RESUMO
Cell type specific delivery of RNAi to T cells has remained to be a challenge. Here we describe an aptamer mediated delivery of shRNA to CD4(+) T cells targeting RORγt to suppress Th17 cells. A cDNA encoding CD4 aptamer and RORγt shRNA was constructed and the chimeric CD4 aptamer-RORγt shRNA (CD4-AshR-RORγt) was generated using in vitro T7 RNA transcription. 2'-F-dCTP and 2'-F-dUTP were incorporated into CD4-AshR-RORγt for RNase resistance. CD4-AshR-RORγt was specifically uptaken by CD4(+) Karpas 299 cells and primary human CD4(+) T cells. The RORγt shRNA moiety of CD4-AshR-RORγt chimera was cleaved and released by Dicer. Furthermore, CD4-AshR-RORγt suppressed RORγt gene expression in Karpas 299 cells and CD4(+) T cells and consequently inhibited Th17 cell differentiation and IL-17 production. These results demonstrate that aptamer-facilitated cell specific delivery of shRNA represents a novel approach for efficient RNAi delivery and is potentially to be developed for therapeutics targeting specific T cells subtypes.
Assuntos
Antígenos CD4/imunologia , Linfócitos T CD4-Positivos/imunologia , Interleucina-17/biossíntese , Membro 3 do Grupo F da Subfamília 1 de Receptores Nucleares/genética , Membro 3 do Grupo F da Subfamília 1 de Receptores Nucleares/imunologia , RNA Interferente Pequeno/genética , Células Th17/imunologia , Linhagem Celular , Humanos , Transfecção/métodosRESUMO
Treatment with partial (p)MHC class II-ß1α1 constructs (also referred to as recombinant T-cell receptor ligands - RTL) linked to antigenic peptides can induce T-cell tolerance, inhibit recruitment of inflammatory cells and reverse autoimmune diseases. Here we demonstrate a novel regulatory pathway that involves RTL binding to CD11b(+) mononuclear cells through a receptor comprised of MHC class II invariant chain (CD74), cell-surface histones and MHC class II itself for treatment of experimental autoimmune encephalomyelitis (EAE). Binding of RTL constructs with CD74 involved a previously unrecognized MHC class II-α1/CD74 interaction that inhibited CD74 expression, blocked activity of its ligand, macrophage migration inhibitory factor, and reduced EAE severity. These findings implicate binding of RTL constructs to CD74 as a key step in both antigen-driven and bystander T-cell tolerance important in treatment of inflammatory diseases.
Assuntos
Antígenos de Diferenciação de Linfócitos B/metabolismo , Doenças Autoimunes/imunologia , Antígenos de Histocompatibilidade Classe II/metabolismo , Monócitos/imunologia , Animais , Antígenos de Diferenciação de Linfócitos B/biossíntese , Antígeno CD11b/metabolismo , Encefalomielite Autoimune Experimental/imunologia , Antígenos de Histocompatibilidade Classe II/biossíntese , Antígenos de Histocompatibilidade Classe II/imunologia , Tolerância Imunológica , Oxirredutases Intramoleculares , Fatores Inibidores da Migração de Macrófagos , Proteínas de Membrana , Camundongos , Receptores de Antígenos de Linfócitos T , Proteínas Recombinantes de Fusão/imunologia , Proteínas Recombinantes de Fusão/metabolismo , Linfócitos T/imunologiaRESUMO
Differentiation of naïve CD4(+) T cells has been considered to be an irreversible event and, in particular, the plasticity is believed to be completely lost in Th1 subset in vitro after multiple stimulations. However, here we demonstrate that highly polarized myelin oligodendrocyte glycoprotein (MOG)- and herpes simples virus-specific Th1 clones were still capable of producing IL-17 upon superantigen stimulation. Anti-MHC class-II and anti-TCR αß chains partially blocked superantigen-induced IL-17 production. These findings suggest that fully differentiated Th1 cells still have capability to produce cytokines of other Th subsets and production of IL-17 by MOG-specific Th1 cells may have implication in initiation and/or exacerbation of neurological autoimmune diseases.
