RESUMO
Programmed cell death-1 (PD-1) is one of the most famous coinhibitory receptors that are expressed on effector T cells to regulate their function. The PD-1 ligands, PD-L1 and PD-L2, are expressed by various cells throughout the body at steady state and their expression was further regulated within different pathological conditions such as tumor-bearing and chronic inflammatory diseases. In recent years, immune checkpoint inhibitor (ICI) therapies with anti-PD-1 or anti-PD-L1 has become a standard treatment for various malignancies and has shown remarkable antitumor effects. Since the discovery of PD-1 in 1992, a huge number of studies have been conducted to elucidate the function of PD-1. Herein, this paper provides an overview of PD-1 biological findings and sheds some light on the current technology for molecular imaging of PD-1.
Assuntos
Neoplasias , Receptor de Morte Celular Programada 1 , Humanos , Receptor de Morte Celular Programada 1/metabolismo , Neoplasias/metabolismo , Linfócitos T/metabolismo , Antígeno B7-H1/metabolismo , Imunoterapia/métodos , Imagem MolecularRESUMO
It is well-established that CD28 co-stimulation is required for the development and the proliferation of thymus-derived regulatory T cells (tTregs). Meanwhile, the role of CD28 co-stimulation in the homeostasis of peripherally derived Tregs (pTregs) remains unclear. To clarify this issue, we analyzed Tregs in small and large intestines (SI and LI), the principle sites of pTreg development. Interestingly, and different from in the thymus, Tregs were abundant in the intestines of CD28-/- mice, and most of them were phenotypically pTregs. We showed that CD28-/- naive T cells differentiated into pTregs in the LI after oral exposure to antigens and that CD28-/- pTregs in the LI had the same highly proliferative activity as CD28+/- cells. CD28-/- pTregs acquired these Treg-specific features at transcriptional and epigenetics levels. On the other hand, some immune suppressive molecules were down-regulated in CD28-/- pTregs. Correspondingly, the suppressive activity of CD28-/- pTregs was weaker than CD28+/+ cells. These results indicate that the homeostasis of pTregs in the intestines is maintained even in the absence of CD28, whereas CD28 is required for the maximal suppressive activity of intestinal pTregs.
Assuntos
Antígenos CD28/imunologia , Homeostase , Mucosa Intestinal/imunologia , Mucosa Intestinal/metabolismo , Linfócitos Intraepiteliais/imunologia , Linfócitos T Reguladores/imunologia , Linfócitos T Reguladores/metabolismo , Animais , Antígenos CD28/genética , Antígenos CD28/metabolismo , Diferenciação Celular/imunologia , Proliferação de Células , Metilação de DNA , Imunomodulação , Linfócitos Intraepiteliais/metabolismo , Camundongos , Camundongos Knockout , Camundongos TransgênicosRESUMO
Foxp3 is the master transcriptional regulator of regulatory T cells (Tregs), and the stabilization of Foxp3 expression is regulated by the demethylation of conserved non-coding sequence 2 (CNS2) in the Foxp3 locus. Recent studies have shown that TCR stimulation is required for the demethylation of Foxp3 CNS2 during Treg development. However, the relationship between the strength of TCR stimulation and the demethylation of Foxp3 CNS2 remains unclear. To address this issue, we compared the frequency of demethylation of the Foxp3 CNS2 among in vitro-induced Tregs (iTreg) that had received a range of TCR stimulation during their development. We found that the frequency of demethylation of the Foxp3 CNS2 was increased with increased TCR stimulation strength, whereas CD28 stimulation had only a limited effect. Mechanistically, the binding of Tet2, a member of the TET family of enzymes involved in DNA demethylation, on the Foxp3 CNS2 was increased by strong TCR stimulation. Furthermore, compared with iTreg induced by weak TCR stimulation, iTreg induced by strong TCR stimulation maintained Foxp3 expression both in vitro and in vivo. These data indicate that the strength of TCR stimulation is a key factor for induction of the demethylation of Foxp3 CNS2 and the generation of stable Tregs.
