RESUMEN
The physiological role of T cell anergy induction as a key mechanism supporting self-tolerance remains undefined, and natural antigens that induce anergy are largely unknown. In this report, we used TCR sequencing to show that the recruitment of CD4+CD44+Foxp3-CD73+FR4+ anergic (Tan) cells expands the CD4+Foxp3+ (Tregs) repertoire. Next, we report that blockade in peripherally-induced Tregs (pTregs) formation due to mutation in CNS1 region of Foxp3 or chronic exposure to a selecting self-peptide result in an accumulation of Tan cells. Finally, we show that microbial antigens from Akkermansia muciniphila commensal bacteria can induce anergy and drive conversion of naive CD4+CD44-Foxp3- T (Tn) cells to the Treg lineage. Overall, data presented here suggest that Tan induction helps the Treg repertoire to become optimally balanced to provide tolerance toward ubiquitous and microbiome-derived epitopes, improving host ability to avert systemic autoimmunity and intestinal inflammation.
Asunto(s)
Linfocitos T CD4-Positivos/inmunología , Microbiota/inmunología , Linfocitos T Reguladores/inmunología , Animales , Antígenos Bacterianos/inmunología , Autoantígenos/inmunología , Diferenciación Celular , Células Cultivadas , Anergia Clonal , Epítopos de Linfocito T/inmunología , Factores de Transcripción Forkhead/metabolismo , Tolerancia Inmunológica , Activación de Linfocitos , Ratones , Ratones TransgénicosRESUMEN
Enteropathogenic bacterial infections are a global health issue associated with high mortality, particularly in developing countries. Efficient host protection against enteropathogenic bacterial infection is characterized by coordinated responses between immune and nonimmune cells. In response to infection in mice, innate immune cells are activated to produce interleukin (IL)-23 and IL-22, which promote antimicrobial peptide (AMP) production and bacterial clearance. IL-36 cytokines are proinflammatory IL-1 superfamily members, yet their role in enteropathogenic bacterial infection remains poorly defined. Using the enteric mouse pathogen, C.rodentium, we demonstrate that signaling via IL-36 receptor (IL-36R) orchestrates a crucial innate-adaptive immune link to control bacterial infection. IL-36R-deficient mice (Il1rl2-/- ) exhibited significant impairment in expression of IL-22 and AMPs, increased intestinal damage, and failed to contain C. rodentium compared to controls. These defects were associated with failure to induce IL-23 and IL-6, two key IL-22 inducers in the early and late phases of infection, respectively. Treatment of Il1rl2-/- mice with IL-23 during the early phase of C. rodentium infection rescued IL-22 production from group 3 innate lymphoid cells (ILCs), whereas IL-6 administration during the late phase rescued IL-22-mediated production from CD4+ T cell, and both treatments protected Il1rl2-/- mice from uncontained infection. Furthermore, IL-36R-mediated IL-22 production by CD4+ T cells was dependent upon NFκB-p65 and IL-6 expression in dendritic cells (DCs), as well as aryl hydrocarbon receptor (AhR) expression by CD4+ T cells. Collectively, these data demonstrate that the IL-36 signaling pathway integrates innate and adaptive immunity leading to host defense against enteropathogenic bacterial infection.
Asunto(s)
Inmunidad Adaptativa , Citrobacter rodentium/inmunología , Infecciones por Enterobacteriaceae/inmunología , Inmunidad Innata , Receptores de Interleucina-1/metabolismo , Animales , Citrobacter rodentium/patogenicidad , Modelos Animales de Enfermedad , Infecciones por Enterobacteriaceae/microbiología , Interleucina-1/metabolismo , Mucosa Intestinal/inmunología , Mucosa Intestinal/metabolismo , Mucosa Intestinal/microbiología , Ratones , Ratones Noqueados , Receptores de Interleucina-1/genética , Transducción de Señal/genética , Transducción de Señal/inmunologíaRESUMEN
Bone morphogenic proteins (BMPs) are members of the transforming growth factor ß (TGF-ß) cytokine family promoting differentiation, homeostasis, and self-renewal of multiple tissues. We show that signaling through the bone morphogenic protein receptor 1α (BMPR1α) sustains expression of FOXP3 in Treg cells in peripheral lymphoid tissues. BMPR1α signaling promotes molecular circuits supporting acquisition and preservation of Treg cell phenotype and inhibiting differentiation of pro-inflammatory effector Th1/Th17 CD4+ T cell. Mechanistically, increased expression of KDM6B (JMJD3) histone demethylase, an antagonist of the polycomb repressive complex 2, underlies lineage-specific changes of T cell phenotypes associated with abrogation of BMPR1α signaling. These results reveal that BMPs are immunoregulatory cytokines mediating maturation and stability of peripheral FOXP3+ regulatory T cells (Treg cells) and controlling generation of iTreg cells. Thus, we establish that BMPs, a large cytokine family, are an essential link between stromal tissues and the adaptive immune system involved in sustaining tissue homeostasis by promoting immunological tolerance.
