RESUMEN
Most T lymphocytes, including regulatory T cells (Treg cells), differentiate in the thymus. The age-dependent involution of this organ leads to decreasing production of T cells. Here we found that the output of new Treg cells from the thymus decreased substantially more than that of conventional T cells. Peripheral mouse and human Treg cells recirculated back to the thymus, where they constituted a large proportion of the pool of Treg cells and displayed an activated and differentiated phenotype. In the thymus, the recirculating cells exerted their regulatory function by inhibiting interleukin 2 (IL-2)-dependent de novo differentiation of Treg cells. Thus, Treg cell development is controlled by a negative feedback loop in which mature progeny cells return to the thymus and restrain development of precursors of Treg cells.
Asunto(s)
Células Precursoras de Linfocitos T/fisiología , Subgrupos de Linfocitos T/fisiología , Linfocitos T Reguladores/fisiología , Timo/inmunología , Envejecimiento/inmunología , Animales , Circulación Sanguínea , Diferenciación Celular/genética , Células Cultivadas , Niño , Retroalimentación Fisiológica , Factores de Transcripción Forkhead/genética , Factores de Transcripción Forkhead/metabolismo , Humanos , Tolerancia Inmunológica , Interleucina-2/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones TransgénicosRESUMEN
BACKGROUND: Pemphigus vulgaris (PV) is characterized by autoantibodies targeting keratinocytes adhesion proteins desmoglein (Dsg) 1 and 3, and by the HLA-DRB1-0402 predisposition allele. Treatment using rituximab (RTX) combined with short-term corticosteroids (CS) allows disease control and long-lasting remission. OBJECTIVE: The principal aim of this study is to evaluate the impact of RTX on the circulating subpopulations of Dsg-3-specific T lymphocytes that specifically regulate B cell responses: follicular helper (Tfh) and follicular regulatory T (Tfr) lymphocytes. METHODS: Using the HLA-DRB1-0402 tetramer loaded with the Dsg-3 immunodominant peptide, we analysed by flow cytometry the frequency, the polarisation and the activation status of blood Dsg-3-specific follicular T cell populations at baseline, Month 6 and long-term follow-up (Month 60-90) from PV patients. RESULTS: At baseline, we observed a predominance of Tfh1* and Tfh17 subsets and an underrepresentation of the Tfh2 subset among autoreactive Dsg-3-specific Tfh cells as compared with non-autoreactive Tfh cells. RTX treatment induced a decrease of autoreactive Tfh cells with no effect on their polarisation during patients' follow-up. In parallel, we observed the emergence of a Dsg-3-specific Tfr subpopulation with a significant overexpression of the surface activation markers PD1, ICOS, and CD25 that was not observed at the surface of autoreactive Tfh and non-autoreactive Tfr cells of the same PV patients. In contrast, a very few Dsg-3 specific Tfr cells were observed in PV patients treated with CS alone. CONCLUSION: Here we show that the emergence of circulating autoreactive Dsg-3-specific Tfr cells is associated with the long-term efficacy of RTX in PV patients.
RESUMEN
Lichen planus (LP) is a cutaneomucosal chronic inflammatory disease characterized by a CD8+ cytotoxic T-lymphocytes (CTL) infiltrate. In erosive oral LP, we found HPV16-specific activated CTL in lesions, supporting a pathogenic contribution of HPV16. Here, we investigated whether a similar scenario occurs in other clinical forms of LP and in lichen sclerosus et atrophicus (LSA), another chronic disease also affecting the mucosa and/or the skin. Blood CTL from LP and LSA patients expressed significant higher levels of granzyme B, perforin and CD107a proteins than healthy donors. Expansions of TCRVß3+ CTL, with presence of TCR clonotypes identical to those previously detected in erosive oral LP, were found both in blood and mucosal/skin lesions of LP, and not of LSA patients. These expansions were enriched with HPV16-specific CD8+ T-cells as shown by their recognition of the E711-20 immunodominant epitope. In LSA patients, the peripheral repertoire of CTL was oligoclonal for TCRVß6+ CTL. Finally, although patients with LP and LSA have developed antibodies against HPV16 capsid L1, antibodies against HPV16 E6 were only observed in patients with LP. Overall, our data collectively suggest an involvement of HPV16-specific CTL in different clinical forms of LP, not only in erosive oral LP, while a different scenario operates in LSA.
