RESUMO
Immune checkpoint blockade antibodies are setting a new standard of care for cancer patients. It is therefore important to assess any new immune-based therapies in the context of immune checkpoint blockade. Here, we evaluate the impact of combining a synthetic consensus TERT DNA vaccine that has improved capacity to break tolerance with immune checkpoint inhibitors. We observed that blockade of CTLA-4 or, to a lesser extent, PD-1 synergized with TERT vaccine, generating more robust anti-tumor activity compared to checkpoint alone or vaccine alone. Despite this anti-tumor synergy, none of these immune checkpoint therapies showed improvement in TERT antigen-specific immune responses in tumor-bearing mice. αCTLA-4 therapy enhanced the frequency of T-bet+/CD44+ effector CD8+ T cells within the tumor and decreased the frequency of regulatory T cells within the tumor, but not in peripheral blood. CTLA-4 blockade synergized more than Treg depletion with TERT DNA vaccine, suggesting that the effect of CTLA-4 blockade is more likely due to the expansion of effector T cells in the tumor rather than a reduction in the frequency of Tregs. These results suggest that immune checkpoint inhibitors function to alter the immune regulatory environment to synergize with DNA vaccines, rather than boosting antigen-specific responses at the site of vaccination.
Assuntos
Antineoplásicos Imunológicos/farmacologia , Vacinas Anticâncer/imunologia , Neoplasias/genética , Neoplasias/imunologia , Telomerase/imunologia , Vacinas de DNA/imunologia , Animais , Biomarcadores Tumorais , Antígeno CTLA-4/antagonistas & inibidores , Vacinas Anticâncer/genética , Linhagem Celular Tumoral , Terapia Combinada , Modelos Animais de Doenças , Feminino , Humanos , Imunoterapia , Camundongos , Neoplasias/patologia , Neoplasias/terapia , Receptor de Morte Celular Programada 1/antagonistas & inibidores , Subpopulações de Linfócitos T/efeitos dos fármacos , Subpopulações de Linfócitos T/imunologia , Subpopulações de Linfócitos T/metabolismo , Telomerase/genética , Vacinas de DNA/genética , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
In this phase 2 multicenter study, we evaluated the efficacy and safety of lifileucel (LN-145), an autologous tumor-infiltrating lymphocyte cell therapy, in patients with metastatic non-small cell lung cancer (mNSCLC) who had received prior immunotherapy and progressed on their most recent therapy. The median number of prior systemic therapies was 2 (range, 1-6). Lifileucel was successfully manufactured using tumor tissue from different anatomic sites, predominantly lung. The objective response rate was 21.4% (6/28). Responses occurred in tumors with profiles typically resistant to immunotherapy, such as PD-L1-negative, low tumor mutational burden, and STK11 mutation. Two responses were ongoing at the time of data cutoff, including one complete metabolic response in a PD-L1-negative tumor. Adverse events were generally as expected and manageable. Two patients died of treatment-emergent adverse events: cardiac failure and multiple organ failure. Lifileucel is a potential treatment option for patients with mNSCLC refractory to prior therapy. Significance: Autologous tumor-infiltrating lymphocyte therapy lifileucel was administered to 28 patients with heavily pretreated metastatic non-small cell lung cancer (mNSCLC). Responses were observed in patients with driver mutations, and various tumor mutational burdens and PD-L1 expression, potentially addressing an unmet medical need in patients with mNSCLC refractory to prior therapy. See related commentary by Lotze et al., p. 1366.