Asunto(s)
Proteínas Morfogenéticas Óseas/inmunología , Citocinas/metabolismo , Factores de Transcripción Forkhead/inmunología , Linfocitos T Reguladores/inmunología , Animales , Humanos , RatonesRESUMEN
The gut microbiome is the largest source of intrinsic non-self-antigens that are continuously sensed by the immune system but typically do not elicit lymphocyte responses. CD4+ T cells are critical to sustain uninterrupted tolerance to microbial antigens and to prevent intestinal inflammation. However, clinical interventions targeting commensal bacteria-specific CD4+ T cells are rare, because only a very limited number of commensal-derived epitopes have been identified. Here, we used a new approach to study epitopes and identify T cell receptors expressed by CD4+Foxp3+ (Treg) cells specific for commensal-derived antigens. Using this approach, we found that antigens from Akkermansia muciniphila reprogram naïve CD4+ T cells to the Treg lineage, expand preexisting microbe specific Tregs, and limit wasting disease in the CD4+ T cell transfer model of colitis. These data suggest that the administration of specific commensal epitopes may help to widen the repertoire of specific Tregs that control intestinal inflammation.
RESUMEN
Thymic central tolerance eliminates most immature T cells with autoreactive T cell receptors (TCR) that recognize self MHC/peptide complexes. Regardless, an unknown number of autoreactive CD4+Foxp3- T cells escape negative selection and in the periphery require continuous suppression by CD4+Foxp3+ regulatory cells (Tregs). Here, we compare immune repertoires of Treg-deficient and Treg-sufficient mice to find Tregs continuously constraining one-third of mature CD4+Foxp3- cells from converting to pathogenic effectors in healthy mice. These dormant pathogenic clones frequently express TCRs activatable by ubiquitous autoantigens presented by class II MHCs on conventional dendritic cells, including self-peptides that select them in the thymus. Our data thus suggest that identification of most potentially autoreactive CD4+ T cells in the peripheral repertoire is critical to harness or redirect these cells for therapeutic advantage.
Asunto(s)
Autoinmunidad/inmunología , Linfocitos T CD4-Positivos/inmunología , Receptores de Antígenos de Linfocitos T/inmunología , Animales , Autoantígenos/inmunología , Tolerancia Central/inmunología , Células Dendríticas/inmunología , Factores de Transcripción Forkhead/genética , Antígenos de Histocompatibilidad Clase II/inmunología , Ratones , Ratones Transgénicos , Receptores de Antígenos de Linfocitos T alfa-beta/inmunología , Linfocitos T Reguladores/inmunología , TimoRESUMEN
The cytokines of the transforming growth factor-ß (TGF-ß) family promote the growth and differentiation of multiple tissues, but the role of only the founding member, TGF-ß, in regulating the immune responses has been extensively studied. TGF-ß is critical to prevent the spontaneous activation of self-reactive T cells and sustain immune homeostasis. In contrast, in the presence of proinflammatory cytokines, TGF-ß promotes the differentiation of effector T helper 17 (TH17) cells. Abrogating TGF-ß receptor signaling prevents the development of interleukin-17 (IL-17)-secreting cells and protects mice from TH17 cell-mediated autoimmunity. We found that the receptor of another member of TGF-ß family, bone morphogenetic protein receptor 1α (BMPR1α), regulates T helper cell activation. We found that the differentiation of TH17 cells from naive CD4+ T cells was inhibited in the presence of BMPs. Abrogation of BMPR1α signaling during CD4+ T cell activation induced a developmental program that led to the generation of inflammatory effector cells expressing large amounts of IL-17, IFN-γ, and TNF family cytokines and transcription factors defining the TH17 cell lineage. We found that TGF-ß and BMPs cooperated to establish effector cell functions and the cytokine profile of activated CD4+ T cells. Together, our data provide insight into the immunoregulatory function of BMPs.
