Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 37
Filtrar
Mais filtros










Base de dados
Intervalo de ano de publicação
1.
Front Cell Dev Biol ; 9: 698503, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34395429

RESUMO

CD30, a member of the TNF receptor superfamily, is selectively expressed on a subset of activated lymphocytes and on malignant cells of certain lymphomas, such as classical Hodgkin Lymphoma (cHL), where it activates critical bystander cells in the tumor microenvironment. Therefore, it is not surprising that the CD30 antibody-drug conjugate Brentuximab Vedotin (BV) represents a powerful, FDA-approved treatment option for CD30+ hematological malignancies. However, BV also exerts a strong anti-cancer efficacy in many cases of diffuse large B cell lymphoma (DLBCL) with poor CD30 expression, even when lacking detectable CD30+ tumor cells. The mechanism remains enigmatic. Because CD30 is released on extracellular vesicles (EVs) from both, malignant and activated lymphocytes, we studied whether EV-associated CD30 might end up in CD30- tumor cells to provide binding sites for BV. Notably, CD30+ EVs bind to various DLBCL cell lines as well as to the FITC-labeled variant of the antibody-drug conjugate BV, thus potentially conferring the BV binding also to CD30- cells. Confocal microscopy and imaging cytometry studies revealed that BV binding and uptake depend on CD30+ EVs. Since BV is only toxic toward CD30- DLBCL cells when CD30+ EVs support its uptake, we conclude that EVs not only communicate within the tumor microenvironment but also influence cancer treatment. Ultimately, the CD30-based BV not only targets CD30+ tumor cell but also CD30- DLBCL cells in the presence of CD30+ EVs. Our study thus provides a feasible explanation for the clinical impact of BV in CD30- DLBCL and warrants confirming studies in animal models.

2.
Artigo em Inglês | MEDLINE | ID: mdl-33749106

RESUMO

BACKGROUND: The activating Natural killer group 2 member D (NKG2D) receptor is typically expressed on NK cells, CD8 T lymphocytes, γδ T cells and small subsets of CD4 T lymphocytes. During the course of an extensive flow cytometry phenotyping of immune cells in the peripheral blood of patients with glioblastoma multiforme (GBM) we noticed an unexpected expression of NKG2D receptor on granulocytes using the phycoerythrin (PE)-conjugated clone 149810 antibody. METHODS: Peripheral blood samples from 35 patients with GBM and 22 age-matched healthy control (HC) donors were analyzed using flow cytometry, imaging cytometry and real-time quantitative reverse transcription PCR to validate the observed expression of NKG2D receptor on myeloid cells. RESULTS: Reactivity with PE-149810 was mostly observed on granulocytes from GBM patients on dexamethasone treatment where it correlated with inferior survival rates. Surprisingly, such NKG2D expression on granulocytes was not observed using the allophycocyanin (APC)-conjugate of the same clone 149810 antibody or an indirect staining procedure with unconjugated clone 149810 antibody. Moreover, the PE-conjugate of a different anti-NKG2D clone (1D11) also did not stain granulocytes. Imaging cytometry indicated cell surface and intracellular localization of PE-149810 but not of PE-1D11 in granulocytes. CONCLUSION: Our results uncover an erroneous and false positive reactivity of PE-labeled (but not of APC-labeled or unconjugated) anti-NKG2D antibody 149810 on granulocytes from dexamethasone-treated GBM patients and raise a note of caution for studies of NKG2D expression on non-lymphoid cells.

3.
Expert Rev Hematol ; 13(11): 1211-1233, 2020 11.
Artigo em Inglês | MEDLINE | ID: mdl-33000968

RESUMO

INTRODUCTION: Blinatumomab, first in a class of bispecific T-cell engagers, revolutionized treatment paradigm of B-cell precursor relapsed/refractory or minimal residual disease positive acute lymphoblastic leukemia (ALL) in adults and children, inducing deep remissions in a proportion of patients. However, significant numbers of patients do not respond or eventually relapse. Strategies for improvement of treatment outcomes are required. AREAS COVERED: This review discusses the main structural and functional features of blinatumomab, and its place in the treatment of ALL. Furthermore, prospects to increase the efficacy of blinatumomab are addressed. The developments in the field of bispecific antibodies and their possible implications for treatment of ALL are reviewed. EXPERT OPINION: Better understanding the mechanisms of response and resistance to blinatumomab might help us to identify the group of patients benefiting most from treatment and to spare potentially toxic subsequent treatment strategies. Data emerging from ongoing clinical trials might change the treatment landscape of ALL and beyond. Early use of blinatumomab in frontline protocols with more advantageous treatment sequences and in combination with other targeted therapies might reduce the failure rates. Exponentially increasing number of novel treatment options and their possible combinations might complicate treatment decision-making without data from randomized trials.