Assuntos
Desmetilação do DNA , Fatores de Transcrição Forkhead/metabolismo , Receptores de Antígenos de Linfócitos T/fisiologia , Linfócitos T Reguladores/metabolismo , Animais , Sequência Conservada , Proteínas de Ligação a DNA/metabolismo , Dioxigenases , Fatores de Transcrição Forkhead/genética , Camundongos , Ligação Proteica , Proteínas Proto-Oncogênicas/metabolismo , Transdução de SinaisRESUMO
It has been reported that splenic immune responses play pivotal roles in the development of allergic diseases; however, the precise role of the spleen remains unclear. Herein, we demonstrated a novel role of the spleen in the pathogenesis of food allergy (FA). We found that mast cells (MCs) developed from progenitor cells present in spleen during an antigen-specific T-cell response in vitro. In a Th2 response-mediated FA model, significant expansion of MCs was also observed in spleen. The incidence of allergic diarrhea was profoundly reduced in splenectomized mice, whereas adoptive transfer of in vitro-induced splenic MCs into these mice restored allergic symptoms, suggesting that the splenic MCs functioned as the pathogenic cells in the development of FA. The in vitro-generated MCs required not only IL-3 but also IFN-γ, and treatment of FA-induced mice with anti-IFN-γ antibody suppressed expansion of MCs in spleen as well as diarrhea development, highlighting that IFN-γ in the spleen orchestrated the development of FA, which was followed by a Th2 response in the local lesion. Overall, we propose that the role of the spleen in the development of FA is to provide a unique site where antigen-specific T cells induce development of pathogenic MCs.
Assuntos
Hipersensibilidade Alimentar/imunologia , Mastócitos/imunologia , Baço/imunologia , Células Th2/imunologia , Animais , Feminino , Hipersensibilidade Alimentar/patologia , Interferon gama/imunologia , Interleucina-3/imunologia , Mastócitos/patologia , Camundongos , Camundongos Endogâmicos BALB C , Baço/patologia , Células Th2/patologiaRESUMO
Suppression of T-cell growth is an important mechanism for establishment of self-tolerance and prevention of unwanted prolonged immune responses that may cause tissue damage. Although negative selection of potentially self-reactive T cells in the thymus as well as in peripheral tissues has been extensively investigated and well documented, regulatory mechanisms to dampen proliferation of antigen-specific effector T cells in response to antigen stimulation remain largely unknown. Thus, in this work, we focus on the identification of growth suppression mechanisms of antigen-specific effector T cells. In order to address this issue, we investigated the cellular and molecular events in growth suppression of an ovalbumin (OVA)-specific T-cell clone after stimulation with a wide range of OVA-peptide concentrations. We observed that while an optimal dose of peptide leads to cell cycle progression and proliferation, higher doses of peptide reduced cell growth, a phenomenon that was previously termed high-dose suppression. Our analysis of this phenomenon indicated that high-dose suppression is a consequence of cell cycle arrest, but not Fas-Fas ligand-dependent apoptosis or T-cell anergy, and that this growth arrest occurs in S phase, accompanied by reduced expression of CDK2 and cyclin A. Importantly, inhibition of MEK/ERK activation eliminated this growth suppression and cell cycle arrest, while it reduced the proliferative response to optimal antigenic stimulation. These results suggest that cell cycle arrest is the major mechanism regulating antigen-specific effector T-cell expansion, and that the MEK/ERK signaling pathway has both positive and negative effects, depending on the strength of antigenic stimulation.
