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Autogeneic RNA-electroporated CD40-ligand activated B-cells from hepatocellular carcinoma patients induce CD8+ T-cell responses ex vivo

Autogeneic RNA-electroporated CD40-ligand activated B-cells from hepatocellular carcinoma patients induce CD8+ T-cell responses ex vivo

Since dendritic cells (DCs) constitute only 0.1–0.5% of human peripheral blood mononuclear cells (PBMCs), and generation of DCs from monocytes or stem cells is difficult and expensive, we choose B-lymphocytes as an alternative, cost-effective source of antigen presenting cells (APC). Aim: To induce...

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Main Authors: Shen, S., Xu, Z., Qian, X., Ding, Y., Yu, L., Liu, B.
Format: Article
Language:English
Published: Інститут експериментальної патології, онкології і радіобіології ім. Р.Є. Кавецького НАН України 2007
Series:Experimental Oncology
Subjects:
Original contributions
Online Access:http://dspace.nbuv.gov.ua/handle/123456789/138573
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id irk-123456789-138573
record_format dspace
institution Digital Library of Periodicals of National Academy of Sciences of Ukraine
collection DSpace DC
language English
topic Original contributions
Original contributions
spellingShingle Original contributions
Original contributions
Shen, S.
Xu, Z.
Qian, X.
Ding, Y.
Yu, L.
Liu, B.
Autogeneic RNA-electroporated CD40-ligand activated B-cells from hepatocellular carcinoma patients induce CD8+ T-cell responses ex vivo
Experimental Oncology
description Since dendritic cells (DCs) constitute only 0.1–0.5% of human peripheral blood mononuclear cells (PBMCs), and generation of DCs from monocytes or stem cells is difficult and expensive, we choose B-lymphocytes as an alternative, cost-effective source of antigen presenting cells (APC). Aim: To induce specific CTLs response ex vivo by CD40L activated B-cells (CD40-B) transfected with hepatocellular carcinoma (HCC) total RNA. Methods: To induce CD40-B PBMCs of patients with HCC were isolated by Ficoll technique and cultured in RMPI 1640 supplemented with 10% fetal calf serum (FCS), sCD40L (2 µg/ml), recombinant human interleukin-4 (IL-4) (4 ng/ml). The expression of CD80 and CD86 was evaluated by flow cytometry. The level of interleukin-12 (IL-12) produced by cultured B-lymphocytes was measured using enzyme-linked immunosorbent assay (ELISA). HCC patient’s T-lymphocytes were obtained from PBMCs cultured in RMPI 1640 supplemented with 10% FCS, 2 ng/mL IL-4 and 10 ng/ml IL-7. CD40-B transfected with tumor total RNA isolated from HCC cells were used to induce specific CTL proliferation. The level of IFN-g was measured using ELISA and the expression of CD8 was determined by FCAS. Specific cytotoxicity was measured using MTT method. Results: The results show that the activated B-lymphocytes were easily expandable and formed large clones, and a high expression of CD80/CD86 and a high IL-12 secretion by CD40-B was registered. CD40-B transfected with tumor total RNA can induce CTLs to express CD8 and generate IFN-g at high levels. Compared to the control group, the specific cytotoxicity of CTLs was up-regulated. Conclusion: These findings demonstrate that CD40-B-cells electroporated with total RNA derived from carcinoma cells can be used as alternative APC for the induction of antigen-specific CD8+ T-cell responses, which might be used in HCC immunotherapy.
format Article
author Shen, S.
Xu, Z.
Qian, X.
Ding, Y.
Yu, L.
Liu, B.
author_facet Shen, S.
Xu, Z.
Qian, X.
Ding, Y.
Yu, L.
Liu, B.
author_sort Shen, S.
title Autogeneic RNA-electroporated CD40-ligand activated B-cells from hepatocellular carcinoma patients induce CD8+ T-cell responses ex vivo
title_short Autogeneic RNA-electroporated CD40-ligand activated B-cells from hepatocellular carcinoma patients induce CD8+ T-cell responses ex vivo
title_full Autogeneic RNA-electroporated CD40-ligand activated B-cells from hepatocellular carcinoma patients induce CD8+ T-cell responses ex vivo
title_fullStr Autogeneic RNA-electroporated CD40-ligand activated B-cells from hepatocellular carcinoma patients induce CD8+ T-cell responses ex vivo
title_full_unstemmed Autogeneic RNA-electroporated CD40-ligand activated B-cells from hepatocellular carcinoma patients induce CD8+ T-cell responses ex vivo
title_sort autogeneic rna-electroporated cd40-ligand activated b-cells from hepatocellular carcinoma patients induce cd8+ t-cell responses ex vivo
publisher Інститут експериментальної патології, онкології і радіобіології ім. Р.Є. Кавецького НАН України
publishDate 2007
topic_facet Original contributions
url http://dspace.nbuv.gov.ua/handle/123456789/138573