Assuntos
Anticorpos Biespecíficos/uso terapêutico , Antineoplásicos Imunológicos/uso terapêutico , Imunoterapia/métodos , Leucemia-Linfoma Linfoblástico de Células Precursoras/terapia , Adulto , Anticorpos Biespecíficos/efeitos adversos , Anticorpos Biespecíficos/economia , Anticorpos Biespecíficos/imunologia , Anticorpos Biespecíficos/farmacocinética , Antígenos CD/análise , Antígenos de Neoplasias/análise , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Criança , Ensaios Clínicos como Assunto , Terapia Combinada , Resistencia a Medicamentos Antineoplásicos , Previsões , Humanos , Imunoterapia Adotiva , Neoplasia Residual , Leucemia-Linfoma Linfoblástico de Células Precursoras B/tratamento farmacológico , Leucemia-Linfoma Linfoblástico de Células Precursoras B/terapia , Leucemia-Linfoma Linfoblástico de Células Precursoras/tratamento farmacológico , Prognóstico , Recidiva , Indução de Remissão , Terapia de Salvação , Subpopulações de Linfócitos T/efeitos dos fármacos , Subpopulações de Linfócitos T/imunologia
4.
PLoS One ; 15(9): e0239369, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32997691

RESUMO

Cancer vaccinations sensitize the immune system to recognize tumor-specific antigens de novo or boosting preexisting immune responses. Dendritic cells (DCs) are regarded as the most potent antigen presenting cells (APCs) for induction of (cancer) antigen-specific CD8+ T cell responses. Chitosan nanoparticles (CNPs) used as delivery vehicle have been shown to improve anti-tumor responses. This study aimed at exploring the potential of CNPs as antigen delivery system by assessing activation and expansion of antigen-specific CD8+ T cells by DCs and subsequent T cell-mediated lysis of pancreatic ductal adenocarcinoma (PDAC) cells. As model antigen the ovalbumin-derived peptide SIINFEKL was chosen. Using imaging cytometry, intracellular uptake of FITC-labelled CNPs of three different sizes and qualities (90/10, 90/20 and 90/50) was demonstrated in DCs and in pro- and anti-inflammatory macrophages to different extents. While larger particles (90/50) impaired survival of all APCs, small CNPs (90/10) were not toxic for DCs. Internalization of SIINFEKL-loaded but not empty 90/10-CNPs promoted a pro-inflammatory phenotype of DCs indicated by elevated expression of pro-inflammatory cytokines. Treatment of murine DC2.4 cells with SIINFEKL-loaded 90/10-CNPs led to a marked MHC-related presentation of SIINFEKL and enabled DC2.4 cells to potently activate SIINFEKL-specific CD8+ OT-1 T cells finally leading to effective lysis of the PDAC cell line Panc-OVA. Overall, our study supports the suitability of CNPs as antigen vehicle to induce potent anti-tumor immune responses by activation and expansion of tumor antigen-specific CD8+ T cells.


Assuntos
Antígenos de Neoplasias/química , Antígenos de Neoplasias/imunologia , Linfócitos T CD8-Positivos/imunologia , Quitosana/química , Portadores de Fármacos/química , Nanopartículas/química , Animais , Linfócitos T CD8-Positivos/citologia , Linhagem Celular , Técnicas de Cocultura , Células Dendríticas/citologia , Células Dendríticas/imunologia , Humanos , Camundongos , Fenótipo , Vacinação
5.
Front Immunol ; 11: 1328, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32695112

RESUMO

Pancreatic ductal adenocarcinoma (PDAC) is characterized by an immunosuppressive tumor microenvironment with a dense desmoplastic stroma. The expression of ß-galactoside-binding protein galectin-3 is regarded as an intrinsic tumor escape mechanism for inhibition of tumor-infiltrating T cell function. In this study, we demonstrated that galectin-3 is expressed by PDAC and by γδ or αß T cells but is only released in small amounts by either cell population. Interestingly, large amounts of galectin-3 were released during the co-culture of allogeneic in vitro expanded or allogeneic or autologous resting T cells with PDAC cells. By focusing on the co-culture of tumor cells and γδ T cells, we observed that knockdown of galectin-3 in tumor cells identified these cells as the source of secreted galectin-3. Galectin-3 released by tumor cells or addition of physiological concentrations of recombinant galectin-3 did neither further inhibit the impaired γδ T cell cytotoxicity against PDAC cells nor did it induce cell death of in vitro expanded γδ T cells. Initial proliferation of resting peripheral blood and tumor-infiltrating Vδ2-expressing γδ T cells was impaired by galectin-3 in a cell-cell-contact dependent manner. The interaction of galectin-3 with α3ß1 integrin expressed by Vδ2 γδ T cells was involved in the inhibition of γδ T cell proliferation. The addition of bispecific antibodies targeting γδ T cells to PDAC cells enhanced their cytotoxic activity independent of the galectin-3 release. These results are of high relevance in the context of an in vivo application of bispecific antibodies which can enhance cytotoxic activity of γδ T cells against tumor cells but probably not their proliferation when galectin-3 is present. In contrast, adoptive transfer of in vitro expanded γδ T cells together with bispecific antibodies will enhance γδ T cell cytotoxicity and overcomes the immunosuppressive function of galectin-3.