Asunto(s)
Liquen Plano Oral , Liquen Plano , Liquen Escleroso y Atrófico , Humanos , Virus del Papiloma Humano , Linfocitos T CD8-positivos/metabolismo , Papillomavirus Humano 16 , Liquen Escleroso y Atrófico/metabolismo , Liquen Escleroso y Atrófico/patología , Liquen Plano/patologíaRESUMEN
Despite genetic and epidemiological evidence strongly supporting an autoimmune basis for narcolepsy type 1, the mechanisms involved have remained largely unknown. Here, we aimed to investigate whether the frequency and function of circulating follicular helper and follicular regulatory T cells are altered in narcolepsy type 1. Peripheral blood mononuclear cells were collected from 32 patients with narcolepsy type 1, including 11 who developed disease after Pandemrix® vaccination, and 32 age-, sex-, and HLA-DQB1*06:02-matched healthy individuals. The frequency and phenotype of the different circulating B cell and follicular T cell subsets were examined by flow cytometry. The function of follicular helper T cells was evaluated by assessing the differentiation of naïve and memory B cells in a co-culture assay. We revealed a notable increase in the frequency of circulating B cells and CD4+CXCR5+ follicular T cells in narcolepsy patients compared to age-, sex- and HLA-matched healthy controls. However, the inducible T-cell costimulator molecule, ICOS, was selectively down-regulated on follicular T cells from patients. Reduced frequency of activated ICOS+ and PD1high blood follicular T cells was also observed in the narcolepsy group. Importantly, follicular T cells isolated from patients with narcolepsy type 1 had a reduced capacity to drive the proliferation/survival and differentiation of memory B cells. Our results provide novel insights into the potential involvement of T cell-dependent B cell responses in the pathogenesis of narcolepsy type 1 in which down-regulation of ICOS expression on follicular helper T cells correlates with their reduced function. We hypothesize that these changes contribute to regulation of the deleterious autoimmune process after disease onset.
Asunto(s)
Linfocitos B/inmunología , Proteína Coestimuladora de Linfocitos T Inducibles/inmunología , Narcolepsia/inmunología , Linfocitos T Colaboradores-Inductores/inmunología , Linfocitos T Reguladores/inmunología , Adolescente , Adulto , Anciano , Linfocitos B/patología , Estudios de Casos y Controles , Diferenciación Celular , Proliferación Celular , Técnicas de Cocultivo , Femenino , Regulación de la Expresión Génica , Cadenas beta de HLA-DQ/genética , Cadenas beta de HLA-DQ/inmunología , Humanos , Memoria Inmunológica , Inmunofenotipificación , Proteína Coestimuladora de Linfocitos T Inducibles/genética , Vacunas contra la Influenza/efectos adversos , Masculino , Persona de Mediana Edad , Narcolepsia/inducido químicamente , Narcolepsia/genética , Narcolepsia/patología , Receptor de Muerte Celular Programada 1/genética , Receptor de Muerte Celular Programada 1/inmunología , Receptores CXCR5/genética , Receptores CXCR5/inmunología , Linfocitos T Colaboradores-Inductores/patología , Linfocitos T Reguladores/patologíaRESUMEN
T-cell polyspecificity, predicting that individual T cells recognize a continuum of related ligands, implies that multiple antigens can tolerize T cells specific for a given self-antigen. We previously showed in C57BL/6 mice that part of the CD4(+) T-cell repertoire specific for myelin oligodendrocyte glycoprotein (MOG) 35-55 also recognizes the neuronal antigen neurofilament medium (NF-M) 15-35. Such bi-specific CD4(+) T cells are frequent and produce inflammatory cytokines after stimulation. Since T cells recognizing two self-antigens would be expected to be tolerized more efficiently, this finding prompted us to study how polyspecificity impacts tolerance. We found that similar to MOG, NF-M is expressed in the thymus by medullary thymic epithelial cells, a tolerogenic population. Nevertheless, the frequency, phenotype, and capacity to transfer experimental autoimmune encephalomyelitis (EAE) of MOG35-55 -reactive CD4(+) T cells were increased in MOG-deficient but not in NF-M-deficient mice. We found that presentation of NF-M15-35 by I-A(b) on dendritic cells is of short duration, suggesting unstable MHC class II binding. Consistently, introducing an MHC-anchoring residue into NF-M15-35 (NF-M15-35 T20Y) increased its immunogenicity, activating a repertoire able to induce EAE. Our results show that in C57BL/6 mice bi-specific encephalitogenic T cells manage to escape tolerization due to inefficient exposure to two self-antigens.
