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1.
Medicine (Baltimore) ; 98(43): e17608, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31651870

RESUMO

This study aims to investigate the changes of cytokines and the effect of programmed death ligand 1 (PD-L1) signaling pathway on T cell function in patients with immune thrombocytopenic purpura (ITP).Totally, 40 untreated ITP patients were recruited and 30 healthy people were recruited as the healthy control. Then whole blood of ITP patients and healthy control was collected, respectively. The sPD-L1/anti-PD-1 was used to activate or block the programmed death (PD-1)/PD-L1 signaling pathway. The expression of PD-1 and PD-L1 on peripheral blood mononuclear cells (PBMCs) were detected by flow cytometry. PBMCs were treated with cluster of differentiation (CD3), cluster of differentiation 28 (CD28), and phytohaemagglutinin (PHA) for 48 hours. Serum levels of sPD-1, sPD-L1, and cytokines were measured by enzyme-linked immunosorbent assay (ELISA).Compared with the healthy control group, the percentages of PD-1+CD3+CD4+ T cells and PD-L1+HLA-DR+CD11c+ DC cells were increased in ITP patients. The levels of interferon-gamma (IFN-γ), interleukin-17 (IL-17), and sPD-1 in the serum of ITP patients were increased, while IL-4 and transforming growth factor-ß (TGF-ß) were decreased. Additionally, the level of sPD-1 was negatively correlated with the platelet count. Consistently, after treatment with CD3, CD28, and PHA, IFN-γ and IL-17 levels in culture supernatant of PBMCs from ITP patients were significantly higher than those from healthy controls whereas IL-4 and TGF-ß levels were significantly lower. Furthermore, IFN-γ and IL-17 levels secreted by PBMCs from ITP patients decreased after sPD-L1 administration, however, IL-4 and TGF-ß levels were increased. The level of IFN-γ in ITP group remained higher after anti-PD-1 blockage, but the levels of IL-4, TGF-ß, and IL-17 were not significantly influenced.sPD-1 may cause the dysfunction of PD-1/PD-L1 signaling pathway, and its level is related to the severity of ITP patients. Activation of PD-1/PD-L1 with sPD-L1 may restore the imbalance of Th1/Th2 and Treg/Th17 cell subtypes in ITP patients but anti-PD-1 may exacerbate disease by enhancing IFN-γ production.


Assuntos
Antígeno B7-H1/imunologia , Púrpura Trombocitopênica Idiopática/imunologia , Linfócitos T Reguladores/imunologia , Equilíbrio Th1-Th2/fisiologia , Células Th17/imunologia , Adulto , Antígenos CD28/imunologia , Complexo CD3/imunologia , Estudos de Casos e Controles , Citocinas/imunologia , Feminino , Humanos , Interferon gama/metabolismo , Leucócitos Mononucleares , Masculino , Pessoa de Meia-Idade , Fito-Hemaglutininas/imunologia , Receptor de Morte Celular Programada 1/fisiologia , Púrpura Trombocitopênica Idiopática/sangue , Transdução de Sinais/imunologia , Linfócitos T/imunologia , Equilíbrio Th1-Th2/efeitos dos fármacos , Fator de Crescimento Transformador beta/metabolismo
2.
Adv Exp Med Biol ; 1172: 63-78, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31628651

RESUMO

The co-stimulation and co-inhibition signal pathways, immune checkpoints, are among the central mechanisms to regulate the T-cell immunity. Optimal signals involve intricate interactions of numerous ligands and receptors. Manipulation of these signals offers great clinical opportunities and has revolutionized the cancer treatment therapies. The 2018 Nobel Prize in Physiology or Medicine was awarded to James P. Allison and Tasuku Honjo in recognition of their discovery of cancer immunotherapy by inhibition of immune checkpoint molecules. Despite the landmark discovery in cancer immunotherapy, the efforts to harness immunity against cancer are also restricted by the limited knowledge on the co-stimulation and co-inhibition signaling networks. Understanding the structures of these molecules, in particular, tackling the interaction paradigms from the structural perspective, help to provide more accurate insights into the signaling mechanisms, which may further facilitate the development of novel biologics and improve the efficacy of the existing biologics against these targets. Here we review our current understanding on the structures of these co-stimulatory and co-inhibitory molecules. Specifically, we focus on the structural basis of several checkpoint molecules among the CD28-B7 family and discuss the therapeutic drugs against these targets for the treatment of human cancers, autoimmune disorders, and transplantation.


Assuntos
Antígenos CD28 , Linfócitos T , Doenças Autoimunes , Antígenos CD28/química , Antígenos CD28/imunologia , Humanos , Imunoterapia , Neoplasias/terapia , Transplante de Órgãos , Transdução de Sinais/imunologia , Linfócitos T/imunologia
3.
Scand J Immunol ; 90(5): e12808, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31322752

RESUMO

CD4+ T cell immunotherapy has potential for treatment in HIV-infected patients. A large number of expanded CD4+ T cells and confirmation of functional-related phenotypes are required for ensuring the successful outcomes of treatment. Freshly isolated CD4+ T cells from healthy donors were activated with anti-CD3/28-coated magnetic beads at different bead-to-cell ratios and cultured in the absence and presence of IL-2 supplementation for 3 weeks. Fold expansion, cell viability, growth kinetic and lymphocyte subset identities were determined. Data demonstrated that a 1:1 bead-to-cell ratio rendered the highest expansion of 1044-fold with 88% viability and 99.5% purity followed by the 2:1 and 0.5:1 ratios. No significant difference in proliferation and phenotypes was found between non-IL-2 and IL-2 supplementation groups. Several specific surface molecule expressions of the expanded cells including chemokine receptors, adhesion molecules, co-stimulatory molecules, activation molecules, maturation markers, cytokine receptors and other molecules were altered when compared to the unexpanded cells. This optimized expansion protocol using the 1:1 bead-to-cell ratio of anti-CD3/28-coated magnetic beads and culture condition without IL-2 supplementation provided the satisfactory yield with good reproducibility. Specific surface molecule expressions of the expanded cells presented potential roles in proliferation, differentiation, homeostasis, apoptosis and organ homing.