Assuntos
Proteínas Sanguíneas/imunologia , Carcinoma Ductal Pancreático/imunologia , Galectinas/imunologia , Linfócitos Intraepiteliais/imunologia , Linfócitos do Interstício Tumoral/imunologia , Neoplasias Pancreáticas/imunologia , Adulto , Linhagem Celular Tumoral , Proliferação de Células , Humanos
6.
Cell Mol Life Sci ; 77(4): 751-764, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-31300870

RESUMO

Dipeptidyl peptidase 4 (DPP4, CD26) is a serine protease detected on several immune cells and on epithelial cells of various organs. Besides the membrane-bound enzyme, a catalytically active soluble form (sCD26/DPP4) is detected in several body fluids. Both variants cleave off dipeptides from the N-termini of various chemokines, neuropeptides, and hormones. CD26/DPP4 plays a fundamental role in the regulation of blood glucose levels by inactivating insulinotropic incretins and CD26/DPP4 inhibitors are thus routinely used in diabetes mellitus type 2 therapy to improve glucose tolerance. Such inhibitors might also prevent the CD26/DPP4-mediated inactivation of the T-cell chemoattractant CXCL10 released by certain tumors and thus improve anti-tumor immunity and immunotherapy. Despite its implication in the regulation of many (patho-)physiological processes and its consideration as a biomarker and therapeutic target, the cellular source of sCD26/DPP4 remains highly debated and mechanisms of its release are so far unknown. In line with recent reports that activated T lymphocytes could be a major source of sCD26/DPP4, we now demonstrate that CD26/DPP4 is stored in secretory granules of several major human cytotoxic lymphocyte populations and co-localizes with effector proteins such as granzymes, perforin, and granulysin. Upon stimulation, vesicular CD26/DPP4 is rapidly translocated to the cell surface in a Ca2+-dependent manner. Importantly, activation-induced degranulation leads to a massive release of proteolytically active sCD26/DPP4. Since activated effector lymphocytes serve as a major source of sCD26/DPP4, these results might explain the observed disease-associated alterations of sCD26/DPP4 serum levels and also indicate a so far unknown role of CD26/DPP4 in lymphocyte-mediated cytotoxicity.


Assuntos
Degranulação Celular , Dipeptidil Peptidase 4/metabolismo , Linfócitos T Citotóxicos/fisiologia , Cálcio/metabolismo , Células Cultivadas , Humanos , Proteólise
7.
Front Immunol ; 10: 569, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30972064

RESUMO

The functional plasticity and anti-tumor potential of human γδ T cells have been widely studied. However, the epigenetic regulation of γδ T-cell/tumor cell interactions has been poorly investigated. In the present study, we show that treatment with the histone deacetylase inhibitor Valproic acid (VPA) significantly enhanced the expression and/or release of the NKG2D ligands MICA, MICB and ULBP-2, but not ULBP-1 in the pancreatic carcinoma cell line Panc89 and the prostate carcinoma cell line PC-3. Under in vitro tumor co-culture conditions, the expression of full length and the truncated form of the NKG2D receptor in γδ T cells was significantly downregulated. Furthermore, using a newly established flow cytometry-based method to analyze histone acetylation (H3K9ac) in γδ T cells, we showed constitutive H3K9aclow and inducible H3K9achigh expression in Vδ2 T cells. The detailed analysis of H3K9aclow Vδ2 T cells revealed a significant reversion of TEMRA to TEM phenotype during in vitro co-culture with pancreatic ductal adenocarcinoma cells. Our study uncovers novel mechanisms of how epigenetic modifiers modulate γδ T-cell differentiation during interaction with tumor cells. This information is important when considering combination therapy of VPA with the γδ T-cell-based immunotherapy for the treatment of certain types of cancer.


Assuntos
Inibidores de Histona Desacetilases/farmacologia , Linfócitos Intraepiteliais/imunologia , Subfamília K de Receptores Semelhantes a Lectina de Células NK/biossíntese , Receptores de Antígenos de Linfócitos T gama-delta/imunologia , Ácido Valproico/farmacologia , Acetilação , Linhagem Celular Tumoral , Proteínas Ligadas por GPI/metabolismo , Antígenos de Histocompatibilidade Classe I/metabolismo , Histonas/metabolismo , Humanos , Memória Imunológica/imunologia , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Ativação Linfocitária/imunologia , Masculino , Células PC-3 , Neoplasias Pancreáticas/imunologia , Neoplasias da Próstata/imunologia
8.
Mol Immunol ; 107: 44-53, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30658247