Asunto(s)
Receptores de Proteínas Morfogenéticas Óseas de Tipo 1/inmunología , Transducción de Señal/inmunología , Células Th17/inmunología , Factor de Crecimiento Transformador beta/inmunología , Animales , Receptores de Proteínas Morfogenéticas Óseas de Tipo 1/genética , Receptores de Proteínas Morfogenéticas Óseas de Tipo 1/metabolismo , Diferenciación Celular/genética , Diferenciación Celular/inmunología , Femenino , Perfilación de la Expresión Génica/métodos , Regulación de la Expresión Génica/inmunología , Activación de Linfocitos/genética , Activación de Linfocitos/inmunología , Masculino , Ratones Endogámicos C57BL , Ratones Noqueados , Transducción de Señal/genética , Linfocitos T Reguladores/inmunología , Linfocitos T Reguladores/metabolismo , Células Th17/metabolismo , Factor de Crecimiento Transformador beta/genética , Factor de Crecimiento Transformador beta/metabolismoRESUMEN
In the gut, various subsets of intraepithelial T cells (IELs) respond to self or non-self-antigens derived from the body, diet, commensal and pathogenic microbiota. Dominant subset of IELs in the small intestine are TCRαßCD8αα+ cells, which are derived from immature thymocytes that express self-reactive TCRs. Although most of TCRαßCD8αα+ IELs are thymus-derived, their repertoire adapts to microbial flora. Here, using high throughput TCR sequencing we examined how clonal diversity of TCRαßCD8αα+ IELs changes upon exposure to commensal-derived antigens. We found that fraction of CD8αα+ IELs and CD4+ T cells express identical αßTCRs and this overlap raised parallel to a surge in the diversity of microbial flora. We also found that an opportunistic pathogen (Staphylococcus aureus) isolated from mouse small intestine specifically activated CD8αα+ IELs and CD4+ derived T cell hybridomas suggesting that some of TCRαßCD8αα+ clones with microbial specificities have extrathymic origin. We also report that CD8ααCD4+ IELs and Foxp3CD4+ T cells from the small intestine shared many αßTCRs, regardless whether the later subset was isolated from Foxp3CNS1 sufficient or Foxp3CNS1 deficient mice that lacks peripherally-derived Tregs. Overall, our results imply that repertoire of TCRαßCD8αα+ in small intestine expends in situ in response to changes in microbial flora.
Asunto(s)
Antígenos Bacterianos/inmunología , Antígenos CD8/metabolismo , Intestino Delgado/inmunología , Linfocitos Intraepiteliales/inmunología , Receptores de Antígenos de Linfocitos T alfa-beta/metabolismo , Infecciones Estafilocócicas/inmunología , Staphylococcus aureus/inmunología , Animales , Antígenos CD8/genética , Linfocitos T CD8-positivos/inmunología , Linfocitos T CD8-positivos/metabolismo , Linfocitos T CD8-positivos/microbiología , Diferenciación Celular , Femenino , Intestino Delgado/metabolismo , Intestino Delgado/microbiología , Linfocitos Intraepiteliales/metabolismo , Linfocitos Intraepiteliales/microbiología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Receptores de Antígenos de Linfocitos T alfa-beta/genética , Infecciones Estafilocócicas/metabolismo , Infecciones Estafilocócicas/microbiología , Subgrupos de Linfocitos T/inmunología , Subgrupos de Linfocitos T/metabolismo , Subgrupos de Linfocitos T/microbiologíaRESUMEN
Regulatory T (Treg) cells expressing Foxp3 transcription factor control homeostasis of the immune system, antigenic responses to commensal and pathogenic microbiota, and immune responses to self and tumour antigens. The Treg cells differentiate in the thymus, along with conventional CD4+ T cells, in processes of positive and negative selection. Another class of Treg cells is generated in peripheral tissues by inducing Foxp3 expression in conventional CD4+ T cells in response to antigenic stimulation. Both thymic and peripheral generation of Treg cells depends on recognition of peptide/MHC ligands by the T-cell receptors (TCR) expressed on thymic Treg precursors or peripheral conventional CD4+ T cells. This review surveys reports describing how thymus Treg cell generation depends on the selecting peptide/MHC ligands and how this process impacts the TCR repertoire expressed by Treg cells. We also describe how Treg cells depend on sustained signalling through the TCR and how they are further regulated by Foxp3 enhancer sequences. Finally, we review the impact of microbiota-derived antigens on the maintenance and functionality of the peripheral pool of Treg cells.
