RESUMO
Antigen presentation by MHC-II proteins in the thymus is central to selection of CD4 T cells, but analysis of the full repertoire of presented peptides responsible for positive and negative selection is complicated by the low abundance of antigen presenting cells. A key challenge in analysis of limiting abundance immunopeptidomes by mass spectrometry is distinguishing true MHC-binding peptides from co-eluting non-specifically bound peptides present in the mixture eluted from immunoaffinity-purified MHC molecules. Herein we tested several approaches to minimize the impact of non-specific background peptides, including analyzing eluates from isotype-control antibody-conjugated beads, considering only peptides present in nested sets, and using predicted binding motif analysis to identify core epitopes. We evaluated these methods using well-understood human cell line samples, and then applied them to analysis of the I-Ab presented immunopeptidome of the thymus of C57BL/6 mice, comparing this to the more easily characterized splenic B cell and dendritic cell populations. We identified a total of 3473 unique peptides eluted from the various tissues, using a data dependent acquisition strategy with a false-discovery rate of <1%. The immunopeptidomes presented in thymus as compared to splenic B cells and DCs identified shared and tissue-specific epitopes. A broader length distribution was observed for peptides presented in the thymus as compared to splenic B cells or DCs. Detailed analysis of 61 differentially presented peptides indicated a wider distribution of I-Ab binding affinities in thymus as compared to splenic B cells. These results suggest different constraints on antigen processing and presentation pathways in central versus peripheral tissues.
Assuntos
Apresentação de Antígeno/imunologia , Mapeamento de Epitopos , Epitopos/imunologia , Peptídeos/imunologia , Timo/imunologia , Motivos de Aminoácidos , Sequência de Aminoácidos , Animais , Linfócitos B/imunologia , Linfócitos B/metabolismo , Linhagem Celular , Biologia Computacional/métodos , Células Dendríticas/imunologia , Células Dendríticas/metabolismo , Mapeamento de Epitopos/métodos , Epitopos/química , Antígenos HLA-DR/química , Antígenos HLA-DR/imunologia , Antígenos de Histocompatibilidade Classe II/química , Antígenos de Histocompatibilidade Classe II/imunologia , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Peptídeos/química , Ligação Proteica , Baço/imunologia , Baço/metabolismo , Timo/metabolismoRESUMO
OPCML, a tumor suppressor gene, is frequently silenced epigenetically in ovarian and other cancers. Here we report, by analysis of databases of tumor sequences, the observation of OPCML somatic missense mutations from various tumor types and the impact of these mutations on OPCML function, by solving the X-ray crystal structure of this glycoprotein to 2.65 Å resolution. OPCML consists of an extended arrangement of three immunoglobulin-like domains and homodimerizes via a network of contacts between membrane-distal domains. We report the generation of a panel of OPCML variants with representative clinical mutations and demonstrate clear phenotypic effects in vitro and in vivo including changes to anchorage-independent growth, interaction with activated cognate receptor tyrosine kinases, cellular migration, invasion in vitro and tumor growth in vivo. Our results suggest that clinically occurring somatic missense mutations in OPCML have the potential to contribute to tumorigenesis in a variety of cancers.
Assuntos
Moléculas de Adesão Celular/genética , Epigênese Genética , Neoplasias Ovarianas/genética , Moléculas de Adesão Celular/química , Transformação Celular Neoplásica , Cristalografia por Raios X , Feminino , Proteínas Ligadas por GPI/química , Proteínas Ligadas por GPI/genética , Glicosilação , Humanos , Mutação de Sentido Incorreto , Invasividade Neoplásica , Agregação Patológica de Proteínas/genética , Estrutura Terciária de ProteínaRESUMO
A common approach to measuring binding constants involves combining receptor and ligand and measuring the distribution of bound and free states after equilibration. For class I major histocompatibility (MHC-I) proteins, which bind short peptides for presentation to T cells, this approach is precluded by instability of peptide-free protein. Here we develop a method wherein a weakly-binding peptide covalently attached to the N-terminus of the MHC-I ß2m subunit is released from the peptide binding site after proteolytic cleavage of the linker. The resultant protein is able to bind added peptide. A direct binding assay and method for estimation of peptide binding constant (Kd) are described, in which fluorescence polarization is used to follow peptide binding. A competition binding assay and method for estimation of inhibitor binding constant (Ki) using the same principle also are also described. The method uses a cubic equation to relate observed binding to probe concentration, probe Kd, inhibitor concentration, and inhibitor Ki under general reaction conditions without assumptions relating to relative binding affinities or concentrations. We also delineate advantages of this approach compared to the Cheng-Prusoff and Munson-Rodbard approaches for estimation of Ki using competition binding data.
