RESUMEN
Insulin-dependent or type 1 diabetes (T1D) is a polygenic autoimmune disease. In humans, more than 60 loci carrying common variants that confer disease susceptibility have been identified by genome-wide association studies, with a low individual risk contribution for most variants excepting those of the major histocompatibility complex (MHC) region (40 to 50% of risk); hence the importance of missing heritability due in part to rare variants. Nonobese diabetic (NOD) mice recapitulate major features of the human disease including genetic aspects with a key role for the MHC haplotype and a series of Idd loci. Here we mapped in NOD mice rare variants arising from genetic drift and significantly impacting disease risk. To that aim we established by selective breeding two sublines of NOD mice from our inbred NOD/Nck colony exhibiting a significant difference in T1D incidence. Whole-genome sequencing of high (H)- and low (L)-incidence sublines (NOD/NckH and NOD/NckL) revealed a limited number of subline-specific variants. Treating age of diabetes onset as a quantitative trait in automated meiotic mapping (AMM), enhanced susceptibility in NOD/NckH mice was unambiguously attributed to a recessive missense mutation of Dusp10, which encodes a dual specificity phosphatase. The causative effect of the mutation was verified by targeting Dusp10 with CRISPR-Cas9 in NOD/NckL mice, a manipulation that significantly increased disease incidence. The Dusp10 mutation resulted in islet cell down-regulation of type I interferon signature genes, which may exert protective effects against autoimmune aggression. De novo mutations akin to rare human susceptibility variants can alter the T1D phenotype.
Asunto(s)
Diabetes Mellitus Tipo 1/genética , Diabetes Mellitus Tipo 1/inmunología , Fosfatasas de Especificidad Dual/genética , Predisposición Genética a la Enfermedad/genética , Mutación de Línea Germinal , Animales , Enfermedades Autoinmunes/genética , Femenino , Estudio de Asociación del Genoma Completo , Haplotipos , Humanos , Islotes Pancreáticos/metabolismo , Complejo Mayor de Histocompatibilidad , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos NOD , Fosfatasas de la Proteína Quinasa Activada por Mitógenos , MutaciónRESUMEN
AIMS/HYPOTHESIS: The CD28/B7 interaction is critical for both effector T cell activation and forkhead box P3 (FOXP3)+ regulatory T cell (Treg) generation and homeostasis, which complicates the therapeutic use of cytotoxic T lymphocyte-associated antigen 4 (CTLA-4)-immunoglobulin fusion protein (CTLA-4Ig) in autoimmunity. Here, we evaluated the impact of a simultaneous and selective blockade of the CD28 and mammalian target of rapamycin (mTOR) pathways in the NOD mouse model of type 1 diabetes. METHODS: NOD mice were treated with PEGylated anti-CD28 Fab' antibody fragments (PV1-polyethylene glycol [PEG], 10 mg/kg i.p., twice weekly), rapamycin (1 mg/kg i.p., twice weekly) or a combination of both drugs. Diabetes incidence, pancreatic islet infiltration and autoreactive T cell responses were analysed. RESULTS: We report that 4 week administration of PV1-PEG combined with rapamycin effectively controlled the progression of autoimmune diabetes in NOD mice at 10 weeks of age by reducing T cell activation and migration into the pancreas. Treatment with rapamycin alone was without effect, as was PV1-PEG monotherapy initiated at 4, 6 or 10 weeks of age. Prolonged PV1-PEG administration (for 10 weeks) accelerated diabetes development associated with impaired peripheral Treg homeostasis. This effect was not observed with the combined treatment. CONCLUSIONS/INTERPRETATION: CD28 antagonist and rapamycin treatment act in a complementary manner to limit T cell activation and infiltration of pancreatic islets and diabetes development. These data provide new perspectives for the treatment of autoimmune diabetes and support the therapeutic potential of protocols combining antagonists of CD28 (presently in clinical development) and the mTOR pathway.