Assuntos
Antígenos CD28/imunologia , Complexo CD3/imunologia , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/transplante , Infecções por HIV/terapia , Imunoterapia Adotiva/métodos , Nanopartículas de Magnetita/uso terapêutico , Adulto , Proliferação de Células , Células Cultivadas , Materiais Revestidos Biocompatíveis , Humanos , Interleucina-2/imunologia , Ativação Linfocitária/imunologia , Resultado do Tratamento
4.
Monoclon Antib Immunodiagn Immunother ; 38(2): 60-69, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-31009338

RESUMO

CD28 superagonist (CD28SA), a therapeutic immunomodulatory monoclonal antibody triggered rapid and exaggerated activation of CD4+ effector memory T cells (TEMs) in humans with unwanted serious adverse effects. It is well known that distinct metabolic programs determine the fate and responses of immune cells. In this study, we show that human CD4+ TEMs stimulated with CD28SA adopt a metabolic program similar to those of tumor cells with enhanced glucose utilization, lipid biosynthesis, and proliferation in hypoxic conditions. Identification of metabolic profiles underlying hyperactive T cell activation would provide a platform to test safety of immunostimulatory antibodies.


Assuntos
Antígenos CD28/imunologia , Linfócitos T CD4-Positivos/imunologia , Glicólise/imunologia , Lipogênese/imunologia , Ativação Linfocitária/imunologia , Neoplasias/metabolismo , Acetilcoenzima A/metabolismo , Anticorpos Monoclonais/imunologia , Antígenos CD28/metabolismo , Proliferação de Células , Glucose/metabolismo , Humanos , Memória Imunológica , Neoplasias/imunologia , Neoplasias/patologia , Proteínas Quinases/metabolismo , Linfócitos T Reguladores/imunologia , Células Tumorais Cultivadas
5.
Nature ; 568(7750): 112-116, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30918399

RESUMO

Chimeric antigen receptors (CARs) are synthetic antigen receptors that reprogram T cell specificity, function and persistence1. Patient-derived CAR T cells have demonstrated remarkable efficacy against a range of B-cell malignancies1-3, and the results of early clinical trials suggest activity in multiple myeloma4. Despite high complete response rates, relapses occur in a large fraction of patients; some of these are antigen-negative and others are antigen-low1,2,4-9. Unlike the mechanisms that result in complete and permanent antigen loss6,8,9, those that lead to escape of antigen-low tumours remain unclear. Here, using mouse models of leukaemia, we show that CARs provoke reversible antigen loss through trogocytosis, an active process in which the target antigen is transferred to T cells, thereby decreasing target density on tumour cells and abating T cell activity by promoting fratricide T cell killing and T cell exhaustion. These mechanisms affect both CD28- and 4-1BB-based CARs, albeit differentially, depending on antigen density. These dynamic features can be offset by cooperative killing and combinatorial targeting to augment tumour responses to immunotherapy.


Assuntos
Antígenos de Neoplasias/imunologia , Antígenos de Neoplasias/metabolismo , Leucemia/imunologia , Receptores de Antígenos Quiméricos/imunologia , Linfócitos T/imunologia , Linfócitos T/metabolismo , Evasão Tumoral/imunologia , Ligante 4-1BB/imunologia , Animais , Antígenos CD28/imunologia , Citotoxicidade Imunológica , Feminino , Imunoterapia Adotiva , Leucemia/patologia , Masculino , Camundongos , Camundongos Endogâmicos NOD , Recidiva Local de Neoplasia/imunologia , Linfócitos T/citologia
6.
Proc Natl Acad Sci U S A ; 116(3): 1007-1016, 2019 01 15.
Artigo em Inglês | MEDLINE | ID: mdl-30598454

RESUMO

T cells proliferate vigorously following acute depletion of CD4+ Foxp3+ T regulatory cells [natural Tregs (nTregs)] and also when naive T cells are transferred to syngeneic, nTreg-deficient Rag1 -/- hosts. Here, using mice raised in an antigen-free (AF) environment, we show that proliferation in these two situations is directed to self ligands rather than food or commensal antigens. In both situations, the absence of nTregs elevates B7 expression on host dendritic cells (DCs) and enables a small subset of naive CD4 T cells with high self affinity to respond overtly to host DCs: bidirectional T/DC interaction ensues, leading to progressive DC activation and reciprocal strong proliferation of T cells accompanied by peripheral Treg (pTreg) formation. Likewise, high-affinity CD4 T cells proliferate vigorously and form pTregs when cultured with autologous DCs in vitro in the absence of nTregs: this anti-self response is MHCII/peptide dependent and elicited by the raised level of B7 on cultured DCs. The data support a model in which self tolerance is imposed via modulation of CD28 signaling and explains the pathological effects of superagonistic CD28 antibodies.