Assuntos
Antígenos de Histocompatibilidade Classe I/metabolismo , Peptídeos/metabolismo , Proteólise , Microglobulina beta-2/metabolismo , Sequência de Aminoácidos , Peptídeos/química , Ligação ProteicaRESUMO
Presentation of antigenic peptides on MHC-II molecules is essential for tolerance to self and for initiation of immune responses against foreign antigens. DO (HLA-DO in humans, H2-O in mice) is a nonclassical MHC-II protein that has been implicated in control of autoimmunity and regulation of neutralizing antibody responses to viruses. These effects likely are related to a role of DO in selecting MHC-II epitopes, but previous studies examining the effect of DO on presentation of selected CD4 T cell epitopes have been contradictory. To understand how DO modulates MHC-II antigen presentation, we characterized the full spectrum of peptides presented by MHC-II molecules expressed by DO-sufficient and DO-deficient antigen-presenting cells in vivo and in vitro using quantitative mass spectrometry approaches. We found that DO controlled the diversity of the presented peptide repertoire, with a subset of peptides presented only when DO was expressed. Antigen-presenting cells express another nonclassical MHC-II protein, DM, which acts as a peptide editor by preferentially catalyzing the exchange of less stable MHC-II peptide complexes, and which is inhibited when bound to DO. Peptides presented uniquely in the presence of DO were sensitive to DM-mediated exchange, suggesting that decreased DM editing was responsible for the increased diversity. DO-deficient mice mounted CD4 T cell responses against wild-type antigen-presenting cells, but not vice versa, indicating that DO-dependent alterations in the MHC-II peptidome could be recognized by circulating T cells. These data suggest that cell-specific and regulated expression of HLA-DO serves to fine-tune MHC-II peptidomes, in order to enhance self-tolerance to a wide spectrum of epitopes while allowing focused presentation of immunodominant epitopes during an immune response.
Assuntos
Antígenos HLA-D/genética , Antígenos de Histocompatibilidade Classe II/química , Peptídeos/metabolismo , Animais , Apresentação de Antígeno , Linhagem Celular , Epitopos de Linfócito T/metabolismo , Antígenos HLA-D/química , Antígenos de Histocompatibilidade Classe II/genética , Humanos , Epitopos Imunodominantes/metabolismo , Camundongos , Camundongos Endogâmicos C57BLRESUMO
Presentation of peptide antigens by MHC-II proteins is prerequisite to effective CD4 T cell tolerance to self and to recognition of foreign antigens. Antigen uptake and processing pathways as well as expression of the peptide exchange factors HLA-DM and HLA-DO differ among the various professional and non-professional antigen-presenting cells and are modulated by cell developmental state and activation. Recent studies have highlighted the importance of these cell-specific factors in controlling the source and breadth of peptides presented by MHC-II under different conditions. During inflammation, increased presentation of selected self-peptides has implications for maintenance of peripheral tolerance and autoimmunity.
Assuntos
Apresentação de Antígeno/imunologia , Células Apresentadoras de Antígenos/imunologia , Antígenos de Histocompatibilidade Classe II/imunologia , Tolerância Imunológica/imunologia , Inflamação/imunologia , Linfócitos T/imunologia , Animais , HumanosRESUMO
We investigated the novel role of HCA2 (GPR109A) and its ligand nicotinic acid in regulating macrophage function. Hca2 expression in the RAW264.7 murine macrophage cell line is strongly induced by LPS treatment and correlates with the expression of TNF-α. Treatment with 300 µM nicotinic acid (reported EC50 3 µM, peak plasma concentration 50-300 µM), significantly inhibited TNF-α, IL-6, IL-12p40, and IL-1ß production (P<0.05) in LPS (1 ng/ml)-stimulated wild-type murine bone marrow-derived macrophages (BMMs) but failed to do so in Hca2(-/-) BMMs. Treatment with nicotinic acid reduced nuclear factor κB (NF-κB) activation levels by 43% (P<0.03) in wild-type BMMs 6 h after LPS stimulation but not in Hca2(-/-) BMMs. Nicotinic acid significantly inhibited wild-type BMM chemotaxis (P<0.001), but had no effect on the chemotaxis of Hca2(-/-) BMMs. A significant increase in low-density lipoprotein uptake by both wild-type (P<0.006) and Hca2(-/-) BMMs (P<0.03) in response to LPS was observed, which was significantly suppressed by nicotinic acid in wild-type BMMs (P<0.04) but not in Hca2(-/-) BMMs. Our results suggest that the nicotinic acid-HCA2 axis is a novel negative regulator of macrophage activation.
