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1.
Arthritis Rheumatol ; 71(3): 431-440, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30277008

RESUMO

OBJECTIVE: Adoptive Treg cell therapy has great potential to treat autoimmune disease. Currently, very little is known about how these cells impact inflamed tissues. This study was undertaken to elucidate how autologous Treg cell therapy influences tissue inflammation in human autoimmune disease. METHODS: We describe a systemic lupus erythematosus (SLE) patient with active skin disease who received adoptive Treg therapy. We comprehensively quantified Treg cells and immune activation in peripheral blood and skin, with data obtained at multiple time points posttreatment. RESULTS: Deuterium tracking of infused Treg cells revealed the transient presence of cells in peripheral blood, accompanied by increased percentages of highly activated Treg cells in diseased skin. Flow cytometric analysis and whole transcriptome RNA sequencing revealed that Treg cell accumulation in skin was associated with a marked attenuation of the interferon-γ pathway and a reciprocal augmentation of the interleukin-17 (IL-17) pathway. This phenomenon was more pronounced in skin relative to peripheral blood. To validate these findings, we investigated Treg cell adoptive transfer of skin inflammation in a murine model and found that it also resulted in a pronounced skewing away from Th1 immunity and toward IL-17 production. CONCLUSION: We report the first case of a patient with SLE treated with autologous adoptive Treg cell therapy. Taken together, our results suggest that this treatment leads to increased activated Treg cells in inflamed skin, with a dynamic shift from Th1 to Th17 responses.


Assuntos
Terapia Baseada em Transplante de Células e Tecidos/métodos , Imunoterapia Adotiva/métodos , Lúpus Eritematoso Sistêmico/terapia , Feminino , Humanos , Lúpus Eritematoso Sistêmico/imunologia , Pessoa de Meia-Idade , Linfócitos T Reguladores/imunologia
2.
Nature ; 559(7714): 405-409, 2018 07.
Artigo em Inglês | MEDLINE | ID: mdl-29995861

RESUMO

Decades of work have aimed to genetically reprogram T cells for therapeutic purposes1,2 using recombinant viral vectors, which do not target transgenes to specific genomic sites3,4. The need for viral vectors has slowed down research and clinical use as their manufacturing and testing is lengthy and expensive. Genome editing brought the promise of specific and efficient insertion of large transgenes into target cells using homology-directed repair5,6. Here we developed a CRISPR-Cas9 genome-targeting system that does not require viral vectors, allowing rapid and efficient insertion of large DNA sequences (greater than one kilobase) at specific sites in the genomes of primary human T cells, while preserving cell viability and function. This permits individual or multiplexed modification of endogenous genes. First, we applied this strategy to correct a pathogenic IL2RA mutation in cells from patients with monogenic autoimmune disease, and demonstrate improved signalling function. Second, we replaced the endogenous T cell receptor (TCR) locus with a new TCR that redirected T cells to a cancer antigen. The resulting TCR-engineered T cells specifically recognized tumour antigens and mounted productive anti-tumour cell responses in vitro and in vivo. Together, these studies provide preclinical evidence that non-viral genome targeting can enable rapid and flexible experimental manipulation and therapeutic engineering of primary human immune cells.


Assuntos
Reprogramação Celular/genética , Edição de Genes , Genoma Humano/genética , Linfócitos T/imunologia , Linfócitos T/metabolismo , Animais , Autoimunidade/genética , Sistemas CRISPR-Cas/genética , Células Cultivadas , Humanos , Subunidade alfa de Receptor de Interleucina-2/genética , Masculino , Camundongos , Transplante de Neoplasias , Engenharia de Proteínas , Receptores de Antígenos de Linfócitos T/genética , Linfócitos T/citologia
3.
Mol Ther Methods Clin Dev ; 4: 178-191, 2017 Mar 17.
Artigo em Inglês | MEDLINE | ID: mdl-28345003