Asunto(s)
Autoantígenos/inmunología , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD4-Positivos/metabolismo , Tolerancia Inmunológica , Proteínas de la Mielina/inmunología , Glicoproteína Mielina-Oligodendrócito/inmunología , Neuronas/inmunología , Animales , Citocinas/biosíntesis , Encefalomielitis Autoinmune Experimental/genética , Encefalomielitis Autoinmune Experimental/inmunología , Encefalomielitis Autoinmune Experimental/patología , Células Epiteliales/inmunología , Células Epiteliales/metabolismo , Expresión Génica , Activación de Linfocitos/inmunología , Ratones , Ratones Noqueados , Glicoproteína Mielina-Oligodendrócito/genética , Proteínas de Neurofilamentos/genética , ARN Mensajero/genética , ARN Mensajero/metabolismo , Especificidad del Receptor de Antígeno de Linfocitos T/genética , Especificidad del Receptor de Antígeno de Linfocitos T/inmunología , Timo/inmunología , Timo/metabolismoRESUMEN
Pemphigus is an autoimmune blistering disease mediated by autoantibodies directed against desmogleins (DSGs). We recently showed that first-line treatment with rituximab (RTX) enables more patients to achieve long-lasting remission off therapy than corticosteroids alone. To understand the immunological mechanisms that mediate long-lasting clinical remission after RTX treatment, we analyzed the phenotype of DSG-specific memory B cells and DSG-specific T follicular helper cells by flow cytometry and measured antibody-secreting cells by enzyme-linked immune absorbent spot in patients treated with corticosteroids alone or RTX. This post hoc analysis of the RITUX3 trial showed that RTX induced a significant decrease of IgG-switched DSG-specific memory B cells. Accordingly, anti-DSG antibody-secreting cells were no longer detected in patients in complete remission after RTX. In contrast, corticosteroids did not modify the frequency or the phenotype of DSG-specific memory B cells, and anti-DSG antibody-secreting cells were still detected after treatment, even in patients in remission. Using peptide-HLADRB1∗0402 tetramer staining, we identified DSG-3-specific T follicular helper cells, which dramatically decreased after RTX, while remaining stable after corticosteroid treatment. Our findings suggest that long-lasting response to RTX in pemphigus relies on the decrease of DSG-specific circulating T follicular helper cells, which correlates with a sustained depletion of IgG-switched memory autoreactive B cells, leading to the disappearance of anti-DSG antibody-secreting cells.
Asunto(s)
Corticoesteroides/uso terapéutico , Subgrupos de Linfocitos B/inmunología , Centro Germinal/inmunología , Inmunosupresores/uso terapéutico , Pénfigo/inmunología , Rituximab/uso terapéutico , Linfocitos T Colaboradores-Inductores/inmunología , Autoinmunidad , Células Cultivadas , Desmogleínas/inmunología , Cadenas HLA-DRB1/metabolismo , Humanos , Memoria Inmunológica , Inmunofenotipificación , Interleucinas/sangre , Pénfigo/tratamiento farmacológicoRESUMEN
The guanine nucleotide exchange factor Vav1 is essential for transducing T cell receptor (TCR) signals and plays an important role in T cell development and activation. Previous genetic studies identified a natural variant of Vav1 characterized by the substitution of an arginine (R) residue by a tryptophane (W) at position 63 (Vav1R63W). This variant impacts Vav1 adaptor functions and controls susceptibility to T cell-mediated neuroinflammation. To assess the implication of this Vav1 variant on the susceptibility to antibody-mediated diseases, we used the animal model of myasthenia gravis, experimental autoimmune myasthenia gravis (EAMG). To this end, we generated a knock-in (KI) mouse model bearing a R to W substitution in the Vav1 gene (Vav1R63W) and immunized it with either torpedo acetylcholine receptor (tAChR) or the α146-162 immunodominant peptide. We observed that the Vav1R63W conferred increased susceptibility to EAMG, revealed by a higher AChR loss together with an increased production of effector cytokines (IFN-γ, IL-17A, GM-CSF) by antigen-specific CD4+ T cells, as well as an increased frequency of antigen-specific CD4+ T cells. This correlated with the emergence of a dominant antigen-specific T cell clone in KI mice that was not present in wild-type mice, suggesting an impact on thymic selection and/or a different clonal selection threshold following antigen encounter. Our results highlight the key role of Vav1 in the pathophysiology of EAMG and this was associated with an impact on the TCR repertoire of AChR reactive T lymphocytes.