Assuntos
Ativação de Macrófagos , Macrófagos/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Receptores Nicotínicos/metabolismo , Animais , Transporte Biológico Ativo/efeitos dos fármacos , Linhagem Celular Tumoral , Quimiotaxia/efeitos dos fármacos , Interleucinas/genética , Interleucinas/metabolismo , Lipopolissacarídeos/farmacologia , Lipoproteínas LDL/metabolismo , Macrófagos/imunologia , Camundongos , NF-kappa B/metabolismo , Niacina/farmacologia , Receptores Acoplados a Proteínas G/agonistas , Receptores Acoplados a Proteínas G/genética , Receptores Nicotínicos/genética , Fator de Necrose Tumoral alfa/farmacologiaRESUMO
Type 1 diabetes (T1D) remains a major health problem worldwide, with a steadily rising incidence yet no cure. Phosphoinositide 3-kinase-γ (PI3Kγ), a member of a family of lipid kinases expressed primarily in leukocytes, has been the subject of substantial research for its role in inflammatory diseases. However, the role of PI3Kγ inhibition in suppressing autoimmune T1D remains to be explored. We tested the role of the PI3Kγ inhibitor AS605240 in preventing and reversing diabetes in NOD mice and assessed the mechanisms by which this inhibition abrogates T1D. Our data indicate that the PI3Kγ pathway is highly activated in T1D. In NOD mice, we found upregulated expression of phosphorylated Akt (PAkt) in splenocytes. Notably, T regulatory cells (Tregs) showed significantly lower expression of PAkt compared with effector T cells. Inhibition of the PI3Kγ pathway by AS605240 efficiently suppressed effector T cells and induced Treg expansion through the cAMP response element-binding pathway. AS605240 effectively prevented and reversed autoimmune diabetes in NOD mice and suppressed T-cell activation and the production of inflammatory cytokines by autoreactive T cells in vitro and in vivo. These studies demonstrate the key role of the PI3Kγ pathway in determining the balance of Tregs and autoreactive cells regulating autoimmune diabetes.
Assuntos
Diabetes Mellitus Tipo 1/tratamento farmacológico , Inibidores de Fosfoinositídeo-3 Quinase , Quinoxalinas/uso terapêutico , Linfócitos T Reguladores/efeitos dos fármacos , Tiazolidinedionas/uso terapêutico , Animais , Diabetes Mellitus Tipo 1/metabolismo , Feminino , Hiperglicemia/tratamento farmacológico , Hiperglicemia/metabolismo , Camundongos , Camundongos Endogâmicos NOD , Fosforilação/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-akt/metabolismo , Quinoxalinas/farmacologia , Baço/efeitos dos fármacos , Baço/metabolismo , Linfócitos T Reguladores/metabolismo , Tiazolidinedionas/farmacologiaRESUMO
Antagonism of CXCR4 disrupts the interaction between the CXCR4 receptor on hematopoietic stem cells (HSCs) and the CXCL12 expressed by stromal cells in the bone marrow, which subsequently results in the shedding of HSCs to the periphery. Because of their profound immunomodulatory effects, HSCs have emerged as a promising therapeutic strategy for autoimmune disorders. We sought to investigate the immunomodulatory role of mobilized autologous HSCs, via target of the CXCR4-CXL12 axis, to promote engraftment of islet cell transplantation. Islets from BALB/c mice were transplanted beneath the kidney capsule of hyperglycemic C57BL/6 mice, and treatment of recipients with CXCR4 antagonist resulted in mobilization of HSCs and in prolongation of islet graft survival. Addition of rapamycin to anti-CXCR4 therapy further promoted HSC mobilization and islet allograft survival, inducing a robust and transferable host hyporesponsiveness, while administration of an ACK2 (anti-CD117) mAb halted CXCR4 antagonist-mediated HSC release and restored allograft rejection. Mobilized HSCs were shown to express high levels of the negative costimulatory molecule programmed death ligand 1 (PD-L1), and HSCs extracted from wild-type mice, but not from PD-L1 knockout mice, suppressed the in vitro alloimmune response. Moreover, HSC mobilization in PD-L1 knockout mice failed to prolong islet allograft survival. Targeting the CXCR4-CXCL12 axis thus mobilizes autologous HSCs and promotes long-term survival of islet allografts via a PD-L1-mediated mechanism.
