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1.
Ann Surg Oncol ; 23(2): 655-62, 2016 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-26289805

RESUMO

BACKGROUND: Complement is a central part of both the innate and adaptive immune response and its activation has traditionally been considered part of the immunosurveillance response against cancer. Its pro-inflammatory role and its contribution to the development of many illnesses associated with inflammatory states implicate complement in carcinogenesis. METHODS: We evaluated the role of three protein inhibitors of complement-cobra venom factor, humanized cobra venom factor, and recombinant staphylococcus aureus superantigen-like protein 7-in the setting of a transplantable murine colon cancer model. Outcomes were evaluated by monitoring tumor growth, and flow cytometry, ELISPOT, and quantitative real-time PCR were used to determine the impact of complement inhibition on the host immune response. RESULTS: Complement inhibitors were effective at depleting complement component C3 in tumor bearing mice and this was temporally correlated with a decreased rate of tumor growth during the establishment of tumors. Treatment with cobra venom factor resulted in increased CD8(+) T cells as a percentage of tumor-infiltrating cells as well as a reduced immunosuppressive environment evidenced by decreased myeloid derived suppressor cells in splenocytes of treated mice. Complement inhibition resulted in increased expression of the chemoattractive cytokines CCL5, CXCL10, and CXCL11. DISCUSSION: Complement depletion represents a promising mode of immunotherapy in cancer by its ability to impair tumor growth by increasing the host's effective immune response to tumor and diminishing the immunosuppressive effect created by the tumor microenvironment and ultimately could be utilized as a component of combination immunotherapy.


Assuntos
Neoplasias do Colo/terapia , Ativação do Complemento/efeitos dos fármacos , Complemento C3/antagonistas & inibidores , Inativadores do Complemento/farmacologia , Imunoterapia , Animais , Western Blotting , Sobrevivência Celular/efeitos dos fármacos , Neoplasias do Colo/imunologia , Neoplasias do Colo/metabolismo , Venenos Elapídicos/farmacologia , Ensaio de Imunoadsorção Enzimática , Exotoxinas/farmacologia , Feminino , Camundongos , Camundongos Endogâmicos C57BL , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Superantígenos/farmacologia , Linfócitos T/efeitos dos fármacos , Linfócitos T/imunologia , Células Tumorais Cultivadas , Microambiente Tumoral/efeitos dos fármacos
2.
J Immunol ; 193(10): 4988-99, 2014 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-25305313

RESUMO

The ultimate goal in transplantation medicine is the promotion of operational tolerance. Although Th cells of the Th17 type have been predominantly associated with rejection of allogeneic solid organ grafts, regulatory T (T(reg)) cells appear to foster operational tolerance. Induced T(reg) and Th17 cells have a higher lineage plasticity than has been recognized thus far. We found that when mesenchymal stem cells (MSCs) were used to induce long-term acceptance of allogeneic heart grafts in mice, the induction of T(reg) cells was preceded by development of a CD11b(hi)Gr1(int) myeloid-derived immunosuppressive cell-mediated Th17 response. Substantial suppression of Foxp3(+) T(reg) cell generation from retinoic acid receptor-related orphan receptor γ(-/-) T cells by MSCs revealed that retinoic acid receptor-related orphan receptor γ is a common factor in the differentiation of T(reg) and Th17 cells. Immunosuppressant mycophenolate mofetil treatment of enriched IL-17A(+) cells from MSC-primed allograft mouse recipients resulted in a reduction of IL-17A production and an increase in the Foxp3(+) T(reg) cell fraction. Furthermore, identification of IL-17A(+) Foxp3(+) double-positive and ex-IL-17-producing IL-17A(neg)Foxp3(+) T cells strongly argues for direct conversion of Th17 cells into T(reg) cells as the underlying mechanism of immune regulation in MSC-mediated allograft survival. The Th17 into T(reg) conversion identified in this study constitutes an important immunological mechanism by which MSC-induced myeloid-derived immunosuppressive cells mediate operational transplant tolerance. The possibility to create T(reg) cell-regulated operational tolerance in the absence of complete immune suppression provides strong clinical implications for cell therapy-assisted minimization protocols.