Assuntos
Carcinoma Pulmonar de Células não Pequenas , Inibidores de Checkpoint Imunológico , Neoplasias Pulmonares , Linfócitos do Interstício Tumoral , Adulto , Idoso , Idoso de 80 Anos ou mais , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Carcinoma Pulmonar de Células não Pequenas/patologia , Resistencia a Medicamentos Antineoplásicos , Inibidores de Checkpoint Imunológico/uso terapêutico , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/patologia , Linfócitos do Interstício Tumoral/imunologia , Linfócitos do Interstício Tumoral/transplanteRESUMO
Mechanistic and therapeutic insights in autoimmune diabetes would benefit from a more complete identification of relevant autoantigens. BDC2.5 TCR transgenic NOD mice express transgenes for TCR Vα1 and Vß4 chains from the highly diabetogenic BDC2.5 CD4(+) T cell clone, which recognizes pancreatic ß cell membrane Ags presented by NOD I-A(g7) MHC class II molecules. The antigenic epitope of BDC2.5 TCR is absent in ß cells that do not express chromogranin A (ChgA) protein. However, characterization of the BDC2.5 epitope in ChgA has given inconclusive results. We have now identified a ChgA29-42 peptide within vasostatin-1, an N-terminal natural derivative of ChgA as the BDC2.5 TCR epitope. Having the necessary motif for binding to I-A(g7), it activates BDC2.5 T cells and induces an IFN-γ response. More importantly, adoptive transfer of naive BDC2.5 splenocytes activated with ChgA29-42 peptide transferred diabetes into NOD/SCID mice.
Assuntos
Cromogranina A/imunologia , Diabetes Mellitus Tipo 1/imunologia , Diabetes Mellitus Tipo 1/metabolismo , Epitopos de Linfócito T/imunologia , Fragmentos de Peptídeos/imunologia , Transferência Adotiva , Sequência de Aminoácidos , Animais , Apresentação de Antígeno/imunologia , Proliferação de Células , Células Cultivadas , Cromogranina A/administração & dosagem , Cromogranina A/metabolismo , Diabetes Mellitus Tipo 1/patologia , Epitopos de Linfócito T/administração & dosagem , Epitopos de Linfócito T/metabolismo , Humanos , Ilhotas Pancreáticas/imunologia , Ilhotas Pancreáticas/patologia , Ativação Linfocitária/imunologia , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Camundongos Transgênicos , Dados de Sequência MolecularRESUMO
We have previously proposed that sequence variation of the CD101 gene between NOD and C57BL/6 mice accounts for the protection from type 1 diabetes (T1D) provided by the insulin-dependent diabetes susceptibility region 10 (Idd10), a <1 Mb region on mouse chromosome 3. In this study, we provide further support for the hypothesis that Cd101 is Idd10 using haplotype and expression analyses of novel Idd10 congenic strains coupled to the development of a CD101 knockout mouse. Susceptibility to T1D was correlated with genotype-dependent CD101 expression on multiple cell subsets, including Foxp3(+) regulatory CD4(+) T cells, CD11c(+) dendritic cells, and Gr1(+) myeloid cells. The correlation of CD101 expression on immune cells from four independent Idd10 haplotypes with the development of T1D supports the identity of Cd101 as Idd10. Because CD101 has been associated with regulatory T and Ag presentation cell functions, our results provide a further link between immune regulation and susceptibility to T1D.
Assuntos
Antígenos CD/genética , Diabetes Mellitus Tipo 1/genética , Diabetes Mellitus Tipo 1/imunologia , Animais , Antígenos CD/biossíntese , Doenças Autoimunes/genética , Doenças Autoimunes/imunologia , Células CHO , Cricetinae , Cricetulus , Modelos Animais de Doenças , Predisposição Genética para Doença , Genótipo , Haplótipos , Camundongos , Camundongos Congênicos , Camundongos Endogâmicos A , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos NOD , Camundongos Knockout , Dados de Sequência MolecularRESUMO
The cytokine transforming growth factor-beta (TGF-beta) is an important negative regulator of adaptive immunity. TGF-beta is secreted by cells as an inactive precursor that must be activated to exert biological effects, but the mechanisms that regulate TGF-beta activation and function in the immune system are poorly understood. Here we show that conditional loss of the TGF-beta-activating integrin alpha(v)beta8 on leukocytes causes severe inflammatory bowel disease and age-related autoimmunity in mice. This autoimmune phenotype is largely due to lack of alpha(v)beta8 on dendritic cells, as mice lacking alpha(v)beta8 principally on dendritic cells develop identical immunological abnormalities as mice lacking alpha(v)beta8 on all leukocytes, whereas mice lacking alpha(v)beta8 on T cells alone are phenotypically normal. We further show that dendritic cells lacking alpha(v)beta8 fail to induce regulatory T cells (T(R) cells) in vitro, an effect that depends on TGF-beta activity. Furthermore, mice lacking alpha(v)beta8 on dendritic cells have reduced proportions of T(R) cells in colonic tissue. These results suggest that alpha(v)beta8-mediated TGF-beta activation by dendritic cells is essential for preventing immune dysfunction that results in inflammatory bowel disease and autoimmunity, effects that are due, at least in part, to the ability of alpha(v)beta8 on dendritic cells to induce and/or maintain tissue T(R) cells.