Assuntos
Antígeno B7-1/fisiologia , Quimiocina CXCL12/antagonistas & inibidores , Marcação de Genes , Sobrevivência de Enxerto/imunologia , Transplante de Células-Tronco Hematopoéticas , Transplante das Ilhotas Pancreáticas/imunologia , Glicoproteínas de Membrana/fisiologia , Peptídeos/fisiologia , Receptores CXCR4/antagonistas & inibidores , Animais , Antígeno B7-1/genética , Antígeno B7-H1 , Benzilaminas , Quimiocina CXCL12/metabolismo , Quimiotaxia de Leucócito/efeitos dos fármacos , Quimiotaxia de Leucócito/imunologia , Ciclamos , Diabetes Mellitus Experimental/imunologia , Diabetes Mellitus Experimental/patologia , Diabetes Mellitus Experimental/cirurgia , Marcação de Genes/métodos , Sobrevivência de Enxerto/efeitos dos fármacos , Sobrevivência de Enxerto/genética , Compostos Heterocíclicos/farmacologia , Transplante das Ilhotas Pancreáticas/patologia , Masculino , Glicoproteínas de Membrana/deficiência , Glicoproteínas de Membrana/genética , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos DBA , Camundongos Knockout , Peptídeos/deficiência , Peptídeos/genética , Receptores CXCR4/metabolismo , Transplante HomólogoRESUMO
Diabetic patients suffer from impaired wound healing, characterized by only modest angiogenesis and cell proliferation. Stem cells may stimulate healing, but little is known about the kinetics of mobilization and function of bone marrow progenitor cells (BM-PCs) during diabetic wound repair. The objective of this study was to investigate the kinetics of BM-PC mobilization and their role during early diabetic wound repair in diabetic db/db mice. After wounding, circulating hematopoietic stem cells (Lin(-)c-Kit(+)Sca-1(+)) stably increased in the periphery and lymphoid tissue of db/db mice compared to unwounded controls. Peripheral endothelial progenitor cells (CD34(+)VEGFR(+)) were 2.5- and 3.5-fold increased on days 6 and 10 after wounding, respectively. Targeting the CXCR4-CXCL12 axis induced an increased release and engraftment of endogenous BM-PCs that was paralleled by an increased expression of CXCL12/SDF-1α in the wounds. Increased levels of peripheral and engrafted BM-PCs corresponded to stimulated angiogenesis and cell proliferation, while the addition of an agonist (GM-CSF) or an antagonist (ACK2) did not further modulate wound healing. Macroscopic histological correlations showed that increased levels of stem cells corresponded to higher levels of wound reepithelialization. After wounding, a natural release of endogenous BM-PCs was shown in diabetic mice, but only low levels of these cells homed in the healing tissue. Higher levels of CXCL12/SDF-1α and circulating stem cells were required to enhance their engraftment and biological effects. Despite controversial data about the functional impairment of diabetic BM-PCs, in this model our data showed a residual capacity of these cells to trigger angiogenesis and cell proliferation.
Assuntos
Células da Medula Óssea/citologia , Diabetes Mellitus Tipo 2/complicações , Células-Tronco/fisiologia , Cicatrização/fisiologia , Animais , Proliferação de Células , Quimiocina CXCL12/metabolismo , Modelos Animais de Doenças , Células Endoteliais/citologia , Fator Estimulador de Colônias de Granulócitos e Macrófagos/farmacologia , Mobilização de Células-Tronco Hematopoéticas , Células-Tronco Hematopoéticas/citologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Receptores CXCR4/metabolismo , Ferimentos e Lesões/complicações , Ferimentos e Lesões/terapiaRESUMO
OBJECTIVE: A number of clinical trials are underway to test whether mesenchymal stem cells (MSCs) are effective in treating various diseases, including type 1 diabetes. Although this cell therapy holds great promise, the optimal source of MSCs has yet to be determined with respect to major histocompatibility complex matching. Here, we examine this question by testing the ability of congenic MSCs, obtained from the NOR mouse strain, to reverse recent-onset type 1 diabetes in NOD mice, as well as determine the immunomodulatory effects of NOR MSCs in vivo. RESEARCH DESIGN AND METHODS: NOR MSCs were evaluated with regard to their in vitro immunomodulatory function in the context of autoreactive T-cell proliferation and dendritic cell (DC) generation. The in vivo effect of NOR MSC therapy on reversal of recent-onset hyperglycemia and on immunogenic cell subsets in NOD mice was also examined. RESULTS: NOR MSCs were shown to suppress diabetogenic T-cell proliferation via PD-L1 and to suppress generation of myeloid/inflammatory DCs predominantly through an IL-6-dependent mechanism. NOR MSC treatment of experimental type 1 diabetes resulted in long-term reversal of hyperglycemia, and therapy was shown to alter diabetogenic cytokine profile, to diminish T-cell effector frequency in the pancreatic lymph nodes, to alter antigen-presenting cell frequencies, and to augment the frequency of the plasmacytoid subset of DCs. CONCLUSIONS: These studies demonstrate the inimitable benefit of congenic MSC therapy in reversing experimental type 1 diabetes. These data should benefit future clinical trials using MSCs as treatment for type 1 diabetes.
Assuntos
Diabetes Mellitus Tipo 1/cirurgia , Transplante de Células-Tronco Mesenquimais/métodos , Animais , Antígenos CD/análise , Antígenos CD/imunologia , Autoantígenos/imunologia , Diferenciação Celular , Citocinas/análise , Células Dendríticas/imunologia , Diabetes Mellitus Tipo 1/imunologia , Diabetes Mellitus Tipo 1/patologia , Modelos Animais de Doenças , Ensaio de Imunoadsorção Enzimática , Citometria de Fluxo , Camundongos , Camundongos Endogâmicos NOD , Receptores de Antígenos de Linfócitos T/genética , Linfócitos T/patologiaRESUMO
OBJECTIVE: To investigate a new clinically relevant immunoregulatory strategy based on treatment with murine Thymoglobulin mATG Genzyme and CTLA4-Ig in NOD mice to prevent allo- and autoimmune activation using a stringent model of islet transplantation and diabetes reversal. RESEARCH DESIGN AND METHODS: Using allogeneic islet transplantation models as well as NOD mice with recent onset type 1 diabetes, we addressed the therapeutic efficacy and immunomodulatory mechanisms associated with a new immunoregulatory protocol based on prolonged low-dose mATG plus CTLA4-Ig. RESULTS: BALB/c islets transplanted into hyperglycemic NOD mice under prolonged mATG+CTLA4-Ig treatment showed a pronounced delay in allograft rejection compared with untreated mice (mean survival time: 54 vs. 8 days, P < 0.0001). Immunologic analysis of mice receiving transplants revealed a complete abrogation of autoimmune responses and severe downregulation of alloimmunity in response to treatment. The striking effect on autoimmunity was confirmed by 100% diabetes reversal in newly hyperglycemic NOD mice and 100% indefinite survival of syngeneic islet transplantation (NOD.SCID into NOD mice). CONCLUSIONS: The capacity to regulate alloimmunity and to abrogate the autoimmune response in NOD mice in different settings confirmed that prolonged mATG+CTLA4-Ig treatment is a clinically relevant strategy to translate to humans with type 1 diabetes.