RESUMO

Granulysin (GNLY) is a cationic antimicrobial, proinflammatory, and cytotoxic effector protein primarily expressed in human cytotoxic T and NK cells. Its two variants, the 15 kDa precursor and the mature 9 kDa protein processed by proteolysis, act on different microbes or infected and transformed target cells and utilize mechanistically different effector activities. In human peripheral blood lymphocytes of healthy individuals, both forms of GNLY are detected in TCR αß+ (CD4+ and CD8+) T cells, TCR γδ+ T cells, and CD3-CD56+ NK cells. In general, classical cytotoxic cells (i.e. CD8+ TCR αß+ T cells, TCR γδ+ T cells, and NK cells) contain effector proteins in higher abundance in more cells of the subset as compared to TCR αß+ CD4+ T cells. Imaging flow cytometry analyses demonstrate that the subcellular localization and internal pools of 9 kDa and 15 kDa GNLY are virtually non-overlapping. The 9 kDa form is enriched in dense granules that also contain granzymes (Grz) and carry CD107a, whereas 15 kDa GNLY is associated with CD107a-negative lysosome-related effector vesicles. We further demonstrate that 15 kDa GNLY serves as an additional indicator for non-classical, PKC-dependent degranulation while the liberation of granules containing 9 kDa GNLY requires calcium mobilization. Our studies provide a deeper insight into the subcellular localization and release mechanisms of the individual GNLY species. This information will not only be useful for the interpretation of GNLY-related pathophysiologies, but also for the development of therapeutic interventions employing distinct GNLY effector functions for microbial targeting or immunoregulation.


Assuntos
Antígenos de Diferenciação de Linfócitos T/metabolismo , Degranulação Celular , Vesículas Citoplasmáticas/metabolismo , Lisossomos/metabolismo , Linfócitos T CD4-Positivos/metabolismo , Linfócitos T CD4-Positivos/fisiologia , Linfócitos T CD8-Positivos/metabolismo , Linfócitos T CD8-Positivos/fisiologia , Proteína Ligante Fas/metabolismo , Humanos , Ativação Linfocitária/imunologia , Subpopulações de Linfócitos/metabolismo , Peso Molecular , Transporte Proteico
9.
Oncoimmunology ; 8(1): e1522471, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30546961

RESUMO

TGF-ß is a pleiotropic cytokine with multiple roles in immunity. Apart from its suppressive activity, TGF-ß is a driving cytokine in the differentiation of induced regulatory T cells (iTreg) but also in the polarization of interleukin-9 (IL-9) producing T helper 9 (Th9) T cells. Human Vδ2 expressing γδ T cells exert potent cytotoxicity towards a variety of solid tumor and leukemia/lymphoma target cells and thus are in the focus of current strategies to develop cell-based immunotherapies. Here we report that TGF-ß unexpectedly augments the cytotoxic effector activity of short-term expanded Vδ2 T cells when purified γδ T cells are activated with specific pyrophosphate antigens and IL-2 or IL-15 in the presence of TGF-ß. TGF-ß up-regulates the expression of CD54, CD103, interferon-γ, IL-9 and granzyme B in γδ T cells while CD56 and CD11a/CD18 are down-regulated. Moreover, we show that CD103 (αE/ß7 integrin) is recruited to the immunological synapse in γδ T cells. Increased cytotoxic activity of TGF-ß-exposed γδ T cells is reduced by anti-CD103 and further diminished upon additional anti-CD11a antibody treatment, pointing to a role of cellular adhesion in the enhanced cytolytic activity. Furthermore, magnetically sorted CD103-positive Vδ2 T cells exhibit superior cytolytic activity. In view of the importance of CD103 for tissue homing of lymphocytes, our results suggest that adoptive transfer of CD103-expressing Vδ2 T cells might favor their homing to solid tumors.

10.
Int Immunol ; 30(5): 215-228, 2018 04 25.
Artigo em Inglês | MEDLINE | ID: mdl-29373679

RESUMO

It is widely accepted that cytotoxic T and NK cells store effector proteins including granzymes, perforin and Fas ligand (FasL) in intracellular granules, often referred to as secretory lysosomes. Upon target cell encounter, these organelles are transported to the cytotoxic immunological synapse, where they fuse with the plasma membrane to release the soluble effector molecules and to expose transmembrane proteins including FasL on the cell surface. We previously described two distinct species of secretory vesicles in T and NK cells that differ in size, morphology and protein loading, most strikingly regarding FasL and granzyme B. We now show that the signal requirements for the mobilization of one or the other granule also differ substantially. We report that prestored FasL can be mobilized independent of extracellular Ca2+, whereas the surface exposure of lysosome-associated membrane proteins (Lamps; CD107a and CD63) and the release of granzyme B are calcium-dependent. The use of selective inhibitors of actin dynamics unequivocally points to different transport mechanisms for individual vesicles. While inhibitors of actin polymerization/dynamics inhibit the surface appearance of prestored FasL, they increase the activation-induced mobilization of CD107a, CD63 and granzyme B. In contrast, inhibition of the actin-based motor protein myosin 2a facilitates FasL-, but impairs CD107a-, CD63- and granzyme B mobilization. From our data, we conclude that distinct cytotoxic effector granules are differentially regulated with respect to signaling requirements and transport mechanisms. We suggest that a T cell might 'sense' which effector proteins it needs to mobilize in a given context, thereby increasing efficacy while minimizing collateral damage.