Assuntos
Proliferação de Células , Células Dendríticas/imunologia , Tolerância Imunológica , Modelos Imunológicos , Linfócitos T Reguladores/imunologia , Animais , Antígenos B7/genética , Antígenos B7/imunologia , Antígenos CD28/genética , Antígenos CD28/imunologia , Células Dendríticas/citologia , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/imunologia , Camundongos , Camundongos Knockout , Linfócitos T Reguladores/citologia
7.
Methods Mol Biol ; 1899: 103-118, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30649768

RESUMO

Alloantigen-specific hyporesponsiveness can be induced in alloreactive T cells contained within the whole peripheral blood mononuclear cell (PBMC) population by stimulating these responder cells ex vivo with HLA-mismatched stimulator PBMC as the antigen presenting cell (APC) source, in the presence of a CD28 costimulation blocking agent. As a result of this approach, specific alloreactivity is markedly decreased (by 1-2 logs), but third-party alloresponses and in vitro responses relying on the activation of pathogen- and tumor-associated antigen T-cell functional activities are not globally impinged upon (Guinan et al. N Engl J Med 340(22):1704-1714, 1999, Davies et al. Transplantation 86(6):854-864, 2008, Davies et al. Cell Transplant 21(9):2047-61, 2012). This method has been used clinically to alloanergize bone marrow and PBMC allografts, creating ex vivo cell therapies for adoptive transfer to blood cancer patients at high risk of disease relapse whose best option was to receive haploidentical hematopoietic cell transplants. These early phase trials consisting of, or containing, alloanergized T-cell infusions show promise in reducing graft-versus-host disease (GvHD), providing more rapid immune reconstitution, and decreasing severe post-transplant infectious complications and disease relapse. Herein, we describe this straightforward technique for generating alloanergized PBMC as it is performed in the research lab setting using belatacept for CD28-mediated costimulatory blockade (CSB) and PBMC isolated by Ficoll Hypaque gradient centrifugation as responders and APC. We also describe methods for evaluating subsequent alloproliferation to first and third party stimulation as well as assessment of cell division, pathogen-specific immunity, or allosuppression. The technique has successfully been transferred to collaborating labs, largely owing to the flexibility of using fresh or frozen PBMC, the lack of a requirement for specially isolated APC populations, and the ability to scale up or scale down the cell numbers that are to be anergized.


Assuntos
Antígenos CD28/imunologia , Tolerância Imunológica , Ativação Linfocitária/imunologia , Teste de Cultura Mista de Linfócitos/métodos , Linfócitos T Reguladores/imunologia , Células Apresentadoras de Antígenos/imunologia , Medula Óssea/imunologia , Transplante de Células-Tronco Hematopoéticas , Humanos , Isoantígenos/imunologia , Leucócitos Mononucleares/imunologia , Transplante Homólogo
8.
Front Immunol ; 9: 2864, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30564247

RESUMO

T cell activation is initiated upon ligand engagement of the T cell receptor (TCR) and costimulatory receptors. The CD28 molecule acts as a major costimulatory receptor in promoting full activation of naive T cells. However, despite extensive studies, why naive T cell activation requires concurrent stimulation of both the TCR and costimulatory receptors remains poorly understood. Here, we explore this issue by analyzing calcium response as a key early signaling event to elicit T cell activation. Experiments using mouse naive CD4+ T cells showed that engagement of the TCR or CD28 with the respective cognate ligand was able to trigger a rise in fluctuating calcium mobilization levels, as shown by the frequency and average response magnitude of the reacting cells compared with basal levels occurred in unstimulated cells. The engagement of both TCR and CD28 enabled a further increase of these two metrics. However, such increases did not sufficiently explain the importance of the CD28 pathways to the functionally relevant calcium responses in T cell activation. Through the autocorrelation analysis of calcium time series data, we found that combined but not separate TCR and CD28 stimulation significantly prolonged the average decay time (τ) of the calcium signal amplitudes determined with the autocorrelation function, compared with its value in unstimulated cells. This increasement of decay time (τ) uniquely characterizes the fluctuating calcium response triggered by concurrent stimulation of TCR and CD28, as it could not be achieved with either stronger TCR stimuli or by co-engaging both TCR and LFA-1, and likely represents an important feature of competent early signaling to provoke efficient T cell activation. Our work has thus provided new insights into the interplay between the TCR and CD28 early signaling pathways critical to trigger naive T cell activation.


Assuntos
Antígenos CD28/metabolismo , Linfócitos T CD4-Positivos/imunologia , Sinalização do Cálcio/imunologia , Ativação Linfocitária , Receptores de Antígenos de Linfócitos T/metabolismo , Animais , Células Apresentadoras de Antígenos , Antígenos CD28/imunologia , Linfócitos T CD4-Positivos/metabolismo , Células COS , Células Cultivadas , Cercopithecus aethiops , Técnicas de Cocultura , Antígeno-1 Associado à Função Linfocitária/imunologia , Antígeno-1 Associado à Função Linfocitária/metabolismo , Camundongos , Camundongos Endogâmicos C3H , Camundongos Endogâmicos CBA , Camundongos Transgênicos , Cultura Primária de Células , Receptores de Antígenos de Linfócitos T/imunologia
9.
Proc Natl Acad Sci U S A ; 115(34): E8027-E8036, 2018 08 21.
Artigo em Inglês | MEDLINE | ID: mdl-30087184