Assuntos
Autoimunidade/imunologia , Imunoconjugados/uso terapêutico , Transplante das Ilhotas Pancreáticas/imunologia , Abatacepte , Animais , Anticorpos Monoclonais/uso terapêutico , Soro Antilinfocitário , Divisão Celular/imunologia , Citocinas/sangue , Diabetes Mellitus Tipo 1/imunologia , Diabetes Mellitus Tipo 1/cirurgia , Ensaio de Imunoadsorção Enzimática , Rejeição de Enxerto/prevenção & controle , Humanos , Imunossupressores/uso terapêutico , Transplante das Ilhotas Pancreáticas/patologia , Camundongos , Camundongos Endogâmicos NOD , Ovalbumina/imunologia , Transplante de Pele/imunologia , Transplante HomólogoRESUMO
Polymeric nanoparticles (NPs), prepared via coprecipitation of drugs and polymers, are promising drug delivery vehicles for treating diseases with improved efficacy and reduced toxicity. Here, we report an unprecedented strategy for preparing polylactide-cyclosporine A (PLA-CsA) NPs (termed CsA-NPs) through CsA-initiated ring-opening polymerization of lactide (LA) followed by nanoprecipitation. The resulting CsA-NPs have sub-100 nm sizes and narrow particle size distributions, and release CsA in a sustained manner without a "burst"-release effect. Both free CsA and CsA-NPs displayed comparable suppression of T-cell proliferation and production of inflammatory cytokines in various T-cell assays in a dose-dependent manner. The IC(50) values for CsA and CsA-NPs were 27.5 and 72.0 ng/ml, respectively. As lymph nodes are the main loci for T-cell activation, we coupled dendritic cells (DCs) with CsA-NPs and successfully delivered CsA selectively to the lymph nodes. Our studies indicated that CsA-NPs could be internalized in the DCs with a sustained release of CsA to the culture medium, suppressing alloreactive T-cell proliferation. Allogeneic DCs loaded with CsA-NPs were able to migrate to the draining lymph nodes where the T-cell priming was significantly reduced without any systemic release. This innovative nanoparticulate CsA delivery technology constitutes a strong basis for future targeted delivery of immunosuppressive drugs with improved efficiency and reduced toxicity.
Assuntos
Ciclosporina/uso terapêutico , Imunossupressores/uso terapêutico , Nanopartículas , Poliésteres/administração & dosagem , Animais , Ensaio de Imunoadsorção Enzimática , Citometria de Fluxo , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BLRESUMO
Ischemic (isc) injury during the course of transplantation enhances the immunogenicity of allografts and thus results in poorer graft outcome. Given the central role of dendritic cells (DCs) in mounting alloimmune responses, activation of donor DCs by ischemia may have a primary function in the increased immunogenicity of isc allografts. In this study, we sought to investigate the effect of ischemia on DC activity in vitro. Following induction of ischemia, bone marrow-derived DCs were shown to augment allogeneic T cell proliferation as well as the IFN-gamma response. Isc DCs produced greater levels of IL-6, and isc insult was concurrent with NF-kappaB activation. TLR4 ligation was also shown to occur in isc DCs, most likely in response to the endogenous ligand heat shock protein 70, which was found to be elevated in DCs following isc injury, and lack of TLR4 abrogated the observed effects of isc DCs. As compared with control DCs, isc DCs injected into the footpads of mice demonstrated enhanced migration, which was concomitant with increased recipient T cell activity. Moreover, isc DCs underwent a greater degree of apoptosis in the lymph nodes of injected mice, which may further demonstrate enhanced immunogenicity of isc DCs. We thus show that isc injury of DCs enhances DC function, augments the allogeneic T cell response, and occurs via ligation of TLR4, followed by activation of NF-kappaB. These data may serve to identify novel therapeutic targets to attenuate graft immunogenicity following ischemia.
