RESUMO
Follicular regulatory T (TFR) cells have specialized roles in modulating follicular helper T (TFH) cell activation of B cells. However, the precise role of TFR cells in controlling antibody responses to foreign antigens and autoantigens in vivo is still unclear due to a lack of specific tools. A TFR cell-deleter mouse was developed that selectively deletes TFR cells, facilitating temporal studies. TFR cells were found to regulate early, but not late, germinal center (GC) responses to control antigen-specific antibody and B cell memory. Deletion of TFR cells also resulted in increased self-reactive immunoglobulin (Ig) G and IgE. The increased IgE levels led us to interrogate the role of TFR cells in house dust mite models. TFR cells were found to control TFH13 cell-induced IgE. In vivo, loss of TFR cells increased house-dust-mite-specific IgE and lung inflammation. Thus, TFR cells control IgG and IgE responses to vaccines, allergens and autoantigens, and exert critical immunoregulatory functions before GC formation.
Assuntos
Linfócitos B/imunologia , Centro Germinativo/imunologia , Hipersensibilidade/imunologia , Pneumonia/imunologia , Linfócitos T Auxiliares-Indutores/imunologia , Linfócitos T Reguladores/imunologia , Animais , Antígenos de Dermatophagoides/imunologia , Autoantígenos/imunologia , Deleção Clonal/genética , Modelos Animais de Doenças , Humanos , Tolerância Imunológica , Imunidade Humoral , Imunoglobulina E/metabolismo , Imunoglobulina G/metabolismo , Memória Imunológica , Interleucina-13/metabolismo , Ativação Linfocitária , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Pyroglyphidae/imunologiaRESUMO
A major breakthrough in cancer treatment occurred with the development of strategies that overcome T-cell tolerance toward tumor cells. These approaches enhance antitumor immunity by overcoming mechanisms that are normally in place to prevent autoimmunity but simultaneously prevent rejection of tumor cells. Although tolerance mechanisms that restrict antitumor immunity take place both in the thymus and periphery, only immunotherapies that target peripheral tolerance mechanisms occurring outside of the thymus are currently available. We review here recent gains in our understanding of how thymic tolerance mediated by the autoimmune regulator (Aire) impedes antitumor immunity. It is now clear that transient depletion of Aire-expressing cells in the thymus can be achieved with RANKL blockade. Finally, we discuss key findings that support the repurposing of anti-RANKL as a cancer immunotherapy with a unique mechanism of action.
Assuntos
Tolerância Central/genética , Tolerância Central/imunologia , Neoplasias/etiologia , Neoplasias/metabolismo , Receptor Ativador de Fator Nuclear kappa-B/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Animais , Doenças Autoimunes/etiologia , Doenças Autoimunes/metabolismo , Autoimunidade/genética , Deleção Clonal/genética , Deleção Clonal/imunologia , Humanos , Imunomodulação , Imunoterapia , Terapia de Alvo Molecular , Neoplasias/patologia , Neoplasias/terapia , Especificidade de Órgãos/genética , Especificidade de Órgãos/imunologia , Ligante RANK/antagonistas & inibidores , Ligante RANK/metabolismo , Linfócitos T/imunologia , Linfócitos T/metabolismo , Linfócitos T Reguladores/imunologia , Linfócitos T Reguladores/metabolismo , Timo/imunologia , Timo/metabolismo , Proteína AIRERESUMO
Delivery of gene therapy as well as of biologic therapeutics is often hampered by the immune response of the subject receiving the therapy. We have reported that effective gene therapy for hemophilia utilizing platelets as a delivery vehicle engenders profound tolerance to the therapeutic product. In this study, we investigated whether this strategy can be applied to induce immune tolerance to a non-coagulant protein and explored the fundamental mechanism of immune tolerance induced by platelet-targeted gene delivery. We used ovalbumin (OVA) as a surrogate non-coagulant protein and constructed a lentiviral vector in which OVA is driven by the platelet-specific αIIb promoter. Platelet-specific OVA expression was introduced by bone marrow transduction and transplantation. Greater than 95% of OVA was stored in platelet α-granules. Control mice immunized with OVA generated OVA-specific IgG antibodies; however, mice expressing OVA in platelets did not. Furthermore, OVA expression in platelets was sufficient to prevent the rejection of skin grafts from CAG-OVA mice, demonstrating that immune tolerance developed in platelet-specific OVA-transduced recipients. To assess the mechanism(s) involved in this tolerance we used OTII mice that express CD4+ effector T cells specific for an OVA-derived peptide. After platelet-specific OVA gene transfer, these mice showed normal thymic maturation of the T cells ruling against central tolerance. In the periphery, tolerance involved elimination of OVA-specific CD4+ effector T cells by apoptosis and expansion of an OVA-specific regulatory T cell population. These experiments reveal the existence of natural peripheral tolerance processes to platelet granule contents which can be co-opted to deliver therapeutically important products.