RESUMO

Activated T cells undergo metabolic reprogramming and effector-cell differentiation but the factors involved are unclear. Utilizing mice lacking DUSP6 (DUSP6-/-), we show that this phosphatase regulates T cell receptor (TCR) signaling to influence follicular helper T (TFH) cell differentiation and T cell metabolism. In vitro, DUSP6-/- CD4+ TFH cells produced elevated IL-21. In vivo, TFH cells were increased in DUSP6-/- mice and in transgenic OTII-DUSP6-/- mice at steady state. After immunization, DUSP6-/- and OTII-DUSP6-/- mice generated more TFH cells and produced more antigen-specific IgG2 than controls. Activated DUSP6-/- T cells showed enhanced JNK and p38 phosphorylation but impaired glycolysis. JNK or p38 inhibitors significantly reduced IL-21 production but did not restore glycolysis. TCR-stimulated DUSP6-/- T cells could not induce phosphofructokinase activity and relied on glucose-independent fueling of mitochondrial respiration. Upon CD28 costimulation, activated DUSP6-/- T cells did not undergo the metabolic commitment to glycolysis pathway to maintain viability. Unexpectedly, inhibition of fatty acid oxidation drastically lowered IL-21 production in DUSP6-/- TFH cells. Our findings suggest that DUSP6 connects TCR signaling to activation-induced metabolic commitment toward glycolysis and restrains TFH cell differentiation via inhibiting IL-21 production.


Assuntos
Diferenciação Celular/fisiologia , Fosfatase 6 de Especificidade Dupla , Glicólise/fisiologia , Receptores de Antígenos de Linfócitos T , Transdução de Sinais/fisiologia , Linfócitos T Auxiliares-Indutores , Animais , Formação de Anticorpos/fisiologia , Antígenos CD28/genética , Antígenos CD28/imunologia , Antígenos CD28/metabolismo , Fosfatase 6 de Especificidade Dupla/genética , Fosfatase 6 de Especificidade Dupla/imunologia , Fosfatase 6 de Especificidade Dupla/metabolismo , Imunoglobulina G/imunologia , Imunoglobulina G/metabolismo , Interleucinas/genética , Interleucinas/imunologia , Interleucinas/metabolismo , MAP Quinase Quinase 4/genética , MAP Quinase Quinase 4/imunologia , MAP Quinase Quinase 4/metabolismo , Camundongos , Camundongos Knockout , Mitocôndrias/genética , Mitocôndrias/imunologia , Mitocôndrias/metabolismo , Consumo de Oxigênio/fisiologia , Receptores de Antígenos de Linfócitos T/genética , Receptores de Antígenos de Linfócitos T/imunologia , Receptores de Antígenos de Linfócitos T/metabolismo , Linfócitos T Auxiliares-Indutores/citologia , Linfócitos T Auxiliares-Indutores/imunologia , Linfócitos T Auxiliares-Indutores/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/genética , Proteínas Quinases p38 Ativadas por Mitógeno/imunologia , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo
10.
Cancer Immunol Immunother ; 67(11): 1695-1707, 2018 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-30128739

RESUMO

Tumor-mediated immunosuppression via regulatory T-cells is a key player among the various immune-escape mechanisms in multiple myeloma. We analyzed the generation, distribution, function and immunophenotype of CD8+CD28- regulatory T-cells in patients with multiple myeloma. Functionality of CD8+CD28- T-cells was assessed by immunological assays using ex vivo generated antigen-specific T-cells from patients with plasma cell dyscrasias and healthy donors. Detailed analysis of distribution, immunophenotype and cytotoxic potential of CD8+CD28- T-cells was performed by flow cytometry and ELISA. We found that the amount of CD8+CD28- T-cells was directly correlated with the suppression of antigen-specific T-cell responses in patients with plasma cell dyscrasia. Analyzing the CD8+CD28- T-cells in detail, increased numbers of these cells were observed in the bone marrow (i.e., tumor microenvironment) of patients with plasma cell dyscrasia. Furthermore, we identified the expression of lymphocyte function-associated antigen 1 (LFA-1) as a marker of immunosuppression and defined the CD8+CD28-CD57+LFA-1high population as the relevant immunosuppressive compartment. These regulatory T-cells act as immunosuppressors via soluble factors and incubation with IL-10 augmented their immunosuppressive capacity. The immunosuppressive regulatory network of IL-10 and the CD8+CD28-CD57+LFA-1high regulatory T-cells show unique characteristics and contribute to the tumor immune escape mechanism in patients with multiple myeloma.


Assuntos
Antígenos CD28/imunologia , Antígenos CD8/imunologia , Linfócitos T CD8-Positivos/imunologia , Interleucina-10/farmacologia , Mieloma Múltiplo/imunologia , Paraproteinemias/imunologia , Subpopulações de Linfócitos T/imunologia , Linfócitos T Reguladores/imunologia , Linfócitos T CD8-Positivos/efeitos dos fármacos , Estudos de Casos e Controles , Células Cultivadas , Humanos , Ativação Linfocitária , Mieloma Múltiplo/metabolismo , Mieloma Múltiplo/patologia , Paraproteinemias/metabolismo , Paraproteinemias/patologia , Subpopulações de Linfócitos T/efeitos dos fármacos , Linfócitos T Citotóxicos/efeitos dos fármacos , Linfócitos T Citotóxicos/imunologia , Linfócitos T Reguladores/efeitos dos fármacos
11.
Biomed Res Int ; 2018: 5647120, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29862277

