- IL-10-producing regulatory T cells (TR1) from conventional T-cell precursors in both murine and human systems. This is, to our knowledge, the first description of an ECM component inducing regulatory T cells. Intact HA, characteristic of healing tissues, promotes induction of TR1 capable of abrogating disease in an IL-10-dependent mouse colitis model whereas fragmentary HA, typical of inflamed tissues, does not, indicating a decisive role for tissue integrity in this system. The TR1 precursor cells in this system are CD4(+)CD62L(-)FoxP3(-), suggesting that effector memory cells assume a regulatory phenotype when they encounter their cognate antigen in the context of intact HA. Matrix integrity cues might thereby play a central role in maintaining peripheral tolerance. This TR1 induction is mediated by CD44 cross-linking and signaling through p38 and ERK1/2. This induction is suppressed, also in a CD44-dependent manner, by osteopontin, a component of chronically inflamed ECM, indicating that CD44 signaling serves as a nexus for fate decisions regarding TR1 induction. Finally, we demonstrate that TR1 induction signals can be recapitulated using synthetic matrices. These results reveal important roles for the matrix microenvironment in immune regulation and suggest unique strategies for immunomodulation.
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Matriz Extracelular/inmunología , Interleucina-10/biosíntesis , Células Precursoras de Linfocitos T/inmunología , Linfocitos T Reguladores/inmunología , Animales , Linfocitos T CD4-Positivos/inmunología , Colitis/inmunología , Factores de Transcripción Forkhead/inmunología , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/inmunología , Humanos , Receptores de Hialuranos/inmunología , Ácido Hialurónico/inmunología , Memoria Inmunológica , Técnicas In Vitro , Interleucina-2/farmacología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Osteopontina/inmunología , Subgrupos de Linfocitos T/inmunologíaRESUMEN
Previous studies suggest regulatory T cells (Tregs) inhibit graft-versus-host disease (GVHD) in mouse and human hematopoietic cell transplant (HCT) recipients. As the gastrointestinal tract represents one of the most common and severe sites of GVHD-related tissue damage, we sought to determine whether a deficit in circulating or gastric mucosal Treg numbers correlates with the clinical onset of gastric GVHD. We used the marker FOXP3 to quantify Tregs in blood and in gastric antral biopsies in a cohort of 60 allogeneic HCT recipients undergoing endoscopy at a single center to evaluate symptoms suspicious for gastrointestinal GVHD. We show for the first time in the gastric mucosa and, contrary to existing reports, in the blood, that the percent of T cells expressing FOXP3 is at least as high in the presence as in the absence of GVHD involving the upper gut. There was no correlation of Treg frequency with the histologic or clinical severity of gastrointestinal GVHD. We conclude that Treg depletion is not a central feature in the pathogenesis of gastric GVHD in humans.