The RNA editing enzyme adenosine deaminase acting on RNA 1 (ADAR1) is essential for correct functioning of innate immune responses. The ADAR1p110 isoform is mainly nuclear and ADAR1p150, which is interferon (IFN) inducible, is predominately cytoplasmic. Using three different methods - co-immunoprecipitation (co-IP) of endogenous ADAR1, Strep-tag co-IP and BioID with individual ADAR1 isoforms - a comprehensive interactome was generated during both homeostasis and the IFN response. Both known and novel interactors as well as editing regulators were identified. Nuclear proteins were detected as stable interactors with both ADAR1 isoforms. In contrast, BioID identified distinct protein networks for each ADAR1 isoform, with nuclear components observed with ADAR1p110 and components of cytoplasmic cellular condensates with ADAR1p150. RNase A digestion distinguished between distal and proximal interactors, as did a double-stranded RNA (dsRNA)-binding mutant of ADAR1 which demonstrated the importance of dsRNA binding for ADAR1 interactions. IFN treatment did not affect the core ADAR1 interactomes but resulted in novel interactions, the majority of which are proximal interactions retained after RNase A treatment. Short treatment with high molecular weight poly(I:C) during the IFN response resulted in dsRNA-binding-dependent changes in the proximal protein network of ADAR1p110 and association of the ADAR1p150 proximal protein network with some components of antiviral stress granules.
- MeSH
- adenosindeaminasa * metabolismus genetika MeSH
- buněčné jádro * metabolismus MeSH
- cytoplazma * metabolismus MeSH
- dvouvláknová RNA metabolismus genetika MeSH
- editace RNA MeSH
- HEK293 buňky MeSH
- HeLa buňky MeSH
- interferony metabolismus genetika MeSH
- lidé MeSH
- mapy interakcí proteinů MeSH
- poly I-C farmakologie MeSH
- protein - isoformy * metabolismus genetika MeSH
- proteiny vázající RNA * metabolismus genetika MeSH
- vazba proteinů MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
Toll-like receptor 3 (TLR3) is an endosomal receptor expressed in several immune and epithelial cells. Recent studies have highlighted its expression also in solid tumors, including prostate cancer (PCa), and have described its role primarily in the proinflammatory response and induction of apoptosis. It is up-regulated in some castration-resistant prostate cancers. However, the role of TLR3 in prostate cancer progression remains largely unknown. The current study experimentally demonstrated that exogenous TLR3 activation in PCa cell lines leads to a significant induction of secretion of the cytokines IL-6, IL-8, and interferon-β, depending on the model and chemoresistance status. Transcriptomic analysis of TLR3-overexpressing cells revealed a functional program that is enriched for genes involved in the regulation of cell motility, migration, and tumor invasiveness. Increased motility, migration, and invasion in TLR3-overexpressing cell line were confirmed by several in vitro assays and using an orthotopic prostate xenograft model in vivo. Furthermore, TLR3-ligand induced apoptosis via cleavage of caspase-3/7 and poly (ADP-ribose) polymerase, predominantly in TLR3-overexpressing cells. These results indicate that TLR3 may be involved in prostate cancer progression and metastasis; however, it might also represent an Achilles heel of PCa, which can be exploited for targeted therapy.
- MeSH
- apoptóza MeSH
- lidé MeSH
- nádorové buněčné linie MeSH
- nádory prostaty * patologie MeSH
- poly I-C farmakologie MeSH
- prostata patologie MeSH
- toll-like receptor 3 * genetika metabolismus MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- mužské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Pancreatic adenocarcinoma is one of the leading causes of cancer-related deaths, and its therapy remains a challenge. Our proposed therapeutic approach is based on the intratumoral injections of mannan-BAM, toll-like receptor ligands, and anti-CD40 antibody (thus termed MBTA therapy), and has shown promising results in the elimination of subcutaneous murine melanoma, pheochromocytoma, colon carcinoma, and smaller pancreatic adenocarcinoma (Panc02). Here, we tested the short- and long-term effects of MBTA therapy in established subcutaneous Panc02 tumors two times larger than in previous study and bilateral Panc02 models as well as the roles of CD4+ and CD8+ T lymphocytes in this therapy. The MBTA therapy resulted in eradication of 67% of Panc02 tumors with the development of long-term memory as evidenced by the rejection of Panc02 cells after subcutaneous and intracranial transplantations. The initial Panc02 tumor elimination is not dependent on the presence of CD4+ T lymphocytes, although these cells seem to be important in long-term survival and resistance against tumor retransplantation. The resistance was revealed to be antigen-specific due to its inability to reject B16-F10 melanoma cells. In the bilateral Panc02 model, MBTA therapy manifested a lower therapeutic response. Despite numerous combinations of MBTA therapy with other therapeutic approaches, our results show that only simultaneous application of MBTA therapy into both tumors has potential for the treatment of the bilateral Panc02 model.