Asunto(s)
Antígenos CD28/antagonistas & inhibidores , Diabetes Mellitus Tipo 1/genética , Diabetes Mellitus Tipo 1/prevención & control , Fragmentos Fab de Inmunoglobulinas/farmacología , Sirolimus/farmacología , Animales , Movimiento Celular , Progresión de la Enfermedad , Sinergismo Farmacológico , Femenino , Homeostasis , Interferón gamma/metabolismo , Islotes Pancreáticos/inmunología , Activación de Linfocitos/inmunología , Ratones , Ratones Endogámicos NOD , Ratones Desnudos , Páncreas/metabolismo , Linfocitos T/citología , Linfocitos T Reguladores/inmunologíaRESUMEN
AIM/HYPOTHESIS: Combination therapy targeting the major actors involved in the immune-mediated destruction of pancreatic beta cells appears to be an indispensable approach to treat type 1 diabetes effectively. We hypothesised that the combination of an orally active pan-histone deacetylase inhibitor (HDACi: givinostat) with subtherapeutic doses of CD3 antibodies may provide ideal synergy to treat ongoing autoimmunity. METHODS: NOD mice transgenic for the human CD3ε (also known as CD3E) chain (NOD-huCD3ε) were treated for recent-onset diabetes with oral givinostat, subtherapeutic doses of humanised CD3 antibodies (otelixizumab, 50 µg/day, 5 days, i.v.) or a combination of both drugs. Disease remission, metabolic profiles and autoreactive T cell responses were analysed in treated mice. RESULTS: We demonstrated that givinostat synergised with otelixizumab to induce durable remission of diabetes in 80% of recently diabetic NOD-huCD3ε mice. Remission was obtained in only 47% of mice treated with otelixizumab alone. Oral givinostat monotherapy did not reverse established diabetes but reduced the in situ production of inflammatory cytokines (IL-1ß, IL-6, TNF-α). Importantly, the otelixizumab + givinostat combination strongly improved the metabolic status of NOD-huCD3ε mice; the mice recovered the capacity to appropriately produce insulin, control hyperglycaemia and sustain glucose tolerance. Finally, diabetes remission induced by the combination therapy was associated with a significant reduction of insulitis and autoantigen-specific CD8+ T cell responses. CONCLUSIONS/INTERPRETATION: HDACi and low-dose CD3 antibodies synergised to abrogate in situ inflammation and thereby improved pancreatic beta cell survival and metabolic function leading to long-lasting diabetes remission. These results support the therapeutic potential of protocols combining these two drugs, both in clinical development, to restore self-tolerance and insulin independence in type 1 diabetes.
Asunto(s)
Diabetes Mellitus Tipo 1/tratamiento farmacológico , Inhibidores de Histona Desacetilasas/uso terapéutico , Inmunoterapia/métodos , Células Secretoras de Insulina/metabolismo , Linfocitos T/fisiología , Administración Oral , Animales , Anticuerpos Monoclonales Humanizados/uso terapéutico , Células Cultivadas , Diabetes Mellitus Tipo 1/metabolismo , Femenino , Citometría de Flujo , Inhibidores de Histona Desacetilasas/sangre , Células Secretoras de Insulina/efectos de los fármacos , Interferón gamma/sangre , Interleucina-10/sangre , Interleucina-1beta/sangre , Interleucina-6/sangre , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos NOD , Factor de Necrosis Tumoral alfa/sangreRESUMEN
CD3-specific monoclonal antibody (mAb) treats autoimmune disease in animal models and has shown promise in clinical trials of type 1 diabetes. Whereas intravenous administration of CD3-specific mAb acts primarily by transient depletion of activated effector T cells, oral CD3-specific mAb acts primarily by the induction Tregs. We investigated whether oral CD3-specific mAb inhibits disease in non obese diabetic (NOD) mice that spontaneously develop autoimmune diabetes, closely resembling human type 1 diabetes. We found that oral CD3-specific mAb treatment delayed onset and reduced incidence of diabetes in NOD mice, inducing changes in both effector and regulatory T cell compartments. The therapeutic effect was associated with decreased T cell proliferation, decreased IFNγ and IL-17 production, and increased TGF-ß and IL-10 production in vitro. In vivo transfer experiments demonstrated that oral CD3-specific mAb decreased diabetogenicity of effector T cells and increased the function of regulatory T cells. Oral OKT3, a monoclonal antibody specific for human CD3 had equivalent effects in transgenic NOD mice expressing the human CD3 epsilon chain which serves as a preclinical model for testing human CD3-specific mAb. These results suggest that oral CD3-specific mAb has the potential for treating autoimmune diabetes in humans.