RESUMO

Umbilical cord blood is a traditional and convenient source of cells for hematopoietic stem cell transplantation. Thymic regulatory T cells (Tregs) are also present in cord blood, and there is growing interest in the use of autologous Tregs to provide a low-risk, fully human leukocyte antigen (HLA)-matched cell product for treating autoimmune diseases, such as type 1 diabetes. Here, we describe a good manufacturing practice (GMP)-compatible Treg expansion protocol using fluorescence-activated cell sorting, resulting in a mean 2,092-fold expansion of Tregs over a 16-day culture for a median yield of 1.26 × 109 Tregs from single-donor cryopreserved units. The resulting Tregs passed prior clinical trial release criteria for Treg purity and sterility, including additional rigorous assessments of FOXP3 and Helios expression and epigenetic analysis of the FOXP3 Treg-specific demethylated region (TSDR). Compared with expanded adult peripheral blood Tregs, expanded cord blood Tregs remained more naive, as assessed by continued expression of CD45RA, produced reduced IFN-γ following activation, and effectively inhibited responder T cell proliferation. Immunosequencing of the T cell receptor revealed a remarkably diverse receptor repertoire within cord blood Tregs that was maintained following in vitro expansion. These data support the feasibility of generating GMP-compliant Tregs from cord blood for adoptive cell transfer therapies and highlight potential advantages in terms of safety, phenotypic stability, autoantigen specificity, and tissue distribution.

4.
Front Immunol ; 8: 1844, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-29379498

RESUMO

Cellular therapies with CD4+ T regulatory cells (Tregs) hold promise of efficacious treatment for the variety of autoimmune and allergic diseases as well as posttransplant complications. Nevertheless, current manufacturing of Tregs as a cellular medicinal product varies between different laboratories, which in turn hampers precise comparisons of the results between the studies performed. While the number of clinical trials testing Tregs is already substantial, it seems to be crucial to provide some standardized characteristics of Treg products in order to minimize the problem. We have previously developed reporting guidelines called minimum information about tolerogenic antigen-presenting cells, which allows the comparison between different preparations of tolerance-inducing antigen-presenting cells. Having this experience, here we describe another minimum information about Tregs (MITREG). It is important to note that MITREG does not dictate how investigators should generate or characterize Tregs, but it does require investigators to report their Treg data in a consistent and transparent manner. We hope this will, therefore, be a useful tool facilitating standardized reporting on the manufacturing of Tregs, either for research purposes or for clinical application. This way MITREG might also be an important step toward more standardized and reproducible testing of the Tregs preparations in clinical applications.

5.
Sci Transl Med ; 7(315): 315ra189, 2015 Nov 25.
Artigo em Inglês | MEDLINE | ID: mdl-26606968

RESUMO

Type 1 diabetes (T1D) is an autoimmune disease that occurs in genetically susceptible individuals. Regulatory T cells (Tregs) have been shown to be defective in the autoimmune disease setting. Thus, efforts to repair or replace Tregs in T1D may reverse autoimmunity and protect the remaining insulin-producing ß cells. On the basis of this premise, a robust technique has been developed to isolate and expand Tregs from patients with T1D. The expanded Tregs retained their T cell receptor diversity and demonstrated enhanced functional activity. We report on a phase 1 trial to assess safety of Treg adoptive immunotherapy in T1D. Fourteen adult subjects with T1D, in four dosing cohorts, received ex vivo-expanded autologous CD4(+)CD127(lo/-)CD25(+) polyclonal Tregs (0.05 × 10(8) to 26 × 10(8) cells). A subset of the adoptively transferred Tregs was long-lived, with up to 25% of the peak level remaining in the circulation at 1 year after transfer. Immune studies showed transient increases in Tregs in recipients and retained a broad Treg FOXP3(+)CD4(+)CD25(hi)CD127(lo) phenotype long-term. There were no infusion reactions or cell therapy-related high-grade adverse events. C-peptide levels persisted out to 2+ years after transfer in several individuals. These results support the development of a phase 2 trial to test efficacy of the Treg therapy.