Assuntos
Interleucina-17/imunologia , Transplante de Células-Tronco Mesenquimais , Linfócitos T Reguladores/citologia , Células Th17/citologia , Tolerância ao Transplante , Animais , Diferenciação Celular , Fatores de Transcrição Forkhead/genética , Fatores de Transcrição Forkhead/imunologia , Regulação da Expressão Gênica , Sobrevivência de Enxerto , Imunossupressores/farmacologia , Interleucina-17/deficiência , Interleucina-17/genética , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Ácido Micofenólico/análogos & derivados , Ácido Micofenólico/farmacologia , Membro 3 do Grupo F da Subfamília 1 de Receptores Nucleares/deficiência , Membro 3 do Grupo F da Subfamília 1 de Receptores Nucleares/genética , Membro 3 do Grupo F da Subfamília 1 de Receptores Nucleares/imunologia , Transdução de Sinais , Linfócitos T Reguladores/efeitos dos fármacos , Linfócitos T Reguladores/imunologia , Células Th17/efeitos dos fármacos , Células Th17/imunologia , Transplante Homólogo
3.
Mediators Inflamm ; 2014: 287531, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-24616553

RESUMO

The immune response to Helicobacter pylori importantly determines the pathogenesis of infection as well as the success of antibiotic eradication of the bacteria. Strains of H. pylori were gathered from 14 patients who failed to eradicate H. pylori infection with antibiotics-therapy resistant strains (TRS)-or from patients who were able to eradicate H. pylori infection-therapy susceptible strains (TSS). The THP-1 cells were stimulated with H. pylori antigens. Cathepsin X expression on THP-1 cells and concentration of cytokines in the supernatant of THP-1 cells were measured with a flow cytometer. TSS H. pylori antigens increased the proportion of cathepsin X positive cells compared to TRS H. pylori antigens. TSS H. pylori antigens induced higher secretion of IL-12 and IL-6 compared to TRS H. pylori antigens (P < 0.001; 0.02). Polymyxin B, a lipid A inhibitor, lowered the secretion of IL-12 and IL-6 in TRS and TSS. We demonstrated a H. pylori strain-dependent cathepsin X and cytokine expression that can be associated with H. pylori resistance to eradication due to lack of effective immune response. Differences in lipid A of H. pylori might have an influence on the insufficient immune response, especially on phagocytosis.


Assuntos
Catepsinas/química , Helicobacter pylori/metabolismo , Imunidade Inata/imunologia , Precursores de Proteínas/química , Antígenos de Bactérias/imunologia , Linhagem Celular , Citocinas/metabolismo , Dispepsia/microbiologia , Citometria de Fluxo , Mucosa Gástrica/microbiologia , Helicobacter pylori/imunologia , Humanos , Interleucina-12/metabolismo , Interleucina-6/metabolismo , Lipídeo A/química , Lipopolissacarídeos/química , Macrófagos/imunologia , Polimixina B/química , Especificidade da Espécie , Estômago/microbiologia
4.
Curr Opin Organ Transplant ; 19(1): 60-4, 2014 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-24231429

RESUMO

PURPOSE OF REVIEW: Despite their potential to supplement the donor organ pool, expanded donor criteria grafts are associated with an elevated risk of graft failure and increased early mortality. Likewise, attempts to promote operational graft tolerance through conventional immunosuppressive therapy have demonstrated significant safety-related drawbacks. Because of their potent regenerative and immunomodulative potential, adjunct mesenchymal stem cell (MSC) therapy represents an innovative approach to both of these clinical problems. RECENT FINDINGS: Recent studies have begun to delineate the benefit and mechanisms of short-term therapy combining MSCs and low-dose immunosuppressive drugs in promoting graft acceptance and potentially regeneration. SUMMARY: The current review presents our rationale for the first-in-man clinical trial in liver transplantation utilizing a mesenchymal cell product (MultiStem, Athersys, Cleveland, Ohio, USA). The long-term objective of this program is to safely minimize the dose of complementary immunosuppressive drugs while achieving long-term allograft survival and operational tolerance. The use of adjunct cellular therapy as a means of reducing long-term pharmacotherapy would represent a major advancement in the field of liver transplantation.


Assuntos
Transplante de Fígado/métodos , Transplante de Células-Tronco Mesenquimais/métodos , Células-Tronco Mesenquimais/citologia , Humanos , Terapia de Imunossupressão/métodos , Imunossupressores/administração & dosagem , Tolerância ao Transplante
5.
Cancer Immunol Res ; 2024 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-38949179

RESUMO

CTL recognition of non-mutated tumor-associated antigens (TAA), present on cancer cells but also in healthy tissues, is an important element of cancer immunity, but the mechanism of its selectivity for cancer cells and opportunities for its enhancement remain elusive. In this study, we found that CTL expression of the NK receptors (NKR) DNAM-1 and NKG2D was associated with the effector status of CD8+ tumor-infiltrating lymphocytes (TIL) and long-term survival of melanoma patients. Using MART-1 and NY-ESO-1 as model TAAs, we demonstrated that DNAM-1 and NKG2D regulate T-cell receptor (TCR) functional avidity and set the threshold for TCR activation of human TAA-specific CTLs. Superior costimulatory effects of DNAM-1 over CD28 involved enhanced TCR signaling, CTL killer function and polyfunctionality. Double transduction of human CTLs with TAA-specific TCR and NKRs resulted in strongly enhanced antigen sensitivity, without a reduction in the antigen specificity and selectivity of killer function. In addition, the elevation of NKR-Ligand expression on cancer cells by chemotherapy also increased CTL recognition of cancer cells expressing low levels of TAA. Our data help to explain the ability of self-antigens to mediate tumor rejection in the absence of autoimmunity and support the development of dual-targeting adoptive T cell therapies that use NKRs to enhance the potency and selectivity of recognition of TAA-expressing cancer cells.