Assuntos
Plaquetas , Deleção Clonal/genética , Técnicas de Transferência de Genes , Terapia Genética/métodos , Tolerância Periférica/genética , Linfócitos T Reguladores/imunologia , Animais , Camundongos , Camundongos Transgênicos , Linfócitos T Reguladores/patologiaRESUMO
Negative selection against highly self-reactive thymocytes is critical for preventing autoimmunity. Thymocyte deletion, anergy induction, and agonist selection are all forms of negative selection that can occur following a high-affinity TCR signal. Of Bim and Nur77, two TCR-induced proteins with proapoptotic function, Bim has been shown to be important for clonal deletion in several model systems, whereas Nur77 was often dispensable. However, Nur77 has been reported to influence other aspects of T cell development by mechanisms that may not be related to its proapoptotic function. In this study, we examined the role of Nur77 during thymocyte development in the presence and absence of Bim to separate apoptotic from nonapoptotic functions of Nur77. Polyclonal Bim-/- and Bim-/-Nur77-/- mice exhibited comparable accumulation of high-affinity signaled CD4+CD8+ double-positive thymocytes and CD8+ and CD4+ single-positive thymocytes. However, combined Bim and Nur77 deficiency increased the frequency of thymic Foxp3+ T regulatory cells and Foxp3-FR4hiCD73hi anergic phenotype CD4+ T cells compared with Bim-/- mice, suggesting that Nur77 expression impairs the development of nonconventional tolerance-inducing cell fates. Using the OT-I RIP-mOVA model, we found that Nur77 deficiency did not substantially impact clonal deletion nor did it exacerbate the defect in clonal deletion in the absence of Bim. However, additional loss of Nur77 in the absence of Bim led to diabetes induction, suggesting that Nur77 promotes tolerance in this context. Together, these data reveal novel nondeletional roles for Nur77 that differ between T cell subsets and have implications for self-tolerance.
Assuntos
Linfócitos T CD8-Positivos/imunologia , Tolerância Imunológica/fisiologia , Membro 1 do Grupo A da Subfamília 4 de Receptores Nucleares/imunologia , Linfócitos T Reguladores/imunologia , Animais , Proteína 11 Semelhante a Bcl-2/genética , Proteína 11 Semelhante a Bcl-2/imunologia , Deleção Clonal/genética , Camundongos , Camundongos Knockout , Membro 1 do Grupo A da Subfamília 4 de Receptores Nucleares/genéticaRESUMO
Neuromyelitis optica (NMO) is an autoimmune disorder of the central nervous system (CNS) mediated by antibodies to the water channel protein AQP4 expressed in astrocytes. The contribution of AQP4-specific T cells to the class switch recombination of pathogenic AQP4-specific antibodies and the inflammation of the blood-brain barrier is incompletely understood, as immunogenic naturally processed T-cell epitopes of AQP4 are unknown. By immunizing Aqp4-/- mice with full-length murine AQP4 protein followed by recall with overlapping peptides, we here identify AQP4(201-220) as the major immunogenic IAb -restricted epitope of AQP4. We show that WT mice do not harbor AQP4(201-220)-specific T-cell clones in their natural repertoire due to deletional tolerance. However, immunization with AQP4(201-220) of Rag1-/- mice reconstituted with the mature T-cell repertoire of Aqp4-/- mice elicits an encephalomyelitic syndrome. Similarly to the T-cell repertoire, the B-cell repertoire of WT mice is "purged" of AQP4-specific B cells, and robust serum responses to AQP4 are only mounted in Aqp4-/- mice. While AQP4(201-220)-specific T cells alone induce encephalomyelitis, NMO-specific lesional patterns in the CNS and the retina only occur in the additional presence of anti-AQP4 antibodies. Thus, failure of deletional T-cell and B-cell tolerance against AQP4 is a prerequisite for clinically manifest NMO.
Assuntos
Aquaporina 4/metabolismo , Astrócitos/metabolismo , Linfócitos B/fisiologia , Epitopos Imunodominantes/metabolismo , Neuromielite Óptica/imunologia , Retina/imunologia , Linfócitos T/imunologia , Animais , Aquaporina 4/genética , Aquaporina 4/imunologia , Autoanticorpos/metabolismo , Autoimunidade , Deleção Clonal/genética , Células Clonais , Mapeamento de Epitopos , Antígenos de Histocompatibilidade Classe II/metabolismo , Humanos , Switching de Imunoglobulina , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Glicoproteína Mielina-Oligodendrócito/imunologia , Fragmentos de Peptídeos/imunologia , Receptores de Antígenos de Linfócitos T/genética , Tolerância a Antígenos PrópriosRESUMO
Studies of repertoires of mouse monoclonal CD4(+) T cells have revealed several mechanisms of self-tolerance; however, which mechanisms operate in normal repertoires is unclear. Here we studied polyclonal CD4(+) T cells specific for green fluorescent protein expressed in various organs, which allowed us to determine the effects of specific expression patterns on the same epitope-specific T cells. Peptides presented uniformly by thymic antigen-presenting cells were tolerated by clonal deletion, whereas peptides excluded from the thymus were ignored. Peptides with limited thymic expression induced partial clonal deletion and impaired effector T cell potential but enhanced regulatory T cell potential. These mechanisms were also active for T cell populations specific for endogenously expressed self antigens. Thus, the immunotolerance of polyclonal CD4(+) T cells was maintained by distinct mechanisms, according to self-peptide expression patterns.