Assuntos
Proteína Ligante Fas/metabolismo , Células Matadoras Naturais/imunologia , Linfócitos T Citotóxicos/imunologia , Citoesqueleto de Actina/metabolismo , Sinalização do Cálcio , Células Cultivadas , Células Clonais , Citotoxicidade Imunológica , Granzimas/metabolismo , Humanos , Ativação Linfocitária , Glicoproteínas de Membrana Associadas ao Lisossomo/metabolismo , Miosinas/metabolismo , Perforina/metabolismo , Vesículas Secretórias/metabolismo
11.
Oncoimmunology ; 6(11): e1358839, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-29147621

RESUMO

Despite aggressive treatment regimens based on surgery and radiochemotherapy, the prognosis of patients with grade IV glioblastoma multiforme (GBM) remains extremely poor, calling for alternative options such as immunotherapy. Immunological mechanisms including the Natural Killer Group 2 member D (NKG2D) receptor-ligand system play an important role in tumor immune surveillance and targeting the NKG2D system might be beneficial. However, before considering any kind of immunotherapy, a precise characterization of the immune system is important, particularly in GBM patients where conventional therapies with impact on the immune system are frequently co-administered. Here we performed an in-depth immunophenotyping of GBM patients and age-matched healthy controls and analyzed NKG2D ligand expression on primary GBM cells ex vivo. We report that GBM patients have a compromised innate immune system irrespective of steroid (dexamethasone) medication. However, dexamethasone drastically reduced the number of immune cells in the blood of GBM patients. Moreover, higher counts of immune cells influenced by dexamethasone like CD45+ lymphocytes and non-Vδ2 γδ T cells were associated with better overall survival. Higher levels of NKG2D ligands on primary GBM tumor cells were observed in patients who received radiochemotherapy, pointing towards increased immunogenic potential of GBM cells following standard radiochemotherapy. This study sheds light on how steroids and radiochemotherapy affect immune cell parameters of GBM patients, a pre-requisite for the development of new therapeutic strategies targeting the immune system in these patients.

12.
F1000Res ; 62017.
Artigo em Inglês | MEDLINE | ID: mdl-28649364

RESUMO

In contrast to conventional T lymphocytes, which carry an αß T-cell receptor and recognize antigens as peptides presented by major histocompatibility complex class I or class II molecules, human γδ T cells recognize different metabolites such as non-peptidic pyrophosphate molecules that are secreted by microbes or overproduced by tumor cells. Hence, γδ T cells play a role in immunosurveillance of infection and cellular transformation. Until recently, it has been unknown how the γδ T-cell receptor senses such pyrophosphates in the absence of known antigen-presenting molecules. Recent studies from several groups have identified a unique role of butyrophilin (BTN) protein family members in this process, notably of BTN3A1. BTNs are a large family of transmembrane proteins with diverse functions in lipid secretion and innate and adaptive immunity. Here we discuss current models of how BTN molecules regulate γδ T-cell activation. We also address the implications of these recent findings on the design of novel immunotherapeutic strategies based on the activation of γδ T cells.

13.
J Immunol ; 197(8): 3059-3068, 2016 10 15.
Artigo em Inglês | MEDLINE | ID: mdl-27619996

RESUMO

Human Vγ9Vδ2 T cells recognize in a butyrophilin 3A/CD277-dependent way microbial (E)-4-hydroxy-3-methyl-but-2-enyl pyrophosphate (HMBPP) or endogenous pyrophosphates (isopentenyl pyrophosphate [IPP]). Nitrogen-bisphosphonates such as zoledronic acid (ZOL) trigger selective γδ T cell activation because they stimulate IPP production in monocytes by inhibiting the mevalonate pathway downstream of IPP synthesis. We performed a comparative analysis of the capacity of purified monocytes, neutrophils, and CD4 T cells to serve as accessory cells for Vγ9Vδ2 T cell activation in response to three selective but mechanistically distinct stimuli (ZOL, HMBPP, agonistic anti-CD277 mAb). Only monocytes supported γδ T cell expansion in response to all three stimuli, whereas both neutrophils and CD4 T cells presented HMBPP but failed to induce γδ T cell expansion in the presence of ZOL or anti-CD277 mAb. Preincubation of accessory cells with the respective stimuli revealed potent γδ T cell-stimulating activity of ZOL- or anti-CD277 mAb-pretreated monocytes, but not neutrophils. In comparison with monocytes, ZOL-pretreated neutrophils produced little, if any, IPP and expressed much lower levels of farnesyl pyrophosphate synthase. Exogenous IL-18 enhanced the γδ T cell expansion with all three stimuli, remarkably also in response to CD4 T cells and neutrophils preincubated with anti-CD277 mAb or HMBPP. Our study uncovers unexpected differences between monocytes and neutrophils in their accessory function for human γδ T cells and underscores the important role of IL-18 in driving γδ T cell expansion. These results may have implications for the design of γδ T cell-based immunotherapeutic strategies.