Assuntos
Diabetes Mellitus Tipo 1/terapia , Imunoterapia , Linfócitos T Reguladores/imunologia , Adulto , Feminino , Humanos , Masculino , Adulto Jovem
6.
J Clin Invest ; 124(3): 1187-98, 2014 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-24569373

RESUMO

The high-affinity IgE receptor FcεRI is constitutively expressed in mast cells and basophils and is required for transmitting stimulatory signals upon engagement of IgE-bound allergens. FcεRI is also constitutively expressed in dendritic cells (DCs) and monocytes in humans; however, the specific functions of the FcεRI expressed by these cells are not completely understood. Here, we found that FcεRI expressed by human blood DC antigen 1-positive (BDCA1+) DCs and monocytes, but not basophils, traffics to endolysosomal compartments under steady-state conditions. Furthermore, IgE bound to FcεRI on BDCA1+ DCs was rapidly endocytosed, transported to the lysosomes, and degraded in vitro. IgE injected into mice expressing human FcεRIα (FCER1A-Tg mice) was endocytosed by conventional DCs and monocytes, and endocytosis was associated with rapid clearance of circulating IgE from these mice. Importantly, this rapid IgE clearance was dependent on monocytes or DCs but not basophils. These findings strongly suggest that constitutive internalization of human FcεRI by DCs and monocytes distinctively contributes to serum IgE clearance.


Assuntos
Imunoglobulina E/sangue , Receptores de IgE/metabolismo , Animais , Basófilos/metabolismo , Células Cultivadas , Células Dendríticas/metabolismo , Endocitose , Endossomos/metabolismo , Humanos , Lisossomos/metabolismo , Camundongos , Camundongos Transgênicos , Monócitos/metabolismo , Transporte Proteico , Proteólise
7.
J Immunol ; 186(7): 3918-26, 2011 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-21368230

RESUMO

Regulatory T cells (Tregs) constitute an attractive therapeutic target given their essential role in controlling autoimmunity. However, recent animal studies provide evidence for functional heterogeneity and lineage plasticity within the Treg compartment. To understand better the plasticity of human Tregs in the context of type 1 diabetes, we characterized an IFN-γ-competent subset of human CD4(+)CD127(lo/-)CD25(+) Tregs. We measured the frequency of Tregs in the peripheral blood of patients with type 1 diabetes by epigenetic analysis of the Treg-specific demethylated region (TSDR) and the frequency of the IFN-γ(+) subset by flow cytometry. Purified IFN-γ(+) Tregs were assessed for suppressive function, degree of TSDR demethylation, and expression of Treg lineage markers FOXP3 and Helios. The frequency of Tregs in peripheral blood was comparable but the FOXP3(+)IFN-γ(+) fraction was significantly increased in patients with type 1 diabetes compared to healthy controls. Purified IFN-γ(+) Tregs expressed FOXP3 and possessed suppressive activity but lacked Helios expression and were predominately methylated at the TSDR, characteristics of an adaptive Treg. Naive Tregs were capable of upregulating expression of Th1-associated T-bet, CXCR3, and IFN-γ in response to IL-12. Notably, naive, thymic-derived natural Tregs also demonstrated the capacity for Th1 differentiation without concomitant loss of Helios expression or TSDR demethylation.


Assuntos
Proliferação de Células , Diabetes Mellitus Tipo 1/imunologia , Diabetes Mellitus Tipo 1/patologia , Interferon gama/biossíntese , Linfócitos T Reguladores/citologia , Linfócitos T Reguladores/imunologia , Adolescente , Adulto , Células Cultivadas , Criança , Metilação de DNA , Diabetes Mellitus Tipo 1/terapia , Feminino , Fatores de Transcrição Forkhead/biossíntese , Fatores de Transcrição Forkhead/genética , Fatores de Transcrição Forkhead/metabolismo , Humanos , Mediadores da Inflamação/metabolismo , Mediadores da Inflamação/fisiologia , Interferon gama/fisiologia , Ativação Linfocitária/imunologia , Masculino , Pessoa de Meia-Idade , Subpopulações de Linfócitos T/citologia , Subpopulações de Linfócitos T/imunologia , Subpopulações de Linfócitos T/patologia , Linfócitos T Reguladores/patologia , Adulto Jovem
8.
PLoS One ; 5(7): e11726, 2010 Jul 22.
Artigo em Inglês | MEDLINE | ID: mdl-20668510