Assuntos
Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/metabolismo , Expressão Gênica , Tolerância Imunológica , Peptídeos/genética , Peptídeos/imunologia , Sequência de Aminoácidos , Animais , Células Apresentadoras de Antígenos/imunologia , Células Apresentadoras de Antígenos/metabolismo , Autoantígenos/química , Autoantígenos/genética , Autoantígenos/imunologia , Autoimunidade , Deleção Clonal/genética , Deleção Clonal/imunologia , Epitopos de Linfócito T/química , Epitopos de Linfócito T/genética , Epitopos de Linfócito T/imunologia , Feminino , Genes Reporter , Camundongos , Camundongos Transgênicos , Peptídeos/química , Subpopulações de Linfócitos T/imunologia , Subpopulações de Linfócitos T/metabolismo , Timo/imunologia , Timo/metabolismoRESUMO
Because regulatory T-cell (Treg) development can be induced by the same agonist self-antigens that induce negative selection, perturbation of apoptosis will affect both negative selection and Treg development. But how the processes of thymocyte deletion versus Treg differentiation bifurcate and their relative importance for tolerance have not been studied in spontaneous organ-specific autoimmune disease. We addressed these questions by removing a critical mediator of thymocyte deletion, BIM, in the NOD mouse model of autoimmune diabetes. Despite substantial defects in the deletion of autoreactive thymocytes, BIM-deficient NOD (NODBim(-/-)) mice developed less insulitis and were protected from diabetes. BIM deficiency did not impair effector T-cell function; however, NODBim(-/-) mice had increased numbers of Tregs, including those specific for proinsulin, in the thymus and peripheral lymphoid tissues. Increased levels of Nur77, CD5, GITR, and phosphorylated IκB-α in thymocytes from NODBim(-/-) mice suggest that autoreactive cells receiving strong T-cell receptor signals that would normally delete them escape apoptosis and are diverted into the Treg pathway. Paradoxically, in the NOD model, reduced thymic deletion ameliorates autoimmune diabetes by increasing Tregs. Thus, modulating apoptosis may be one of the ways to increase antigen-specific Tregs and prevent autoimmune disease.
Assuntos
Proteínas Reguladoras de Apoptose/genética , Deleção Clonal/genética , Diabetes Mellitus Tipo 1/genética , Proteínas de Membrana/genética , Proteínas Proto-Oncogênicas/genética , Linfócitos T Reguladores/imunologia , Timócitos/imunologia , Animais , Proteínas Reguladoras de Apoptose/imunologia , Proteína 11 Semelhante a Bcl-2 , Antígenos CD5/metabolismo , Deleção Clonal/imunologia , Diabetes Mellitus Tipo 1/imunologia , Modelos Animais de Doenças , Proteína Relacionada a TNFR Induzida por Glucocorticoide/metabolismo , Proteínas I-kappa B/metabolismo , Proteínas de Membrana/imunologia , Camundongos , Camundongos Endogâmicos NOD , Inibidor de NF-kappaB alfa , Membro 1 do Grupo A da Subfamília 4 de Receptores Nucleares/metabolismo , Fosforilação , Proteínas Proto-Oncogênicas/imunologia , Timócitos/metabolismoRESUMO
Thymic involution and the subsequent amplified release of autoreactive T cells increase the susceptibility toward developing autoimmunity, but whether they induce chronic inflammation with advanced age remains unclear. The presence of chronic low-level proinflammatory factors in elderly individuals (termed inflammaging) is a significant risk factor for morbidity and mortality in virtually every chronic age-related disease. To determine how thymic involution leads to the persistent release and activation of autoreactive T cells capable of inducing inflammaging, we used a Foxn1 conditional knockout mouse model that induces accelerated thymic involution while maintaining a young periphery. We found that thymic involution leads to T cell activation shortly after thymic egress, which is accompanied by a chronic inflammatory phenotype consisting of cellular infiltration into non-lymphoid tissues, increased TNF-α production, and elevated serum IL-6. Autoreactive T cell clones were detected in the periphery of Foxn1 conditional knockout mice. A failure of negative selection, facilitated by decreased expression of Aire rather than impaired regulatory T cell generation, led to autoreactive T cell generation. Furthermore, the young environment can reverse age-related regulatory T cell accumulation in naturally aged mice, but not inflammatory infiltration. Taken together, these findings identify thymic involution and the persistent activation of autoreactive T cells as a contributing source of chronic inflammation (inflammaging).