Assuntos
Células Dendríticas/imunologia , Células Dendríticas/patologia , Isquemia/induzido quimicamente , Isquemia/imunologia , Óleo Mineral/toxicidade , NF-kappa B/metabolismo , Receptor 4 Toll-Like/metabolismo , Regulação para Cima/imunologia , Animais , Células Cultivadas , Técnicas de Cocultura , Células Dendríticas/efeitos dos fármacos , Injeções Intraperitoneais , Isquemia/patologia , Ligantes , Ativação Linfocitária/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , NF-kappa B/fisiologia , Linfócitos T/imunologia , Linfócitos T/metabolismo , Linfócitos T/transplante , Receptor 2 Toll-Like/biossíntese , Receptor 2 Toll-Like/deficiência , Receptor 2 Toll-Like/genética , Receptor 4 Toll-Like/biossíntese , Receptor 4 Toll-Like/deficiência , Regulação para Cima/genéticaRESUMO
Clinical and experimental studies that discuss the different immune functions of the renin-angiotensin system (RAS) in kidney diseases were reviewed, with emphasis on studies of kidney transplantation. The RAS has been shown to affect both the innate and adaptive immune responses and has a well-established role in fibrinogenesis. Of special clinical interest is the ability of the RAS to activate the transforming growth factor beta(1) and the Smad pathways leading to fibrinogenesis. In addition to the RAS enhancing effect on the activity of T cells, several components of the RAS have also been shown to be chemotactic to macrophages, T cells, and natural killer cells. Experimental studies have found that RAS blockade decreases the histologic lesions of chronic allograft nephropathy but can enhance acute graft vasculopathy. Although the blockade of RAS has been commonly practiced to reduce posttransplantation hypertension, proteinuria, and erythrocytosis, however, its role in prolonging graft survival is not well established.
Assuntos
Rejeição de Enxerto/imunologia , Transplante de Rim , Sistema Renina-Angiotensina/imunologia , Angiotensina II/imunologia , Angiotensina II/fisiologia , Animais , Quimiotaxia de Leucócito , Células Dendríticas/imunologia , Sobrevivência de Enxerto/imunologia , Humanos , Nefropatias/imunologia , Transplante de Rim/efeitos adversos , Células Matadoras Naturais/imunologia , Peptidil Dipeptidase A/genética , Polimorfismo Genético , Disfunção Primária do Enxerto/imunologia , Sistema Renina-Angiotensina/genética , Sistema Renina-Angiotensina/fisiologia , Linfócitos T/imunologia , Fator de Crescimento Transformador beta1/imunologiaRESUMO
Human clinical trials in type 1 diabetes (T1D) patients using mesenchymal stem cells (MSC) are presently underway without prior validation in a mouse model for the disease. In response to this void, we characterized bone marrow-derived murine MSC for their ability to modulate immune responses in the context of T1D, as represented in NOD mice. In comparison to NOD mice, BALB/c-MSC mice were found to express higher levels of the negative costimulatory molecule PD-L1 and to promote a shift toward Th2-like responses in treated NOD mice. In addition, transfer of MSC from resistant strains (i.e., nonobese resistant mice or BALB/c), but not from NOD mice, delayed the onset of diabetes when administered to prediabetic NOD mice. The number of BALB/c-MSC trafficking to the pancreatic lymph nodes of NOD mice was higher than in NOD mice provided autologous NOD-MSC. Administration of BALB/c-MSC temporarily resulted in reversal of hyperglycemia in 90% of NOD mice (p = 0.002). Transfer of autologous NOD-MSC imparted no such therapeutic benefit. We also noted soft tissue and visceral tumors in NOD-MSC-treated mice, which were uniquely observed in this setting (i.e., no tumors were present with BALB/c- or nonobese resistant mice-MSC transfer). The importance of this observation remains to be explored in humans, as inbred mice such as NOD may be more susceptible to tumor formation. These data provide important preclinical data supporting the basis for further development of allogeneic MSC-based therapies for T1D and, potentially, for other autoimmune disorders.