Asunto(s)
Linfocitos T CD4-Positivos/inmunología , Factores de Transcripción Forkhead/inmunología , Linfocitos T Reguladores/inmunología , Animales , Antígenos/inmunología , Homeostasis/inmunología , Complejo Mayor de Histocompatibilidad/inmunología , Receptores de Antígenos de Linfocitos T/inmunologíaRESUMEN
Crosslinking of glucocorticoid-induced TNF family-related receptor (GITR) with agonist antibodies restores cancer immunity by enhancing effector T cell (Teff) responses while interfering with intra-tumor regulatory T cell (Treg) stability and/or accumulation. However, how anti-GITR antibody infusion changes T cell receptor (TCR) repertoire of Teffs and Tregs engaged in anti-tumor immune response is unclear. Here, we used a transgenic mouse model (TCRmini) where T cells express naturally generated but limited TCR repertoire to trace the fate of individual T cells recognizing B16 melanoma in tumor-bearing mice, treated or non-treated with an anti-GITR monoclonal antibody DTA-1. Analysis of TCRs of CD4+ T cells from these mice revealed that the TCR repertoire of dominant tumor-reactive Teff clones remained rather similar in treated and non-treated mice. In contrast, both tumor-associated and peripheral TCR repertoire of Tregs, which were mostly distinct from that of Teffs, underwent DTA-1 mediated remodeling characterized by depletion of dominant clones and an emergence of more diverse, low-frequency clones bearing increased numbers of TCRs shared with Teffs. We conclude that the DTA-1 infusion eliminates activated Tregs engaged in the initial maintenance of tolerogenic niche for tumor growth, but over time, it favors tumor replenishment by Tregs expressing an array of TCRs able to compete with Teffs for recognition of the same tumor antigens which may prevent its complete eradication.
Asunto(s)
Anticuerpos Monoclonales/uso terapéutico , Proteína Relacionada con TNFR Inducida por Glucocorticoide/inmunología , Inmunoterapia/métodos , Neoplasias Experimentales/inmunología , Receptores de Antígenos de Linfocitos T/genética , Linfocitos T Colaboradores-Inductores/fisiología , Linfocitos T Reguladores/fisiología , Animales , Variación Genética , Depleción Linfocítica , Melanoma Experimental , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Microambiente TumoralRESUMEN
Helios transcription factor and semaphorin receptor Nrp-1 were originally described as constitutively expressed at high levels on CD4+Foxp3+ T regulatory cells of intrathymic origin (tTregs). On the other hand, CD4+Foxp3+ Tregs generated in the periphery (pTregs) or induced ex vivo (iTregs) were reported to express low levels of Helios and Nrp-1. Soon afterwards the reliability of Nrp-1 and Helios as markers discriminating between tTregs and pTregs was questioned and until now no consensus has been reached. Here, we used several genetically modified mouse strains that favor pTregs or tTregs formation and analyzed the TCR repertoire of these cells. We found that Tregs with variable levels of Nrp-1 and Helios were abundant in mice with compromised ability to support natural differentiation of tTregs or pTregs. We also report that TCR repertoires of Treg clones expressing high or low levels of Nrp-1 or Helios are similar and more alike repertoire of CD4+Foxp3+ than repertoire of CD4+Foxp3- thymocytes. These results show that high vs. low expression of Nrp-1 or Helios does not unequivocally identify Treg clones of thymic or peripheral origin.