Asunto(s)
Anticuerpos Monoclonales/farmacología , Complejo CD3/antagonistas & inhibidores , Complejo CD3/genética , Diabetes Mellitus Tipo 1/genética , Diabetes Mellitus Tipo 1/inmunología , Administración a través de la Mucosa , Animales , Anticuerpos Monoclonales/administración & dosificación , Anticuerpos Monoclonales/inmunología , Complejo CD3/inmunología , Diabetes Mellitus Tipo 1/tratamiento farmacológico , Modelos Animales de Enfermedad , Femenino , Tolerancia Inmunológica , Inmunidad Mucosa , Interleucina-10/metabolismo , Activación de Linfocitos/inmunología , Ratones , Ratones Endogámicos NOD , Ratones Transgénicos , Sustancias Protectoras/farmacología , Linfocitos T Reguladores/inmunología , Linfocitos T Reguladores/metabolismo , Factor de Crecimiento Transformador beta/metabolismoRESUMEN
Cell therapy and the use of mAbs that interfere with T cell effector functions constitute promising approaches for the control of allograft rejection. In the current study, we investigated a novel approach combining administration of autologous tolerogenic dendritic cells with short-term treatment with CD3-specific Abs. Permanent acceptance of pancreatic islet allografts was achieved in mice treated with the combination therapy the day before transplantation but not in recipients treated with either therapy alone. The combination treatment induced a marked decrease in T cells infiltrating the allografts and a sustained reduction of antidonor responses. Importantly, CD4(+)Foxp3(+) regulatory T cells appeared to play a crucial role in the long-term graft acceptance. Their frequency increased significantly in the spleen, draining lymph nodes, and transplanted islets and remained elevated over the long term; they exhibited increased donor-specific suppressive functions; and their removal at the time of transplantation abrogated the therapeutic effect of the combined therapy. These results support the therapeutic potential of protocols combining autologous dendritic cells and low-dose CD3 Abs, both currently in clinical development, and that act in synergy to control allogeneic immune responses and favor graft survival in a full-mismatch situation.
Asunto(s)
Anticuerpos Monoclonales/farmacología , Complejo CD3/metabolismo , Células Dendríticas/inmunología , Células Dendríticas/trasplante , Trasplante de Islotes Pancreáticos , Linfocitos T Reguladores/efectos de los fármacos , Linfocitos T Reguladores/inmunología , Aloinjertos , Animales , Epítopos/inmunología , Supervivencia de Injerto/efectos de los fármacos , Supervivencia de Injerto/inmunología , Inmunomodulación/efectos de los fármacos , Inmunomodulación/inmunología , Trasplante de Islotes Pancreáticos/métodos , Ratones , Modelos Animales , Subgrupos de Linfocitos T/inmunología , Subgrupos de Linfocitos T/metabolismo , Tolerancia al Trasplante/efectos de los fármacos , Tolerancia al Trasplante/inmunología , Trasplante AutólogoRESUMEN
Genetic association studies of type 1 diabetes (T1D) in humans, and in congenic non-obese diabetic (NOD) mice harboring DNA segments from T1D-resistant mice, face the challenge of assigning causation to specific gene variants among many within loci that affect disease risk. Here, we created random germline mutations in NOD/NckH mice and used automated meiotic mapping to identify mutations modifying T1D incidence and age of onset. In contrast with association studies in humans or congenic NOD mice, we analyzed a relatively small number of genetic changes in each pedigree, permitting implication of specific mutations as causative. Among 844 mice from 14 pedigrees bearing 594 coding/splicing changes, we identified seven mutations that accelerated T1D development, and five that delayed or suppressed T1D. Eleven mutations affected genes not previously known to influence T1D (Xpnpep1, Herc1, Srrm2, Rapgef1, Ppl, Zfp583, Aldh1l1, Col6a1, Ccdc13, Cd200r1, Atrnl1). A suppressor mutation in Coro1a validated the screen. Mutagenesis coupled with automated meiotic mapping can detect genes in which allelic variation influences T1D susceptibility in NOD mice. Variation of some of the orthologous/paralogous genes may influence T1D susceptibility in humans.