Assuntos
Autoimunidade , Seleção Clonal Mediada por Antígeno , Inflamação/imunologia , Subpopulações de Linfócitos T/imunologia , Timo/imunologia , Fatores Etários , Animais , Microambiente Celular , Doença Crônica , Deleção Clonal/genética , Deleção Clonal/imunologia , Modelos Animais de Doenças , Fatores de Transcrição Forkhead/genética , Técnicas de Silenciamento de Genes , Imunofenotipagem , Inflamação/genética , Inflamação/metabolismo , Ativação Linfocitária/imunologia , Camundongos , Camundongos Knockout , Fenótipo , Subpopulações de Linfócitos T/metabolismo , Timócitos/citologia , Timócitos/imunologia , Timócitos/metabolismoRESUMO
Negative selection, primarily mediated through clonal deletion of self-reactive thymocytes, is critical for establishing self-tolerance and preventing autoimmunity. Recent studies suggest that the molecular mechanisms of negative selection differ depending on the thymic compartment and developmental stage at which thymocytes are deleted. Using the physiological HY(cd4) TCR transgenic model of negative selection against ubiquitous self-antigen, we previously found that one of the principal mediators implicated in clonal deletion, Bim, is required for caspase-3 activation but is ultimately dispensable for negative selection. On the basis of these data, we hypothesized that Nur77, another molecule thought to be a key mediator of clonal deletion, could be responsible for Bim-independent deletion. Despite comparable Nur77 induction in thymocytes during negative selection, Bim deficiency resulted in an accumulation of high-affinity-signaled thymocytes as well as impairment in caspase-mediated and caspase-independent cell death. Although these data suggested that Bim may be required for Nur77-mediated cell death, we found that transgenic Nur77 expression was sufficient to induce apoptosis independently of Bim. However, transgenic Nur77-induced apoptosis was significantly inhibited in the context of TCR signaling, suggesting that endogenous Nur77 could be similarly regulated during negative selection. Although Nur77 deficiency alone did not alter positive or negative selection, combined deficiency in Bim and Nur77 impaired clonal deletion efficiency and significantly increased positive selection efficiency. Collectively, these data shed light on the different roles for Bim and Nur77 during ubiquitous Ag-mediated clonal deletion and highlight potential differences from their reported roles in tissue-restricted Ag-mediated clonal deletion.
Assuntos
Proteínas Reguladoras de Apoptose/imunologia , Autoantígenos/imunologia , Deleção Clonal/imunologia , Proteínas de Membrana/imunologia , Membro 1 do Grupo A da Subfamília 4 de Receptores Nucleares/imunologia , Proteínas Proto-Oncogênicas/imunologia , Timócitos/imunologia , Animais , Antígenos CD/imunologia , Antígenos CD/metabolismo , Antígenos de Diferenciação de Linfócitos T/imunologia , Antígenos de Diferenciação de Linfócitos T/metabolismo , Apoptose/genética , Apoptose/imunologia , Proteínas Reguladoras de Apoptose/deficiência , Proteínas Reguladoras de Apoptose/genética , Proteína 11 Semelhante a Bcl-2 , Caspase 3/imunologia , Caspase 3/metabolismo , Células Cultivadas , Deleção Clonal/genética , Ativação Enzimática/imunologia , Feminino , Citometria de Fluxo , Lectinas Tipo C/imunologia , Lectinas Tipo C/metabolismo , Masculino , Proteínas de Membrana/deficiência , Proteínas de Membrana/genética , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Membro 1 do Grupo A da Subfamília 4 de Receptores Nucleares/deficiência , Membro 1 do Grupo A da Subfamília 4 de Receptores Nucleares/genética , Proteínas Proto-Oncogênicas/deficiência , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas c-bcl-2/imunologia , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Receptores de Antígenos de Linfócitos T/genética , Receptores de Antígenos de Linfócitos T/imunologia , Receptores de Antígenos de Linfócitos T/metabolismo , Transdução de Sinais/genética , Transdução de Sinais/imunologia , Timócitos/citologia , Timócitos/metabolismoRESUMO
While surrogate light chain (SLC) expression is normally terminated in differentiating pre-B cells, co-expression of SLC and conventional light chains has been reported in a small population of autoreactive peripheral human B cells that accumulate in arthritic joints. Despite this association with autoimmunity the contribution of SLC expressing mature B cells to disease development is still unknown. We studied the pathogenicity of SLC(+) B cells in a panel of mice that transgenically express the SLC components VpreB and λ5 throughout B cell development. Here we report that although VpreB or λ5 expression mildly activated mature B cells, only moderate VpreB expression levels - in the absence of λ5 - enhanced IgG plasma cell formation. However, no autoantibody production was detectable in VpreB or λ5 transgenic mice and VpreB expression could not accelerate autoimmunity. Instead, moderate VpreB expression partially protected mice from induced autoimmune arthritis. In support of a tolerogenic role of SLC-transgenic B cells, we observed that in a dose-dependent manner SLC expression beyond the pre-B cell stage enhanced clonal deletion among immature and transitional B cells and rendered mature B cells anergic. These findings suggest that SLC expression does not propagate autoimmunity, but instead may impose tolerance.