RESUMO

Primary nephrotic syndrome (PNS) is a devastating pediatric disorder. However, its mechanism remains unclear. Previous studies detected B7-1 in podocytes; meanwhile, γδT cells play pivotal roles in immune diseases. Therefore, this study aimed to assess whether and how γδT cells impact podocytes via the CD28/B7-1 pathway. WT and TCRδ-/- mice were assessed. LPS was used to induce nephropathy. Total γδT and CD28+γδT cells were quantitated in mouse spleen and kidney samples. B7-1 and phosphor-SRC levels in the kidney were detected as well. In vitro, γδT cells from the mouse spleen were cocultured with mouse podocytes, and apoptosis rate and phosphor-SRC expression in podocytes were assessed. Compared with control mice, WT mice with LPS nephropathy showed increased amounts of γδT cells in the kidney. Kidney injury was alleviated in TCRδ-/- mice. Meanwhile, B7-1 and phosphor-SRC levels were increased in the kidney from WT mice with LPS nephropathy. CD28+γδT cells were decreased, indicating CD28 may play a role in LPS nephropathy. Immunofluorescence colocalization analysis revealed a tight association of γδT cells with B7-1 in the kidney. High B7-1 expression was detected in podocytes treated with LPS. Podocytes cocultured with γδT cells showed higher phosphor-SRC and apoptosis rate than other cell groups. Furthermore, CD28/B7-1 blockage with CTLA4-Ig in vitro relieved podocyte injury. γδT cells exacerbate podocyte injury via CD28/B7-1 signaling, with downstream involvement of phosphor-SRC. The CD28/B7-1 blocker CTLA4-Ig prevented progressive podocyte injury, providing a potential therapeutic tool for PNS.


Assuntos
Antígeno B7-1/imunologia , Antígenos CD28/imunologia , Síndrome Nefrótica/imunologia , Podócitos/imunologia , Receptores de Antígenos de Linfócitos T gama-delta/imunologia , Transdução de Sinais/imunologia , Linfócitos T/imunologia , Quinases da Família src/imunologia , Animais , Feminino , Camundongos , Síndrome Nefrótica/patologia , Podócitos/patologia , Linfócitos T/patologia
12.
Int J Mol Sci ; 19(1)2018 Jan 17.
Artigo em Inglês | MEDLINE | ID: mdl-29342108

RESUMO

Lipid metabolism is altered in several cancer settings leading to different ratios of intermediates. Ovarian cancer is the most lethal gynecological malignancy. Cancer cells disperse in the abdominal space and ascites occurs. T cells obtained from ascites are unable to proliferate after an antigenic stimulus. The proliferation of ascites-derived T cells can be restored after culturing the cells for ten days in normal culture medium. No pathway aberrancies were detected. The acellular fraction of ascites can inhibit the proliferation of autologous as well as allogeneic peripheral blood lymphocytes, indicating the presence of soluble factors that interfere with T cell functionality. Therefore, we analyzed 109 lipid mediators and found differentially regulated lipids in suppressive ascitic fluid compared to normal abdominal fluid. Our study indicates the presence of lipid intermediates in ascites of ovarian cancer patients, which coincidences with T cell dysfunctionality. Since the immune system in the abdominal cavity is compromised, this may explain the high seeding efficiency of disseminated tumor cells. Further research is needed to fully understand the correlation between the various lipids and T cell proliferation, which could lead to new treatment options.


Assuntos
Metabolismo dos Lipídeos , Neoplasias Ovarianas/metabolismo , Ascite/imunologia , Antígenos CD28/imunologia , Antígenos CD28/metabolismo , Complexo CD3/imunologia , Complexo CD3/metabolismo , Citocinas/metabolismo , Feminino , Humanos , Sistema Imunitário/citologia , Sistema Imunitário/imunologia , Sistema Imunitário/metabolismo , Imunomodulação , Ativação Linfocitária/imunologia , Neoplasias Ovarianas/imunologia , Neoplasias Ovarianas/patologia , Receptores de Interleucina-2/metabolismo , Transdução de Sinais , Subpopulações de Linfócitos T/imunologia , Subpopulações de Linfócitos T/metabolismo , Linfócitos T/imunologia , Linfócitos T/metabolismo , Microambiente Tumoral/imunologia
13.
Int Immunopharmacol ; 54: 1-11, 2018 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-29100032

RESUMO

First infusion reactions along with severe anaphylactic responses can occur as a result of systemic administration of therapeutic antibodies. The underlying mechanisms by which monoclonal antibodies induce cytokine release syndrome (CRS) can involve direct agonistic effects via the drug target, or a combination of target-engagement along with innate receptor interactions. Despite the wide variety of pathways and cells that can play a role in CRS, many currently used assays are devoid of one or more components that must be present for these responses to occur. One assay that has not been assessed for its capacity to predict CRS is the modified Chandler loop model. Herein we evaluate a plethora of commercially available monoclonal antibodies to evaluate the modified Chandler loop model's potential in CRS prediction. We demonstrate that in a 4-hour loop assay, both the superagonistic antibodies, anti-CD3 (OKT3) and anti-CD28 (ANC28.1), display a clear cytokine response with a mixed adaptive/innate cytokine source. OKT3 induce TNFα and IFN-γ release in 20 out of 23 donors tested, whereas ANC28.1 induce TNF-α, IL-2 and IFN-γ release in all donors tested (n=18-22). On the other hand, non-agonistic antibodies associated with no or low infusion reactions in the clinic, namely cetuximab and natalizumab, neither induce cytokine release nor cause false positive responses. A TGN1412-like antibody also display a clear cytokine release with an adaptive cytokine profile (IFN-γ and IL-2) and all donors (n=9) induce a distinct IL-2 response. Additionally, the value of an intact complement system in the assay is highlighted by the possibility to dissect out the mechanism-of-action of alemtuzumab and rituximab. The loop assay can either complement lymph node-like assays or stand-alone to investigate drug/blood interactions during preclinical development, or for individual safety screening prior to first-in-man clinical trial.