RESUMO

BACKGROUND: Therapies directed at augmenting regulatory T cell (Treg) activities in vivo as a systemic treatment for autoimmune disorders and transplantation may be associated with significant off-target effects, including a generalized immunosuppression that may compromise beneficial immune responses to infections and cancer cells. Adoptive cellular therapies using purified expanded Tregs represents an attractive alternative to systemic treatments, with results from animal studies noting increased therapeutic potency of antigen-specific Tregs over polyclonal populations. However, current methodologies are limited in terms of the capacity to isolate and expand a sufficient quantity of endogenous antigen-specific Tregs for therapeutic intervention. Moreover, FOXP3+ Tregs fall largely within the CD4+ T cell subset and are thus routinely MHC class II-specific, whereas class I-specific Tregs may function optimally in vivo by facilitating direct tissue recognition. METHODOLOGY/PRINCIPAL FINDINGS: To overcome these limitations, we have developed a novel means for generating large numbers of antigen-specific Tregs involving lentiviral T cell receptor (TCR) gene transfer into in vitro expanded polyclonal natural Treg populations. Tregs redirected with a high-avidity class I-specific TCR were capable of recognizing the melanoma antigen tyrosinase in the context of HLA-A*0201 and could be further enriched during the expansion process by antigen-specific reactivation with peptide loaded artificial antigen presenting cells. These in vitro expanded Tregs continued to express FOXP3 and functional TCRs, and maintained the capacity to suppress conventional T cell responses directed against tyrosinase, as well as bystander T cell responses. Using this methodology in a model tumor system, murine Tregs designed to express the tyrosinase TCR effectively blocked antigen-specific effector T cell (Teff) activity as determined by tumor cell growth and luciferase reporter-based imaging. CONCLUSIONS/SIGNIFICANCE: These results support the feasibility of class I-restricted TCR transfer as a promising strategy to redirect the functional properties of Tregs and provide for a more efficacious adoptive cell therapy.


Assuntos
Receptores de Antígenos de Linfócitos T/imunologia , Linfócitos T Reguladores/imunologia , Adulto , Animais , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/metabolismo , Células Cultivadas , Feminino , Citometria de Fluxo , Vetores Genéticos/genética , Humanos , Lentivirus/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Receptores de Antígenos de Linfócitos T/genética , Adulto Jovem
9.
Diabetes ; 58(3): 652-62, 2009 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-19074986

RESUMO

OBJECTIVE: Regulatory T-cells (Tregs) have catalyzed the field of immune regulation. However, translating Treg-based therapies from animal models of autoimmunity to human clinical trials requires robust methods for the isolation and expansion of these cells-a need forming the basis for these studies. RESEARCH DESIGN AND METHODS: Tregs from recent-onset type 1 diabetic patients and healthy control subjects were isolated by fluorescence-activated cell sorting and compared for their capacity to expand in vitro in response to anti-CD3-anti-CD28-coated microbeads and IL-2. Expanded cells were examined for suppressive function, lineage markers and FOXP3, and cytokine production. RESULTS: Both CD4+CD127(lo/-) and CD4+CD127(lo/-)CD25+ T-cells could be expanded and used as Tregs. However, expansion of CD4+CD127(lo/-) cells required the addition of rapamycin to maintain lineage purity. In contrast, expansion of CD4+CD127(lo/-)CD25+ T-cells, especially the CD45RA+ subset, resulted in high yield, functional Tregs that maintained higher FOXP3 expression in the absence of rapamycin. Tregs from type 1 diabetic patients and control subjects expanded similarly and were equally capable of suppressing T-cell proliferation. Regulatory cytokines were produced by Tregs after culture; however, a portion of FOXP3+ cells were capable of producing interferon (IFN)-gamma after reactivation. IFN-gamma production was observed from both CD45RO+ and CD45RA+ Treg populations. CONCLUSIONS: The results support the feasibility of isolating Tregs for in vitro expansion. Based on expansion capacity, FOXP3 stability, and functional properties, the CD4+CD127(lo/-)CD25+ T-cells represent a viable cell population for cellular therapy in this autoimmune disease.