Asunto(s)
Proteínas de Unión al ADN/genética , Neuropilina-1/genética , Linfocitos T Reguladores/citología , Timocitos/citología , Timo/citología , Factores de Transcripción/genética , Animales , Diferenciación Celular , Linaje de la Célula/inmunología , Células Clonales , Cruzamientos Genéticos , Proteínas de Unión al ADN/inmunología , Femenino , Citometría de Flujo , Factores de Transcripción Forkhead/genética , Factores de Transcripción Forkhead/inmunología , Regulación de la Expresión Génica/inmunología , Inmunofenotipificación , Activación de Linfocitos , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Neuropilina-1/inmunología , Linfocitos T Reguladores/inmunología , Timocitos/inmunología , Timo/inmunología , Factores de Transcripción/inmunologíaRESUMEN
Type 1 diabetes is one of the most extensively studied autoimmune diseases, but the cellular and molecular mechanisms leading to T cell-mediated destruction of insulin-producing ß cells are still not well understood. In this study, we show that regulatory T cells (T(regs)) in NOD mice undergo age-dependent loss of suppressor functions exacerbated by the decreased ability of activated effector T cells to upregulate Foxp3 and generate T(regs) in the peripheral organs. This age-dependent loss is associated with reduced intercellular communication mediated by gap junctions, which is caused by impaired upregulation and decreased expression of connexin 43. Regulatory functions can be corrected, even in T cells isolated from aged, diabetic mice, by a synergistic activity of retinoic acid, TGF-ß, and IL-2, which enhance connexin 43 and Foxp3 expression in T(regs) and restore the ability of conventional CD4(+) T cells to upregulate Foxp3 and generate peripherally derived T(regs). Moreover, we demonstrate that suppression mediated by T(regs) from diabetic mice is enhanced by a novel reagent, which facilitates gap junction aggregation. In summary, our report identifies gap junction-mediated intercellular communication as an important component of the T(reg) suppression mechanism compromised in NOD mice and suggests how T(reg) mediated immune regulation can be improved.
Asunto(s)
Comunicación Celular/inmunología , Conexina 43/biosíntesis , Diabetes Mellitus Tipo 1/inmunología , Uniones Comunicantes/metabolismo , Linfocitos T Reguladores/citología , Factores de Edad , Animales , Diferenciación Celular/inmunología , Conexina 43/genética , Diabetes Mellitus Tipo 1/genética , Femenino , Factores de Transcripción Forkhead/biosíntesis , Inmunosupresores/inmunología , Interleucina-2/farmacología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos NOD , Factor de Transcripción STAT5/genética , Factor de Transcripción STAT5/metabolismo , Proteína Smad2/metabolismo , Proteína smad3/metabolismo , Linfocitos T Reguladores/inmunología , Factor de Crecimiento Transformador beta/farmacología , Tretinoina/farmacología , Regulación hacia ArribaRESUMEN
The role of the T-cell receptor (TCR) in commitment of thymocytes to regulatory CD4(+)Foxp3(+) and conventional CD4(+)Foxp3(-) T-cell lineages remains controversial. According to the prevailing view, commitment to the former lineage, in contrast to the latter, requires that high affinity TCRs bind rare class II MHC/peptide complexes presented in 'thymic niches', which could explain differences between their TCR repertoires. Here we challenge this view and show that the binding of identical TCRs to the same ubiquitously expressed MHC/peptide complex often directs thymocytes to both CD4(+) lineages, indicating that the TCR affinity does not play the instructive role, and that restricted presentation of peptides in 'thymic niches' is not necessary for selection of CD4(+)Foxp3(+) T cells. However, depending on whether immature thymocytes bound the ligand predominantly with low or high affinity, the repertoires of regulatory and conventional CD4(+) T cells were correspondingly similar or mostly different, suggesting that negative rather than positive selection sets them apart.
Asunto(s)
Linfocitos T CD4-Positivos/inmunología , Péptidos/inmunología , Receptores de Antígenos de Linfocitos T/inmunología , Animales , Factores de Transcripción Forkhead/genética , Factores de Transcripción Forkhead/inmunología , Antígenos de Histocompatibilidad Clase I/química , Antígenos de Histocompatibilidad Clase I/genética , Antígenos de Histocompatibilidad Clase I/inmunología , Ratones , Ratones Transgénicos , Péptidos/genética , Unión Proteica , Receptores de Antígenos de Linfocitos T/genéticaRESUMEN
How commensal microbiota contributes to immune cell homeostasis at barrier surfaces is poorly understood. Lamina propria (LP) T helper 17 (Th17) cells participate in mucosal protection and are induced by commensal segmented filamentous bacteria (SFB). Here we show that MHCII-dependent antigen presentation of SFB antigens by intestinal dendritic cells (DCs) is crucial for Th17 cell induction. Expression of MHCII on CD11c(+) cells was necessary and sufficient for SFB-induced Th17 cell differentiation. Most SFB-induced Th17 cells recognized SFB in an MHCII-dependent manner. SFB primed and induced Th17 cells locally in the LP and Th17 cell induction occurred normally in mice lacking secondary lymphoid organs. The importance of other innate cells was unveiled by the finding that MHCII deficiency in group 3 innate lymphoid cells (ILCs) resulted in an increase in SFB-independent Th17 cell differentiation. Our results outline the complex role of DCs and ILCs in the regulation of intestinal Th17 cell homeostasis.