Assuntos
Autoimunidade/imunologia , Colite/metabolismo , Células Dendríticas/metabolismo , Integrinas/deficiência , Integrinas/metabolismo , Leucócitos/metabolismo , Envelhecimento/imunologia , Animais , Colite/imunologia , Colo/citologia , Colo/imunologia , Imunoglobulinas/sangue , Memória Imunológica , Integrinas/genética , Interferon gama/metabolismo , Interleucina-4/metabolismo , Leucócitos/imunologia , Leucócitos/patologia , Ativação Linfocitária , Contagem de Linfócitos , Camundongos , Fenótipo , Linfócitos T Reguladores/citologia , Linfócitos T Reguladores/imunologia , Linfócitos T Reguladores/metabolismo , Linfócitos T Reguladores/patologia , Fator de Crescimento Transformador beta/metabolismoRESUMO
The low number of CD4+ CD25+ regulatory T cells (Tregs), their anergic phenotype, and diverse antigen specificity present major challenges to harnessing this potent tolerogenic population to treat autoimmunity and transplant rejection. In this study, we describe a robust method to expand antigen-specific Tregs from autoimmune-prone nonobese diabetic mice. Purified CD4+ CD25+ Tregs were expanded up to 200-fold in less than 2 wk in vitro using a combination of anti-CD3, anti-CD28, and interleukin 2. The expanded Tregs express a classical cell surface phenotype and function both in vitro and in vivo to suppress effector T cell functions. Most significantly, small numbers of antigen-specific Tregs can reverse diabetes after disease onset, suggesting a novel approach to cellular immunotherapy for autoimmunity.
Assuntos
Autoantígenos/imunologia , Antígenos CD4/análise , Diabetes Mellitus Tipo 1/terapia , Receptores de Interleucina-2/análise , Linfócitos T Reguladores/imunologia , Transferência Adotiva , Animais , Diabetes Mellitus Tipo 1/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos NOD , Camundongos Transgênicos , Receptores de Antígenos de Linfócitos T/fisiologiaRESUMO
Forkhead transcription factors play critical roles in leukocyte homeostasis. To study further the immunological functions of Foxo1, we generated mice that selectively lack Foxo1 in T cells (Foxo1(flox/flox) Lck.cre(+)conditional knockout mice (cKO)). Although thymocyte development appeared relatively normal, Foxo1 cKO mice harbored significantly increased percentages of mature single positive T cells in the thymus as compared with WT mice, yet possessed smaller lymph nodes and spleens that contained fewer T cells. Foxo1 cKO T cells were not more prone to apoptosis, but instead were characterized by a CD62L(lo) CCR7(lo) CD44(hi) surface phenotype, a poorly populated lymphoid compartment in the periphery, and were relatively refractory to TCR stimulation, all of which were associated with reduced expression of Sell, Klf2, Ccr7, and S1pr1. Thus, Foxo1 is critical for naïve T cells to populate the peripheral lymphoid organs by coordinating a molecular program that maintains homeostasis and regulates trafficking.