Assuntos
Polaridade Celular/imunologia , Interleucina-17/biossíntese , Superantígenos/imunologia , Células Th1/citologia , Células Th1/imunologia , Polaridade Celular/efeitos dos fármacos , Antígenos de Histocompatibilidade Classe II/metabolismo , Humanos , Mediadores da Inflamação/farmacologia , Interferon gama/biossíntese , Receptores de Antígenos de Linfócitos T alfa-beta/metabolismo , Células Th1/efeitos dos fármacosRESUMO
Antigen-presenting cell-associated four-domain MHC class II (MHC-II) molecules play a central role in activating autoreactive CD4(+) T cells involved in multiple sclerosis (MS) and type 1 diabetes (T1D). In contrast, two-domain MHC-II structures with the same covalently attached self-peptide (recombinant T-cell receptor ligands (RTLs)) can regulate pathogenic CD4(+) T cells and reverse clinical signs of experimental autoimmune diseases. RTL1000, which is composed of the ß1α1 domains of human leukocyte antigen (HLA)-DR2 linked to the encephalitogenic human myelin oligodendrocyte glycoprotein (MOG)-35-55 peptide, was recently shown to be safe and well tolerated in a phase I clinical trial in MS. To evaluate the opposing biological effects of four- versus two-domain MHC-II structures, we screened phage Fab antibodies (Abs) for the neutralizing activity of RTL1000. Five different TCR-like Abs were identified that could distinguish between the two- versus four-domain MHC-peptide complexes while the cognate TCR was unable to make such a distinction. Moreover, Fab detection of native two-domain HLA-DR structures in human plasma implies that there are naturally occurring regulatory MHC-peptide complexes. These results demonstrate for the first time distinct conformational determinants characteristic of activating versus tolerogenic MHC-peptide complexes involved in human autoimmunity.
Assuntos
Células Apresentadoras de Antígenos/imunologia , Linfócitos T CD4-Positivos/imunologia , Antígenos de Histocompatibilidade Classe II/imunologia , Receptores de Antígenos de Linfócitos T/imunologia , Animais , Autoimunidade/imunologia , Diabetes Mellitus Tipo 1/imunologia , Encefalomielite Autoimune Experimental/imunologia , Encefalomielite Autoimune Experimental/terapia , Ensaio de Imunoadsorção Enzimática , Citometria de Fluxo , Glicoproteínas , Antígeno HLA-DR2/química , Antígeno HLA-DR2/genética , Antígeno HLA-DR2/imunologia , Humanos , Tolerância Imunológica , Fragmentos Fab das Imunoglobulinas/imunologia , Complexo Principal de Histocompatibilidade/imunologia , Camundongos , Camundongos Transgênicos , Esclerose Múltipla/imunologia , Esclerose Múltipla/terapia , Glicoproteína Mielina-Oligodendrócito , Fragmentos de Peptídeos , Proteínas Recombinantes de Fusão/sangue , Proteínas Recombinantes de Fusão/imunologia , Proteínas Recombinantes/imunologiaRESUMO
Therapeutic vaccination using T-cell receptor (TCR) peptides from V genes commonly expressed by potentially pathogenic T cells remains an approach of interest for treatment of multiple sclerosis (MS) and other autoimmune diseases. We developed a trivalent TCR vaccine containing complementarity determining region (CDR) 2 peptides from BV5S2, BV6S5 and BV13S1 emulsified in incomplete Freund's adjuvant that reliably induced high frequencies of TCR-specific T cells. To evaluate induction of regulatory T-cell subtypes, immunological and clinical parameters were followed in 23 treatment-naïve subjects with relapsing-remitting or progressive MS who received 12 monthly injections of the trivalent peptide vaccine over 1 year in an open-label study design. Prior to vaccination, subjects had reduced expression of forkhead box (Fox) P3 message and protein, and reduced recognition of the expressed TCR repertoire by TCR-reactive cells compared with healthy control donors. After three or four injections, most vaccinated MS subjects developed high frequencies of circulating interleukin (IL)-10-secreting T cells specific for the injected TCR peptides and significantly enhanced expression of FoxP3 by regulatory T cells present in both 'native' CD4+ CD25+ and 'inducible' CD4+ CD25- peripheral blood mononuclear cells (PBMC). At the end of the trial, PBMC from vaccinated MS subjects retained or further increased FoxP3 expression levels, exhibited significantly enhanced recognition of the TCR V gene repertoire apparently generated by perturbation of the TCR network, and significantly suppressed neuroantigen but not recall antigen responses. These findings demonstrate that therapeutic vaccination using only three commonly expressed BV gene determinants can induce an expanded immunoregulatory network in vivo that may optimally control complex autoreactive responses that characterize the inflammatory phase of MS.