6.
Eur J Immunol ; 42(2): 364-73, 2012 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-22101830

RESUMO

The generation of effective type 1 T helper (Th1)-cell responses is required for immunity against intracellular bacteria. However, some intracellular bacteria require interleukin (IL)-17 to drive Th1-cell immunity and subsequent protective host immunity. Here, in a model of Mycobacterium bovis Bacille Calmette-Guerin (BCG) vaccination in mice, we demonstrate that the dependence on IL-17 to drive Th1-cell responses is a host mechanism to overcome bacteria-induced IL-10 inhibitory effects. We show that BCG-induced prostaglandin-E2 (PGE2) promotes the production of IL-10 which limits Th1-cell responses, while simultaneously inducing IL-23 and Th17-cell differentiation. The ability of IL-17 to downregulate IL-10 and induce IL-12 production allows the generation of subsequent Th1-cell responses. Accordingly, BCG-induced Th17-cell responses precede the generation of Th1-cell responses in vivo, whereas the absence of the IL-23 pathway decreases BCG vaccine-induced Th17 and Th1-cell immunity and subsequent vaccine-induced protection upon M. tuberculosis challenge. Importantly, in the absence of IL-10, BCG-induced Th1-cell responses occur in an IL-17-independent manner. These novel data demonstrate a role for the IL-23/IL-17 pathway in driving Th1-cell responses, specifically to overcome IL-10-mediated inhibition and, furthermore, show that in the absence of IL-10, the generation of BCG-induced Th1-cell immunity is IL-17 independent.


Assuntos
Interferon gama/metabolismo , Interleucina-17/metabolismo , Mycobacterium bovis/imunologia , Células Th1/metabolismo , Tuberculose/imunologia , Animais , Carga Bacteriana/genética , Dinoprostona/imunologia , Dinoprostona/metabolismo , Modelos Animais de Doenças , Humanos , Imunomodulação , Interferon gama/imunologia , Interleucina-10/genética , Interleucina-17/genética , Interleucina-17/imunologia , Interleucina-23/imunologia , Interleucina-23/metabolismo , Ativação Linfocitária/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Mycobacterium bovis/crescimento & desenvolvimento , Mycobacterium bovis/patogenicidade , Comunicação Parácrina/genética , Células Th1/imunologia , Células Th1/patologia , Vacinação
7.
Blood ; 118(20): 5498-505, 2011 Nov 17.
Artigo em Inglês | MEDLINE | ID: mdl-21972293

RESUMO

Dendritic cells (DCs) and myeloid-derived suppressor cells (MDSCs) show opposing roles in the immune system. In the present study, we report that the establishment of a positive feedback loop between prostaglandin E(2) (PGE(2)) and cyclooxygenase 2 (COX2), the key regulator of PGE(2) synthesis, represents the determining factor in redirecting the development of CD1a(+) DCs to CD14(+)CD33(+)CD34(+) monocytic MDSCs. Exogenous PGE(2) and such diverse COX2 activators as lipopolysaccharide, IL-1ß, and IFNγ all induce monocyte expression of COX2, blocking their differentiation into CD1a(+) DCs and inducing endogenous PGE(2), IDO1, IL-4Rα, NOS2, and IL-10, typical MDSC-associated suppressive factors. The addition of PGE(2) to GM-CSF/IL-4-supplemented monocyte cultures is sufficient to induce the MDSC phenotype and cytotoxic T lymphocyte (CTL)-suppressive function. In accordance with the key role of PGE(2) in the physiologic induction of human MDSCs, the frequencies of CD11b(+)CD33(+) MDSCs in ovarian cancer are closely correlated with local PGE(2) production, whereas the cancer-promoted induction of MDSCs is strictly COX2 dependent. The disruption of COX2-PGE(2) feedback using COX2 inhibitors or EP2 and EP4 antagonists suppresses the production of MDSC-associated suppressive factors and the CTL-inhibitory function of fully developed MDSCs from cancer patients. The central role of COX2-PGE(2) feedback in the induction and persistence of MDSCs highlights the potential for its manipulation to enhance or suppress immune responses in cancer, autoimmunity, or transplantation.