Assuntos
Antígenos CD/metabolismo , Butirofilinas/metabolismo , Linfócitos T CD4-Positivos/imunologia , Monócitos/imunologia , Neutrófilos/imunologia , Linfócitos T/imunologia , Anticorpos Bloqueadores/imunologia , Antígenos CD/imunologia , Butirofilinas/imunologia , Células Cultivadas , Difosfonatos/imunologia , Geraniltranstransferase/metabolismo , Hemiterpenos/imunologia , Humanos , Imidazóis/imunologia , Interleucina-18/metabolismo , Ativação Linfocitária , Ácido Mevalônico/metabolismo , Organofosfatos/imunologia , Compostos Organofosforados/imunologia , Receptores de Antígenos de Linfócitos T gama-delta/metabolismo , Ácido Zoledrônico
14.
Oncoimmunology ; 5(4): e1093276, 2016 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-27141377

RESUMO

The interaction of the MHC class I-related chain molecules A and B (MICA and MICB) and UL-16 binding protein (ULBP) family members expressed on tumor cells with the corresponding NKG2D receptor triggers cytotoxic effector functions in NK cells and γδ T cells. However, as a mechanism of tumor immune escape, NKG2D ligands (NKG2DLs) can be released from the cell surface. In this study, we investigated the NKG2DL system in different human glioblastoma (GBM) cell lines, the most lethal brain tumor in adults. Flow cytometric analysis and ELISA revealed that despite the expression of various NKG2DLs only ULBP2 is released as a soluble protein via the proteolytic activity of "a disintegrin and metalloproteases" (ADAM) 10 and 17. Moreover, we report that temozolomide (TMZ), a chemotherapeutic agent in clinical use for the treatment of GBM, increases the cell surface expression of NKG2DLs and sensitizes GBM cells to γδ T cell-mediated lysis. Both NKG2D and the T-cell receptor (TCR) are involved. The cytotoxic activity of γδ T cells toward GBM cells is strongly enhanced in a TCR-dependent manner by stimulation with pyrophosphate antigens. These data clearly demonstrate the complexity of mechanisms regulating NKG2DL expression in GBM cells and further show that treatment with TMZ can increase the immunogenicity of GBM. Thus, TMZ might enhance the potential of the adoptive transfer of ex vivo expanded γδ T cells for the treatment of malignant glioblastoma.

15.
Mol Immunol ; 65(2): 416-28, 2015 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-25745808

RESUMO

The "A Disintegrin And Metalloproteinases" (ADAMs) form a subgroup of the metzincin endopeptidases. Proteolytically active members of this protein family act as sheddases and govern key processes in development and inflammation by regulating cell surface expression and release of cytokines, growth factors, adhesion molecules and their receptors. In T lymphocytes, ADAM10 sheds the death factor Fas Ligand (FasL) and thereby regulates T cell activation, death and effector function. Although FasL shedding by ADAM10 was confirmed in several studies, its regulation is still poorly defined. We recently reported that ADAM10 is highly abundant on T cells whereas its close relative ADAM17 is expressed at low levels and transiently appears at the cell surface upon stimulation. Since FasL is also stored intracellularly and brought to the plasma membrane upon stimulation, we addressed where the death factor gets exposed to ADAM proteases. We report for the first time that both ADAM10 and ADAM17 are associated with FasL-containing secretory lysosomes. Moreover, we demonstrate that TCR/CD3/CD28-stimulation induces a partial positioning of both proteases and FasL to lipid rafts and only the activation-induced raft-positioning results in FasL processing. TCR/CD3/CD28-induced FasL proteolysis is markedly affected by reducing both ADAM10 and ADAM17 protein levels, indicating that in human T cells also ADAM17 is implicated in FasL processing. Since FasL shedding is affected by cholesterol depletion and by inhibition of Src kinases or palmitoylation, we conclude that it requires mobilization and co-positioning of ADAM proteases in lipid raft-like platforms associated with an activation of raft-associated Src-family kinases.


Assuntos
Proteínas ADAM/imunologia , Secretases da Proteína Precursora do Amiloide/imunologia , Proteína Ligante Fas/imunologia , Regulação Enzimológica da Expressão Gênica/imunologia , Proteínas de Membrana/imunologia , Proteólise , Linfócitos T/imunologia , Proteína ADAM10 , Proteína ADAM17 , Antígenos CD28/imunologia , Complexo CD3/imunologia , Linhagem Celular , Ativação Enzimática/imunologia , Feminino , Humanos , Ativação Linfocitária , Lisossomos/imunologia , Masculino , Microdomínios da Membrana/imunologia , Receptores de Antígenos de Linfócitos T/imunologia , Vesículas Secretórias/imunologia
16.
Eur J Immunol ; 45(2): 551-61, 2015 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-25359136