Assuntos
Fatores de Transcrição Forkhead/sangue , Esclerose Múltipla/terapia , Receptores de Antígenos de Linfócitos T/imunologia , Vacinas de Subunidades Antigênicas/uso terapêutico , Adulto , Idoso , Autoantígenos/imunologia , Autoimunidade/imunologia , Regiões Determinantes de Complementaridade/imunologia , Feminino , Genes Codificadores dos Receptores de Linfócitos T/imunologia , Humanos , Tolerância Imunológica/imunologia , Memória Imunológica , Masculino , Pessoa de Meia-Idade , Esclerose Múltipla/imunologia , Proteínas do Tecido Nervoso/imunologia , Linfócitos T Reguladores/imunologia , Vacinação/métodosRESUMO
Alpha B-crystallin (alphaB) is a small heat shock protein that is strongly up-regulated in multiple sclerosis (MS) brain tissue, and can induce strong T cell responses. Assessing a potential encephalitogenic function for alphaB protein in MS and experimental autoimmune encephalomyelitis (EAE) has been challenging due to its ubiquitous expression that likely maintains central and peripheral tolerance to this protein in mice. To address this issue, we obtained alphaB-knockout (alphaB-KO) mice in H-2b background that lack immune tolerance to alphaB protein, and thus are capable of developing alphaB-specific T cells that could be tested for encephalitogenic activity after transfer into alphaB-expressing wild type (WT) mice. We found that T cell lines from spleens of alphaB protein-immunized alphaB-KO mice proliferated strongly to alphaB protein itself, and the majority of T cells were CD4+ and capable of secreting pro-inflammatory Th1 cytokines upon restimulation. However, transfer of such alphaB-reactive T cells back into WT recipients was not sufficient to induce EAE, compared to the transfer of mouse MOG-35-55 peptide-reactive T cells from the same donors that induced severe EAE in recipients. Moreover, alphaB-specific T cells failed to augment severity of actively induced EAE in WT mice that were expressing high levels of alphaB message in the CNS at the time of transfer. These results suggest that alphaB-specific T cells are immunocompetent but not encephalitogenic in 129SvEv mice, and that immune tolerance may not be the main factor that limits the encephalitogenic potential of alphaB.
Assuntos
Encefalomielite Autoimune Experimental/etiologia , Linfócitos T/imunologia , Cadeia B de alfa-Cristalina/imunologia , Sequência de Aminoácidos , Animais , Encefalomielite Autoimune Experimental/imunologia , Glicoproteínas/imunologia , Linfonodos/imunologia , Ativação Linfocitária , Camundongos , Camundongos Knockout , Dados de Sequência Molecular , Glicoproteína Mielina-Oligodendrócito , Fragmentos de Peptídeos/imunologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Baço/imunologia , Cadeia B de alfa-Cristalina/genética , Cadeia B de alfa-Cristalina/fisiologiaRESUMO
Single-chain human recombinant T cell receptor ligands derived from the peptide binding/TCR recognition domain of human HLA-DR2b (DRA*0101/DRB1*1501) produced in Escherichia coli with and without amino-terminal extensions containing antigenic peptides have been described previously. While molecules with the native sequence retained biological activity, they formed higher order aggregates in solution. In this study, we used site-directed mutagenesis to modify the ß-sheet platform of the DR2-derived RTLs, obtaining two variants that were monomeric in solution by replacing hydrophobic residues with polar (serine) or charged (aspartic acid) residues. Size exclusion chromatography and dynamic light scattering demonstrated that the modified RTLs were monomeric in solution, and structural characterization using circular dichroism demonstrated the highly ordered secondary structure of the RTLs. Peptide binding to the `empty' RTLs was quantified using biotinylated peptides, and functional studies showed that the modified RTLs containing covalently tethered peptides were able to inhibit antigen-specific T cell proliferation in vitro, as well as suppress experimental autoimmune encephalomyelitis in vivo. These studies demonstrated that RTLs encoding the Ag-binding/TCR recognition domain of MHC class II molecules are innately very robust structures, capable of retaining potent biological activity separate from the Ig-fold domains of the progenitor class II structure, with prevention of aggregation accomplished by modification of an exposed surface that was buried in the progenitor structure.