Assuntos
Linfócitos B/imunologia , Tolerância Imunológica/imunologia , Cadeias Leves Substitutas da Imunoglobulina/imunologia , Células Precursoras de Linfócitos B/imunologia , Animais , Artrite Experimental/genética , Artrite Experimental/imunologia , Artrite Experimental/metabolismo , Autoimunidade/genética , Autoimunidade/imunologia , Linfócitos B/metabolismo , Células Cultivadas , Deleção Clonal/genética , Deleção Clonal/imunologia , Citometria de Fluxo , Expressão Gênica/imunologia , Humanos , Tolerância Imunológica/genética , Cadeias Leves Substitutas da Imunoglobulina/genética , Cadeias Leves Substitutas da Imunoglobulina/metabolismo , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Plasmócitos/imunologia , Plasmócitos/metabolismo , Células Precursoras de Linfócitos B/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Análise de Sequência de RNA/métodosRESUMO
This study examines the loss of peripherally induced B cell immune tolerance in Rheumatoid arthritis (RA) and establishes a novel signaling-based measure of activation in a subset of autoreactive B cells--the Induced tolerance status index (ITSI). Naturally occurring naïve autoreactive B cells can escape the "classical" tolerogenic mechanisms of clonal deletion and receptor editing, but remain peripherally tolerized through B cell receptor (BCR) signaling inhibition (postdevelopmental "receptor tuning" or anergy). ITSI is a statistical index that numerically determines the level of homology between activation patterns of BCR signaling intermediaries in B cells that are either tolerized or activated by auto antigen exposure, and thus quantifies the level of peripheral immune tolerance. The index is based on the logistic regression analysis of phosphorylation levels in a panel of BCR signaling proteins. Our results demonstrate a new approach to identifying autoreactive B cells based on their BCR signaling features.
Assuntos
Artrite Reumatoide/imunologia , Linfócitos B/imunologia , Tolerância Periférica/genética , Receptores de Antígenos de Linfócitos B/imunologia , Índice de Gravidade de Doença , Adulto , Artrite Reumatoide/genética , Artrite Reumatoide/metabolismo , Artrite Reumatoide/patologia , Autoantígenos/genética , Autoantígenos/imunologia , Autoimunidade , Linfócitos B/metabolismo , Linfócitos B/patologia , Biomarcadores/metabolismo , Anergia Clonal/genética , Deleção Clonal/genética , Feminino , Regulação da Expressão Gênica , Humanos , Modelos Logísticos , Ativação Linfocitária , Masculino , Pessoa de Meia-Idade , Fosforilação , Receptores de Antígenos de Linfócitos B/genética , Receptores de Antígenos de Linfócitos B/metabolismo , Transdução de Sinais/imunologiaRESUMO
Autoreactive CD4(+) CD8(-) (CD4SP) thymocytes can be subjected to deletion when they encounter self-peptide during their development, but they can also undergo selection to become CD4SPFoxp3(+) Treg cells. We have analyzed the relationship between these distinct developmental fates using mice in which signals transmitted by the TCR have been attenuated by mutation of a critical tyrosine residue of the adapter protein SLP-76. In mice containing polyclonal TCR repertoires, the mutation caused increased frequencies of CD4SPFoxp3(+) thymocytes. CD4SP thymocytes expressing TCR Vß-chains that are subjected to deletion by endogenous retroviral superantigens were also present at increased frequencies, particularly among Foxp3(+) thymocytes. In transgenic mice in which CD4SP thymocytes expressing an autoreactive TCR undergo both deletion and Treg-cell formation in response to a defined self-peptide, SLP-76 mutation abrogated deletion of autoreactive CD4SP thymocytes. Notably, Foxp3(+) Treg-cell formation still occurred, albeit with a reduced efficiency, and the mutation was also associated with decreased Nur77 expression by the autoreactive CD4SP thymocytes. These studies provide evidence that the strength of the TCR signal can play a direct role in directing the extent of both thymocyte deletion and Treg-cell differentiation, and suggest that distinct TCR signaling thresholds and/or pathways can promote CD4SP thymocyte deletion versus Treg-cell formation.