RESUMO

Noncatalytic region of tyrosine kinase (Nck) is an adapter protein that comprises one SH2 (Src homology) domain and three SH3 domains. Nck links receptors and receptor-associated tyrosine kinases or adapter proteins to proteins that regulate the actin cytoskeleton. Whereas the SH2 domain binds to phosphorylated receptors or associated phosphoproteins, individual interactions of the SH3 domains with proline-based recognition motifs result in the formation of larger protein complexes. In T cells, changes in cell polarity and morphology during T-cell activation and effector function require the T-cell receptor-mediated recruitment and activation of actin-regulatory proteins to initiate cytoskeletal reorganization at the immunological synapse. We previously identified the adapter protein HS1 as a putative Nck-interacting protein. We now demonstrate that the SH2 domain of Nck specifically interacts with HS1 upon phosphorylation of its tyrosine residue 378. We report that in human T cells, ligation of the chemokine receptor CXCR4 by stromal cell-derived factor 1α (SDF1α) induces a rapid and transient phosphorylation of tyrosine 378 of HS1 resulting in an increased association with Nck. Consequently, siRNA-mediated downregulation of HS1 and/or Nck impairs SDF1α-induced actin polymerization and T-cell migration.


Assuntos
Citoesqueleto de Actina/metabolismo , Actinas/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas Sanguíneas/metabolismo , Quimiocina CXCL12/metabolismo , Proteínas Oncogênicas/metabolismo , Linfócitos T/metabolismo , Citoesqueleto de Actina/química , Citoesqueleto de Actina/genética , Actinas/genética , Proteínas Adaptadoras de Transdução de Sinal/genética , Sítios de Ligação , Proteínas Sanguíneas/antagonistas & inibidores , Proteínas Sanguíneas/genética , Movimento Celular , Quimiocina CXCL12/genética , Quimiocina CXCL12/farmacologia , Regulação da Expressão Gênica , Células HEK293 , Humanos , Interleucina-2/farmacologia , Células Jurkat , Proteínas Oncogênicas/genética , Fosforilação , Polimerização , Cultura Primária de Células , Ligação Proteica , Estrutura Terciária de Proteína , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Receptores CXCR4/genética , Receptores CXCR4/metabolismo , Transdução de Sinais , Linfócitos T/citologia , Linfócitos T/efeitos dos fármacos
17.
PLoS One ; 9(7): e102899, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-25036101

RESUMO

The a disintegrin and metalloproteases (ADAMs) play a pivotal role in the control of development, adhesion, migration, inflammation and cancer. Although numerous substrates of ADAM10 have been identified, the regulation of its surface expression and proteolytic activity is still poorly defined. One current hypothesis is that both processes are in part modulated by protein-protein interactions mediated by the intracellular portion of the protease. For related proteases, especially proline-rich regions serving as docking sites for Src homology domain 3 (SH3) domain-containing proteins proved to be important for mediating regulatory interactions. In order to identify ADAM10-binding SH3 domain proteins, we screened the All SH3 Domain Phager library comprising 305 human SH3 domains using a GST fusion protein with the intracellular region of human ADAM10 as a bait for selection. Of a total of 291 analyzed phage clones, we found 38 SH3 domains that were precipitated with the ADAM10-derived fusion protein but not with GST. We verified the binding to the cytosolic portion of ADAM10 for several candidates by co-immunoprecipitation and/or pull down analyses. Intriguingly, several of the identified proteins have been implicated in regulating surface appearance and/or proteolytic activity of related ADAMs. Thus, it seems likely that they also play a role in ADAM10 biology.


Assuntos
Proteínas ADAM/metabolismo , Secretases da Proteína Precursora do Amiloide/metabolismo , Citoplasma/metabolismo , Desintegrinas/metabolismo , Proteínas de Membrana/metabolismo , Mapas de Interação de Proteínas/fisiologia , Domínios de Homologia de src/fisiologia , Proteína ADAM10 , Linhagem Celular , Linhagem Celular Tumoral , Células HEK293 , Humanos , Imunoprecipitação/métodos , Ligação Proteica/fisiologia , Transdução de Sinais/fisiologia
18.
Mol Immunol ; 60(1): 72-9, 2014 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-24769494

RESUMO

Nck adapter proteins link receptor and receptor-associated tyrosine kinases with proteins implicated in the regulation of the actin cytoskeleton. Nck is involved in a multitude of receptor-initiated signaling pathways and its physiological role thus covers aspects of tissue development and homeostasis, malignant transformation/invasiveness of tumour cells and also immune cell function. In T cells, changes of cell polarity and morphology associated with cellular activation and effector function crucially rely on the T cell receptor-mediated recruitment and activation of different actin-regulatory proteins to orchestrate and drive cytoskeletal reorganization at the immunological synapse. In a former approach to determine the interactome of Nck in human T cells, we identified the adapter protein ADAP as a Nck-interacting protein. This adhesion and degranulation-promoting adapter protein had already been implicated in the inside-out activation of integrins. Employing co-immunoprecipitations, we demonstrate that both Nck family members Nck1 and Nck2 coprecipitate with ADAP. Specifically, Nck interacts via its Src homology 2 domain with phosphorylated tyrosine Y595DDV and Y651DDV sites of ADAP. Moreover, we show that endogenous ADAP is phosphorylated in primary human T cell blasts and thus associates with Nck. At the functional level, ADAP and Nck adapter proteins cooperatively facilitate T cell adhesion to the LFA-1 ligand ICAM-1. Our data indicate that the ADAP/Nck complex might provide a means to link integrin activation with the actin cytoskeleton.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas Oncogênicas/metabolismo , Linfócitos T/imunologia , Citoesqueleto de Actina/imunologia , Proteínas Adaptadoras de Transdução de Sinal/genética , Adesão Celular/imunologia , Linhagem Celular , Ativação Enzimática/imunologia , Células HEK293 , Humanos , Integrinas/imunologia , Molécula 1 de Adesão Intercelular/imunologia , Antígeno-1 Associado à Função Linfocitária/imunologia , Proteínas Oncogênicas/genética , Fosforilação , Estrutura Terciária de Proteína , Interferência de RNA , RNA Interferente Pequeno , Transdução de Sinais
19.
Exp Cell Res ; 320(2): 290-301, 2014 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-24291222