Asunto(s)
Antígenos Bacterianos/inmunología , Infecciones por Clostridium/inmunología , Clostridium/inmunología , Células Dendríticas/inmunología , Antígenos de Histocompatibilidad Clase II/metabolismo , Intestinos/inmunología , Linfocitos/inmunología , Células Th17/inmunología , Animales , Presentación de Antígeno , Diferenciación Celular , Células Cultivadas , Células Dendríticas/microbiología , Antígenos de Histocompatibilidad Clase II/genética , Intestinos/microbiología , Activación de Linfocitos , Ratones , Ratones Noqueados , Ratones Transgénicos , Microbiota/inmunología , Miembro 3 del Grupo F de la Subfamilia 1 de Receptores Nucleares/genética , Miembro 3 del Grupo F de la Subfamilia 1 de Receptores Nucleares/metabolismoRESUMEN
Peripheral mechanisms preventing autoimmunity and maintaining tolerance to commensal microbiota involve CD4(+) Foxp3(+) regulatory T (Treg) cells generated in the thymus or extrathymically by induction of naive CD4(+) Foxp3(-) T cells. Previous studies suggested that the T-cell receptor repertoires of thymic Treg cells and induced Treg cells are biased towards self and non-self antigens, respectively, but their relative contribution in controlling immunopathology, such as colitis and other untoward inflammatory responses triggered by different types of antigens, remains unresolved. The intestine, and especially the colon, is a particularly suitable organ to study this question, given the variety of self-, microbiota- and food-derived antigens to which Treg cells and other T-cell populations are exposed. Intestinal environments can enhance conversion to a regulatory lineage and favour tolerogenic presentation of antigens to naive CD4(+) T cells, suggesting that intestinal homeostasis depends on microbiota-specific induced Treg cells. Here, to identify the origin and antigen-specificity of intestinal Treg cells, we performed single-cell and high-throughput sequencing of the T-cell receptor repertoires of CD4(+) Foxp3(+) and CD4(+) Foxp3(-) T cells, and analysed their reactivity against specific commensal species. We show that thymus-derived Treg cells constitute most Treg cells in all lymphoid and intestinal organs, including the colon, where their repertoire is heavily influenced by the composition of the microbiota. Our results suggest that thymic Treg cells, and not induced Treg cells, dominantly mediate tolerance to antigens produced by intestinal commensals.
Asunto(s)
Colon/microbiología , Tolerancia Inmunológica/inmunología , Simbiosis/inmunología , Linfocitos T Reguladores/inmunología , Timo/inmunología , Animales , Antibacterianos/farmacología , Antígenos Bacterianos/inmunología , Colon/efectos de los fármacos , Colon/inmunología , Femenino , Factores de Transcripción Forkhead/metabolismo , Secuenciación de Nucleótidos de Alto Rendimiento , Homeostasis/efectos de los fármacos , Homeostasis/inmunología , Tolerancia Inmunológica/efectos de los fármacos , Tejido Linfoide/citología , Tejido Linfoide/inmunología , Masculino , Ratones , Ratones Transgénicos , Receptores de Antígenos de Linfocitos T/genética , Receptores de Antígenos de Linfocitos T/metabolismo , Análisis de la Célula Individual , Simbiosis/efectos de los fármacos , Linfocitos T Reguladores/citología , Linfocitos T Reguladores/efectos de los fármacos , Linfocitos T Reguladores/metabolismo , Timocitos/citología , Timocitos/efectos de los fármacos , Timocitos/inmunología , Timocitos/metabolismo , Timo/citologíaRESUMEN
A major feature of an adaptive immune system is its ability to generate B- and T-cell clones capable of recognizing and neutralizing specific antigens. These clones recognize antigens with the help of the surface molecules, called antigen receptors, acquired individually during the clonal development process. In order to ensure a response to a broad range of antigens, the number of different receptor molecules is extremely large, resulting in a huge clonal diversity of both B- and T-cell receptor populations and making their experimental comparisons statistically challenging. To facilitate such comparisons, we propose a flexible parametric model of multivariate count data and illustrate its use in a simultaneous analysis of multiple antigen receptor populations derived from mammalian T-cells. The model relies on a representation of the observed receptor counts as a multivariate Poisson abundance mixture (m PAM). A Bayesian parameter fitting procedure is proposed, based on the complete posterior likelihood, rather than the conditional one used typically in similar settings. The new procedure is shown to be considerably more efficient than its conditional counterpart (as measured by the Fisher information) in the regions of m PAM parameter space relevant to model T-cell data.