Assuntos
Autoantígenos/imunologia , Deleção Clonal/imunologia , Peptídeos/imunologia , Receptores de Antígenos de Linfócitos T/metabolismo , Linfócitos T Reguladores/imunologia , Linfócitos T Reguladores/metabolismo , Timócitos/imunologia , Timócitos/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Apresentação de Antígeno , Autoantígenos/química , Autoimunidade , Deleção Clonal/genética , Fatores de Transcrição Forkhead/metabolismo , Expressão Gênica , Subunidade alfa de Receptor de Interleucina-2/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Transgênicos , Mutação , Peptídeos/química , Fenótipo , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Receptores de Antígenos de Linfócitos T alfa-beta/metabolismo , Transdução de SinaisRESUMO
Members of the galectin family of proteins have been shown to regulate the development and the function of immune cells. We previously identified the increased expression of galectin-1 and galectin-3 mRNA and protein in anergic B cells relative to their naïve counterparts. To investigate the role of these galectins in maintaining B cell tolerance, we crossed mice deficient in galectin-1 or galectin-3 with mice bearing a lupus autoantigen-binding transgenic (Tg) B cell receptor, using a model with a well-characterized B cell tolerance phenotype of deletion, receptor editing and anergy. Here, we present data showing that the global knockout of galectin-1 or galectin-3 yields subtle alterations in B cell fate in autoantibody Tg mice. The absence of galectin-3 leads to a significant increase in the number of Tg spleen B cells, with the recovery of anti-laminin antibodies from a subset of mice. The B cell number increases further in antibody Tg mice with the dual deficiency of both galectin-1 and galectin-3. Isolated galectin-1 deficiency significantly enhances the proliferation of Tg B cells in response to lipopolysaccharide stimulation. These findings add to the growing body of evidence indicating a role for the various galectin family members, and for galectins 1 and 3 in particular, in the regulation of autoimmunity.
Assuntos
Autoanticorpos/imunologia , Linfócitos B/imunologia , Anergia Clonal/genética , Deleção Clonal/genética , Galectina 1/genética , Galectina 3/genética , Animais , Laminina/imunologia , Camundongos , Camundongos Knockout , TransgenesRESUMO
The duration of signaling through the MAP kinase (or ERK pathway) cascade has been implicated in thymic development, particularly positive and negative selection. In T cells, two isoforms of the MAP kinase kinase kinase Raf function to transmit signals from the T-cell receptor to ERK: C-Raf and B-Raf. In this study, we conditionally ablated B-Raf expression within thymocytes to assess the effects on ERK activation and thymocyte development. The complete loss of B-Raf is accompanied by a dramatic loss of ERK activation in both the double positive (DP) and single positive (SP) thymocytes, as well as peripheral splenocytes. There was a significant decrease in the cellularity of KO thymi, largely due to a loss of pre-selected DP cells, a decrease in DP cells undergoing positive selection, and a defect in SP maturation. B-Raf plays significant roles in survival of DP thymocytes and function of SP cells in the periphery. Surprisingly, we saw no effect of B-Raf deficiency on negative selection of autoreactive SP thymocytes, despite the greatly reduced ERK activation in these cells.
Assuntos
Proteínas Proto-Oncogênicas B-raf/metabolismo , Receptores de Antígenos de Linfócitos T/metabolismo , Subpopulações de Linfócitos T/imunologia , Linfócitos T/imunologia , Timo/imunologia , Animais , Antígenos CD4/metabolismo , Antígenos CD8/metabolismo , Diferenciação Celular/genética , Sobrevivência Celular/genética , Células Cultivadas , Deleção Clonal/genética , Ativação Enzimática/genética , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Sistema de Sinalização das MAP Quinases/genética , Sistema de Sinalização das MAP Quinases/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas Proto-Oncogênicas B-raf/genética , Proteínas Proto-Oncogênicas B-raf/imunologiaRESUMO
The autoimmune regulator (Aire), mediates central tolerance of peripheral self. Its activity in thymic epithelial cells (TECs) directs the ectopic expression of thousands of tissue-restricted antigens (TRAs), causing the deletion of autoreactive thymocytes. The molecular mechanisms orchestrating the breadth of transcriptional regulation by Aire remain unknown. One prominent model capable of explaining both the uniquely high number of Aire-dependent targets and their specificity posits that tissue-specific transcription factors induced by Aire directly activate their canonical targets, exponentially adding to the total number of Aire-dependent TRAs. To test this "Hierarchical Transcription" model, we analysed mice deficient in the pancreatic master transcription factor pancreatic and duodenal homeobox 1 (Pdx1), specifically in TECs (Pdx1(ΔFoxn1) ), for the expression and tolerance of pancreatic TRAs. Surprisingly, we found that lack of Pdx1 in TECs did not reduce the transcription of insulin or somatostatin, or alter glucagon expression. Moreover, in a model of thymic deletion driven by a neo-TRA under the control of the insulin promoter, Pdx1 in TECs was not required to affect thymocyte deletion or the generation of regulatory T (Treg) cells. These findings suggest that the capacity of Aire to regulate expression of a huge array of TRAs relies solely on an unconventional transcriptional mechanism, without intermediary transcription factors.