RESUMO

Fas ligand (FasL) is a death factor of the tumor necrosis factor superfamily. Like other members of this family of type II transmembrane proteins, FasL is subject to ectodomain shedding by a disintegrin and metalloproteinases (ADAMs) liberating soluble FasL and leaving membrane-integral N-terminal fragments (NTFs). These NTFs are further processed by intramembrane proteolysis through signal peptide peptidase-like 2a (SPPL2a), releasing intracellular domains (ICDs) which might translocate to the nucleus to regulate transcription. Previous work established that the proline-rich domain within the cytosolic N-terminus of FasL is required for protein-protein interactions with different Src homology 3 (SH3) or WW domain proteins. Distinct binding partners regulate FasL storage and surface appearance or are involved in other aspects of FasL biology. Given the large number of FasL interactors, we asked whether proteolytically processed FasL fragments associate with the same or distinct sets of SH3 domain proteins. To address this, we performed co-precipitation experiments using a monoclonal antibody directed against the FasL N-terminus for subsequent protein detection of full length FasL and NTFs/ICDs in Western blots. We demonstrate that members of the sorting nexin (SNX) family bind full length FasL and its N-terminal fragments whereas members of the Pombe Cdc15 homology (PCH) protein family bind full length FasL, but fail to associate with processed FasL. Thus, we provide first evidence that full length FasL and FasL fragments display selectivity regarding their association with intracellular binding partners. The differential binding most likely governs the fate and function of the intracellular FasL fragments.


Assuntos
Proteína Ligante Fas/química , Proteína Ligante Fas/metabolismo , Fragmentos de Peptídeos/metabolismo , Domínios e Motivos de Interação entre Proteínas/fisiologia , Proteólise , Animais , Células Cultivadas , Células HEK293 , Humanos , Células Jurkat , Células K562 , Camundongos , Camundongos Endogâmicos BALB C , Fragmentos de Peptídeos/química , Ligação Proteica/fisiologia , Mapeamento de Interação de Proteínas , Especificidade por Substrato
20.
Cell Commun Signal ; 11(1): 41, 2013 Jun 10.
Artigo em Inglês | MEDLINE | ID: mdl-23758873

RESUMO

BACKGROUND: Due to their restricted expression in male germ cells and certain tumors, cancer/testis (CT) antigens are regarded as promising targets for tumor therapy. CT45 is a recently identified nuclear CT antigen that was associated with a severe disease score in Hodgkin's lymphoma and poor prognosis in multiple myeloma. As for many CT antigens, the biological function of CT45 in developing germ cells and in tumor cells is largely unknown. METHODS: CT45 expression was down-regulated in CT45-positive Hodgkin's lymphoma (L428), fibrosarcoma (HT1080) and myeloma (U266B1) cells using RNA interference. An efficient CT45 knock-down was confirmed by immunofluorescence staining and/or Western blotting. These cellular systems allowed us to analyze the impact of CT45 down-regulation on proliferation, cell cycle progression, morphology, adhesion, migration and invasive capacity of tumor cells. RESULTS: Reduced levels of CT45 did not coincide with changes in cell cycle progression or proliferation. However, we observed alterations in cell adherence, morphology and migration/invasion after CT45 down-regulation. Significant changes in the distribution of cytoskeleton-associated proteins were detected by confocal imaging. Changes in cell adherence were recorded in real-time using the xCelligence system with control and siRNA-treated cells. Altered migratory and invasive capacity of CT45 siRNA-treated cells were visualized in 3D migration and invasion assays. Moreover, we found that CT45 down-regulation altered the level of the heterogeneous nuclear ribonucleoprotein syncrip (hnRNP-Q1) which is known to be involved in the control of focal adhesion formation and cell motility. CONCLUSIONS: Providing first evidence of a cell biological function of CT45, we suggest that this cancer/testis antigen is involved in the modulation of cell morphology, cell adherence and cell motility. Enhanced motility and/or invasiveness of CT45-positive cells could contribute to the more severe disease progression that is correlated to CT45-positivity in several malignancies.

SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA
...