Assuntos
Ciclo-Oxigenase 2/metabolismo , Células Dendríticas/imunologia , Dinoprostona/metabolismo , Células Mieloides/imunologia , Neoplasias Ovarianas/imunologia , Antígenos CD/metabolismo , Antígenos CD34/metabolismo , Antígenos de Diferenciação Mielomonocítica/metabolismo , Ascite/imunologia , Linfócitos T CD8-Positivos/citologia , Linfócitos T CD8-Positivos/imunologia , Diferenciação Celular/imunologia , Células Dendríticas/citologia , Células Dendríticas/metabolismo , Retroalimentação Fisiológica/fisiologia , Feminino , Humanos , Imunoterapia/métodos , Receptores de Lipopolissacarídeos/metabolismo , Células Mieloides/citologia , Células Mieloides/metabolismo , Neoplasias Ovarianas/terapia , Receptores de Prostaglandina E Subtipo EP2/metabolismo , Receptores de Prostaglandina E Subtipo EP4/metabolismo , Lectina 3 Semelhante a Ig de Ligação ao Ácido Siálico , Linfócitos T Citotóxicos/citologia , Linfócitos T Citotóxicos/imunologia
8.
Biochem J ; 443(2): 439-50, 2012 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-22257123

RESUMO

γ-Enolase, a glycolytic enzyme, is expressed specifically in neurons. It exerts neurotrophic activity and has been suggested to regulate growth, differentiation, survival and regeneration of neurons. In the present study, we investigated the involvement of γ-enolase in PI3K (phosphoinositide 3-kinase)/Akt and MAPK (mitogen-activated protein kinase)/ERK (extracellular-signal-regulated kinase) signalling, the two pathways triggered predominantly by neurotrophic factors. Whereas the PI3K/Akt pathway, rather than the MAPK/ERK pathway, is involved in γ-enolase-enhanced cell survival, γ-enolase-stimulated neurite outgrowth requires both pathways, i.e. the activation of both PI3K and ERK1/2, leading to subsequent expression of the growth-cone-specific protein GAP-43 (growth-associated protein of 43 kDa). MEK (MAPK/ERK kinase) and PI3K inhibition blocked or attenuated the neurite outgrowth associated with dynamic remodelling of the actin-based cytoskeleton. We show that γ-enolase-mediated PI3K activation regulates RhoA kinase, a key regulator of actin cytoskeleton organization. Moreover, the inhibition of RhoA downstream effector ROCK (Rho-associated kinase) results in enhanced γ-enolase-induced neurite outgrowth, accompanied by actin polymerization and its redistribution to growth cones. Our results show that γ-enolase controls neuronal survival, differentiation and neurite regeneration by activating the PI3K/Akt and MAPK/ERK signalling pathways, resulting in downstream regulation of the molecular and cellular processes of cytoskeleton reorganization and cell remodelling, activation of transcriptional factors and regulation of the cell cycle.


Assuntos
Neuritos/metabolismo , Fragmentos de Peptídeos/farmacologia , Fosfopiruvato Hidratase/farmacologia , Transdução de Sinais/efeitos dos fármacos , Diferenciação Celular , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Humanos , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Neuritos/efeitos dos fármacos , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo
9.
Res Sq ; 2023 Oct 19.
Artigo em Inglês | MEDLINE | ID: mdl-37886562

RESUMO

CD28-driven "signal 2" is critical for naïve CD8+ T cell responses to dendritic cell (DC)-presented weak antigens, including non-mutated tumor-associated antigens (TAAs). However, it is unclear how DC-primed cytotoxic T lymphocytes (CTLs) respond to the same TAAs presented by cancer cells which lack CD28 ligands. Here, we show that NK receptors (NKRs) DNAM-1 and NKG2D replace CD28 during CTL re-activation by cancer cells presenting low levels of MHC I/TAA complexes, leading to enhanced proximal TCR signaling, immune synapse formation, CTL polyfunctionality, release of cytolytic granules and antigen-specific cancer cell killing. Double-transduction of T cells with recombinant TCR and NKR constructs or upregulation of NKR-ligand expression on cancer cells by chemotherapy enabled effective recognition and killing of poorly immunogenic tumor cells by CTLs. Operational synergy between TCR and NKRs in CTL recognition explains the ability of cancer-expressed self-antigens to serve as tumor rejection antigens, helping to develop more effective therapies.