RESUMO
Chronic beryllium disease (CBD) is characterized pathologically by granulomatous inflammation in the lung, composed of a large core of epithelioid cells surrounded by a dense shell of CD4+ T cells. Using beryllium-specific CD4+ T cell lines derived from the bronchoalveolar lavage (BAL) fluid of CBD patients, we show that purified CD4+ T cells produced significant amounts of IFN-gamma and TNF-alpha upon exposure to beryllium in the absence of antigen-presenting cells (APC). However, unlike BAL T cells stimulated by beryllium in the presence of APC, self-presentation by BAL T cells did not induce detectable IL-2 production, and in its absence these activated T cells die from programmed cell death. Resting BAL CD4+ T cells constitutively express high levels of HLA-DP, lymphocyte function-associated antigen 1 (LFA-1) and ICAM-3. When stimulated with beryllium/APC, the adhesion molecule ICAM-1 was up-regulated, as well as several costimulation molecules including CD28, OX-40 (CD134), 4-1-BB (CD137) and B7-1 (CD80). Notably, CD28 was not up-regulated during self-presentation by BAL T cells, and these cells do not express OX-40L, suggesting that lack of appropriate costimulation was responsible for programmed cell death observed upon beryllium self-presentation. Restricting anti-MHC class II mAb completely eliminated beryllium-induced T cell proliferation during self-presentation and significantly reduced IFN-gamma and TNF-alpha production. Our data demonstrate for the first time that self-presentation by BAL T cells in response to beryllium can occur ex vivo, in the absence of professional APC, with a specific dependence on T cell-expressed MHC class II molecules and exogenous IL-2 for survival.
Assuntos
Apresentação de Antígeno/imunologia , Beriliose/imunologia , Berílio/imunologia , Líquido da Lavagem Broncoalveolar/citologia , Linfócitos T CD4-Positivos/imunologia , Comunicação Celular/imunologia , Líquido da Lavagem Broncoalveolar/imunologia , Antígenos de Histocompatibilidade Classe II/imunologia , Humanos , Interferon gama/imunologia , Interferon gama/metabolismo , Interleucina-2/imunologia , Interleucina-2/metabolismo , Ativação Linfocitária/imunologia , Fator de Necrose Tumoral alfa/imunologia , Fator de Necrose Tumoral alfa/metabolismoRESUMO
Chronic beryllium disease is a lung disorder caused by beryllium exposure in the workplace and is characterized by granulomatous inflammation and the accumulation of beryllium-specific, HLA-DP2-restricted CD4+ T lymphocytes in the lung that proliferate and secrete Th1-type cytokines. To characterize the interaction among HLA-DP2, beryllium, and CD4+ T cells, we constructed rHLA-DP2 and rHLA-DP4 molecules consisting of the alpha-1 and beta-1 domains of the HLA-DP molecules genetically linked into single polypeptide chains. Peptide binding to rHLA-DP2 and rHLA-DP4 was consistent with previously published peptide-binding motifs for these MHC class II molecules, with peptide binding dominated by aromatic residues in the P1 pocket. 9Be nuclear magnetic resonance spectroscopy showed that beryllium binds to the HLA-DP2-derived molecule, with no binding to the HLA-DP4 molecule that differs from DP2 by four amino acid residues. Using beryllium-specific CD4+ T cell lines derived from the lungs of chronic beryllium disease patients, beryllium presentation to those cells was independent of Ag processing because fixed APCs were capable of presenting BeSO4 and inducing T cell proliferation. Exposure of beryllium-specific CD4+ T cells to BeSO4 -pulsed, plate-bound rHLA-DP2 molecules induced IFN-gamma secretion. In addition, pretreatment of beryllium-specific CD4+ T cells with BeSO4-pulsed, plate-bound HLA-DP2 blocked proliferation and IL-2 secretion upon re-exposure to beryllium presented by APCs. Thus, the rHLA-DP2 molecules described herein provide a template for engineering variants that retain the ability to tolerize pathogenic CD4+ T cells, but do so in the absence of the beryllium Ag.
Assuntos
Beriliose/imunologia , Berílio/metabolismo , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/metabolismo , Antígenos HLA-DP/metabolismo , Proteínas Recombinantes/metabolismo , Sequência de Aminoácidos , Animais , Beriliose/terapia , Berílio/imunologia , Linhagem Celular , Doença Crônica , Antígenos HLA-DP/genética , Cadeias beta de HLA-DP , Humanos , Ligantes , Camundongos , Dados de Sequência Molecular , Ratos , Receptores de Antígenos de Linfócitos T/metabolismo , Proteínas Recombinantes/genéticaRESUMO
Autoimmune diseases such as multiple sclerosis (MS) may result from the failure of tolerance mechanisms to prevent expansion of pathogenic T cells. Our study is the first to establish that MS patients have abnormalities in FOXP3 message and protein expression levels in peripheral CD4+ CD25+ T cells (Tregs) that are quantitatively related to a reduction in functional suppression induced during suboptimal T-cell receptor (TCR) ligation. Of importance, this observation links a defect in functional peripheral immunoregulation to an established genetic marker that has been unequivocally shown to be involved in maintaining immune tolerance and preventing autoimmune diseases. Diminished FOXP3 levels thus indicate impaired immunoregulation by Tregs that may contribute to MS. Future studies will evaluate the effects of therapies known to influence Treg cell function and FOXP3 expression, including TCR peptide vaccination and supplemental estrogen.