Assuntos
Antígenos/imunologia , Pontos de Checagem do Ciclo Celular/imunologia , Sistema de Sinalização das MAP Quinases/imunologia , Linfócitos T/imunologia , Animais , Camundongos , Camundongos Endogâmicos BALB CRESUMO
In patients receiving allogeneic hematopoietic cell transplantation, chronic graft-versus-host disease (cGVHD) remains a frequent complication and resembles autoimmune diseases such as systemic lupus erythematosus and systemic sclerosis. Our previous work demonstrated the critical role of CD28 costimulation of donor T cells for GVHD induction. In this study, we investigate the role of CD28 costimulation of host T cells in cGVHD. CD28-intact mice as hosts showed systemic lupus erythematosus-type cGVHD, whereas CD28-deficient mice developed a distinct phenotype of cGVHD, with fibrotic damage in skin and internal organs, resembling systemic sclerosis. This phenotype was due to a lack of signaling through the C-terminal proline-rich motif within host CD28's cytoplasmic tail, a motif previously shown to be required for development of regulatory T cells (Tregs) and function of conventional T cells. Adoptive transfer experiments demonstrated that a defect in host CD4(+)CD25(+) Tregs, but not in conventional T cells, was responsible for disease phenotype. Host Treg deficiency altered the cytokine pattern of donor CD4(+) T cells and the Ag specificity of autoantibodies, and these might lead to phenotypic change. Thus, host CD28 signaling controlled the pathogenesis of cGVHD through effects on host Tregs, whose status impacts qualitatively on the allogeneic immune responses.
Assuntos
Antígenos CD28/deficiência , Doença Enxerto-Hospedeiro/imunologia , Linfócitos T Reguladores/imunologia , Animais , Antígenos CD28/genética , Antígenos CD28/imunologia , Doença Crônica , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Linfócitos T Reguladores/metabolismo , Transplante HomólogoRESUMO
Costimulatory molecules of the CD28 family on T lymphocytes integrate cues from innate immune system sensors and modulate activation responses in conventional CD4(+) T cells (Tconv) and their FoxP3(+) regulatory counterparts (Treg). To better understand how costimulatory and coinhibitory signals might be integrated, we profiled the changes in gene expression elicited in the hours and days after engagement of Treg and Tconv by anti-CD3 and either anti-CD28, -CTLA4, -ICOS, -PD1, -BLA, or -CD80. In Tconv, a shared "main response" was induced by CD28, ICOS, and, surprisingly, BTLA and CD80, with very limited CD28-specific (primarily Il2) or ICOS-specific elements (including Th1 and Th2 but not the follicular T signature). CTLA4 and PD1 had a very subtle impact in this system, similarly inhibiting the response to anti-CD3. Treg responded to the same costimulatory hierarchy and to the same extent as Tconv, but inducing different clusters of genes. In this reductionist system, costimulatory or coinhibitory engagement mainly elicits generic responses, suggesting that the variability of their effects in vivo result from temporal or anatomical differences in their engagement, rather than from inherently different wiring.
Assuntos
Linfócitos T CD4-Positivos/imunologia , Linfócitos T Reguladores/imunologia , Animais , Antígeno B7-1/imunologia , Antígenos CD28/imunologia , Complexo CD3/imunologia , Antígeno CTLA-4/imunologia , Fatores de Transcrição Forkhead/genética , Fatores de Transcrição Forkhead/imunologia , Perfilação da Expressão Gênica , Imunidade Inata/genética , Proteína Coestimuladora de Linfócitos T Induzíveis/imunologia , Ativação Linfocitária/genética , Camundongos , Camundongos Transgênicos , Receptor de Morte Celular Programada 1/imunologia , Receptores Imunológicos/imunologiaRESUMO
Much of the knowledge about cell differentiation and function in the immune system has come from studies in mice, but the relevance to human immunology, diseases, and therapy has been challenged, perhaps more from anecdotal than comprehensive evidence. To this end, we compare two large compendia of transcriptional profiles of human and mouse immune cell types. Global transcription profiles are conserved between corresponding cell lineages. The expression patterns of most orthologous genes are conserved, particularly for lineage-specific genes. However, several hundred genes show clearly divergent expression across the examined cell lineages, and among them, 169 genes did so even with highly stringent criteria. Finally, regulatory mechanisms--reflected by regulators' differential expression or enriched cis-elements--are conserved between the species but to a lower degree, suggesting that distinct regulation may underlie some of the conserved transcriptional responses.