10.
Immunol Invest ; 41(6-7): 635-57, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-23017139

RESUMO

Myeloid-derived suppressor cells (MDSCs) are critical mediators of tumor-associated immune suppression, with their numbers and activity strongly increased in most human cancers and animal models. MDSCs suppress anti-tumor immunity through multiple mechanisms, including the manipulation of arginine and tryptophan metabolism by such factors as arginase (Arg), inducible nitric oxide synthase (iNOS/NOS2), and indoleamine-2,3-dioxygenase (IDO). Prostaglandin E(2) (PGE(2)), a mediator of chronic inflammation and tumor progression, has emerged as a key molecule in MDSC biology. PGE(2) promotes MDSC development and their induction by additional factors, directly suppresses T cell immune responses and participates in the induction of other MDSC-associated suppressive factors, including Arg, iNOS and IDO. It further promotes MDSC recruitment to tumor environments through the local induction of CXCL12/SDF-1 and the induction and stabilization of the CXCL12 receptor, CXCR4, on tumor-associated MDSCs. The establishment of a positive feedback loop between PGE(2) and cyclooxygenase 2 (COX-2), the key regulator of PGE(2) synthesis, stabilizes the MDSC phenotype and is required for their suppressive function. The central role of PGE(2) in MDSC biology provides for a feasible target for counteracting MDSC-mediated immune suppression in cancer.


Assuntos
Arginase/imunologia , Arginina/metabolismo , Ciclo-Oxigenase 2/imunologia , Dinoprostona/metabolismo , Células Progenitoras Mieloides/metabolismo , Neoplasias/metabolismo , Animais , Arginase/genética , Quimiocina CXCL12/genética , Quimiocina CXCL12/imunologia , Ciclo-Oxigenase 2/genética , Humanos , Indolamina-Pirrol 2,3,-Dioxigenase/genética , Indolamina-Pirrol 2,3,-Dioxigenase/imunologia , Camundongos , Células Progenitoras Mieloides/imunologia , Células Progenitoras Mieloides/patologia , Neoplasias/imunologia , Neoplasias/patologia , Óxido Nítrico Sintase Tipo II/genética , Óxido Nítrico Sintase Tipo II/imunologia , Receptores CXCR4/genética , Receptores CXCR4/imunologia , Transdução de Sinais , Linfócitos T/imunologia , Linfócitos T/metabolismo , Linfócitos T/patologia
11.
IUBMB Life ; 63(9): 686-93, 2011 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-21796748

RESUMO

The adhesion molecule lymphocyte function-associated antigen (LFA)-1 plays a key role in immune surveillance and response. Its conformation is spatially and temporally regulated, enabling adhesion and deadhesion during T-cell migration. LFA-1 adhesion to its major ligand intercellular adhesion molecule 1 is controlled by adaptor proteins which bind the cytoplasmic tail of the ß (2) subunit. Cathepsin X, a cysteine carboxypeptidase, promotes T-cell migration and morphological changes by cleaving the ß (2) cytoplasmic tail of LFA-1. In this way, it modulates the affinity of LFA-1 for structural adaptors talin-1 and α-actinin-1 and enables the stepwise transition between intermediate and high-affinity conformations of LFA-1, an event that is necessary for effective T-cell function. Cathepsin X regulation that would allow precise modulation of LFA-1 affinity has a great potential for anti-LFA-1 therapy.


Assuntos
Catepsinas/metabolismo , Movimento Celular/imunologia , Vigilância Imunológica/imunologia , Antígeno-1 Associado à Função Linfocitária/metabolismo , Conformação Proteica , Linfócitos T/imunologia , Actinina/metabolismo , Adesão Celular , Antígeno-1 Associado à Função Linfocitária/imunologia , Ligação Proteica , Talina/metabolismo
12.
Mol Divers ; 15(2): 347-60, 2011 May.
Artigo em Inglês | MEDLINE | ID: mdl-21076980

RESUMO

In this article, we describe the design, synthesis and activity evaluation of glycomimetic DC-SIGN antagonists, that use a mannose residue to anchor to the protein carbohydrate recognition domain (CRD). The molecules were designed from the structure of the known pseudo-mannobioside antagonist 1, by including additional hydrophobic groups, which were expected to engage lipophilic areas of DC-SIGN CRD. The results demonstrate that the synthesized compounds potently inhibit DC-SIGN-mediated adhesion to mannan coated plates. Additionally, in silico docking studies were performed to rationalize the results and to suggest further optimization.