Asunto(s)
Variación Genética , Miastenia Gravis Autoinmune Experimental/etiología , Miastenia Gravis Autoinmune Experimental/metabolismo , Proteínas Proto-Oncogénicas c-vav/genética , Proteínas Proto-Oncogénicas c-vav/metabolismo , Receptores de Antígenos de Linfocitos T/metabolismo , Transducción de Señal , Linfocitos T/inmunología , Linfocitos T/metabolismo , Animales , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD4-Positivos/metabolismo , Citocinas/metabolismo , Susceptibilidad a Enfermedades , Ratones , Miastenia Gravis Autoinmune Experimental/patología , Fenotipo , Receptores Nicotínicos/metabolismo , Especificidad del Receptor de Antígeno de Linfocitos TRESUMEN
Follicular helper T cells regulate high-affinity antibody production. Memory follicular helper T cells can be local in draining lymphoid organs and circulate in the blood, but the underlying mechanisms of this subdivision are unresolved. Here we show that both memory follicular helper T subsets sustain B-cell responses after reactivation. Local cells promote more plasma cell differentiation, whereas circulating cells promote more secondary germinal centers. In parallel, local memory B cells are homogeneous and programmed to become plasma cells, whereas circulating memory B cells are able to rediversify. Local memory follicular helper T cells have higher affinity T-cell receptors, which correlates with expression of peptide MHC-II at the surface of local memory B cells only. Blocking T-cell receptor-peptide MHC-II interactions induces the release of local memory follicular helper T cells in the circulating compartment. Our studies show that memory follicular helper T localization is highly intertwined with memory B cells, a finding that has important implications for vaccine design.Tfh cells can differentiate into memory cells. Here the authors describe distinct functional and phenotypic profiles of these memory Tfh cells dependent on their anatomical localization to the lymphoid organs or to the circulation.
Asunto(s)
Memoria Inmunológica , Linfocitos T Colaboradores-Inductores/fisiología , Animales , Ganglios Linfáticos/citología , Ratones Endogámicos C57BL , Ratones Transgénicos , Fenotipo , Bazo/citologíaRESUMEN
Cyclin-dependent kinases constitute attractive pharmacological targets for cancer therapeutics, yet inhibitors in clinical trials target the ATP-binding pocket of the CDK and therefore suffer from limited selectivity and emergence of resistance. The more recent development of allosteric inhibitors targeting conformational plasticity of protein kinases offers promising perspectives for therapeutics. In particular tampering with T-loop dynamics of CDK2 kinase would provide a selective means of inhibiting this kinase, by preventing its conformational activation. To this aim we engineered a fluorescent biosensor that specifically reports on conformational changes of CDK2 activation loop and is insensitive to ATP or ATP-competitive inhibitors, which constitutes a highly sensitive probe for identification of selective T-loop modulators. This biosensor was successfully applied to screen a library of small chemical compounds leading to discovery of a family of quinacridine analogs, which potently inhibit cancer cell proliferation, and promote accumulation of cells in S phase and G2. These compounds bind CDK2/ Cyclin A, inhibit its kinase activity, compete with substrate binding, but not with ATP, and dock onto the T-loop of CDK2. The best compound also binds CDK4 and CDK4/Cyclin D1, but not CDK1. The strategy we describe opens new doors for the discovery of a new class of allosteric CDK inhibitors for cancer therapeutics.
Asunto(s)
Proliferación Celular/efectos de los fármacos , Quinasa 2 Dependiente de la Ciclina/química , Neoplasias/tratamiento farmacológico , Quinacrina/administración & dosificación , Adenosina Trifosfato/química , Regulación Alostérica/efectos de los fármacos , Técnicas Biosensibles , Quinasa 2 Dependiente de la Ciclina/antagonistas & inhibidores , Humanos , Neoplasias/química , Neoplasias/patología , Conformación Proteica/efectos de los fármacos , Inhibidores de Proteínas Quinasas/química , Quinacrina/química , Quinacrina/aislamiento & purificación , Bibliotecas de Moléculas Pequeñas/administración & dosificación , Bibliotecas de Moléculas Pequeñas/aislamiento & purificación , Relación Estructura-ActividadRESUMEN
Escherichia coli FtsK is an essential cell division protein, which is thought to pump chromosomal DNA through the closing septum in an oriented manner by following DNA sequence polarity. Here, we perform single-molecule measurements of translocation by FtsK50C, a derivative that functions as a DNA translocase in vitro. FtsK50C translocation follows Michaelis-Menten kinetics, with a maximum speed of approximately 6.7 kbp/s. We present results on the effect of applied force on the speed, distance translocated, and the mean times during and between protein activity. Surprisingly, we observe that FtsK50C can spontaneously reverse its translocation direction on a fragment of E. coli chromosomal DNA, indicating that DNA sequence is not the sole determinant of translocation direction. We conclude that in vivo polarization of FtsK translocation could require the presence of cofactors; alternatively, we propose a model in which tension in the DNA directs FtsK translocation.