Assuntos
Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Esclerose Múltipla/genética , Esclerose Múltipla/imunologia , Subpopulações de Linfócitos T/metabolismo , Adulto , Antígenos CD4/imunologia , Feminino , Fatores de Transcrição Forkhead , Marcadores Genéticos/imunologia , Marcadores Genéticos/fisiologia , Humanos , Masculino , Pessoa de Meia-Idade , Esclerose Múltipla/metabolismo , RNA Mensageiro/análise , Receptores de Interleucina-2/imunologia , Valores de Referência , Subpopulações de Linfócitos T/imunologiaRESUMO
CD4(+) T cells play a key role in granulomatous inflammation in the lung of patients with chronic beryllium disease. The goal of this study was to characterize activation pathways of beryllium-responsive bronchoalveolar lavage (BAL) CD4(+) T cells from chronic beryllium disease patients to identify possible therapeutic interventional strategies. Our results demonstrate that in the presence of APCs, beryllium induced strong proliferation responses of BAL CD4(+) T cells, production of superoptimal concentrations of secreted proinflammatory cytokines, IFN-gamma, TNF-alpha,and IL-2, and up-regulation of numerous T cell surface markers that would promote T-T Ag presentation. Ab blocking experiments revealed that anti-HLA-DP or anti-LFA-1 Ab strongly reduced proliferation responses and cytokine secretion by BAL CD4(+) T cells. In contrast, anti-HLA-DR or anti-OX40 ligand Ab mainly affected beryllium-induced proliferation responses with little impact on cytokines other than IL-2, thus implying that nonproliferating BAL CD4(+) T cells may still contribute to inflammation. Blockade with CTLA4-Ig had a minimal effect on proliferation and cytokine responses, confirming that activation was independent of B7/CD28 costimulation. These results indicate a prominent role for HLA-DP and LFA-1 in BAL CD4(+) T cell activation and further suggest that specific Abs to these molecules could serve as a possible therapy for chronic beryllium disease.
Assuntos
Anticorpos Monoclonais/uso terapêutico , Beriliose/tratamento farmacológico , Antígenos HLA-DP/imunologia , Antígeno-1 Associado à Função Linfocitária/imunologia , Transdução de Sinais , Berílio/imunologia , Linfócitos T CD4-Positivos/imunologia , Doença Crônica , Antígenos HLA-DP/genética , Cadeias beta de HLA-DP , Heterozigoto , Humanos , Imunoterapia/métodos , Ativação LinfocitáriaRESUMO
Of potential importance to multiple sclerosis (MS), oligodendroglial alpha B-crystallin is expressed and associated with the myelin sheath at the earliest stage of MS lesion development. We selected T-cell lines specific for human alpha B-crystallin from peripheral blood mononuclear cells (PBMC) of HLA-DR2 homozygous MS patients and found that the alpha B-crystallin-specific T-cells were CD4+ and restricted by DRB1*1501, and expressed Th1 cytokines. The CD4 T-cell epitopes of human alpha B-crystallin were determined by proliferation of alpha B-crystallin-specific T-cell lines to 17 20-mer synthetic overlapping peptides spanning the entire molecule of human alpha B-crystallin. It was found that the HLA-DR2 donor-derived alpha B-crystallin-specific T-cell lines proliferated to alpha B-crystallin peptides 21-40, 41-60, and to a lesser extent, 131-150. These T-cell proliferation responses were associated with intracellular expression of interleukin-2 (IL-2) and secretion of interferon-gamma (IFN-gamma), and tumor necrosis factor-alpha (TNF-alpha). The amino acid sequences of these peptides were compatible with predicted HLA-DR2-restricted binding motifs. PBMC of an early active MS patient proliferated to the epitope-containing peptides significantly better than did those of later stage MS patients or healthy controls. Taken together, these findings suggest that autoreactive alpha B-crystallin-specific Th1 cells may have the potential to contribute to MS pathogenesis.