Assuntos
Moléculas de Adesão Celular/antagonistas & inibidores , Desenho de Fármacos , Lectinas Tipo C/antagonistas & inibidores , Manose , Receptores de Superfície Celular/antagonistas & inibidores , Bioensaio , Manose/química , Modelos Moleculares , Ligação Proteica/fisiologia , Reprodutibilidade dos Testes , Relação Estrutura-Atividade
13.
Cancer Gene Ther ; 28(1-2): 98-111, 2021 02.
Artigo em Inglês | MEDLINE | ID: mdl-32632271

RESUMO

Adoptive cell therapy (ACT) using tumor-specific tumor-infiltrating lymphocytes (TILs) has demonstrated success in patients where tumor-antigen specific TILs can be harvested from the tumor, expanded, and re-infused in combination with a preparatory regimen and IL2. One major issue for non-immunogenic tumors has been that the isolated TILs lack tumor specificity and thus possess limited in vivo therapeutic function. An oncolytic virus (OV) mediates an immunogenic cell death for cancer cells, leading to elicitation and dramatic enhancement of tumor-specific TILs. We hypothesized that the tumor-specific TILs elicited and promoted by an OV would be a great source for ACT for solid cancer. In this study, we show that a local injection of oncolytic poxvirus in MC38 tumor with low immunogenicity in C57BL/6 mice, led to elicitation and accumulation of tumor-specific TILs in the tumor tissue. Our analyses indicated that IL2-armed OV-elicited TILs contain lower quantities of exhausted PD-1hiTim-3+ CD8+ T cells and regulatory T cells. The isolated TILs from IL2-expressing OV-treated tumor tissue retained high tumor specificity after expansion ex vivo. These TILs resulted in significant tumor regression and improved survival after adoptive transfer in mice with established MC38 tumor. Our study showcases the feasibility of using an OV to induce tumor-reactive TILs that can be expanded for ACT.


Assuntos
Terapia Baseada em Transplante de Células e Tecidos/métodos , Imunoterapia Adotiva/métodos , Linfócitos do Interstício Tumoral/imunologia , Vírus Oncolíticos/imunologia , Animais , Feminino , Humanos , Camundongos
14.
Biochim Biophys Acta ; 1790(3): 173-81, 2009 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-19100814

RESUMO

BACKGROUND: Lectins are a diverse group of carbohydrate-binding proteins exhibiting numerous biological activities and functions. METHODS: Two-step serial carbohydrate affinity chromatography was used to isolate a lectin from the edible mushroom clouded agaric (Clitocybe nebularis). It was characterized biochemically, its gene and cDNA cloned and the deduced amino acid sequence analyzed. Its activity was tested by hemagglutination assay and carbohydrate-binding specificity determined by glycan microarray analysis. Its effect on proliferation of several human cell lines was determined by MTS assay. RESULTS: A homodimeric lectin with 15.9-kDa subunits agglutinates human group A, followed by B, O, and bovine erythrocytes. Hemagglutination was inhibited by glycoprotein asialofetuin and lactose. Glycan microarray analysis revealed that the lectin recognizes human blood group A determinant GalNAcalpha1-3(Fucalpha1-2)Galbeta-containing carbohydrates, and GalNAcbeta1-4GlcNAc (N,N'-diacetyllactosediamine). The lectin exerts antiproliferative activity specific to human leukemic T cells. CONCLUSIONS: The protein belongs to the ricin B-like lectin superfamily, and has been designated as C. nebularis lectin (CNL). Its antiproliferative effect appears to be elicited by binding to carbohydrate receptors on human leukemic T cells. GENERAL SIGNIFICANCE: CNL is one of the few mushroom ricin B-like lectins that have been identified and the only one so far shown to possess immunomodulatory properties.


Assuntos
Agaricales/química , Proliferação de Células/efeitos dos fármacos , Lectinas/isolamento & purificação , Leucemia de Células T/patologia , Sequência de Aminoácidos , Sequência de Carboidratos , Linhagem Celular Tumoral , Cromatografia em Gel , DNA Complementar , Eletroforese em Gel de Poliacrilamida , Humanos , Concentração de Íons de Hidrogênio , Ponto Isoelétrico , Lectinas/química , Lectinas/genética , Lectinas/farmacologia , Dados de Sequência Molecular , Peso Molecular , Homologia de Sequência de Aminoácidos , Espectrometria de Massas por Ionização por Electrospray
15.
Immunology ; 129(4): 525-35, 2010 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-20002210

RESUMO

Resveratrol is a polyphenol that acts on multiple molecular targets important for cell differentiation and activation. Dendritic cells (DCs) are a functionally diverse cell type and represent the most potent antigen-presenting cells of the immune system. In this study, we investigated resveratrol-induced effects on DCs during their differentiation and maturation. Our results show that resveratrol induces DC-associated tolerance, particularly when applied during DC differentiation. Costimulatory molecules CD40, CD80 and CD86 were down-regulated, as was the expression of major histocompatibility complex (MHC) class II molecules. Surface expression of inhibitory immunoglobulin-like transcript 3 (ILT3) and ILT4 molecules was induced, while human leucocyte antigen (HLA)-G expression was not affected. Resveratrol-treated DCs lost the ability to produce interleukin (IL)-12p70 after activation, but had an increased ability to produce IL-10. Such DCs were poor stimulators of allogeneic T cells and had lowered ability to induce CD4(+) T-cell migration. Furthermore, treated cells were able to generate allogeneic IL-10-secreting T cells, but were not competent in inducing FoxP3 expression These tolerogenic effects are probably associated with the effect of resveratrol on multiple molecular targets through which it interferes with DC differentiation and nuclear factor (NF)-kappaB translocation. Our data provide new insights into the molecular and functional mechanisms of the tolerogenic effects that resveratrol exerts on DCs.