Assuntos
Anafilaxia/imunologia , Anticorpos Monoclonais/uso terapêutico , Citocinas/metabolismo , Efeitos Colaterais e Reações Adversas Relacionados a Medicamentos/imunologia , Imunoterapia/métodos , Imunidade Adaptativa , Anticorpos Monoclonais/metabolismo , Antígenos CD28/imunologia , Complexo CD3/imunologia , Circulação Extracorpórea , Humanos , Imunidade Inata , Infusões Intravenosas , Microfluídica , Modelos Moleculares , Prognóstico
14.
Fish Shellfish Immunol ; 72: 95-103, 2018 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-29074133

RESUMO

Interaction of CD28 with CD80 or CD86 molecules provides a costimulatory signals required in T cell activation. In this study, we cloned and analyzed a CD28 gene (On-CD28) and a CD80/86 gene (On-CD80/86) from Nile tilapia (Oreochromis niloticus). Sequence analysis revealed the typical characteristics of On-CD28 protein; for instance, the proline-based motif (117TYPPPL122) is essential in binding of CD28 to CD80/86 ligands. Moreover, an extracellular Ig domain was found in On-CD80/86; this domain is responsible in binding of CD28 to CD80/86 receptors. Subcellular localization analysis showed that both On-CD28 and On-CD80/86 were distributed predominantly in the cytomembrane. Yeast two-hybrid assay showed that On-CD28 directly interacted with On-CD80/86. On-CD28 and On-CD80/86 transcripts were detected in all the examined tissues of healthy Nile tilapia, and the highest expression levels of On-CD28 and On-CD80/86 were detected in the brain and heart, respectively. Following a bacterial challenge using Streptococcus agalactiae in vivo, On-CD28 and On-CD80/86 were upregulated in head kidney, spleen, intestines, and brain. However, they showed different expression profiles in response to stimulation with inactivated S. agalactiae in vitro. These findings indicated that the interaction of On-CD28 with On-CD80/86 provides a costimulatory signals that possibly play an important role in T cell activation during S. agalactiae infection.


Assuntos
Imunidade Adaptativa/genética , Ciclídeos/genética , Ciclídeos/imunologia , Doenças dos Peixes/imunologia , Regulação da Expressão Gênica/imunologia , Proteínas de Membrana/genética , Proteínas de Membrana/imunologia , Sequência de Aminoácidos , Animais , Antígeno B7-1/química , Antígeno B7-1/genética , Antígeno B7-1/imunologia , Antígeno B7-2/química , Antígeno B7-2/genética , Antígeno B7-2/imunologia , Antígenos CD28/química , Antígenos CD28/genética , Antígenos CD28/imunologia , Proteínas de Peixes/química , Proteínas de Peixes/genética , Proteínas de Peixes/imunologia , Perfilação da Expressão Gênica/veterinária , Proteínas de Membrana/química , Filogenia , Alinhamento de Sequência/veterinária , Infecções Estreptocócicas/imunologia , Streptococcus agalactiae/fisiologia
15.
J Cell Physiol ; 233(2): 759-770, 2018 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-28608562

RESUMO

Malignant glioma is the most fatal of astrocytic lineage tumors despite therapeutic advances. Onset and progression of gliomas is accompanied by severe debilitation of T-cell defense and T-cell survival. One of the chief contributors to T-cell survival downstream of activation is the PI3K-AKT pathway. Our prior studies showed that the novel immunotherapeutic molecule T11-target structure (T11TS) blocks T-cell apoptosis in glioma. We also showed activation of immunological synapse components and calcineurin-NFAT pathway following T11TS immunotherapy of glioma-bearing rats. This lead to investigations whether such T-cell activation upon T11TS therapy translates into activation of downstream PI3K/AKT signals which may be related to observed blockade of T-cell apoptosis. For the purpose, we assessed by flowcytometry and immunoblotting, expressions of PI3K, PDK1, AKT, p-AKT, and PTEN in splenic T-cells of normal, experimentally-induced glioma-bearing rats and glioma-bearing rats receiving first, second and third doses of T11TS. We also determined comparative nuclear translocation of NF-κB across groups. We found significant increases in T-cell expressions of PDK1, PI3K, and p-AKT in T11TS-treated animal groups compared to sharp downregulations in glioma. AKT levels remained unchanged across groups. PTEN levels declined sharply after T11TS immunotherapy. T11TS also caused enhanced NF-κB translocation to the T-cell nucleus compared to glioma group. Results showed heightened activation of the PI3K-AKT pathway in glioma-bearing rats following T11TS immunotherapy. These results illustrate the novel role of T11TS immunotherapy in ameliorating the PI3K pathway in T-cells in glioma-bearing animals to enhance T-cell survival, according greater defense against glioma. The study thus has far-reaching clinical outcomes.