Assuntos
Diabetes Mellitus Tipo 1/imunologia , Linfócitos T Reguladores/imunologia , Adulto , Idade de Início , Antígenos CD/imunologia , Linfócitos T CD4-Positivos/imunologia , Divisão Celular , Proliferação de Células , Citocinas/metabolismo , Feminino , Citometria de Fluxo , Fatores de Transcrição Forkhead/análise , Humanos , Imunossupressão , Ionomicina/farmacologia , Masculino , Fenótipo , Valores de Referência , Linfócitos T Reguladores/citologia , Linfócitos T Reguladores/efeitos dos fármacos , Acetato de Tetradecanoilforbol/farmacologia , Adulto Jovem
10.
Immunol Rev ; 223: 371-90, 2008 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-18613848

RESUMO

SUMMARY: The importance of regulatory T lymphocytes (Tregs) in the control of autoimmunity is now well established in a variety of experimental animal models. In addition, there are numerous studies suggesting that Treg deficits may be an underlying cause of human autoimmune diseases. The emergence of Tregs as an essential component of immune homeostasis provides a potential therapeutic opportunity for active immune regulation and long-term tolerance induction. In this article, we summarize the core basic science and animal model studies of Tregs, review the status of multiple biologic and small molecule chemical compounds to promote Treg development in vivo, and discuss recent advances for the identification and expansion of polyclonal and antigen-specific Tregs for adoptive immunotherapy. In summary, the review provides an in-depth analysis and highlights the challenges and opportunities for immune intervention with Treg-based therapeutics.


Assuntos
Doenças Autoimunes/imunologia , Imunoterapia Adotiva , Linfócitos T Reguladores/imunologia , Animais , Anticorpos Bloqueadores/imunologia , Anticorpos Bloqueadores/uso terapêutico , Apresentação do Antígeno , Antígenos de Superfície/imunologia , Doenças Autoimunes/terapia , Células Dendríticas/imunologia , Fatores de Transcrição Forkhead/imunologia , Doença Enxerto-Hospedeiro/prevenção & controle , Humanos , Tolerância Imunológica , Subunidade alfa de Receptor de Interleucina-2/imunologia , Camundongos , Especificidade do Receptor de Antígeno de Linfócitos T
11.
J Exp Med ; 203(7): 1701-11, 2006 Jul 10.
Artigo em Inglês | MEDLINE | ID: mdl-16818678

RESUMO

Regulatory T (T reg) cells are critical regulators of immune tolerance. Most T reg cells are defined based on expression of CD4, CD25, and the transcription factor, FoxP3. However, these markers have proven problematic for uniquely defining this specialized T cell subset in humans. We found that the IL-7 receptor (CD127) is down-regulated on a subset of CD4(+) T cells in peripheral blood. We demonstrate that the majority of these cells are FoxP3(+), including those that express low levels or no CD25. A combination of CD4, CD25, and CD127 resulted in a highly purified population of T reg cells accounting for significantly more cells that previously identified based on other cell surface markers. These cells were highly suppressive in functional suppressor assays. In fact, cells separated based solely on CD4 and CD127 expression were anergic and, although representing at least three times the number of cells (including both CD25(+)CD4(+) and CD25(-)CD4(+) T cell subsets), were as suppressive as the "classic" CD4(+)CD25(hi) T reg cell subset. Finally, we show that CD127 can be used to quantitate T reg cell subsets in individuals with type 1 diabetes supporting the use of CD127 as a biomarker for human T reg cells.


Assuntos
Fatores de Transcrição Forkhead/biossíntese , Receptores de Interleucina-7/biossíntese , Linfócitos T Reguladores/imunologia , Adolescente , Adulto , Biomarcadores , Contagem de Linfócito CD4 , Células Cultivadas , Diabetes Mellitus Tipo 1/metabolismo , Fatores de Transcrição Forkhead/genética , Humanos , Memória Imunológica , Pessoa de Meia-Idade , Receptores de Interleucina-2/biossíntese , Receptores de Interleucina-2/genética , Receptores de Interleucina-7/genética , Linfócitos T Reguladores/metabolismo
12.
J Pharmacol Exp Ther ; 313(3): 1263-71, 2005 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-15761110