Asunto(s)
Interpretación Estadística de Datos , Modelos Inmunológicos , Receptores de Antígenos de Linfocitos T/inmunología , Linfocitos T/inmunología , Teorema de Bayes , Humanos , Activación de Linfocitos , Recuento de Linfocitos/estadística & datos numéricos , Análisis Multivariante , Distribución de Poisson , Linfocitos T/citologíaRESUMEN
We have investigated the gross, microscopic and molecular effects of carnitine deficiency in the neonatal gut using a mouse model with a loss-of-function mutation in the OCTN2 (SLC22A5) carnitine transporter. The tissue carnitine content of neonatal homozygous (OCTN2(-/-)) mouse small intestine was markedly reduced; the intestine displayed signs of stunted villous growth, early signs of inflammation, lymphocytic and macrophage infiltration and villous structure breakdown. Mitochondrial ß-oxidation was active throughout the GI tract in wild type newborn mice as seen by expression of 6 key enzymes involved in ß-oxidation of fatty acids and genes for these 6 enzymes were up-regulated in OCTN2(-/-) mice. There was increased apoptosis in gut samples from OCTN2(-/-) mice. OCTN2(-/-) mice developed a severe immune phenotype, where the thymus, spleen and lymph nodes became atrophied secondary to increased apoptosis. Carnitine deficiency led to increased expression of CD45-B220(+) lymphocytes with increased production of basal and anti-CD3-stimulated pro-inflammatory cytokines in immune cells. Real-time PCR array analysis in OCTN2(-/-) mouse gut epithelium demonstrated down-regulation of TGF-ß/BMP pathway genes. We conclude that carnitine plays a major role in neonatal OCTN2(-/-) mouse gut development and differentiation, and that severe carnitine deficiency leads to increased apoptosis of enterocytes, villous atrophy, inflammation and gut injury.
Asunto(s)
Apoptosis , Carnitina/inmunología , Tracto Gastrointestinal/patología , Ganglios Linfáticos/patología , Proteínas de Transporte de Catión Orgánico/genética , Bazo/patología , Timo/patología , Animales , Atrofia/genética , Atrofia/inmunología , Atrofia/patología , Proteínas Morfogenéticas Óseas/genética , Carnitina/análisis , Carnitina/metabolismo , Citocinas/inmunología , Regulación hacia Abajo , Enterocitos/metabolismo , Femenino , Tracto Gastrointestinal/inmunología , Tracto Gastrointestinal/metabolismo , Eliminación de Gen , Ganglios Linfáticos/inmunología , Ganglios Linfáticos/metabolismo , Linfocitos/inmunología , Linfocitos/metabolismo , Ratones , Mitocondrias/enzimología , Mitocondrias/genética , Mitocondrias/metabolismo , Mitocondrias/patología , Oxidación-Reducción , Fenotipo , Miembro 5 de la Familia 22 de Transportadores de Solutos , Bazo/inmunología , Bazo/metabolismo , Timo/inmunología , Timo/metabolismo , Factor de Crecimiento Transformador beta/genéticaRESUMEN
Dysregulated CD4(+) T cell responses and alterations in T regulatory cells (T(reg) cells) play a critical role in autoimmune diseases, including inflammatory bowel disease (IBD). The current study demonstrates that removal of Bcl11b at the double-positive stage of T cell development or only in T(reg) cells causes IBD because of proinflammatory cytokine-producing CD4(+) T cells infiltrating the colon. Provision of WT T(reg) cells prevented IBD, demonstrating that alterations in T(reg) cells are responsible for the disease. Furthermore, Bcl11b-deficient T(reg) cells had reduced suppressor activity with altered gene expression profiles, including reduced expression of the genes encoding Foxp3 and IL-10, and up-regulation of genes encoding proinflammatory cytokines. Additionally, the absence of Bcl11b altered the induction of Foxp3 expression and reduced the generation of induced T(reg) cells (iT(reg) cells) after Tgf-ß treatment of conventional CD4(+) T cells. Bcl11b bound to Foxp3 and IL-10 promoters, as well as to critical conserved noncoding sequences within the Foxp3 and IL-10 loci, and mutating the Bcl11b binding site in the Foxp3 promoter reduced expression of a luciferase reporter gene. These experiments demonstrate that Bcl11b is indispensable for T(reg) suppressor function and for maintenance of optimal Foxp3 and IL-10 gene expression, as well as for the induction of Foxp3 expression in conventional CD4(+) T cells in response to Tgf-ß and generation of iT(reg) cells.