Assuntos
Quimiotaxia de Leucócito/imunologia , Fatores de Transcrição Forkhead/imunologia , Tecido Linfoide/imunologia , Subpopulações de Linfócitos T/imunologia , Linfócitos T/imunologia , Animais , Apoptose/imunologia , Proliferação de Células , Citometria de Fluxo , Proteína Forkhead Box O1 , Tecido Linfoide/citologia , Camundongos , Camundongos Knockout , Fenótipo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Subpopulações de Linfócitos T/citologia , Linfócitos T/citologiaRESUMO
Overcoming tolerance to tumor-associated antigens remains a hurdle for cancer vaccine-based immunotherapy. A strategy to enhance the anti-tumor immune response is the inclusion of adjuvants to cancer vaccine protocols. In this report, we generated and systematically screened over twenty gene-based molecular adjuvants composed of cytokines, chemokines, and T cell co-stimulators for the ability to increase anti-tumor antigen T cell immunity. We identified several robust adjuvants whose addition to vaccine formulations resulted in enhanced T cell responses targeting the cancer antigens STEAP1 and TERT. We further characterized direct T cell stimulation through CD80-Fc and indirect T cell targeting via the dendritic cell activator Flt3L-Fc. Mechanistically, intramuscular delivery of Flt3L-Fc into mice was associated with a significant increase in infiltration of dendritic cells at the site of administration and trafficking of activated dendritic cells to the draining lymph node. Gene expression analysis of the muscle tissue confirmed a significant up-regulation in genes associated with dendritic cell signaling. Addition of CD80-Fc to STEAP1 vaccine formulation mimicked the engagement provided by DCs and increased T cell responses to STEAP1 by 8-fold, significantly increasing the frequency of antigen-specific cells expressing IFNγ, TNFα, and CD107a for both CD8+ and CD4+ T cells. CD80-Fc enhanced T cell responses to multiple tumor-associated antigens including Survivin and HPV, indicating its potential as a universal adjuvant for cancer vaccines. Together, the results of our study highlight the adjuvanting effect of T cell engagement either directly, CD80-Fc, or indirectly, Flt3L-Fc, for cancer vaccines.
Assuntos
Adjuvantes Imunológicos/farmacologia , Antígeno B7-1/imunologia , Vacinas Anticâncer/imunologia , Proteínas de Membrana/imunologia , Neoplasias/terapia , Linfócitos T/imunologia , Tetraspanina 28/imunologia , Animais , Antígenos de Neoplasias , Antígeno B7-1/genética , Movimento Celular/imunologia , Citocinas/metabolismo , DNA/genética , Células Dendríticas/imunologia , Feminino , Humanos , Imunoterapia/métodos , Ativação Linfocitária , Proteínas de Membrana/genética , Camundongos , Camundongos Endogâmicos BALB C , Neoplasias/imunologia , Plasmídeos/genética , Vacinas de DNA/genética , Vacinas de DNA/imunologiaRESUMO
Antibody-based immune therapies targeting the T-cell checkpoint molecules CTLA-4 and PD-1 have affected cancer therapy. However, this immune therapy requires complex manufacturing and frequent dosing, limiting the global use of this treatment. Here, we focused on the development of a DNA-encoded monoclonal antibody (DMAb) approach for delivery of anti-CTLA-4 monoclonal antibodies in vivo With this technology, engineered and formulated DMAb plasmids encoding IgG inserts were directly injected into muscle and delivered intracellularly by electroporation, leading to in vivo expression and secretion of the encoded IgG. DMAb expression from a single dose can continue for several months without the need for repeated administration. Delivery of an optimized DMAb encoding anti-mouse CTLA-4 IgG resulted in high serum levels of the antibody as well as tumor regression in Sa1N and CT26 tumor models. DNA-delivery of the anti-human CTLA-4 antibodies ipilimumab and tremelimumab in mice achieved potent peak levels of approximately 85 and 58 µg/mL, respectively. These DMAb exhibited prolonged expression, with maintenance of serum levels at or above 15 µg/mL for over a year. Anti-human CTLA-4 DMAbs produced in vivo bound to human CTLA-4 protein expressed on stimulated human peripheral blood mononuclear cells and induced T-cell activation in a functional assay ex vivo In summary, direct in vivo expression of DMAb encoding checkpoint inhibitors serves as a novel tool for immunotherapy that could significantly improve availability and provide broader access to such therapies.Significance: DNA-encoded monoclonal antibodies represent a novel technology for delivery and expression of immune checkpoint blockade antibodies, thus expanding patient access to, and possible clinical applications of, these therapies. Cancer Res; 78(22); 6363-70. ©2018 AACR.