Assuntos
Tolerância Central , Proteínas de Homeodomínio/metabolismo , Pâncreas/imunologia , Linfócitos T/imunologia , Timo/imunologia , Transativadores/metabolismo , Fatores de Transcrição/metabolismo , Animais , Autoantígenos/genética , Autoantígenos/metabolismo , Autoimunidade , Células Cultivadas , Deleção Clonal/genética , Células Epiteliais/imunologia , Proteínas de Homeodomínio/genética , Insulina/genética , Insulina/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Modelos Imunológicos , Especificidade de Órgãos , Regiões Promotoras Genéticas/genética , Transativadores/genética , Fatores de Transcrição/genética , Transcrição Gênica , Proteína AIRERESUMO
The autoimmune regulator (Aire) plays a critical role in central tolerance by promoting the display of tissue-specific antigens in the thymus. To study the influence of Aire on thymic selection in a physiological setting, we used tetramer reagents to detect autoreactive T cells specific for the Aire-dependent tissue-specific antigen interphotoreceptor retinoid-binding protein (IRBP), in the polyclonal repertoire. Two class II tetramer reagents were designed to identify T cells specific for two different peptide epitopes of IRBP. Analyses of the polyclonal T-cell repertoire showed a high frequency of activated T cells specific for both IRBP tetramers in Aire(-/-) mice, but not in Aire(+/+) mice. Surprisingly, although one tetramer-binding T-cell population was efficiently deleted in the thymus in an Aire-dependent manner, the second tetramer-binding population was not deleted and could be detected in both the Aire(-/-) and Aire(+/+) T-cell repertoires. We found that Aire-dependent thymic deletion of IRBP-specific T cells relies on intercellular transfer of IRBP between thymic stroma and bone marrow-derived antigen-presenting cells. Furthermore, our data suggest that Aire-mediated deletion relies not only on thymic expression of IRBP, but also on proper antigen processing and presentation of IRBP by thymic antigen-presenting cells.
Assuntos
Células Apresentadoras de Antígenos/imunologia , Deleção Clonal/imunologia , Proteínas do Olho/imunologia , Proteínas de Ligação ao Retinol/imunologia , Linfócitos T/imunologia , Timo/imunologia , Fatores de Transcrição/imunologia , Uveíte/imunologia , Animais , Células Apresentadoras de Antígenos/metabolismo , Deleção Clonal/genética , Proteínas do Olho/metabolismo , Imunização , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas de Ligação ao Retinol/metabolismo , Estatísticas não Paramétricas , Linfócitos T/metabolismo , Timo/citologia , Fatores de Transcrição/genética , Uveíte/patologia , Proteína AIRERESUMO
The absence of regulatory T cells (Tregs) results in significant immune dysregulation that includes autoimmunity. The mechanism(s) by which Tregs suppress autoimmunity remains unclear. We have shown that B cell anergy, a major mechanism of B cell tolerance, is broken in the absence of Tregs. In this study, we identify a unique subpopulation of CD4(+) Th cells that are highly supportive of Ab production and promote loss of B cell anergy. Notably, this novel T cell subset was shown to express the germinal center Ag GL7 and message for the B cell survival factor BAFF, yet failed to express markers of the follicular Th cell lineage. We propose that the absence of Tregs results in the expansion of a unique nonfollicular Th subset of helper CD4(+) T cells that plays a pathogenic role in autoantibody production.
Assuntos
Subpopulações de Linfócitos B/imunologia , Linfócitos T CD4-Positivos/imunologia , Diferenciação Celular/imunologia , Anergia Clonal/imunologia , Deleção Clonal/imunologia , Linfócitos T Reguladores/imunologia , Linfócitos T Reguladores/patologia , Animais , Subpopulações de Linfócitos B/citologia , Subpopulações de Linfócitos B/metabolismo , Linfócitos T CD4-Positivos/citologia , Linfócitos T CD4-Positivos/metabolismo , Diferenciação Celular/genética , Linhagem da Célula/genética , Linhagem da Célula/imunologia , Células Cultivadas , Anergia Clonal/genética , Deleção Clonal/genética , Técnicas de Cocultura , Camundongos , Camundongos Endogâmicos C57BL , Camundongos TransgênicosRESUMO
Negative selection plays a key role in the clonal deletion of autoreactive T cells in the thymus. However, negative selection is incomplete; as high numbers of autoreactive T cells can be detected in normal individuals, mechanisms that regulate negative selection must exist. In this regard, we previously reported that CD24, a GPI-anchored glycoprotein, is required for thymic generation of autoreactive T lymphocytes. The CD24-deficient 2D2 TCR transgenic mice (2D2(+) CD24(-/-) ), whose TCR recognizes myelin oligodendrocyte glycoprotein (MOG), fail to generate functional 2D2 T cells. However, it was unclear if CD24 regulated negative selection, and if so, what cellular mechanisms were involved. Here, we show that elimination of MOG or Aire gene expression in 2D2(+) CD24(-/-) mice - through the creation of 2D2(+) CD24(-/-) MOG(-/-) or 2D2(+) CD24(/) â¼Aire(-/-) mice - completely restores thymic cellularity and function of 2D2 T cells. Restoration of CD24 expression on DCs, but not on thymocytes also partially restores 2D2 T-cell generation in 2D2(+) CD24(-/-) mice. Taken together, we propose that CD24 expression on thymic antigen-presenting cells (mTECs, DCs) down-regulates autoantigen-mediated clonal deletion of autoreactive thymocytes.