Asunto(s)
Enfermedades Inflamatorias del Intestino/inmunología , Enfermedades Inflamatorias del Intestino/prevención & control , Proteínas Represoras/inmunología , Linfocitos T Reguladores/inmunología , Proteínas Supresoras de Tumor/inmunología , Animales , Linfocitos T CD4-Positivos/inmunología , Colon/citología , Colon/inmunología , Colon/patología , Factores de Transcripción Forkhead/genética , Factores de Transcripción Forkhead/inmunología , Perfilación de la Expresión Génica , Regulación de la Expresión Génica , Humanos , Enfermedades Inflamatorias del Intestino/patología , Enfermedades Inflamatorias del Intestino/fisiopatología , Integrinas/inmunología , Interleucina-10/genética , Interleucina-10/inmunología , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Receptores CCR/inmunología , Proteínas Represoras/genética , Proteínas Supresoras de Tumor/genéticaRESUMEN
Generation of regulatory T cells (or Treg) derived hybridomas offers a tool to study their antigen specificity. T cells hybridomas are produced by fusing TCR α-ß-thymoma BW5147 with highly dividing T cell population. In vitro anergy of Tregs is an obstacle in generation of highly dividing Treg population for their fusion. In this chapter, we describe a simple and efficient method to generate large number of blasting Treg and their successful fusion with thymoma BW5147. The resultant hybridomas lose Treg-specific transcription factor FoxP3, respond to antigenic stimulation by producing IL-2, and thus allow the evaluation of antigen specific, Tregs-derived TCRs.
Asunto(s)
Factores de Transcripción Forkhead/metabolismo , Hibridomas/citología , Linfocitos T Reguladores/citología , Animales , Antígenos/inmunología , Ratones , Linfocitos T Reguladores/inmunología , Linfocitos T Reguladores/metabolismo , Timoma/metabolismoRESUMEN
In modern molecular biology one of the standard ways of analyzing a vertebrate immune system is to sequence and compare the counts of specific antigen receptor clones (either immunoglobulins or T-cell receptors) derived from various tissues under different experimental or clinical conditions. The resulting statistical challenges are difficult and do not fit readily into the standard statistical framework of contingency tables primarily due to the serious under-sampling of the receptor populations. This under-sampling is caused, on one hand, by the extreme diversity of antigen receptor repertoires maintained by the immune system and, on the other, by the high cost and labor intensity of the receptor data collection process. In most of the recent immunological literature the differences across antigen receptor populations are examined via non-parametric statistical measures of the species overlap and diversity borrowed from ecological studies. While this approach is robust in a wide range of situations, it seems to provide little insight into the underlying clonal size distribution and the overall mechanism differentiating the receptor populations. As a possible alternative, the current paper presents a parametric method that adjusts for the data under-sampling as well as provides a unifying approach to a simultaneous comparison of multiple receptor groups by means of the modern statistical tools of unsupervised learning. The parametric model is based on a flexible multivariate Poisson-lognormal distribution and is seen to be a natural generalization of the univariate Poisson-lognormal models used in the ecological studies of biodiversity patterns. The procedure for evaluating a model's fit is described along with the public domain software developed to perform the necessary diagnostics. The model-driven analysis is seen to compare favorably vis a vis traditional methods when applied to the data from T-cell receptors in transgenic mice populations.