Assuntos
Diabetes Mellitus Tipo 1/terapia , Transplante de Células-Tronco Mesenquimais/métodos , Células-Tronco Mesenquimais/imunologia , Animais , Células da Medula Óssea , Movimento Celular , Diabetes Mellitus Experimental/complicações , Diabetes Mellitus Experimental/imunologia , Diabetes Mellitus Experimental/terapia , Diabetes Mellitus Tipo 1/complicações , Diabetes Mellitus Tipo 1/imunologia , Hiperglicemia/terapia , Fatores Imunológicos/imunologia , Transplante de Células-Tronco Mesenquimais/efeitos adversos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos NOD , Neoplasias/etiologia , Resultado do TratamentoRESUMO
Little is known about heart tissue/donor dendritic cells, which play a key role in mounting alloimmune responses. In this report, we focus on three primary features of donor dendritic cells: their generation, their trafficking after transplantation, and their role in regulating tolerance versus rejection. Using transgenic mice as donors of heart allografts enabled us to monitor trafficking of donor dendritic cells after transplantation. Donor dendritic cells rapidly migrated into secondary lymphoid tissues within 3 h of transplantation. We found that the chemokine receptor CX3CR1 regulates the generation of heart tissue dendritic cells constitutively. Compared with wild-type hearts, CX3CR1(-/-) hearts contained fewer dendritic cells, and heart allografts from CX3CR1(-/-) donors survived significantly longer without immunosuppression. Unexpectedly, though, co-stimulatory blockade with anti-CD154 or CTLA4-Ig induced long-term survival for wild-type heart allografts but not for CX3CR1(-/-) heart allografts. Increasing the dendritic cell frequency in CX3CR1(-/-) hearts by treatment with Flt3L restored the anti-CD154-induced prolongation of CX3CR1(-/-) heart allograft survival. Compared with wild-type donors, depleting transgenic donors of dendritic cells before heart transplantation also markedly worsened chronic rejection under anti-CD154 treatment. These data indicate the importance of the CX3CR1 pathway in the generation of heart tissue dendritic cells and the divergent role of tissue/dendritic cells in rejection versus tolerance.
Assuntos
Células Dendríticas/imunologia , Células Dendríticas/transplante , Rejeição de Enxerto/imunologia , Transplante de Coração/imunologia , Tolerância ao Transplante/imunologia , Animais , Ligante de CD40/antagonistas & inibidores , Proteínas de Fluorescência Verde/genética , Isoanticorpos/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Receptores de Interleucina-8A/deficiência , Receptores de Interleucina-8A/genética , Receptores de Interleucina-8A/fisiologia , Proteínas Recombinantes/genética , Linfócitos T/imunologia , Doadores de Tecidos , Transplante HomólogoRESUMO
OBJECTIVES: To investigate a B-cell-depleting strategy to reverse diabetes in naïve NOD mice. RESEARCH DESIGN AND METHODS: We targeted the CD22 receptor on B-cells of naïve NOD mice to deplete and reprogram B-cells to effectively reverse autoimmune diabetes. RESULTS: Anti-CD22/cal monoclonal antibody (mAb) therapy resulted in early and prolonged B-cell depletion and delayed disease in pre-diabetic mice. Importantly, when new-onset hyperglycemic mice were treated with the anti-CD22/cal mAb, 100% of B-cell-depleted mice became normoglycemic by 2 days, and 70% of them maintained a state of long-term normoglycemia. Early therapy after onset of hyperglycemia and complete B-cell depletion are essential for optimal efficacy. Treated mice showed an increase in percentage of regulatory T-cells in islets and pancreatic lymph nodes and a diminished immune response to islet peptides in vitro. Transcriptome analysis of reemerging B-cells showed significant changes of a set of proinflammatory genes. Functionally, reemerging B-cells failed to present autoantigen and prevented diabetes when cotransferred with autoreactive CD4(+) T-cells into NOD.SCID hosts. CONCLUSIONS: Targeting CD22 depletes and reprograms B-cells and reverses autoimmune diabetes, thereby providing a blueprint for development of novel therapies to cure autoimmune diabetes.
Assuntos
Anticorpos Monoclonais/uso terapêutico , Linfócitos B/imunologia , Diabetes Mellitus Tipo 1/tratamento farmacológico , Estado Pré-Diabético/imunologia , Lectina 2 Semelhante a Ig de Ligação ao Ácido Siálico/imunologia , Animais , Linfócitos B/citologia , Antígenos CD4/imunologia , Diabetes Mellitus Tipo 1/imunologia , Fatores de Transcrição Forkhead/imunologia , Subunidade alfa de Receptor de Interleucina-2/imunologia , Linfonodos/efeitos dos fármacos , Linfonodos/imunologia , Camundongos , Camundongos Endogâmicos NOD , Pâncreas/efeitos dos fármacos , Pâncreas/imunologiaRESUMO
BACKGROUND: Trafficking of dendritic cells (DC), the primary regulators of alloimmune responses, is controlled by chemokines. Here, we provide evidence that lack of CCR2 could lead to the generation of functionally and phenotypically different DC, which in part could explain the benefits observed in transplanting islets in CCR2 recipients. METHODS AND RESULTS: We show that, in contrast to the in vitro DC maturation model, in vivo DC maturation is accompanied by an increase in the expression of CCR2. Compared with wild-type (WT), DC generated in vitro from CCR2 mice, and DC extracted from CCR2 naïve mice or from CCR2 recipients of islet allografts, display lesser allostimulatory capacity. Compared with WT DC, CCR2 DC produce more IL-4 and induce more IL-4-producing T cells. CCR2 DC also promote the generation of regulatory T cells that more efficiently suppress T cell proliferative responses by mixed leukocyte reaction. Similarly, the percentage of CD4CD25FoxP3 cells were found to be higher in CCR2 recipients of islet allografts than in WT recipients. CONCLUSIONS: In summary, lack of CCR2 interferes with the allostimulatory capacity of DC and promotes the generation of regulatory T cells. This is the first demonstration of a mechanistic link between targeting a specific chemokine pathway and the DC-regulatory T cell axis in alloimmunity.
