RESUMO
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.
Assuntos
Diabetes Mellitus Tipo 1/genética , Diabetes Mellitus Tipo 1/imunologia , Fosfatases de Especificidade Dupla/genética , Predisposição Genética para Doença/genética , Mutação em Linhagem Germinativa , Animais , Doenças Autoimunes/genética , Feminino , Estudo de Associação Genômica Ampla , Haplótipos , Humanos , Ilhotas Pancreáticas/metabolismo , Complexo Principal de Histocompatibilidade , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos NOD , Fosfatases da Proteína Quinase Ativada por Mitógeno , MutaçãoRESUMO
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.
Assuntos
Antígenos CD28/antagonistas & inibidores , Diabetes Mellitus Tipo 1/genética , Diabetes Mellitus Tipo 1/prevenção & controle , Fragmentos Fab das Imunoglobulinas/farmacologia , Sirolimo/farmacologia , Animais , Movimento Celular , Progressão da Doença , Sinergismo Farmacológico , Feminino , Homeostase , Interferon gama/metabolismo , Ilhotas Pancreáticas/imunologia , Ativação Linfocitária/imunologia , Camundongos , Camundongos Endogâmicos NOD , Camundongos Nus , Pâncreas/metabolismo , Linfócitos T/citologia , Linfócitos T Reguladores/imunologiaRESUMO
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.
Assuntos
Diabetes Mellitus Tipo 1/tratamento farmacológico , Inibidores de Histona Desacetilases/uso terapêutico , Imunoterapia/métodos , Células Secretoras de Insulina/metabolismo , Linfócitos T/fisiologia , Administração Oral , Animais , Anticorpos Monoclonais Humanizados/uso terapêutico , Células Cultivadas , Diabetes Mellitus Tipo 1/metabolismo , Feminino , Citometria de Fluxo , Inibidores de Histona Desacetilases/sangue , Células Secretoras de Insulina/efeitos dos fármacos , Interferon gama/sangue , Interleucina-10/sangue , Interleucina-1beta/sangue , Interleucina-6/sangue , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos NOD , Fator de Necrose Tumoral alfa/sangueRESUMO
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.
Assuntos
Anticorpos Monoclonais/farmacologia , Complexo CD3/antagonistas & inibidores , Complexo CD3/genética , Diabetes Mellitus Tipo 1/genética , Diabetes Mellitus Tipo 1/imunologia , Administração através da Mucosa , Animais , Anticorpos Monoclonais/administração & dosagem , Anticorpos Monoclonais/imunologia , Complexo CD3/imunologia , Diabetes Mellitus Tipo 1/tratamento farmacológico , Modelos Animais de Doenças , Feminino , Tolerância Imunológica , Imunidade nas Mucosas , Interleucina-10/metabolismo , Ativação Linfocitária/imunologia , Camundongos , Camundongos Endogâmicos NOD , Camundongos Transgênicos , Substâncias Protetoras/farmacologia , Linfócitos T Reguladores/imunologia , Linfócitos T Reguladores/metabolismo , Fator de Crescimento Transformador beta/metabolismoRESUMO
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.
Assuntos
Anticorpos Monoclonais/farmacologia , Complexo CD3/metabolismo , Células Dendríticas/imunologia , Células Dendríticas/transplante , Transplante das Ilhotas Pancreáticas , Linfócitos T Reguladores/efeitos dos fármacos , Linfócitos T Reguladores/imunologia , Aloenxertos , Animais , Epitopos/imunologia , Sobrevivência de Enxerto/efeitos dos fármacos , Sobrevivência de Enxerto/imunologia , Imunomodulação/efeitos dos fármacos , Imunomodulação/imunologia , Transplante das Ilhotas Pancreáticas/métodos , Camundongos , Modelos Animais , Subpopulações de Linfócitos T/imunologia , Subpopulações de Linfócitos T/metabolismo , Tolerância ao Transplante/efeitos dos fármacos , Tolerância ao Transplante/imunologia , Transplante AutólogoRESUMO
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.
Assuntos
Diabetes Mellitus Experimental , Diabetes Mellitus Tipo 1 , Animais , Diabetes Mellitus Tipo 1/genética , Etilnitrosoureia , Predisposição Genética para Doença , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos NOD , Mutação/genética , Ubiquitina-Proteína Ligases/genéticaRESUMO
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.
Assuntos
Rejeição de Enxerto/metabolismo , Interferon gama/metabolismo , Transplante das Ilhotas Pancreáticas/efeitos adversos , Ilhotas Pancreáticas/cirurgia , Proteínas com Domínio T/metabolismo , Linfócitos T/metabolismo , Transferência Adotiva , Aloenxertos , Animais , Feminino , Rejeição de Enxerto/genética , Rejeição de Enxerto/imunologia , Rejeição de Enxerto/prevenção & controle , Sobrevivência de Enxerto , Imunidade Celular , Interferon gama/deficiência , Interferon gama/genética , Ilhotas Pancreáticas/imunologia , Ilhotas Pancreáticas/metabolismo , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fenótipo , Proteínas com Domínio T/deficiência , Proteínas com Domínio T/genética , Linfócitos T/imunologia , Linfócitos T/transplante , Fatores de TempoRESUMO
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.
RESUMO
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.
Assuntos
Antígeno B7-H1/biossíntese , Linfócitos T CD8-Positivos/imunologia , Anergia Clonal , Tolerância Imunológica , Receptor de Morte Celular Programada 1/biossíntese , Fator de Crescimento Transformador beta/metabolismo , Transplante , Aloenxertos/imunologia , Animais , Anticorpos/administração & dosagem , Complexo CD3/metabolismo , Ilhotas Pancreáticas/imunologia , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Modelos AnimaisRESUMO
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.
Assuntos
Antibacterianos/efeitos adversos , Diabetes Mellitus Tipo 1/microbiologia , Microbioma Gastrointestinal/efeitos dos fármacos , Vancomicina/efeitos adversos , Animais , Antibacterianos/uso terapêutico , Diabetes Mellitus Tipo 1/imunologia , Feminino , Incidência , Linfócitos/imunologia , Masculino , Camundongos Endogâmicos NOD , Mucosa/imunologia , Baço/imunologia , Vancomicina/uso terapêuticoRESUMO
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.
Assuntos
Anticorpos/farmacologia , Diabetes Mellitus Tipo 1/tratamento farmacológico , Hipoglicemiantes/farmacologia , Receptores de Lisoesfingolipídeo/imunologia , Tiazóis/farmacologia , Administração Oral , Transferência Adotiva , Animais , Linfócitos B/efeitos dos fármacos , Linfócitos B/imunologia , Linfócitos B/patologia , Complexo CD3/genética , Complexo CD3/imunologia , Diabetes Mellitus Tipo 1/genética , Diabetes Mellitus Tipo 1/imunologia , Diabetes Mellitus Tipo 1/patologia , Esquema de Medicação , Expressão Gênica , Glucose-6-Fosfatase/antagonistas & inibidores , Glucose-6-Fosfatase/genética , Glucose-6-Fosfatase/imunologia , Linfonodos/efeitos dos fármacos , Linfonodos/imunologia , Linfonodos/patologia , Contagem de Linfócitos , Camundongos , Camundongos Endogâmicos NOD , Receptores de Lisoesfingolipídeo/antagonistas & inibidores , Receptores de Lisoesfingolipídeo/genética , Recidiva , Tolerância a Antígenos Próprios , Baço/efeitos dos fármacos , Baço/imunologia , Baço/patologia , Linfócitos T/efeitos dos fármacos , Linfócitos T/imunologia , Linfócitos T/patologiaRESUMO
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.