Assuntos
Anticorpos Monoclonais/química , Antígeno CTLA-4/imunologia , DNA/química , Neoplasias/imunologia , Neoplasias/terapia , Animais , Anticorpos Monoclonais/farmacologia , Anticorpos Monoclonais Humanizados , Antígeno CTLA-4/antagonistas & inibidores , Linhagem Celular Tumoral , Células HEK293 , Humanos , Imunoglobulina G/química , Imunoterapia , Concentração Inibidora 50 , Ipilimumab/farmacologia , Leucócitos Mononucleares/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Transplante de Neoplasias , Plasmídeos/metabolismo , Linfócitos T/metabolismoRESUMO
Type 1 diabetes mellitus is caused by the T cell mediated autoimmune destruction of insulin-producing beta cells of the islets of Langerhans within the pancreas. Current immunotherapy strategies are aimed at directly inactivating the autoreactive T cells and/or inducing T cells with regulatory capabilities. At the preclinical level, several strategies that employ TCR antagonists -- including monoclonal antibodies, autoantigen-specific peptides and soluble TCR ligands -- are showing promise and being developed for clinical application. Several of these approaches employing monoclonal antibodies against the TCR-CD3 complex or soluble peptide antigens are producing favorable results in the clinic.
Assuntos
Anticorpos Monoclonais/uso terapêutico , Diabetes Mellitus Tipo 1/terapia , Imunoterapia , Animais , Complexo CD3/imunologia , Humanos , Transplante das Ilhotas Pancreáticas , Complexo Receptor-CD3 de Antígeno de Linfócitos T/imunologia , Receptores de Antígenos de Linfócitos T/metabolismoRESUMO
BACKGROUND: CSF-1 or IL-34 stimulation of CSF1R promotes macrophage differentiation, activation and osteoclastogenesis, and pharmacological inhibition of CSF1R is beneficial in animal models of arthritis. The objective of this study was to determine the relative contributions of CSF-1 and IL-34 signaling to CSF1R in RA. METHODS: CSF-1 and IL-34 were detected by immunohistochemical and digital image analysis in synovial tissue from 15 biological-naïve rheumatoid arthritis (RA) , 15 psoriatic arthritis (PsA) and 7 osteoarthritis (OA) patients . Gene expression in CSF-1- and IL-34-differentiated human macrophages was assessed by FACS analysis and quantitative PCR. RA synovial explants were incubated with CSF-1, IL-34, control antibody (Ab), or neutralizing/blocking Abs targeting CSF-1, IL-34, or CSF1R. The effect of a CSF1R-blocking Ab was examined in murine collagen-induced arthritis (CIA). RESULTS: CSF-1 (also known as M-CSF) and IL-34 expression was similar in RA and PsA synovial tissue, but lower in controls (P < 0.05). CSF-1 expression was observed in the synovial sublining, and IL-34 in the sublining and the intimal lining layer. CSF-1 and IL-34 differentially regulated the expression of 17 of 336 inflammation-associated genes in macrophages, including chemokines, extra-cellular matrix components, and matrix metalloproteinases. Exogenous CSF-1 or IL-34, or their independent neutralization, had no effect on RA synovial explant IL-6 production. Anti-CSF1R Ab significantly reduced IL-6 and other inflammatory mediator production in RA synovial explants, and paw swelling and joint destruction in CIA. CONCLUSIONS: Simultaneous inhibition of CSF1R interactions with both CSF-1 and IL-34 suppresses inflammatory activation of RA synovial tissue and pathology in CIA, suggesting a novel therapeutic strategy for RA.