Assuntos
Perfilação da Expressão Gênica , Sistema Imunitário/metabolismo , Transcrição Gênica , Animais , Humanos , Ativação Linfocitária , Camundongos , Linfócitos T/imunologiaRESUMO
Blockade of immune checkpoint receptors has shown outstanding efficacy for tumor immunotherapy. Promising treatment with anti-lymphocyte-activation gene-3 (LAG-3) has already been recognized as the next efficacious treatment, but there is still limited understanding of the mechanism of LAG-3-mediated immune suppression. Here, utilizing high-resolution molecular imaging, we find a mechanism of CD4 T cell suppression via LAG-3, in which LAG-3-bound major histocompatibility complex (MHC) class II molecules on antigen-presenting cells (APCs) gather at the central region of an immunological synapse and are trans-endocytosed by T cell receptor-driven internalization motility toward CD4 and CD8 T cells expressing LAG-3. Downregulation of MHC class II molecules on APCs thus results in the attenuation of their antigen-presentation function and impairment of CD4 T cell activation. From these data, anti-LAG-3 treatment is suggested to have potency to directly block the inhibitory signaling via LAG-3 and simultaneously reduce MHC class II expression on APCs by LAG-3-mediated trans-endocytosis for recovery from T cell exhaustion.
RESUMO
OBJECTIVE: To clarify the role of T-bet in the pathogenesis of collagen-induced arthritis (CIA). METHODS: T-bet-transgenic (Tg) mice under the control of the CD2 promoter were generated. CIA was induced in T-bet-Tg mice and wild-type C57BL/6 (B6) mice. Levels of type II collagen (CII)-reactive T-bet and retinoic acid receptor-related orphan nuclear receptor γt (RORγt) messenger RNA expression were analyzed by real-time polymerase chain reaction. Criss-cross experiments using CD4+ T cells from B6 and T-bet-Tg mice, as well as CD11c+ splenic dendritic cells (DCs) from B6 and T-bet-Tg mice with CII were performed, and interleukin-17 (IL-17) and interferon-γ (IFNγ) in the supernatants were measured by enzyme-linked immunosorbent assay. CD4+ T cells from B6, T-bet-Tg, or T-bet-Tg/IFNγ-/- mice were cultured for Th17 cell differentiation, then the proportions of cells producing IFNγ and IL-17 were analyzed by fluorescence-activated cell sorting. RESULTS: Unlike the B6 mice, the T-bet-Tg mice did not develop CIA. T-bet-Tg mice showed overexpression of Tbx21 and down-regulation of Rorc in CII-reactive T cells. Criss-cross experiments with CD4+ T cells and splenic DCs showed a significant reduction in IL-17 production by CII-reactive CD4+ T cells in T-bet-Tg mice, even upon coculture with DCs from B6 mice, indicating dysfunction of IL-17-producing CD4+ T cells. Inhibition of Th17 cell differentiation under an in vitro condition favoring Th17 cell differentiation was observed in both T-bet-Tg mice and T-bet-Tg/IFNγ-/- mice. CONCLUSION: Overexpression of T-bet in T cells suppressed the development of autoimmune arthritis. The regulatory mechanism of arthritis might involve dysfunction of CII-reactive Th17 cell differentiation by overexpression of T-bet via IFNγ-independent pathways.
Assuntos
Artrite Experimental/genética , Artrite Reumatoide/genética , Expressão Gênica , Proteínas com Domínio T/genética , Animais , Artrite Experimental/imunologia , Artrite Experimental/patologia , Artrite Reumatoide/imunologia , Artrite Reumatoide/patologia , Autoimunidade/genética , Autoimunidade/imunologia , Linfócitos T CD4-Positivos/metabolismo , Células Cultivadas , Técnicas de Cocultura , Células Dendríticas/metabolismo , Feminino , Interferon gama/metabolismo , Interleucina-17/metabolismo , Linfonodos/imunologia , Linfonodos/patologia , Linfócitos/imunologia , Linfócitos/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Membro 3 do Grupo F da Subfamília 1 de Receptores Nucleares/genética , Membro 3 do Grupo F da Subfamília 1 de Receptores Nucleares/metabolismo , Joelho de Quadrúpedes/patologia , Membrana Sinovial/patologia , Proteínas com Domínio T/metabolismoRESUMO
With recent advances in immune checkpoint inhibitors (ICIs), immunotherapy has become the standard treatment for various malignant tumors. Their indications and dosages have been determined empirically, taking individually conducted clinical trials into consideration, but without a standard method to evaluate them. Here we establish an advanced imaging system to visualize human PD-1 microclusters, in which a minimal T cell receptor (TCR) signaling unit co-localizes with the inhibitory co-receptor PD-1 in vitro. In these microclusters PD-1 dephosphorylates both the TCR/CD3 complex and its downstream signaling molecules via the recruitment of a phosphatase, SHP2, upon stimulation with the ligand hPD-L1. In this system, blocking antibodies for hPD-1-hPD-L1 binding inhibits hPD-1 microcluster formation, and each therapeutic antibody (pembrolizumab, nivolumab, durvalumab and atezolizumab) is characterized by a proprietary optimal concentration and combinatorial efficiency enhancement. We propose that our imaging system could digitally evaluate PD-1-mediated T cell suppression to evaluate their clinical usefulness and to develop the most suitable combinations among ICIs or between ICIs and conventional cancer treatments.