Assuntos
Diferenciação Celular , Células Dendríticas/efeitos dos fármacos , Células Dendríticas/imunologia , Tolerância Imunológica/efeitos dos fármacos , Monócitos/citologia , Estilbenos/farmacologia , Biomarcadores/metabolismo , Linfócitos T CD4-Positivos/efeitos dos fármacos , Linfócitos T CD4-Positivos/imunologia , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/imunologia , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Células Dendríticas/citologia , Humanos , Tolerância Imunológica/imunologia , Interleucina-10/biossíntese , Interleucina-10/imunologia , Interleucina-12/biossíntese , Interleucina-12/imunologia , Lipopolissacarídeos/farmacologia , Glicoproteínas de Membrana/antagonistas & inibidores , Glicoproteínas de Membrana/imunologia , NF-kappa B/imunologia , Receptores de Superfície Celular/antagonistas & inibidores , Receptores de Superfície Celular/imunologia , Receptores Imunológicos/antagonistas & inibidores , Receptores Imunológicos/imunologia , Valores de Referência , Resveratrol
16.
Eur J Immunol ; 39(11): 3217-27, 2009 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-19750481

RESUMO

The motility of T cells depends on the dynamic spatial regulation of integrin-mediated adhesion and de-adhesion. Cathepsin X, a cysteine protease, has been shown to regulate T-cell migration by interaction with lymphocyte function associated antigen-1 (LFA-1). LFA-1 adhesion to the ICAM-1 is controlled by the association of actin-binding proteins with the cytoplasmic tail of the beta(2) chain of LFA-1. Cleavage by cathepsin X of the amino acid residues S(769), E(768) and A(767) from the C-terminal of the beta(2) cytoplasmic tail of LFA-1 is shown to promote binding of the actin-binding protein alpha-actinin-1. Furthermore, cathepsin X overexpression reduced LFA-1 clustering and induced an intermediate affinity LFA-1 conformation that is known to associate with alpha-actinin-1. Increased levels of intermediate affinity LFA-1 resulted in augmented cell spreading due to reduced attachment of T cells to the ICAM-1-coated surface. Gradual cleavage of LFA-1 by cathepsin X enables the transition between intermediate and high affinity LFA-1, an event that is crucial for effective T-cell migration.


Assuntos
Actinina/metabolismo , Catepsinas/metabolismo , Quimiotaxia de Leucócito/imunologia , Antígeno-1 Associado à Função Linfocitária/metabolismo , Actinina/imunologia , Catepsinas/imunologia , Imunofluorescência , Humanos , Molécula 1 de Adesão Intercelular/imunologia , Molécula 1 de Adesão Intercelular/metabolismo , Células Jurkat , Antígeno-1 Associado à Função Linfocitária/imunologia , Linfócitos T/imunologia
17.
Appl Environ Microbiol ; 76(20): 6928-32, 2010 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-20802083

RESUMO

We have optimized the display of the B domain of staphylococcal protein A on the surface of Lactococcus lactis. The maximum binding capacity was estimated at 0.146 µg of antibody per 108 cells and was sustained at 86% after treatment with simulated gastric juice. A tumor necrosis factor alpha (TNF-α)-binding affibody was also displayed and bound TNF-α, which could be useful in the treatment of inflammatory bowel disease.


Assuntos
Anticorpos/metabolismo , Lactococcus lactis/genética , Lactococcus lactis/metabolismo , Proteína Estafilocócica A/genética , Proteína Estafilocócica A/metabolismo , Fator de Necrose Tumoral alfa/metabolismo , Ligação Proteica , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo
18.
Anal Biochem ; 406(2): 222-9, 2010 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-20667443

RESUMO

We report a new dendritic cell adhesion assay, using either immature or mature dendritic cells, for identifying functional dendritic cell-specific ICAM-3-grabbing nonintegrin (DC-SIGN) inhibitors. Because immature dendritic cells are responsible for pathogen binding and invasion, this in vitro assay provides an important link between in vitro results and pathogen-based in vivo assays. Furthermore, this assay does not require laborious expression, refolding, and purification of DC-SIGN carbohydrate recognition domain or extracellular domain as receptor-based assays. The assay power evaluated with Z and Z' parameters enables screening of compound libraries and determination of IC(50) values in the first stage of DC-SIGN inhibitor development.