RESUMO

The CXC chemokine receptor 3 (CXCR3) is predominantly expressed on T helper type 1 (Th1) cells that are involved in inflammatory diseases. The three CXCR3 ligands CXCL9, CXCL10, and CXCL11 are produced at sites of inflammation and elicit migration of pathological Th1 cells. Here, we are the first to characterize the pharmacological potencies and specificity of a CXCR3 antagonist, N-1R-[3-(4-ethoxy-phenyl)-4-oxo-3,4-dihydro-pyrido[2,3-d]pyrimidin-2-yl]-ethyl-N-pyridin-3-ylmethyl-2-(4-fluoro-3-trifluoromethyl-phenyl)-acetamide (NBI-74330), from the T487 small molecule series. NBI-74330 demonstrated potent inhibition of [(125)I]CXCL10 and [(125)I]CXCL11 specific binding (K(i) of 1.5 and 3.2 nM, respectively) and of functional responses mediated by CXCR3, such as ligand-induced guanosine 5'-O-(3-[(35)S]thio)triphosphate ([(35)S]GTPgammaS) binding, calcium mobilization, and cellular chemotaxis (IC(50) of 7 to 18 nM). NBI-74330 was selective for CXCR3 because it showed no significant inhibition of chemotactic responses to other chemokines and did not inhibit radioligand binding to a panel of nonchemokine G-protein coupled receptors. There was a striking difference in potencies among the three CXCR3 ligands, with CXCL11 >> CXCL10 > CXCL9. A comparison of the rank order of K(i) values with the rank order of monocyte production levels of these three ligands revealed a precise inverse correlation, suggesting that the weaker receptor affinities of CXCL9 and CXCL10 were physiologically compensated for by an elevated expression, perhaps to maintain effectiveness of each ligand under physiological conditions.


Assuntos
Acetamidas/farmacologia , Quimiocinas CXC/metabolismo , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Pirimidinas/farmacologia , Receptores de Quimiocinas/antagonistas & inibidores , Receptores de Quimiocinas/metabolismo , Acetamidas/metabolismo , Linhagem Celular Tumoral , Quimiocina CXCL10 , Quimiocina CXCL11 , Quimiocina CXCL9 , Guanosina 5'-O-(3-Tiotrifosfato)/metabolismo , Humanos , Ligantes , Pirimidinas/metabolismo , Receptores CXCR3
13.
J Autoimmun ; 24(1): 55-62, 2005 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-15725577

RESUMO

In mouse models, CD4+CD25+ T cells are involved in maintenance of peripheral tolerance. In humans, a subset of CD4+CD25+ T cells expressing high levels of CD25 (CD4+CD25high) with characteristics identical to murine CD4+CD25+ was recently described. We evaluated the characteristics of CD4+CD25high T cells in peripheral blood of type 1 diabetic subjects (T1D) and normal controls (NC). In contrast to a previous report, we found no difference in the number of CD4+CD25high and CD4+CD25+ T cells between T1D and NC. We confirmed previous studies that demonstrated that human CD4+CD25high cells can suppress the proliferation of co-cultured CD4+CD25- cells stimulated in conditions of sub-maximal cross-linking by anti-CD3 either with or without anti-CD28. However, we did not observe statistical differences between the normal controls and the chronic diabetic subjects we tested. Culturing of these cell populations did not appear to affect their ability to suppress proliferation in both groups. In conclusion, we found no significant differences in number or in vitro regulatory function of CD4+CD25high in chronic human T1D subjects.


Assuntos
Antígenos CD4/metabolismo , Linfócitos T CD4-Positivos/imunologia , Diabetes Mellitus Tipo 1/imunologia , Receptores de Interleucina-2/metabolismo , Adulto , Antígenos CD4/imunologia , Linfócitos T CD4-Positivos/citologia , Linfócitos T CD4-Positivos/metabolismo , Proliferação de Células , Células Cultivadas , Citocinas/metabolismo , Diabetes Mellitus Tipo 1/metabolismo , Diabetes Mellitus Tipo 1/patologia , Humanos , Pessoa de Meia-Idade , Receptores de Interleucina-2/imunologia
14.
J Clin Immunol ; 24(4): 327-39, 2004 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-15163889

RESUMO

Human type 1 diabetes is thought to be mediated by autoreactive T cells specific for antigens expressed by pancreatic beta cells. However, it is unclear which autoantigens and determinants thereof are the targets of the autoimmune attack. Using comprehensive peptide libraries that cover the entire sequence of two major candidate autoantigens, GAD65 and proinsulin, we measured the in vivo frequencies of peptide-specific, IFN-gamma-producing memory T cells in 27 diabetic patients, 14 high risk individuals, and 15 partially HLA-matched healthy controls. Compared to the controls, both a higher number of determinants on the islet cell antigens were recognized and the frequencies of peptide specific cells were increased in patients and high risk individuals. Inclusion of signal enhancing anti-CD28 antibody further accentuated this difference. Considerable heterogeneity in peptide recognition was seen even in DRB1*04, DQB1*0302 matched individuals. Unlike its peptides, the GAD protein antigen did not recall a T cell memory response. The highly heterogeneous recognition of a multitude of peptide determinants on both autoantigens, occurring in the absence of protein recognition, and the low functional avidity of the memory cells involved jointly suggest that the autoimmune T cell repertoire in human type 1 diabetes primarily targets cryptic determinants engaged by determinant spreading.