Assuntos
Artrite Reumatoide/imunologia , Artrite Reumatoide/patologia , Fator Estimulador de Colônias de Macrófagos/antagonistas & inibidores , Animais , Artrite Experimental/imunologia , Artrite Experimental/metabolismo , Artrite Experimental/patologia , Artrite Reumatoide/metabolismo , Benzo(a)Antracenos , Feminino , Citometria de Fluxo , Humanos , Processamento de Imagem Assistida por Computador , Imuno-Histoquímica , Inflamação/imunologia , Inflamação/metabolismo , Inflamação/patologia , Interleucinas/biossíntese , Fator Estimulador de Colônias de Macrófagos/imunologia , Macrófagos/imunologia , Macrófagos/metabolismo , Masculino , Camundongos , Camundongos SCID , Reação em Cadeia da Polimerase em Tempo Real , Receptor de Fator Estimulador de Colônias de Macrófagos/imunologia , Receptor de Fator Estimulador de Colônias de Macrófagos/metabolismoRESUMO
A second ligand for colony-stimulating factor-1 receptor (CSF-1R) with distinct biologic activities had long been implicated but not appreciated until the recent discovery of interleukin (IL)-34. IL-34 and CSF-1 signal through this common receptor to mediate the biology of mononuclear phagocytic cells. Aberrant macrophage activation by CSF-1 and/or IL-34 is associated with numerous diseases, and clinical therapies targeting this pathway are being tested. Although IL-34 and CSF-1 have distinct activities under physiologic conditions, they appear functionally redundant in various disease states. Thus, blocking the activity of both might be necessary for maximal efficacy.
Assuntos
Anti-Inflamatórios/farmacologia , Anti-Inflamatórios/uso terapêutico , Inflamação/tratamento farmacológico , Inflamação/metabolismo , Interleucinas/metabolismo , Animais , Humanos , Receptor de Fator Estimulador de Colônias de Macrófagos/metabolismo , Transdução de Sinais/efeitos dos fármacosRESUMO
CD4(+)CD25(+)Foxp3(+) regulatory T cells (Treg) are critical for the regulation of tolerance and have shown enormous potential in suppressing pathological immune responses in autoimmune disease, transplantation, and graft-versus-host disease (GVHD). Recent data indicate that suppression of organ-specific autoimmunity is critically dependent on the antigen specificity of the Treg. An emerging model of Treg action is that organ-specific Treg acquire suppressive activity through activation by dendritic cells expressing organ-derived antigens. Thus, efficacy of Treg-based therapy should be increased by using organ-specific Treg rather than polyclonal Treg. This necessitates the ability to identify relevant antigens and to expand rare antigen-specific Treg from diverse polyclonal populations. Here, we consider the importance of antigen specificity in Treg function and discuss recent advances for the expansion of antigen-specific Treg and the therapeutic potential of Treg in controlling autoimmunity and GVHD.
Assuntos
Proliferação de Células , Epitopos de Linfócito T/imunologia , Imunoterapia Adotiva , Linfócitos T Reguladores/imunologia , Linfócitos T Reguladores/transplante , Animais , Epitopos de Linfócito T/metabolismo , Humanos , Imunoterapia Adotiva/métodos , Linfócitos T Reguladores/metabolismoRESUMO
CD4+CD25+Foxp3+ regulatory T cells (T(reg)) are critical for controlling autoimmunity. Evidence suggests that T(reg) development, peripheral maintenance, and suppressive function are dependent on Ag specificity. However, there is little direct evidence that the T(reg) responsible for controlling autoimmunity in NOD mice or other natural settings are Ag specific. In fact, some investigators have argued that polyclonal Ag-nonspecific T(reg) are efficient regulators of immunity. Thus, the goal of this study was to identify, expand, and characterize islet Ag-specific T(reg) in NOD mice. Ag-specific T(reg) from NOD mice were efficiently expanded in vitro using IL-2 and beads coated with recombinant islet peptide mimic-MHC class II and anti-CD28 mAb. The expanded Ag-specific T(reg) expressed prototypic surface markers and cytokines. Although activated in an Ag-specific fashion, the expanded T(reg) were capable of bystander suppression both in vitro and in vivo. Importantly, the islet peptide mimic-specific T(reg) were more efficient than polyclonal T(reg) in suppressing autoimmune diabetes. These results provide a direct demonstration of the presence of autoantigen-specific T(reg) in the natural setting that can be applied as therapeutics for organ-specific autoimmunity.