Asunto(s)
Diabetes Mellitus Experimental , Diabetes Mellitus Tipo 1 , Animales , Diabetes Mellitus Tipo 1/genética , Etilnitrosourea , Predisposición Genética a la Enfermedad , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos NOD , Mutación/genética , Ubiquitina-Proteína Ligasas/genéticaRESUMEN
BACKGROUND: T cell-mediated graft rejection is mostly correlated with potent Th1 responses. However, because IFNγ mice reject their graft as efficiently as wild-type (WT) mice, the exact contribution of IFNγ and its transcription factor T-bet remains a matter of debate. Here, we address this question in the context of pancreatic islet allograft to better inform the molecular pathways that hampers islet survival in vivo. METHODS: Pancreatic islets from BALB/c mice were transplanted in WT, IFNγ, or T-bet C57BL/6 mice. Graft survival and the induction of effector and cytotoxic T-cell responses were monitored. RESULTS: Rejection of fully mismatched islet allografts correlated with high expression of both IFNγ and T-bet in WT recipients. However, allogeneic islets were permanently accepted in T-bet mice, in contrast to IFNγ hosts. Long-term survival correlated with decreased CD4 and CD8 T-cell infiltrates, drastically reduced donor-specific IFNγ and tumor necrosis factor tumor necrosis factor α responses and very low expression of the cytotoxic markers granzyme B, perforin, and FasLigand. In addition, in vitro and in vivo data pointed to an increased susceptibility of T-bet CD8 T cell to apoptosis. These observations were not reported in IFNγ mice, which have set up compensatory effector mechanisms comprising an increased expression of the transcription factor Eomes and cytolytic molecules as well as tumor necrosis factor α-mediated but not IL-4 nor IL-17-mediated allogeneic responses. CONCLUSIONS: Anti-islet T-cell responses require T-bet but not IFNγ-dependent programs. Our results provide new clues on the mechanisms dictating islet rejection and may help refine the therapeutic/immunosuppressive regimens applied in diabetic patients receiving islets or pancreas allografts.
Asunto(s)
Rechazo de Injerto/metabolismo , Interferón gamma/metabolismo , Trasplante de Islotes Pancreáticos/efectos adversos , Islotes Pancreáticos/cirugía , Proteínas de Dominio T Box/metabolismo , Linfocitos T/metabolismo , Traslado Adoptivo , Aloinjertos , Animales , Femenino , Rechazo de Injerto/genética , Rechazo de Injerto/inmunología , Rechazo de Injerto/prevención & control , Supervivencia de Injerto , Inmunidad Celular , Interferón gamma/deficiencia , Interferón gamma/genética , Islotes Pancreáticos/inmunología , Islotes Pancreáticos/metabolismo , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Noqueados , Fenotipo , Proteínas de Dominio T Box/deficiencia , Proteínas de Dominio T Box/genética , Linfocitos T/inmunología , Linfocitos T/trasplante , Factores de TiempoRESUMEN
Therapeutic tolerance to self-antigens or foreign antigens is thought to depend on constant vigilance by Foxp3+ regulatory T cells (Tregs). Previous work using a pancreatic islet allograft model and a short pulse of CD3 antibody therapy has shown that CD8+ T cells become anergic and use TGFß and coinhibitory signaling as their contribution to the tolerance process. Here, we examine the role of CD4+ T cells in tolerization by CD3 antibodies. We show that both Foxp3+ Tregs and CD4+ T cell anergy play a role in the induction of tolerance and its maintenance. Foxp3+ Tregs resisted CD3 antibody-mediated depletion, unlike intragraft Th1 CD4+ lymphocytes coexpressing granzyme B and Tbx21, which were selectively eliminated. Tregs were mandatory for induction of tolerance as their depletion at the time of CD3 antibody therapy or for a short time thereafter, by an antibody to CD25 (PC61), led to graft rejection. Early treatment with CTLA-4 antibody gave the same outcome. In contrast, neither PC61 nor anti-CTLA-4 given late, at day 100 posttransplant, reversed tolerance once established. Ablation of Foxp3 T cells after diphtheria toxin injection in tolerant Foxp3DTR recipient mice provided the same outcome. Alloreactive T cells had been rendered intrinsically unresponsive as total CD4+ or Treg-deprived CD4+ T cells from tolerant recipients were unable to mount donor-specific IFN-γ responses. In addition, intragraft Treg-deprived CD4+ T cells lacked proliferative capacities, expressed high levels of the inhibitory receptor PD-1, and exhibited a CD73hiFR4hi phenotype, thus reflecting a state of T cell anergy. We conclude that Tregs play a substantive and critical role in guiding the immune system toward tolerance of the allograft, when induced by CD3 antibody, but are less important for maintenance of the tolerant state, where T cell anergy appears sufficient.