Assuntos
Antineoplásicos/farmacologia , Neoplasias Encefálicas/tratamento farmacológico , Antígenos CD58/farmacologia , Glioma/tratamento farmacológico , Imunoterapia/métodos , Fosfatidilinositol 3-Quinase/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Linfócitos T/efeitos dos fármacos , Evasão Tumoral/efeitos dos fármacos , Proteínas Quinases Dependentes de 3-Fosfoinositídeo/metabolismo , Transporte Ativo do Núcleo Celular , Animais , Neoplasias Encefálicas/enzimologia , Neoplasias Encefálicas/imunologia , Neoplasias Encefálicas/patologia , Antígenos CD28/imunologia , Antígenos CD28/metabolismo , Sobrevivência Celular , Etilnitrosoureia , Feminino , Glioma/enzimologia , Glioma/imunologia , Glioma/patologia , Masculino , NF-kappa B/metabolismo , PTEN Fosfo-Hidrolase/metabolismo , Fosforilação , Ratos , Transdução de Sinais/efeitos dos fármacos , Linfócitos T/enzimologia , Linfócitos T/imunologia
16.
Eur J Immunol ; 48(1): 106-119, 2018 01.
Artigo em Inglês | MEDLINE | ID: mdl-28891065

RESUMO

Amino-acid catabolizing enzymes produced by mononuclear phagocytes play a central role in regulating the immune response. The mammalian phenylalanine-catabolizing enzyme IL4-induced gene 1 (IL4I1) inhibits effector T lymphocyte proliferation and facilitates regulatory T-cell development. IL4I1 expression by macrophages of various human tumors may affect patient prognosis as it facilitates tumor escape from the T-cell response in murine models. Its enzymatic activity appears to participate in its effects, but some actions of IL4I1 remain unclear. Here, we show that the presence of IL4I1 during T-cell activation decreases early signaling events downstream of TCR stimulation, resulting in global T-cell inhibition which is more pronounced when there is CD28 costimulation. Surprisingly, the enzymatic activity of IL4I1 is not involved. Focal secretion of IL4I1 into the immune synaptic cleft and its binding to CD3+ lymphocytes could be important in IL4I1 immunosuppressive mechanism of action.


Assuntos
Interleucina-4/genética , L-Aminoácido Oxidase/genética , Ativação Linfocitária/imunologia , Receptores de Antígenos de Linfócitos T/imunologia , Linfócitos T/imunologia , Antígenos CD28/imunologia , Comunicação Celular/imunologia , Diferenciação Celular/imunologia , Proliferação de Células , Humanos , Imunossupressão , Interleucina-4/imunologia , L-Aminoácido Oxidase/metabolismo , Macrófagos/imunologia , Neoplasias/imunologia , Neoplasias/patologia , Neutrófilos/imunologia , Fenilalanina/metabolismo , Transdução de Sinais/imunologia , Evasão Tumoral/imunologia , Proteína-Tirosina Quinase ZAP-70/metabolismo
17.
J Immunol ; 199(12): 3972-3980, 2017 12 15.
Artigo em Inglês | MEDLINE | ID: mdl-29093061

RESUMO

Follicular regulatory T (TFR) cells are a newly defined regulatory T cell (Treg) subset that suppresses follicular helper T cell-mediated B cell responses in the germinal center reaction. The precise costimulatory signal requirements for proper TFR cell differentiation and function are still not known. Using conditional knockout strategies of CD28, we previously demonstrated that loss of CD28 signaling in Tregs caused autoimmunity in mice (termed CD28-ΔTreg mice), characterized by lymphadenopathy, accumulation of activated T cells, and cell-mediated inflammation of the skin and lung. In this study, we show that CD28 signaling is required for TFR cell differentiation. Treg-specific deletion of CD28 caused a reduction in TFR cell numbers and function, which resulted in increased germinal center B cells and Ab production. Moreover, residual CD28-deficient TFR cells showed a diminished suppressive capacity as assessed by their ability to inhibit Ab responses in vitro. Surprisingly, genetic deletion of B cells in CD28-ΔTreg mice prevented the development of lymphadenopathy and CD4+ T cell activation, and autoimmunity that mainly targeted skin and lung tissues. Thus, autoimmunity occurring in mice with CD28-deficient Tregs appears to be driven primarily by loss of TFR cell differentiation and function with resulting B cell-driven inflammation.


Assuntos
Doenças Autoimunes/imunologia , Autoimunidade/imunologia , Linfócitos B/imunologia , Antígenos CD28/deficiência , Subpopulações de Linfócitos T/imunologia , Linfócitos T Reguladores/imunologia , Animais , Formação de Anticorpos , Doenças Autoimunes/etiologia , Doenças Autoimunes/patologia , Antígenos CD28/imunologia , Linfócitos T CD4-Positivos/imunologia , Citocinas/biossíntese , Feminino , Centro Germinativo/imunologia , Tolerância Imunológica/imunologia , Pulmão/imunologia , Pulmão/patologia , Linfadenopatia/etiologia , Linfadenopatia/imunologia , Linfadenopatia/prevenção & controle , Depleção Linfocítica , Linfopoese , Camundongos , Especificidade de Órgãos , Pele/imunologia , Pele/patologia , Organismos Livres de Patógenos Específicos , Linfócitos T Reguladores/classificação
18.
Proc Natl Acad Sci U S A ; 114(46): 12190-12195, 2017 11 14.
Artigo em Inglês | MEDLINE | ID: mdl-29087297

RESUMO

Antigen discrimination by T cells occurs at the junction between a T cell and an antigen-presenting cell. Juxtacrine binding between numerous adhesion, signaling, and costimulatory molecules defines both the topographical and lateral geometry of this cell-cell interface, within which T cell receptor (TCR) and peptide major histocompatibility complex (pMHC) interact. These physical constraints on receptor and ligand movement have significant potential to modulate their molecular binding properties. Here, we monitor individual ligand:receptor binding and unbinding events in space and time by single-molecule imaging in live primary T cells for a range of different pMHC ligands and surface densities. Direct observations of pMHC:TCR and CD80:CD28 binding events reveal that the in situ affinity of both pMHC and CD80 ligands for their respective receptors is modulated by the steady-state number of agonist pMHC:TCR interactions experienced by the cell. By resolving every single pMHC:TCR interaction it is evident that this cooperativity is accomplished by increasing the kinetic on-rate without altering the off-rate and has a component that is not spatially localized. Furthermore, positive cooperativity is observed under conditions where the T cell activation probability is low. This TCR-mediated feedback is a global effect on the intercellular junction. It is triggered by the first few individual pMHC:TCR binding events and effectively increases the efficiency of TCR scanning for antigen before the T cell is committed to activation.