- MeSH
- adenokarcinom imunologie patologie MeSH
- antigeny CD40 antagonisté a inhibitory MeSH
- imidazoly farmakologie MeSH
- imunoterapie MeSH
- kyseliny teichoové farmakologie MeSH
- ligandy MeSH
- lipopolysacharidy farmakologie MeSH
- mannany farmakologie MeSH
- myši MeSH
- nádory slinivky břišní imunologie patologie MeSH
- poly I-C farmakologie MeSH
- protokoly protinádorové kombinované chemoterapie farmakologie MeSH
- T-lymfocyty účinky léků imunologie MeSH
- toll-like receptory MeSH
- zvířata MeSH
- Check Tag
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
BACKGROUND: Dendritic cells (DCs) are professional antigen-presenting cells that are capable of inducing immune responses. DC-based vaccines are normally generated using a standard 5- to 7-day protocol. To shorten the DC-based vaccine production for use in cancer immunotherapy, we have developed a fast DC protocol by comparing standard DCs (Day 5 DCs) and fast DCs (Day 3 DCs). METHODS: We tested the generation of Day 5 versus Day 3 DCs using CellGro media and subsequent activation by two activation stimuli: Poly (I:C) and LPS. We evaluated DC morphology, viability, phagocyte activity, cytokine production and ability to stimulate antigen-specific T cells. RESULTS: Day 5 and Day 3 DCs exhibited similar phagocytic capacity. Poly (I:C)-activated Day 5 DCs expressed higher levels of the costimulatory and surface molecules CD80, CD86 and HLA-DR compared to Poly (I:C)-activated Day 3 DCs. Nevertheless, LPS-activated Day 5 and Day 3 DCs were phenotypically similar. Cytokine production was generally stronger when LPS was used as the maturation stimulus, and there were no significant differences between Day 5 and Day 3 DCs. Importantly, Day 5 and Day 3 DCs were able to generate comparable numbers of antigen-specific CD8(+) T cells. The number of Tregs induced by Day 5 and Day 3 DCs was also comparable. CONCLUSION: We identified monocyte-derived DCs generated in CellGro for 3 days and activated using Poly (I:C) similarly potent in most functional aspects as DCs produced by the standard 5 day protocol. These results provide the rationale for the evaluation of faster protocols for DC generation in clinical trials.
- MeSH
- antigen prezentující buňky účinky léků imunologie metabolismus MeSH
- antigenní specifita receptorů T-buněk imunologie MeSH
- buněčná diferenciace účinky léků imunologie MeSH
- buněčné kultury * MeSH
- časové faktory MeSH
- cytokiny biosyntéza MeSH
- dendritické buňky cytologie účinky léků imunologie metabolismus MeSH
- fagocytóza imunologie MeSH
- fenotyp MeSH
- imunofenotypizace MeSH
- imunoterapie MeSH
- lidé MeSH
- nádory imunologie metabolismus terapie MeSH
- poly I-C imunologie farmakologie MeSH
- protinádorové vakcíny imunologie MeSH
- T-lymfocyty - podskupiny imunologie metabolismus MeSH
- viabilita buněk MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
The expression of the transcription factor encoded by the Wilms tumor gene 1 (WT1) is associated with a variety of human cancers. WT1 protein has been reported to serve as a target antigen for tumor-specific immune responses. We observed that the immunization of mice with peptide vaccines derived from WT1 in a mixture with the CpG adjuvant (ODN 1826) by tattoo administration was superior to subcutaneous delivery of the peptides in combination with CpG formulated with the mineral oil adjuvant or a DNA vaccine or a recombinant vaccinia virus vaccine expressing the truncated WT1 protein. Tattooing with the WT1122-140 and WT1126-134 peptide elicited the response of WT1-specific interferon-γ-producing T cells. Peptide vaccine administered with a tattoo device had an antitumor effect on the growth of the prostate tumor cell line TRAMP-C2, provided that the transforming growth factor-β produced by tumor cells was neutralized by anti-TGFβ monoclonal antibody. The treatment of the tumor-bearing mice with 5-azadeoxycytidine or poly IC did not work in synergy with the peptide vaccine.