Assuntos
Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/metabolismo , Epitopos de Linfócito T/imunologia , Epitopos de Linfócito T/metabolismo , Esclerose Múltipla/metabolismo , Cadeia B de alfa-Cristalina/imunologia , Cadeia B de alfa-Cristalina/metabolismo , Sequência de Aminoácidos , Sítios de Ligação/imunologia , Linfócitos T CD4-Positivos/citologia , Divisão Celular/imunologia , Linhagem Celular , Epitopos de Linfócito T/genética , Humanos , Ativação Linfocitária/imunologia , Dados de Sequência Molecular , Esclerose Múltipla/genética , Esclerose Múltipla/imunologia , Análise de Sequência de Proteína/métodos , Cadeia B de alfa-Cristalina/genéticaRESUMO
Susceptibility to multiple sclerosis (MS) is associated genetically with human leucocyte antigen (HLA) class II alleles, including DRB1*1501, DRB5*0101, and DQB1*0602, and it is possible that these alleles contribute to MS through an enhanced ability to present encephalitogenic myelin peptides to pathogenic T cells. HLA-DRB1*1502, which contains glycine instead of valine at position 86 of the P1 peptide-binding pocket, is apparently not genetically associated with MS. To identify possible differences between these alleles in their antigen-presenting function, we determined if T-cell responses to known DRB1*1501-restricted myelin peptides might be diminished or absent in transgenic (Tg) DRB1*1502-expressing mice. We found that Tg DRB1*1502 mice had moderate to strong T-cell responses to several myelin peptides with favorable DRB1*1501 binding motifs, notably myelin oligodendrocyte glycoprotein (MOG)-35-55 (which was also encephalitogenic), proteolipid protein (PLP)-95-116, and MOG-194-208, as well as other PLP and MOG peptides. These peptides, with the exception of MOG-194-208, were also immunogenic in healthy human donors expressing either DRB1*1502 or DRB1*1501. In contrast, the DRB1*1502 mice had weak or absent responses to peptides with unfavorable DRB1*1501 binding motifs. Overall, none of the DRB1*1501-restricted myelin peptides tested selectively lacked immunogenicity in association with DRB1*1502. These results indicate that the difference in risk association with MS of DRB1*1501 versus DRB1*1502 is not due to a lack of antigen presentation by DRB1*1502, at least for this set of myelin peptides, and suggest that other mechanisms involving DRB1*1501 may account for increased susceptibility to MS.
Assuntos
Apresentação de Antígeno , Antígenos HLA-DR/metabolismo , Epitopos Imunodominantes/metabolismo , Esclerose Múltipla/imunologia , Proteína Básica da Mielina/metabolismo , Glicoproteína Associada a Mielina/metabolismo , Sequência de Aminoácidos , Animais , Apresentação de Antígeno/genética , Linhagem Celular , Células Cultivadas , Feminino , Antígenos HLA-DR/biossíntese , Antígenos HLA-DR/genética , Cadeias HLA-DRB1 , Humanos , Epitopos Imunodominantes/administração & dosagem , Epitopos Imunodominantes/genética , Ativação Linfocitária/genética , Masculino , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Dados de Sequência Molecular , Esclerose Múltipla/genética , Esclerose Múltipla/metabolismo , Proteína Básica da Mielina/administração & dosagem , Proteína Básica da Mielina/genética , Proteínas da Mielina , Glicoproteína Associada a Mielina/genética , Glicoproteína Associada a Mielina/imunologia , Glicoproteína Mielina-Oligodendrócito , Fatores de RiscoRESUMO
Recombinant TCR ligands (RTLs) consisting of covalently linked alpha(1) and beta(1) domains of MHC class II molecules tethered to specific antigenic peptides represent minimal TCR ligands. In a previous study we reported that the rat RTL201 construct, containing RT1.B MHC class II domains covalently coupled to the encephalitogenic guinea pig myelin basic protein (Gp-MBP(72-89)) peptide, could prevent and treat actively and passively induced experimental autoimmune encephalomyelitis in vivo by selectively inhibiting Gp-MBP(72-89) peptide-specific CD4(+) T cells. To evaluate the inhibitory signaling pathway, we tested the effects of immobilized RTL201 on T cell activation of the Gp-MBP(72-89)-specific A1 T cell hybridoma. Activation was exquisitely Ag-specific and could not be induced by RTL200 containing the rat MBP(72-89) peptide that differed by a threonine for serine substitution at position 80. Partial activation by RTL201 included a CD3zeta p23/p21 ratio shift, ZAP-70 phosphorylation, calcium mobilization, NFAT activation, and transient IL-2 production. In comparison, anti-CD3epsilon treatment produced stronger activation of these cellular events with additional activation of NF-kappaB and extracellular signal-regulated kinases as well as long term increased IL-2 production. These results demonstrate that RTLs can bind directly to the TCR and modify T cell behavior through a partial activation mechanism, triggering specific downstream signaling events that deplete intracellular calcium stores without fully activating T cells. The resulting Ag-specific activation of the transcription factor NFAT uncoupled from the activation of NF-kappaB or extracellular signal-regulated kinases constitutes a unique downstream activation pattern that accounts for the inhibitory effects of RTL on encephalitogenic CD4(+) T cells.