Assuntos
Diabetes Mellitus Tipo 1/imunologia , Glutamato Descarboxilase/imunologia , Isoenzimas/imunologia , Proinsulina/imunologia , Linfócitos T/imunologia , Variação Antigênica , Autoantígenos/imunologia , Estudos de Casos e Controles , Epitopos/imunologia , Teste de Histocompatibilidade , Humanos , Memória Imunológica , Contagem de Linfócitos , Fragmentos de Peptídeos/imunologia , Subpopulações de Linfócitos T/citologia , Subpopulações de Linfócitos T/imunologia
15.
J Immunol ; 172(6): 3955-62, 2004 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-15004204

RESUMO

Susceptibility to type 1A autoimmune diabetes is linked to expression of particular MHC class II molecules, notably HLA-DQ8 in man and the orthologous I-Ag7 in the nonobese diabetic mouse. In the present study, we analyzed two peptide epitopes (peptides 2 and 7) from the diabetes autoantigen phogrin (IA-2beta), in the context of their presentation by the I-Ag7 and HLA-DQ8 molecules and their role as potential T cell antigenic epitopes in human diabetes. Both of these peptides are targets of diabetogenic CD4+ T cell clones in the nonobese diabetic mouse. Transgenic mice expressing HLA-DQ8 as the sole class II molecule generated a robust T cell-proliferative response when primed with peptide 2 or peptide 7 in CFA. Analysis of the IL-2 secretion from peptide 2-reactive T cell hybridomas stimulated with alanine-substituted peptides identified three residues that were crucial to the response. Among 41 islet cell Ag-positive prediabetic human subjects, 36.5% showed PBMC-proliferative responses to peptide 7, 17.1% to peptide 2, and 17.1% to both peptides; no response was seen among 20 matched healthy controls. Stratification of the data based upon HLA haplotype suggested that peptide 7 could be presented by at least one HLA-DR molecule in addition to HLA-DQ8, a finding that was supported by blocking studies with monomorphic mAbs. The results indicate that common phogrin peptides are targeted by autoreactive T cells in human and murine type 1A diabetes, and that the responses may in part be associated with the similar peptide-binding specificities of I-Ag7 and HLA-DQ8.


Assuntos
Autoantígenos/imunologia , Diabetes Mellitus Tipo 1/imunologia , Antígenos HLA-DQ/imunologia , Proteínas de Membrana/imunologia , Estado Pré-Diabético/imunologia , Proteínas Tirosina Fosfatases/imunologia , Adolescente , Adulto , Animais , Autoanticorpos/biossíntese , Autoantígenos/administração & dosagem , Divisão Celular/genética , Divisão Celular/imunologia , Linhagem Celular , Criança , Pré-Escolar , Células Clonais , Diabetes Mellitus Tipo 1/genética , Epitopos de Linfócito T/administração & dosagem , Epitopos de Linfócito T/imunologia , Humanos , Hibridomas , Interleucina-2/farmacologia , Linfonodos/citologia , Linfonodos/imunologia , Proteínas de Membrana/administração & dosagem , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos CBA , Camundongos Endogâmicos NOD , Camundongos Transgênicos , Pessoa de Meia-Idade , Fragmentos de Peptídeos/administração & dosagem , Fragmentos de Peptídeos/imunologia , Estado Pré-Diabético/genética , Proteína Tirosina Fosfatase não Receptora Tipo 1 , Proteínas Tirosina Fosfatases/administração & dosagem , Proteínas Tirosina Fosfatases Classe 8 Semelhantes a Receptores , Subpopulações de Linfócitos T/citologia , Subpopulações de Linfócitos T/imunologia , Subpopulações de Linfócitos T/metabolismo
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