Assuntos
Autoantígenos/imunologia , Diabetes Mellitus Tipo 1/imunologia , Receptores de Interleucina-2/fisiologia , Linfócitos T Reguladores/fisiologia , Animais , Antígenos CD28/fisiologia , Citocinas/biossíntese , Diabetes Mellitus Tipo 1/prevenção & controle , Tolerância Imunológica , Camundongos , Camundongos Endogâmicos NOD , Receptores de Antígenos de Linfócitos T alfa-beta/análise , Receptores de Interleucina-2/análiseRESUMO
Self peptide-MHC ligands create and maintain the mature T cell repertoire by positive selection in the thymus and by homeostatic proliferation in the periphery. A low affinity/avidity interaction among T cells, self peptides, and MHC molecules has been suggested for these events, but it remains unknown whether or how this self-interaction is involved in tolerance and/or autoimmunity. Several lines of evidence implicate the glutamic acid decarboxylase 65 (GAD-65) peptide, p524-543, as a specific, possibly low affinity, stimulus for the spontaneously arising, diabetogenic T cell clone BDC2.5. Interestingly, BDC2.5 T cells, which normally are unresponsive to p524-543 stimulation, react to the peptide when provided with splenic APC obtained from mice immunized with the same peptide, p524-543, but not, for example, with hen egg white lysozyme. Immunization with p524-543 increases the susceptibility of the NOD mice to type 1 diabetes induced by the adoptive transfer of BDC2.5 T cells. In addition, very few CFSE-labeled BDC2.5 T cells divide in the recipient's pancreas after transfer into a transgenic mouse that overexpresses GAD-65 in B cells, whereas they divide vigorously in the pancreas of normal NOD recipients. A special relationship between the BDC2.5 clone and the GAD-65 molecule is further demonstrated by generation of a double-transgenic mouse line carrying both the BDC2.5 TCR and GAD-65 transgenes, in which a significant reduction of BDC2.5 cells in the pancreas has been observed, presumably due to tolerance induction. These data suggest that unique and/or altered processing of self Ags may play an essential role in the development and expansion of autoreactive T cells.
Assuntos
Diabetes Mellitus Tipo 1/imunologia , Glutamato Descarboxilase/imunologia , Camundongos Endogâmicos NOD/imunologia , Pâncreas/imunologia , Fragmentos de Peptídeos/imunologia , Linfócitos T/imunologia , Transferência Adotiva , Animais , Autoimunidade , Proliferação de Células , Quimiotaxia de Leucócito/imunologia , Células Clonais/imunologia , Tolerância Imunológica , Camundongos , Camundongos TransgênicosRESUMO
Many autoimmune diseases have been linked to the class II region of the major histocompatibility complex (MHC). The linkage is thought to be a result of autoreactive T cells that recognize self-peptides bound to a product of this locus. For example, T cells from non-obese diabetic mice recognize specific 'diabetogenic' peptides bound to a class II MHC allele called I-A(g7). The I-A(g7) molecule is noted for being unstable and difficult to work with, especially in soluble form. In this work, yeast surface display combined with fluorescence-activated cell sorting was used as a means of directed evolution to engineer stabilized variants of a single-chain form of I-A(g7). A library containing mutations at two residues (positions 56 and 57 of the I-A(g7) beta-chain) that are important in the class II disease associations yielded stabilized mutants with preferences for a glutamic acid at residue 56 and a leucine at residue 57. Random mutation of I-A(g7) followed by selection with an anti-I-A(g7) antibody also yielded stabilized variants with mutations in other residues. The methods described here allow the discovery of novel MHC complexes that could facilitate structural studies and provide new opportunities in the development of diagnostics or antagonists of class II MHC-associated diseases.