RESUMEN
CD8(+) T cell anergy is a critical mechanism of peripheral tolerance, poorly investigated in response to immunotherapy. Here, using a pancreatic islet allograft model and CD3 antibody therapy, we showed, by single cell gene profiling, that intragraft CD8(+) lymphocytes coexpressing granzyme B and perforin were selectively depleted through the Fas/FasL pathway. This step led to long-standing anergy of the remaining CD8(+) T cells marked by the absence of cytotoxic/inflammatory gene expression also confirmed by transcriptome analysis. This sustained unresponsiveness required the presence of the alloantigens. Furthermore, tissue-resident CD8(+) lymphocytes produced TGFß and expressed the inhibitory receptors PD-1 and PD-L1. Blockade of TGFß downregulated PD-1 and PD-L1 expression and precipitated graft rejection. Neutralizing PD-1, PD-L1 or TGFßRII signaling in T cells also abrogated CD3 antibody-induced tolerance. These studies unravel novel mechanisms underlying CD8(+) T cell anergy and reveal a cell intrinsic regulatory link between the TGFß and the PD-1/PD-L1 pathways.
Asunto(s)
Antígeno B7-H1/biosíntesis , Linfocitos T CD8-positivos/inmunología , Anergia Clonal , Tolerancia Inmunológica , Receptor de Muerte Celular Programada 1/biosíntesis , Factor de Crecimiento Transformador beta/metabolismo , Trasplante , Aloinjertos/inmunología , Animales , Anticuerpos/administración & dosificación , Complejo CD3/metabolismo , Islotes Pancreáticos/inmunología , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Modelos AnimalesRESUMEN
Insulin-dependent or type 1 diabetes is a prototypic autoimmune disease whose incidence steadily increased over the past decades in industrialized countries. Recent evidence suggests the importance of the gut microbiota to explain this trend. Here, non-obese diabetic (NOD) mice that spontaneously develop autoimmune type 1 diabetes were treated with different antibiotics to explore the influence of a targeted intestinal dysbiosis in the progression of the disease. A mixture of wide spectrum antibiotics (i.e. streptomycin, colistin and ampicillin) or vancomycin alone were administered orally from the moment of conception, treating breeding pairs, and during the postnatal and adult life until the end of follow-up at 40 weeks. Diabetes incidence significantly and similarly increased in male mice following treatment with these two antibiotic regimens. In NOD females a slight yet not significant trend towards an increase in disease incidence was observed. Changes in gut microbiota composition were assessed by sequencing the V3 region of bacterial 16S rRNA genes. Administration of the antibiotic mixture resulted in near complete ablation of the gut microbiota. Vancomycin treatment led to increased Escherichia, Lactobacillus and Sutterella genera and decreased members of the Clostridiales order and Lachnospiraceae, Prevotellaceae and Rikenellaceae families, as compared to control mice. Massive elimination of IL-17-producing cells, both CD4+TCRαß+ and TCRγδ+ T cells was observed in the lamina propria of the ileum and the colon of vancomycin-treated mice. These results show that a directed even partial ablation of the gut microbiota, as induced by vancomycin, significantly increases type 1 diabetes incidence in male NOD mice thus prompting for caution in the use of antibiotics in pregnant women and newborns.