Assuntos
Moléculas de Adesão Celular/antagonistas & inibidores , Células Dendríticas/citologia , Células Dendríticas/metabolismo , Imunoensaio/métodos , Lectinas Tipo C/antagonistas & inibidores , Receptores de Superfície Celular/antagonistas & inibidores , Especificidade de Anticorpos/imunologia , Adesão Celular , Moléculas de Adesão Celular/metabolismo , Diferenciação Celular , Colorimetria , Citometria de Fluxo , Fluoresceínas/metabolismo , Fluorescência , Humanos , Concentração Inibidora 50 , Lectinas Tipo C/metabolismo , Ligantes , Receptores de Superfície Celular/metabolismo , Reprodutibilidade dos Testes , Succinimidas/metabolismo , Fatores de Tempo
19.
Neurosignals ; 18(4): 246-58, 2010.
Artigo em Inglês | MEDLINE | ID: mdl-21358174

RESUMO

Syntrophins are scaffold proteins that can bind several signaling molecules and localize them to the plasma membrane. We demonstrate here that in neuroblastoma SH-SY5Y cells, brain-specific γ1-syntrophin binds the neurotrophic factor γ-enolase through its PDZ domain, and translocates it to the plasma membrane, as shown by immunoprecipitation, surface plasmon resonance, fluorescence colocalization and flow cytometry. Extensive colocalization of γ1-syntrophin and γ-enolase was observed in neurite growth cones in differentiated SH-SY5Y cells. Silencing of the γ1-syntrophin gene by RNA interference significantly reduced the re-distribution of γ-enolase to the plasma membrane and impaired its neurotrophic effects. We demonstrated that an intact C-terminal end of γ-enolase is essential for its γ1-syntrophin-assisted trafficking. The cleavage of two amino acids at the C-terminal end of γ-enolase by the carboxypeptidase cathepsin X prevents binding with the γ1-syntrophin PDZ domain. Collectively, these data demonstrate that γ1-syntrophin participates in γ-enolase translocation towards the plasma membrane, a pre-requisite for its neurotrophic activity. By disrupting this γ1-syntrophin-guided subcellular distribution, cathepsin X reduces γ-enolase-induced neurotrophic signaling.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/fisiologia , Membrana Celular/metabolismo , Cones de Crescimento/metabolismo , Fatores de Crescimento Neural/metabolismo , Fosfopiruvato Hidratase/metabolismo , Regulação para Cima/fisiologia , Sequência de Aminoácidos , Diferenciação Celular/fisiologia , Linhagem Celular Tumoral , Membrana Celular/enzimologia , Membrana Celular/patologia , Cones de Crescimento/enzimologia , Cones de Crescimento/patologia , Humanos , Dados de Sequência Molecular , Fatores de Crescimento Neural/fisiologia , Neuroblastoma/enzimologia , Neuroblastoma/metabolismo , Neuroblastoma/patologia , Fosfopiruvato Hidratase/fisiologia , Transporte Proteico/fisiologia
20.
Cell Motil Cytoskeleton ; 66(11): 1030-40, 2009 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-19670215

RESUMO

T cells migrate through restrictive barriers in a protease-independent, amoeboid fashion that is characterized by morphological cell polarization. The interaction of cysteine-dependent carboxypeptidase cathepsin X with beta(2) integrin LFA-1 (lymphocyte function associated antigen 1) induces T-cell morphological changes, displaying into a 3D extracellular matrix a cytoplasmic projection termed a uropod. In the present study we show that inhibition of cathepsin X and a cysteine-dependent endopeptidase, cathepsin L, markedly inhibits T-cell actin polymerization, shape polarization, and chemotaxis. We propose that cathepsin L promotes T-cell migration associated processes by activating procathepsin X in the endolysosomal vesicles near the cell membrane and at the peak of the uropod, where both proteases were colocalized. We show that active cathepsin X modifies the beta(2) cytoplasmic tail of LFA-1 in the uropod, promoting its high affinity conformation. We suggest that LFA-1 cleavage contributes to the conformational change in the cytoplasmic tail, promoting the binding of the cytoskeletal protein talin. This interaction is restricted to the uropod and results in the stabilization of this region, promoting LFA-1-mediated cell uropod elongation.


Assuntos
Actinas/metabolismo , Catepsinas/metabolismo , Cisteína Proteases/metabolismo , Antígeno-1 Associado à Função Linfocitária/metabolismo , Linfócitos T/ultraestrutura , Polaridade Celular , Extensões da Superfície Celular/metabolismo , Quimiotaxia , Humanos , Células Jurkat , Multimerização Proteica , Linfócitos T/enzimologia , Talina/metabolismo
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