Assuntos
Evolução Molecular Direcionada , Antígenos de Histocompatibilidade Classe II/genética , Saccharomyces cerevisiae/genética , Sequência de Aminoácidos , Animais , Anticorpos Monoclonais/química , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/metabolismo , Especificidade de Anticorpos , Linhagem Celular , Estabilidade de Medicamentos , Citometria de Fluxo , Antígenos de Histocompatibilidade Classe II/química , Antígenos de Histocompatibilidade Classe II/imunologia , Antígenos de Histocompatibilidade Classe II/metabolismo , Camundongos , Modelos Moleculares , Dados de Sequência Molecular , Biblioteca de Peptídeos , Proteínas Proto-Oncogênicas c-myc/imunologia , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Proteínas Recombinantes/metabolismo , Saccharomyces cerevisiae/imunologia , Saccharomyces cerevisiae/metabolismo , Análise de Sequência de Proteína , TransfecçãoRESUMO
NOD mice spontaneously develop insulitis and type 1 diabetes (T1D) mellitus similar to humans. Insulitis without overt disease occurs in the BDC2.5 TCR-transgenic NOD mice that express the rearranged TCR alpha- and beta-chain genes of a diabetogenic T cell clone reactive to an unknown beta cell autoantigen. A previous study identified an extensive panel of peptides that are highly active in stimulating T cells from transgenic BDC2.5 mice in culture. However, none of these peptides cause active disease in NOD and BDC2.5 animals or in NOD recipients of adoptively transferred BDC2.5 T cells following direct immunization in vivo. We show that direct immunization of transgenic BDC2.5 mice causes many BDC2.5 T cells to become activated and apoptotic. Strikingly, soluble peptides administered to recipients of activated, highly pathogenic BDC2.5 T cells results in protection from disease. These results suggest that high affinity peptide analogues of autoimmune epitopes might be useful as therapeutic modulators in active autoimmune disease.
Assuntos
Diabetes Mellitus Tipo 1/prevenção & controle , Peptídeos/farmacologia , Linfócitos T/efeitos dos fármacos , Vacinas/farmacologia , Animais , Apoptose/fisiologia , Glicemia/metabolismo , Diabetes Mellitus Tipo 1/imunologia , Modelos Animais de Doenças , Tolerância Imunológica/efeitos dos fármacos , Tolerância Imunológica/imunologia , Ilhotas Pancreáticas/efeitos dos fármacos , Ilhotas Pancreáticas/imunologia , Camundongos , Camundongos Endogâmicos NOD , Linfócitos T/imunologia , Linfócitos T/fisiologiaRESUMO
Type 1 diabetes is an autoimmune disorder caused by autoreactive T cells that mediate destruction of insulin-producing beta cells of the pancreas. Studies have shown that T cell tolerance can be restored by inducing a partial or altered signal through the TCR. To investigate the potential of bivalent peptide-MHC class II/Ig fusion proteins as therapeutics to restore Ag-specific tolerance, we have developed soluble peptide I-A(g7) dimers for use in the nonobese diabetic mouse model of diabetes. I-A(g7) dimers with a linked peptide specific for islet-reactive BDC2.5 TCR transgenic CD4(+) T cells were shown to specifically bind BDC2.5 T cells as well as a small population of Ag-specific T cells in nonobese diabetic mice. In vivo treatment with BDC2.5 peptide I-A(g7) dimers protected mice from diabetes mediated by the adoptive transfer of diabetogenic BDC2.5 CD4(+) T cells. The dimer therapy resulted in the activation and increased cell death of transferred BDC2.5 CD4(+) T cells. Surviving cells were hypoproliferative to challenge by Ag and produced increased levels of IL-10 and decreased levels of IFN-gamma compared with cells from control I-A(g7) dimer-treated mice. Anti-IL-10R therapy reversed the tolerogenic effects of the dimer. Thus, peptide I-A(g7) dimers induce tolerance of BDC2.5 TCR T cells through a combination of the induction of clonal anergy and anti-inflammatory cytokines.