- MeSH
- adjuvancia imunologická MeSH
- azacytidin analogy a deriváty farmakologie terapeutické užití MeSH
- injekce intradermální MeSH
- monoklonální protilátky imunologie MeSH
- myši inbrední BALB C MeSH
- myši MeSH
- nádorové buněčné linie MeSH
- nádory prostaty terapie MeSH
- poly I-C farmakologie terapeutické užití MeSH
- proteiny WT1 aplikace a dávkování imunologie MeSH
- protinádorové látky farmakologie terapeutické užití MeSH
- protinádorové vakcíny aplikace a dávkování MeSH
- subjednotkové vakcíny aplikace a dávkování MeSH
- tetování MeSH
- transformující růstový faktor beta antagonisté a inhibitory imunologie MeSH
- zvířata MeSH
- Check Tag
- mužské pohlaví MeSH
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
BACKGROUND: For clinical applications, dendritic cells (DCs) need to be generated using GMP-approved reagents. In this study, we tested the characteristics of DCs generated in two clinical grade culture media and activated by three maturation stimuli, Poly I: C, LPS and the mixture of proinflammatory cytokines in order to identify the optimal combination of culture media and activation stimulus for the clinical use. METHOD: We tested DCs generation using two GMP-certified culture media, CellGro and RPMI+5% human AB serum and evaluated DCs morphology, viability and capapability to mature. We tested three maturation stimuli, PolyI:C, LPS and the mixture of proinflammatory cytokines consisting of IL-1, IL-6, TNF and prostaglandin E2. We evaluated the capacity of activated DCs to induce antigen-specific T cells and regulatory T lymphocytes. RESULTS: Cell culture in CellGro resulted in a higher yield of immature DCs resulting from increased number of adherent monocytes. DCs that were generated in CellGro and activated using Poly I:C were the most efficient in expanding antigen-specific T cells compared to the DCs that were generated in other media and activated using LPS or the cocktail of proinflammatory cytokines. A comparison of all tested combinations revealed that DCs that were generated in CellGro and activated using Poly I:C induced low numbers of regulatory T cells. CONCLUSION: In this study, we identified monocyte-derived DCs that were generated in CellGro and activated using Poly I:C as the most potent clinical-grade DCs for the induction of antigen-specific T cells.
- MeSH
- buněčná diferenciace účinky léků MeSH
- dendritické buňky cytologie účinky léků MeSH
- epitopy imunologie MeSH
- fenotyp MeSH
- imunoterapie metody MeSH
- klinické zkoušky jako téma MeSH
- kultivační média farmakologie MeSH
- lidé MeSH
- nádory imunologie terapie MeSH
- poly I-C farmakologie MeSH
- proliferace buněk účinky léků MeSH
- regulační T-lymfocyty cytologie účinky léků imunologie MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Dendritic cells (DC) have been shown to be efficient antigen-presenting cells (APC) and, as such, could be considered ideal candidates for cancer immunotherapy. Immature DC (iDC) efficiently capture surrounding antigens; however, only mature DC (mDC) prime naive T lymphocytes. Clinical trials using DC-based tumor vaccines have achieved encouraging, but limited, success, possibly due to the use of immature or incompletely mature DC. Thus, it was apparent that a method capable of generating large numbers of fully functional iDC, their pulsing with desired form of tumor antigens and the subsequent complete and reproducible maturation of iDC is needed. Therefore, we compared two different methods of producing large numbers of iDC. Both protocols yielded comparable numbers of cells with an iDC phenotype with phagocytic function. We next determined which of the clinically applicable activators could induce the complete and reproducible maturation of DC, in order to define the most suitable combination for future clinical trials. Only a combination of TNFalpha + Poly (I:C), or a previously described cytokine cocktail of TNFalpha + IL-1beta + IL-6 + prostaglandin E2, induced the complete activation of the whole DC population, as assessed by the cell surface expression of CD83 and costimulatory molecules. The matured DC were functionally superior to iDC in their ability to stimulate the proliferation of allogeneic lymphocytes and autologous keyhole limpet hemocyanin (KLH)-specific T lymphocytes. Furthermore, only the combination of TNFalpha + Poly (L:C) activated DC to produce large amounts of biologically active p70 IL-12. Thus DC maturation by TNFalpha + Poly (I:C) could efficiently bias T cell response towards Th1 response. Implementation of our results into clinical protocols used for DC generation could be beneficial for future immunotherapy trials.
- MeSH
- apoptóza MeSH
- buněčná diferenciace MeSH
- buněčné dělení MeSH
- buněčné kultury * metody MeSH
- dendritické buňky * cytologie sekrece účinky léků MeSH
- dinoproston farmakologie MeSH
- fagocytóza MeSH
- faktor stimulující granulocyto-makrofágové kolonie farmakologie MeSH
- hemokyanin imunologie MeSH
- imunofenotypizace MeSH
- interleukin-1 farmakologie MeSH
- interleukin-12 chemie sekrece MeSH
- interleukin-13 farmakologie MeSH
- interleukin-6 farmakologie MeSH
- kultivované buňky cytologie sekrece účinky léků MeSH
- lidé MeSH
- melanom patologie MeSH
- nádorové buňky kultivované MeSH
- poly I-C farmakologie MeSH
- průtoková cytometrie MeSH
- rekombinantní proteiny farmakologie MeSH
- reprodukovatelnost výsledků MeSH
- terciární struktura proteinů MeSH
- test smíšené lymfocytární kultury MeSH
- TNF-alfa farmakologie MeSH
- viabilita buněk MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- práce podpořená grantem MeSH
- MeSH
- centrifugace - gradient hustoty MeSH
- cytosin MeSH
- dextrany farmakologie MeSH
- ethylaminy MeSH
- interferony biosyntéza MeSH
- L buňky (buněčná linie) metabolismus MeSH
- molekulová hmotnost MeSH
- nukleosidy MeSH
- poly I-C farmakologie MeSH
- polynukleotidy MeSH
- ribonukleasy MeSH
- sacharosa MeSH
- spektrofotometrie MeSH
- ultracentrifugace MeSH
- virus vezikulární stomatitidy, kmen Indiana MeSH