Assuntos
Sinalização do Cálcio/imunologia , Antígeno HLA-DR2/fisiologia , Ativação Linfocitária , Proteína Básica da Mielina/farmacologia , Proteínas Nucleares , Fragmentos de Peptídeos/farmacologia , Receptores de Antígenos de Linfócitos T alfa-beta/fisiologia , Linfócitos T/imunologia , Linfócitos T/metabolismo , Sequência de Aminoácidos , Animais , Complexo CD3/metabolismo , Sinalização do Cálcio/genética , Proteínas de Ligação a DNA/metabolismo , Cobaias , Antígeno HLA-DR2/metabolismo , Antígenos de Histocompatibilidade/metabolismo , Antígenos de Histocompatibilidade/fisiologia , Humanos , Hibridomas , Interleucina-2/biossíntese , Ligantes , Ativação Linfocitária/genética , Camundongos , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Dados de Sequência Molecular , Proteína Básica da Mielina/metabolismo , NF-kappa B/metabolismo , Fatores de Transcrição NFATC , Fragmentos de Peptídeos/metabolismo , Fosforilação , Proteínas Tirosina Quinases/metabolismo , Ratos , Ratos Endogâmicos Lew , Receptores de Antígenos de Linfócitos T/fisiologia , Receptores de Antígenos de Linfócitos T alfa-beta/metabolismo , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/farmacologia , Linfócitos T/enzimologia , Fatores de Transcrição/metabolismo , Células Tumorais Cultivadas , Proteína-Tirosina Quinase ZAP-70RESUMO
The goal of this study was to establish an unlimited and standardized source of humanized myelin peptide-specific T cells for in vitro testing of biological function. Thus, we perpetuated myelin oligodendrocyte glycoprotein (MOG)-35-55 peptide-specific T cells obtained from immunized HLA-DRB1*1501-transgenic (Tg) mice by somatic fusions with BW5147 thymoma cells or BW5147 T-cell receptor (TCR) alpha(-)beta(-) variant (BW5147 variant) cells. The resulting T-cell hybridomas responded strongly to both mouse MOG-35-55 (42S) and human MOG-35-55 peptide (42P), regardless of which peptide was used for initial immunization, and were DRB1*1501 restricted. The MOG-35-55-reactive T-cell hybridomas were CD3(+)CD4(+)CD8(-) and expressed intracellular Th1 cytokines upon concanavalin A stimulation. Clones from either human MOG-35-55- or mouse MOG-35-55-selected hybridomas uniquely expressed the TCR BV8 gene in combination with AV17 and AV11 genes. V gene analyses confirmed the expression of TCR AV1, AV11, AV16, BV1, and BV5 gene segments in the widely used fusion partner BW5147 and demonstrated deletion of TCR AV1, AV11, and BV1 in the BW5147 variant. T-cell hybridomas were positively stained with anti-TCR beta-chain antibody on the cell surface, whereas neither BW5147 nor its variant had positive TCR surface expression. For functional application, we found that a monomeric form of the human HLA-DR2-derived recombinant T-cell receptor ligand (RTL) covalently linked to human MOG-35-55 peptide specifically inhibited proliferation of a hybridoma clone selected with human MOG-35-55 but not a different hybridoma clone selected with myelin basic protein. The RTL-induced inhibition in vitro of the human MOG-35-55 peptide-specific hybridoma reflected the ability of the RTL to inhibit experimental autoimmune encephalomyelitis induced by human MOG-35-55 peptide in HLA-DR2 transgenic mice. Thus, the MOG-35-55 peptide-specific T-cell hybridoma from DR2-Tg mice represents a novel humanized T-cell reagent useful for standardized biological screening of both DR2-restricted stimulation and RTL-dependent inhibition of response to human MOG-35-55 peptide.