Assuntos
Antígenos/imunologia , Antígeno B7-1/imunologia , Antígenos CD28/imunologia , Complexo Principal de Histocompatibilidade , Receptores de Antígenos de Linfócitos T/imunologia , Linfócitos T/imunologia , Animais , Antígenos/metabolismo , Antígeno B7-1/genética , Antígenos CD28/genética , Expressão Gênica , Ligantes , Bicamadas Lipídicas/química , Ativação Linfocitária , Camundongos , Cultura Primária de Células , Ligação Proteica , Receptores de Antígenos de Linfócitos T/genética , Transdução de Sinais/imunologia , Análise de Célula Única , Linfócitos T/citologia
19.
Blood ; 130(22): 2410-2419, 2017 11 30.
Artigo em Inglês | MEDLINE | ID: mdl-29042364

RESUMO

Acute myeloid leukemia (AML), the most common adult acute leukemia in the United States, has the poorest survival rate, with 26% of patients surviving 5 years. Adoptive immunotherapy with T cells genetically modified to recognize tumors is a promising and evolving treatment option. However, antitumor activity, particularly in the context of progressive leukemia, can be dampened both by limited costimulation and triggering of immunoregulatory checkpoints that attenuate T-cell responses. Expression of CD200 (OX2), a negative regulator of T-cell function that binds CD200 receptor (CD200R), is commonly increased in leukemia and other malignancies and is associated with poor prognosis in leukemia patients. To appropriate and redirect the inhibitory effects of CD200R signaling on transferred CD8+ T cells, we engineered CD200R immunomodulatory fusion proteins (IFPs) with the cytoplasmic tail replaced by the signaling domain of the costimulatory receptor, CD28. An analysis of a panel of CD200R-CD28 IFP constructs revealed that the most effective costimulation was achieved in IFPs containing a dimerizing motif and a predicted tumor-T-cell distance that facilitates localization to the immunological synapse. T cells transduced with the optimized CD200R-CD28 IFPs exhibited enhanced proliferation and effector function in response to CD200+ leukemic cells in vitro. In adoptive therapy of disseminated leukemia, CD200R-CD28-transduced leukemia-specific CD8 T cells eradicated otherwise lethal disease more efficiently than wild-type cells and bypassed the requirement for interleukin-2 administration to sustain in vivo activity. The transduction of human primary T cells with the equivalent human IFPs increased proliferation and cytokine production in response to CD200+ leukemia cells, supporting clinical translation. This trial was registered at www.clinicaltrials.gov as #NCT01640301.


Assuntos
Antígenos de Superfície/genética , Antígenos CD28/genética , Imunoterapia Adotiva/métodos , Leucemia/terapia , Glicoproteínas de Membrana/genética , Receptores de Superfície Celular/genética , Linfócitos T/transplante , Animais , Antígenos de Superfície/imunologia , Antígenos CD28/imunologia , Proliferação de Células , Humanos , Imunomodulação , Leucemia/genética , Leucemia/imunologia , Glicoproteínas de Membrana/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Receptores de Superfície Celular/imunologia , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/imunologia , Linfócitos T/imunologia , Linfócitos T/metabolismo , Transdução Genética
20.
Nano Lett ; 17(11): 7045-7054, 2017 11 08.
Artigo em Inglês | MEDLINE | ID: mdl-28994285

RESUMO

Particles engineered to engage and interact with cell surface ligands and to modulate cells can be harnessed to explore basic biological questions as well as to devise cellular therapies. Biology has inspired the design of these particles, such as artificial antigen-presenting cells (aAPCs) for use in immunotherapy. While much has been learned about mimicking antigen presenting cell biology, as we decrease the size of aAPCs to the nanometer scale, we need to extend biomimetic design to include considerations of T cell biology-including T-cell receptor (TCR) organization. Here we describe the first quantitative analysis of particle size effect on aAPCs with both Signals 1 and 2 based on T cell biology. We show that aAPCs, larger than 300 nm, activate T cells more efficiently than smaller aAPCs, 50 nm. The 50 nm aAPCs require saturating doses or require artificial magnetic clustering to activate T cells. Increasing ligand density alone on the 50 nm aAPCs did not increase their ability to stimulate CD8+ T cells, confirming the size-dependent phenomenon. These data support the need for multireceptor ligation and activation of T-cell receptor (TCR) nanoclusters of similar sizes to 300 nm aAPCs. Quantitative analysis and modeling of a nanoparticle system provides insight into engineering constraints of aAPCs for T cell immunotherapy applications and offers a case study for other cell-modulating particles.


Assuntos
Células Apresentadoras de Antígenos/química , Células Artificiais/química , Imunomodulação , Ativação Linfocitária , Nanopartículas/química , Células Artificiais/imunologia , Células Artificiais/ultraestrutura , Materiais Biomiméticos/química , Materiais Biomiméticos/uso terapêutico , Biomimética/métodos , Antígenos CD28/imunologia , Antígenos CD8/imunologia , Humanos , Imunoterapia , Ligantes , Complexo Principal de Histocompatibilidade , Nanopartículas/uso terapêutico , Nanopartículas/ultraestrutura , Neoplasias/terapia , Tamanho da Partícula , Receptores de Antígenos de Linfócitos T/imunologia
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