Asunto(s)
Antibacterianos/efectos adversos , Diabetes Mellitus Tipo 1/microbiología , Microbioma Gastrointestinal/efectos de los fármacos , Vancomicina/efectos adversos , Animales , Antibacterianos/uso terapéutico , Diabetes Mellitus Tipo 1/inmunología , Femenino , Incidencia , Linfocitos/inmunología , Masculino , Ratones Endogámicos NOD , Membrana Mucosa/inmunología , Bazo/inmunología , Vancomicina/uso terapéuticoRESUMEN
In the present study, we investigated the therapeutic potential of a selective S1P1 receptor modulator, ponesimod, to protect and reverse autoimmune diabetes in non-obese diabetic (NOD) mice. Ponesimod was administered orally to NOD mice starting at 6, 10, 13 and 16 weeks of age up to 35 weeks of age or to NOD mice showing recent onset diabetes. Peripheral blood and spleen B and T cell counts were significantly reduced after ponesimod administration. In pancreatic lymph nodes, B lymphocytes were increased and expressed a transitional 1-like phenotype. Chronic oral ponesimod treatment efficiently prevented autoimmune diabetes in 6, 10 and 16 week-old pre-diabetic NOD mice. Treatment withdrawal led to synchronized disease relapse. Ponesimod did not inhibit the differentiation of autoreactive T cells as assessed by adoptive transfer of lymphocytes from treated disease-free NOD mice. In addition, it did not affect the migration, proliferation and activation of transgenic BDC2.5 cells into the target tissue. However, ponesimod inhibited spreading of the T cell responses to islet-specific glucose-6-phosphatase catalytic subunit-related protein (IGRP). Treatment of diabetic NOD mice with ponesimod induced disease remission. However, here again, upon treatment cessation, the disease rapidly recurred. This recurrence was effectively prevented by combination treatment with a CD3 antibody leading to the restoration of self-tolerance. In conclusion, treatment with a selective S1P1 modulator in combination with CD3 antibody represents a promising therapeutic approach for the treatment of autoimmune diabetes.
Asunto(s)
Anticuerpos/farmacología , Diabetes Mellitus Tipo 1/tratamiento farmacológico , Hipoglucemiantes/farmacología , Receptores de Lisoesfingolípidos/inmunología , Tiazoles/farmacología , Administración Oral , Traslado Adoptivo , Animales , Linfocitos B/efectos de los fármacos , Linfocitos B/inmunología , Linfocitos B/patología , Complejo CD3/genética , Complejo CD3/inmunología , Diabetes Mellitus Tipo 1/genética , Diabetes Mellitus Tipo 1/inmunología , Diabetes Mellitus Tipo 1/patología , Esquema de Medicación , Expresión Génica , Glucosa-6-Fosfatasa/antagonistas & inhibidores , Glucosa-6-Fosfatasa/genética , Glucosa-6-Fosfatasa/inmunología , Ganglios Linfáticos/efectos de los fármacos , Ganglios Linfáticos/inmunología , Ganglios Linfáticos/patología , Recuento de Linfocitos , Ratones , Ratones Endogámicos NOD , Receptores de Lisoesfingolípidos/antagonistas & inhibidores , Receptores de Lisoesfingolípidos/genética , Recurrencia , Autotolerancia , Bazo/efectos de los fármacos , Bazo/inmunología , Bazo/patología , Linfocitos T/efectos de los fármacos , Linfocitos T/inmunología , Linfocitos T/patologíaRESUMEN
Monoclonal antibodies have proven to be potent agents to promote immunological tolerance in animal models of autoimmune disease and transplantation. However, optimal clinical application and pharmaceutical development have been limited by the species specificity of therapeutic antibodies, as well exemplified in the case of anti-CD3 antibodies. Compelling evidence in the nonobese diabetic (NOD) mouse, recently translated to clinical autoimmune insulin-dependent diabetes, demonstrates that a short CD3 antibody treatment effectively and durably controls disease progression. We established transgenic mice expressing the human ε chain of the CD3 complex bred onto the NOD background. These mice developed a high incidence of spontaneous autoimmune diabetes and harbored T cells sensitive both in vitro and in vivo to anti-human CD3 antibodies. Treatment of diabetic transgenic mice with otelixizumab, an anti-human CD3 antibody that has proven effective in the clinic, resulted in durable disease remission dependent on transferable T cell-mediated tolerance. This model should enable the evaluation of anti-human CD3 antibodies to determine their potential clinical utility.