Assuntos
Neoplasias , Receptor de Morte Celular Programada 1 , Humanos , Imagem Individual de Molécula , Nivolumabe/farmacologia , Nivolumabe/uso terapêutico , Neoplasias/diagnóstico por imagem , Neoplasias/tratamento farmacológico , Receptores de Antígenos de Linfócitos T , Antígeno B7-H1/metabolismo , Imunoterapia/métodosRESUMO
The coinhibitory receptor, PD-1, is of major importance for the suppression of T cell activation in various types of immune responses. A high-resolution imaging study showed that PD-1 forms a coinhibitory signalosome, "PD-1 microcluster", with the phosphatase, SHP2, to dephosphorylate the TCR/CD3 complex and its downstream signaling molecules. Such a consecutive reaction entirely depended on PD-1-PD-L1/2 binding. PD-L2 is expressed on professional antigen-presenting cells and also on some tumor cells, which possibly explains the discrepant efficacy of immune checkpoint therapy for PD-L1-negative tumors. Here, we performed precise imaging analysis of PD-L2 forming PD-1-PD-L2 clusters associating with SHP2. PD-L2 could compete with PD-L1 for binding to PD-1, occupying the same space at TCR microclusters. The PD-1 microcluster formation was inhibited by certain mAbs with functional consequences. Thus, PD-1 microcluster formation provides a visible index for the effectiveness of anti-PD-1- or anti-PD-L1/2-mediated T cell suppression. PD-L2 may exert immune suppressive responses cooperatively with PD-L1 on the microcluster scale.
Assuntos
Linfócitos T CD8-Positivos/imunologia , Células Dendríticas/imunologia , Ativação Linfocitária/imunologia , Proteína 2 Ligante de Morte Celular Programada 1/metabolismo , Receptor de Morte Celular Programada 1/fisiologia , Proteína Tirosina Fosfatase não Receptora Tipo 11/metabolismo , Animais , Linfócitos T CD8-Positivos/metabolismo , Células Cultivadas , Células Dendríticas/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteína 2 Ligante de Morte Celular Programada 1/genética , Proteína Tirosina Fosfatase não Receptora Tipo 11/genética , Transdução de SinaisRESUMO
The aim of this study was to clarify the role of the immune response to muscarinic type 3 receptor (M3R) in the pathogenesis of Sjögren's syndrome (SS). M3R(-/-) mice were immunized with murine M3R peptides and their splenocytes were inoculated into Rag1(-/-) (M3R(-/-)âRag1(-/-)) mice. M3R(-/-)âRag1(-/-) mice had high serum levels of anti-M3R antibodies and low saliva volume. Histological examination showed marked infiltration of mononuclear cells in the salivary glands and immunohistochemistry demonstrated that the majority of these cells were CD4(+) T cells with a few B cells and several IFN-γ- and IL-17-producing cells. Apoptotic cells were present in the salivary glands of M3R(-/-)âRag1(-/-) mice. Moreover, transfer of only CD3(+) T cells from M3R(-/-) immunized with M3R peptides into Rag1(-/-) mice resulted in cell infiltration and destruction of epithelial cells in the salivary glands, suggesting that M3R reactive CD3(+) T cells play a pathogenic role in the development of autoimmune sialoadenitis. Our findings support the notion that the immune response to M3R plays a crucial role in the pathogenesis of SS-like autoimmune sialoadenitis.