Assuntos
Linfócitos T CD4-Positivos/imunologia , Diabetes Mellitus Experimental/cirurgia , Rejeição de Enxerto/imunologia , Sobrevivência de Enxerto/imunologia , Transplante das Ilhotas Pancreáticas/imunologia , Receptores CCR2/deficiência , Receptores CCR2/genética , Linfócitos T Reguladores/imunologia , Animais , Movimento Celular , Diabetes Mellitus Experimental/imunologia , Citometria de Fluxo , Subunidade alfa de Receptor de Interleucina-2/análise , Interleucina-4/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Knockout , Fenótipo , Transplante HomólogoRESUMO
Acceptance of the fetus expressing allogeneic paternal Ags by the mother is a physiologic model of transplantation tolerance. Various mechanisms contribute to fetal evasion from immune attack by maternal leukocytes. We have recently demonstrated that the inhibitory costimulatory molecule PDL1 plays a critical role in fetomaternal tolerance in that PDL1 blockade or deficiency resulted in decreased allogeneic fetal survival rates. CD4(+)CD25(+) T regulatory cells (Tregs) have also been demonstrated to play an important role in fetomaternal tolerance. Since PDL1 is expressed on Tregs, we explored the interactions between PDL1 and Tregs in vivo in a mouse model of fetomaternal tolerance. Depletion of CD25(+) T cells abrogated the effect of anti-PDL1 Ab indicating that the effect of PDL1 is possibly mediated by CD25(+) Tregs. Adoptive transfer of Tregs from wild-type but not PDL1-deficient mice into PDL1-deficient recipients significantly improved fetal survival. The frequency, phenotype and placental trafficking of Tregs from PDL1-deficient mice were similar to those of wild-type controls, but were defective in inhibiting alloreactive Th1 cells in vitro. This is the first report providing evidence for a link between PDL1 and T regulatory cells in mediating fetomaternal tolerance.
Assuntos
Antígeno B7-1/imunologia , Tolerância Imunológica , Imunidade Materno-Adquirida/imunologia , Glicoproteínas de Membrana/imunologia , Peptídeos/imunologia , Linfócitos T Reguladores/imunologia , Linfócitos T Reguladores/metabolismo , Animais , Antígeno B7-1/genética , Antígeno B7-H1 , Movimento Celular/genética , Movimento Celular/imunologia , Pai , Feminino , Feto/imunologia , Tolerância Imunológica/genética , Imunidade Materno-Adquirida/genética , Subunidade alfa de Receptor de Interleucina-2/deficiência , Subunidade alfa de Receptor de Interleucina-2/genética , Subunidade alfa de Receptor de Interleucina-2/imunologia , Depleção Linfocítica , Masculino , Glicoproteínas de Membrana/antagonistas & inibidores , Glicoproteínas de Membrana/deficiência , Glicoproteínas de Membrana/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos CBA , Camundongos Knockout , Peptídeos/antagonistas & inibidores , Peptídeos/deficiência , Peptídeos/genética , Placenta/citologia , Placenta/imunologia , Placenta/metabolismo , Gravidez , Resultado da Gravidez , Linfócitos T Reguladores/citologiaRESUMO
BACKGROUND AND OBJECTIVES: Beta3 Integrin may play a role in the process of acute rejection by increasing leukocyte adhesion to the endothelium, cytotoxic T lymphocyte activation, and platelet aggregation. DESIGN, SETTING, PARTICIPANTS, & MEASUREMENTS: For investigation of the role of beta3 integrin in the pathogenesis of acute rejection, this study examined the surface expression of beta3 integrin on leukocyte subsets and analyzed a common single-nucleotide polymorphism in exon 2 of the gene encoding the beta3 subunit that generates two beta3 integrin isoforms, termed Pl(A1) and Pl(A2). Pl(A) genotype was determined in blood samples from 445 renal allograft recipients at two centers. Patients were then grouped by Pl(A) genotype, and clinical outcomes as recorded in a preexisting database were analyzed. RESULTS: Although almost all monocytes express beta3 integrin, its expression was also found on all leukocyte subsets, including T, B, and NK cells. The percentage of patients who experienced acute rejection was noted to be significantly higher in those with Pl(A1)/Pl(A1) (TT) genotype versus patients with the Pl(A1)/Pl(A2) or Pl(A2)/Pl(A2) (CT or CC) genotypes (33% for TT versus 20% for CT or CC). In a multivariate analysis, the Pl(A1)/Pl(A1) (TT) genotype remained significantly associated with acute rejection. Patients with Pl(A1)/Pl(A1) (TT) genotype also exhibited a higher number of acute rejection episodes per patient. CONCLUSIONS: The Pl(A1)/Pl(A1) (TT) genotype is associated with an increased incidence of acute renal allograft rejection in humans, supporting a role for beta3 integrin in the pathophysiology of acute rejection.