Assuntos
Antígeno CD24/imunologia , Células Dendríticas/imunologia , Proteínas da Mielina/imunologia , Timócitos/imunologia , Timo/imunologia , Animais , Autoantígenos/genética , Autoantígenos/imunologia , Autoantígenos/metabolismo , Antígeno CD24/genética , Antígeno CD24/metabolismo , Deleção Clonal/genética , Deleção Clonal/imunologia , Células Dendríticas/metabolismo , Regulação da Expressão Gênica/imunologia , Camundongos , Camundongos Knockout , Proteínas da Mielina/genética , Proteínas da Mielina/metabolismo , Bainha de Mielina/imunologia , Bainha de Mielina/metabolismo , Glicoproteína Mielina-Oligodendrócito , Receptores de Antígenos de Linfócitos T/genética , Receptores de Antígenos de Linfócitos T/imunologia , Receptores de Antígenos de Linfócitos T/metabolismo , Timócitos/metabolismo , Timo/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/imunologia , Fatores de Transcrição/metabolismo , Proteína AIRERESUMO
In his clonal selection theory, Frank Macfarlane Burnet predicted that autoreactive lymphocytes are deleted to prevent autoimmunity. This and other principles of lymphocyte behavior outlined by Burnet guided many studies that lead to our current understanding of thymic selection. Thus, when the genetic mutation responsible for autoimmune polyglandular syndrome type 1 was mapped to the autoimmune regulator (AIRE) gene, and Aire was found to be highly expressed in thymic epithelium, studying the role of Aire in negative selection made sense in the context of modern models of thymic selection. We now know Aire is a transcription factor required for the expression of many tissue-specific antigens (TSAs) in the thymus. In the absence of functional Aire, human patients and mice develop multi-organ autoimmune disease because of a defect in thymic negative selection. In addition to its role in the thymus, recent work in our lab suggests that extrathymic Aire-expressing cells have an important role in the clonal deletion of autoreactive CD8+ T cells. In this review, we summarize the latest studies on thymic and peripheral Aire-expressing cells, as well as other TSA-expressing stromal cell populations in peripheral lymphoid organs. We also discuss theoretical differences in thymic and peripheral Aire function that warrant further studies.
Assuntos
Deleção Clonal/imunologia , Fatores de Transcrição/metabolismo , Animais , Autoimunidade/imunologia , Deleção Clonal/genética , Humanos , Tolerância Imunológica/genética , Tolerância Imunológica/imunologia , Poliendocrinopatias Autoimunes/genética , Linfócitos T/imunologia , Linfócitos T/metabolismo , Timo/imunologia , Timo/metabolismo , Fatores de Transcrição/genética , Proteína AIRERESUMO
In mice expressing a transgenic T-cell receptor (TCR; TCRP1A) of DBA/2 origin with reactivity towards a cancer-germline antigen P1A, the number of TCRP1A CD8+ T cells in lymphoid organs is lower in DBA/2 than in B10.D2 or B10.D2(x DBA/2)F1 mice. This reduction results from haemopoietic cell autonomous differences in the differentiation of the major histocompatibility complex class I-restricted TCRP1A thymocytes controlled by DBA/2 versus B10.D2-encoded genes. We report here that the lower number of TCRP1A CD8+ T cells in DBA/2 mice correlated with their poor resistance to P1A-expressing mastocytoma solid tumours. Functional potency of CD8+ cytolytic T lymphocytes (CTL) from the above strains was not compromised, but their number after expansion appeared to be influenced by their genetic background. Intriguingly, non-transgenic DBA/ 2 mice resisted P1A+ tumours more efficiently despite poor representation of P1A-specific CTL. This was partly the result of their more heterogeneous TCR repertoire, including reactivity to non-P1A tumour antigens because mice that had rejected a P1A+ tumour became resistant to a P1A) variant of the tumour. Such 'cross-resistance' did not develop in the TCRP1A transgenic mice. Nonetheless, reconstitution of RAGo/o mice with TCRP1A CD8+ T cells, with or without CD4+ T cells, or exclusive representation of TCRP1A CD8+ T cells in RAGo/o TCRP1A transgenic mice efficiently resisted the growth of P1A-expressing tumours. Natural killer cells present at a higher number in RAGo/o mice also contributed to tumour resistance, in part through an NKG2D-dependent mechanism. Hence, in the absence of a polyclonal T-cell repertoire, precursor frequencies of natural killer cells and tumour-specific CTL affect tumour resistance.