Assuntos
Linfócitos T CD4-Positivos/metabolismo , Fragmentos de Peptídeos/metabolismo , Receptor Muscarínico M3/metabolismo , Sialadenite/imunologia , Síndrome de Sjogren/imunologia , Transferência Adotiva , Animais , Apoptose , Autoanticorpos/sangue , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/patologia , Células Cultivadas , Modelos Animais de Doenças , Células Epiteliais/imunologia , Células Epiteliais/patologia , Humanos , Imunização , Interferon gama/metabolismo , Interleucina-17/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/imunologia , Receptor Muscarínico M3/genética , Receptor Muscarínico M3/imunologia , Glândulas Salivares/patologia , Sialadenite/sangueRESUMO
It is thought that CD28 plays a crucial role in the maintenance of regulatory T cell (Treg) pool size through promoting the development and proliferation of these cells. However, recently we found that the dependency on CD28 co-stimulation for their development is different between Treg subsets, thymus-derived Tregs (tTregs, CD28-dependent) and peripherally-derived Tregs (pTregs, CD28-independent), suggesting that CD28 may also have differential influences on the homeostasis of each Treg subset. Here, we demonstrated that both Treg subsets were reduced in secondary lymphoid organs of CD28 deficient mice, and that this reduction was due to impaired proliferation in both Treg subsets by the intrinsic CD28 defect. However, we found that the massive proliferation of both Treg subsets under lymphopenic condition was regulated by CD28, whereas the proliferative activity of tTregs but not pTregs in the steady state was dependent on CD28. Also, experiments using mutant CD28 knock-in mice revealed that proliferation of pTregs under lymphopenic condition required only the Lck-NFκB pathway of CD28, whereas tTregs required an additional unknown pathway. These findings indicate that the dependency on CD28 for proliferation in each Treg subset differs depending on the environment.
Assuntos
Antígenos CD28/metabolismo , Linfócitos T Reguladores/citologia , Linfócitos T Reguladores/metabolismo , Animais , Apoptose , Proliferação de Células , Subpopulações de Linfócitos/metabolismo , Camundongos Endogâmicos C57BL , Transdução de Sinais , Baço/citologia , Timo/citologiaRESUMO
The objective of this study was to examine the clinical and immunological factors influencing the efficacy of cevimeline hydrochloride hydrate (cevimeline) for the treatment of xerostomia in patients with Sjögren's syndrome (SS). Thirty primary SS patients who were medicated with cevimeline were enrolled in this study. Whole stimulated sialometry (WSS) was compared between pre- and posttreatment points (4 weeks after oral cevimeline administration) and the increment rate of WSS was calculated. Multiple regression was employed to examine the relative contributions of the clinical and immunological factors, including age, pretreatment WSS, duration of disease, sialography, minor salivary gland biopsy, anti-Ro/SS-A antibodies, anti-La/SS-B antibodies, and antibodies to muscarinic type 3 receptors to the posttreatment WSS. Patients with normal sialography findings, negative minor salivary gland biopsy, and absence of anti-La/SS-B antibodies had significantly higher increment rates of WSS compared with those with positive findings (p=0.042, 0.002, and 0.018, respectively). Results of the multiple regression analysis showed that sialography (coefficient=-0.867, p=0.004) and minor salivary gland biopsy (coefficient=-0.869, p=0.003) had significant associations with the posttreatment WSS. Our preliminary results demonstrated the relationship between the effect of cevimeline on saliva secretion and the degree of salivary gland destruction evaluated by sialography and histopathological findings in the labial minor salivary glands. These diagnostic approaches could provide useful prognostic information on the efficacy of cevimeline in SS patients.