Assuntos
Glicoproteínas/imunologia , Antígenos HLA-DR/metabolismo , Hibridomas/metabolismo , Fragmentos de Peptídeos/imunologia , Linfócitos T/metabolismo , Animais , Formação de Anticorpos/fisiologia , Southern Blotting/métodos , Linhagem Celular Tumoral , Técnicas de Cocultura/métodos , Relação Dose-Resposta Imunológica , Citometria de Fluxo/métodos , Antígenos HLA-DR/genética , Cadeias HLA-DRB1 , Humanos , Interleucina-2/metabolismo , Camundongos , Camundongos Endogâmicos , Camundongos Transgênicos , Glicoproteína Mielina-Oligodendrócito , RNA Mensageiro/biossíntese , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Especificidade do Receptor de Antígeno de Linfócitos T/fisiologia , Fatores de TempoRESUMO
CD4+CD25+ regulatory T cells (Treg cells) prevent T cell-mediated autoimmune diseases in rodents. To develop a functional Treg assay for human blood cells, we used FACS- or bead-sorted CD4+CD25+ T cells from healthy donors to inhibit anti-CD3/CD28 activation of CD4+CD25- indicator T cells. The data clearly demonstrated classical Treg suppression of CD4+CD25- indicator cells by both CD4+CD25(+high) and CD4+CD25(+low) T cells obtained by FACS or magnetic bead sorting. Suppressive activity was found in either CD45RO- (naive) or CD45RO+ (memory) subpopulations, was independent of the TCR signal strength, required cell-cell contact, and was reversible by interleukin-2 (IL-2). Of general interest is that a wider sampling of 27 healthy donors revealed an age- but not gender-dependent loss of suppressive activity in the CD4+CD25+ population. The presence or absence of suppressive activity in CD4+CD25+ T cells from a given donor could be demonstrated consistently over time, and lack of suppression was not due to method of sorting, strength of signal, or sensitivity of indicator cells. Phenotypic markers did not differ on CD4+CD25+ T cells tested ex vivo from suppressive vs. nonsuppressive donors, although, upon activation in vitro, suppressive CD4+CD25+ T cells had significantly higher expression of both CTLA-4 and GITR than CD4+CD25- T cells from the same donors. Moreover, antibody neutralization of CTLA-4, GITR, IL-10, or IL-17 completely reversed Treg-induced suppression. Our results are highly consistent with those reported for murine Treg cells and are the first to demonstrate that suppressive activity of human CD4+CD25+ T cells declines with age.
Assuntos
Envelhecimento/imunologia , Bioensaio/métodos , Linfócitos T CD4-Positivos/citologia , Tolerância Imunológica/imunologia , Receptores de Interleucina-2/imunologia , Linfócitos T/imunologia , Adulto , Anticorpos , Antígenos CD , Antígenos de Diferenciação/imunologia , Biomarcadores , Linfócitos T CD4-Positivos/imunologia , Antígeno CTLA-4 , Regulação para Baixo/imunologia , Feminino , Citometria de Fluxo/métodos , Proteína Relacionada a TNFR Induzida por Glucocorticoide , Humanos , Tolerância Imunológica/efeitos dos fármacos , Separação Imunomagnética , Interleucina-2/farmacologia , Antígenos Comuns de Leucócito/genética , Antígenos Comuns de Leucócito/imunologia , Masculino , Pessoa de Meia-Idade , Receptores de Fator de Crescimento Neural/imunologia , Receptores do Fator de Necrose Tumoral/imunologia , Fatores Sexuais , Linfócitos T/efeitos dos fármacosRESUMO
Although the phenotypic and regulatory properties of the CD4(+)CD25(+) T cell lineage (Treg cells) have been well described, the specificities remain largely unknown. We demonstrate here that the CD4(+)CD25(+) Treg population includes the recognition of a broad spectrum of human TCR CDR2 determinants found in the germline V gene repertoire as well as that of a clonotypic nongermline-encoded CDR3beta sequence present in a recombinant soluble T cell receptor (TCR) protein. Regulatory activity was demonstrated in T cell lines responsive to TCR but not in T cell lines responsive to control antigens. Inhibitory activity of TCR-reactive T cells required cell-cell contact and involved CTLA-4, GITR, IL-10, and IL-17. Thus, the T-T regulatory network includes Treg cells with specificity directed toward self-TCR determinants.