Assuntos
Parassimpatomiméticos/uso terapêutico , Quinuclidinas/uso terapêutico , Receptores Muscarínicos/efeitos dos fármacos , Salivação/efeitos dos fármacos , Síndrome de Sjogren/tratamento farmacológico , Tiofenos/uso terapêutico , Xerostomia/tratamento farmacológico , Adulto , Idoso , Feminino , Humanos , Pessoa de Meia-Idade , Glândula Parótida/efeitos dos fármacos , Glândula Parótida/patologia , Prognóstico , Sialografia , Síndrome de Sjogren/complicações , Resultado do Tratamento , Xerostomia/etiologiaRESUMO
Anti-glucose-6-phosphate isomerase (GPI) antibodies (Abs) solely induce arthritis in mice. High titers of anti-GPI Abs are found in some patients with rheumatoid arthritis (RA), but their pathogenic role remains elusive. The aim of this study was to evaluate the pathogenic role of anti-GPI Abs in cynomolgus monkeys. IgG fractions were separated from sera of anti-GPI Abs-positive RA patients and healthy subjects and directly injected into the metacarpophalangeal joints of 4 cynomolgus monkeys. At day 16, the joints were harvested and examined histologically and immunohistochemically. The expression of C5a receptor (C5aR) molecule in the synovium was quantified by real-time PCR using cDNA from monkey joints. In monkey joints, IgG including anti-GPI Abs resulted in recruitment of granulocytes and mononuclear cells, strong deposition of human IgG on the articular surface, and overexpression of C5aR, but no joint swelling. No infiltrated cells or IgG deposition were observed in monkeys injected with IgGs from healthy subjects. Our results suggest that IgG fraction from RA patients including anti-GPI Abs may play a crucial role in the generation of synovitis in monkeys, although the pathogenesis of anti-GPI Abs in RA patients is still uncertain.
Assuntos
Artrite Reumatoide/imunologia , Autoanticorpos/administração & dosagem , Glucose-6-Fosfato Isomerase/imunologia , Imunoglobulina G/administração & dosagem , Sinovite/imunologia , Animais , Complexo Antígeno-Anticorpo/metabolismo , Complexo Antígeno-Anticorpo/fisiologia , Artrite Reumatoide/enzimologia , Autoanticorpos/metabolismo , Cartilagem Articular/imunologia , Cartilagem Articular/metabolismo , Cartilagem Articular/patologia , Humanos , Imunoglobulina G/metabolismo , Macaca fascicularis , Receptor da Anafilatoxina C5a/biossíntese , Receptor da Anafilatoxina C5a/genética , Líquido Sinovial/imunologia , Líquido Sinovial/metabolismo , Sinovite/enzimologia , Sinovite/metabolismoRESUMO
Kit is a receptor-type tyrosine kinase found on the plasma membrane. It can transform mast cells through activating mutations. Here, we show that a mutant Kit from neoplastic mast cells from mice, Kit(D814Y), is permanently active and allows cells to proliferate autonomously. It does so by activating two signalling pathways from different intracellular compartments. Mutant Kit from the cell surface accumulates on endolysosomes through clathrin-mediated endocytosis, which requires Kit's kinase activity. Kit(D814Y) is constitutively associated with phosphatidylinositol 3-kinase, but the complex activates Akt only on the cytoplasmic surface of endolysosomes. It resists destruction because it is under-ubiquitinated. Kit(D814Y) also appears in the endoplasmic reticulum soon after biosynthesis, and there, can activate STAT5 aberrantly. These mechanisms of oncogenic signalling are also seen in rat and human mast cell leukemia cells. Thus, oncogenic Kit signalling occurs from different intracellular compartments, and the mutation acts by altering Kit trafficking as well as activation.
RESUMO
AIM: It is reported that in salivary glands of Sjögren's syndrome (SS), interferon gamma (IFNγ) and IFNγ-inducible genes containing signal transducers and activators of transcription 1 (STAT1) are upregulated and play a crucial role in the pathogenesis of SS. The aim of this study is to clarify which phosphorylation of STAT1, serine727 (Ser(727)) or tyrosine701 (Tyr(701)) of STAT1, is important for IFNγ signaling and IFNγ-induced apoptosis in salivary gland cells. METHODS: We established STAT1 Tyr(701) variant (tyrosine to phenylalanine; Y701F) and STAT1 Ser(727) variant (serine to alanine; S727A), which were transfected into human salivary gland (HSG) cells. HSG cells transfected with these mutant-STAT1 were analyzed on the expression of IFNγ-inducible genes and apoptosis after stimulation with IFNγ. RESULTS: In Y701F mutant-STAT1 transfected HSG cells (Ser(727)-dominant HSG cells), IFNγ-inducible genes such as IP10, IRF1, and Fas expression were increased after stimulation with IFNγ. In Ser(727)-dominant HSG cells, the induction of apoptosis after stimulation with IFNγ was also increased compared with S727A mutant-STAT1 transfected HSG cells (Tyr(701)-dominant HSG cells). CONCLUSION: Phosphorylation of Ser(727) in STAT1 might be more important in IFNγ signaling and the induction of apoptosis in HSG cells than phosphorylation of Tyr(701). Accordingly, we propose that phosphorylation of Ser(727) in STAT1 could be a potentially suitable new therapeutic target for SS patients to prevent the destruction of salivary glands.
Assuntos
Apoptose/efeitos dos fármacos , Interferon gama/farmacologia , Fator de Transcrição STAT1/genética , Glândulas Salivares/efeitos dos fármacos , Células Cultivadas , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Mutação , Fosforilação , Fator de Transcrição STAT1/metabolismo , Glândulas Salivares/metabolismo , Serina/metabolismo , Transdução de Sinais , Síndrome de Sjogren/genética , Síndrome de Sjogren/metabolismo , Transfecção , Tirosina/metabolismoRESUMO
Sjögren's syndrome (SS) is a chronic autoimmune disease characterized by lymphocytic infiltration of salivary glands, in which CD4(+) T cells are predominant. These infiltrating T cells play a crucial role in the generation of SS. Previous studies showed that autoantibodies and auto-reactive T cells against M3 muscarinic acethylcholine receptor (M3R) were detected in patients with SS. In this study, to reveal the pathological mechanisms underlying immune response against M3R, we tried to induce SS like sialoadenitis. M3R knockout (M3R(-/-)) mice were immunized with murine M3R peptides. Their splenocytes were isolated and transferred into Rag1 knockout (Rag1(-/-)) mice. Mononuclear cells infiltration was detected in salivary glands of Rag1(-/-) mice inoculated splenocytes of M3R(-/-) mice immunized with M3R peptides. Moreover we transferred CD3(+) cells from splenocytes of M3R(-/-) mice immunized with M3R peptides into Rag1(-/-) mice. In their salivary glands, mononuclear infiltration was also detected. These findings suggest that the immune response to M3R plays a crucial role in the generation of SS like sialoadenitis.
Assuntos
Autoanticorpos , Receptor Muscarínico M3/imunologia , Síndrome de Sjogren/imunologia , Linfócitos T/imunologia , Animais , Modelos Animais de Doenças , Humanos , Camundongos , Camundongos Knockout , Glândulas Salivares/imunologiaRESUMO
We reported that peripheral blood mononuclear cells from HLA-DRB1*0101 Japanese patients with rheumatoid arthritis (RA) were highly reactive to 256-271 peptide of type II collagen (CII). Similar to RA, T cells reactive to CII (AA256-271) play a crucial role in the generation of arthritis in CII-induced arthritis mouse (I-A(q)). In the present study, we regulated the CII reactivity of T cells from CIA mouse with I-A(q) by altered peptide ligand (APL). Eight different APLs were designed and screened for their antagonistic activity using CII reactive cytokine production assay. Four APLs of CII 256-271 exhibited antagonistic activity in CII-reactive T cells. Moreover, intraperitoneally injected APL-5 (G262A) significantly suppressed CII-induced arthritis in mice, whereas the other three APLs did not. Compared with the control, APL-5 suppressed interleukin (IL)-17 production by T cells from CII-induced arthritis mice. These results suggest that CII APL is a potentially suitable therapeutic strategy for the control of RA.