citation_txt Autogeneic RNA-electroporated CD40-ligand activated B-cells from hepatocellular carcinoma patients induce CD8+ T-cell responses ex vivo / S. Shen, Z. Xu, X. Qian, Y. Ding, L. Yu, B. Liu // Experimental Oncology. — 2007. — Т. 29, № 2. — С. 137-143. — Бібліогр.: 49 назв. — англ.
series Experimental Oncology
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spelling irk-123456789-1385732018-06-20T03:10:05Z Autogeneic RNA-electroporated CD40-ligand activated B-cells from hepatocellular carcinoma patients induce CD8+ T-cell responses ex vivo Shen, S. Xu, Z. Qian, X. Ding, Y. Yu, L. Liu, B. Original contributions Since dendritic cells (DCs) constitute only 0.1–0.5% of human peripheral blood mononuclear cells (PBMCs), and generation of DCs from monocytes or stem cells is difficult and expensive, we choose B-lymphocytes as an alternative, cost-effective source of antigen presenting cells (APC). Aim: To induce specific CTLs response ex vivo by CD40L activated B-cells (CD40-B) transfected with hepatocellular carcinoma (HCC) total RNA. Methods: To induce CD40-B PBMCs of patients with HCC were isolated by Ficoll technique and cultured in RMPI 1640 supplemented with 10% fetal calf serum (FCS), sCD40L (2 µg/ml), recombinant human interleukin-4 (IL-4) (4 ng/ml). The expression of CD80 and CD86 was evaluated by flow cytometry. The level of interleukin-12 (IL-12) produced by cultured B-lymphocytes was measured using enzyme-linked immunosorbent assay (ELISA). HCC patient’s T-lymphocytes were obtained from PBMCs cultured in RMPI 1640 supplemented with 10% FCS, 2 ng/mL IL-4 and 10 ng/ml IL-7. CD40-B transfected with tumor total RNA isolated from HCC cells were used to induce specific CTL proliferation. The level of IFN-g was measured using ELISA and the expression of CD8 was determined by FCAS. Specific cytotoxicity was measured using MTT method. Results: The results show that the activated B-lymphocytes were easily expandable and formed large clones, and a high expression of CD80/CD86 and a high IL-12 secretion by CD40-B was registered. CD40-B transfected with tumor total RNA can induce CTLs to express CD8 and generate IFN-g at high levels. Compared to the control group, the specific cytotoxicity of CTLs was up-regulated. Conclusion: These findings demonstrate that CD40-B-cells electroporated with total RNA derived from carcinoma cells can be used as alternative APC for the induction of antigen-specific CD8+ T-cell responses, which might be used in HCC immunotherapy. Поскольку дендритные клетки (ДК) составляют только 0,1–0,5% мононуклеаров периферической крови (МПК) человека, и получение ДК из моноцитов и стволовых кроветворных клеток является трудоемкой и дорогостоящей процедурой, выбраны В-лимфоциты в качестве альтернативного источника антигенпрезентирующих клеток (АПК). Цель: изучить возможность активированных CD40-лигандом В-лимфоцитов (CD40-B), трансфецированных суммарной РНК, выделенной из клеток гепатоцеллюлярной карциномы, индуцировать специфический ответ цитотоксических Т-лимфоцитов ex vivo Методы: МПК пациентов с гепатоцеллюлярной карциномой изолировали на градиенте фиколла и культивировали в среде RMPI 1640 с 10% эмбриональной сыворотки телят (ЭСТ), sCD40 (2 мкг/мл) и рекомбинантным человеческим интерлейкином-4 (ИЛ-4, 4 нг/мл). Экспрессию СD80 и CD86 оценивали на проточном цитофлюориметре. Концентрацию интерлейкина-12, продуцируемого культивируемыми В-лимфоцитами, оценивали с помощью иммуноферментного анализа (ИФА). Т-лимфоциты получали из МПК пациентов с гепатоцеллюлярной карциномой и культивировали в среде RMPI 1640 с 10% ЭСТ, 2 нг/мл ИЛ-4 и 10 нг/мл ИЛ-7 и культивированы совместно с CD40- клетками, трансфецированными опухолевой РНК, для индукции пролиферации специфических цитотоксических Т-лимфоцитов. Уровень интерферона- (ИНФ-γ) определяли с помощью ИФА, а экспрессию CD8 на проточном цитофлуориметре. Специфическую цитотоксичность оценивали в МТТ тесте. Результаты: показано, что активированные in В-лимфоциты формируют большие колонии, на поверхности клеток отмечается высокая экспрессия антигенов CD80/CD86, и ими в повышенных количествах секретируется ИЛ-12. CD40-B-лимфоциты, трансфецированные опухолевой РНК, могут активировать CD + цитотоксические лимфоциты, которые продуцируют ИНФ-γ. Выводы: CD40-B-клетки, трансфецированные суммарной РНК, выделенной из опухолевых клеток больных с гепатоцеллюлярной карциномой, — это альтернативные АПК для индукции антигенспецифического CD + T-клеточного ответа, что может быть использовано для иммунотерапии пациентов с гепатоцеллюлярной карциномой. 2007 Article Autogeneic RNA-electroporated CD40-ligand activated B-cells from hepatocellular carcinoma patients induce CD8+ T-cell responses ex vivo / S. Shen, Z. Xu, X. Qian, Y. Ding, L. Yu, B. Liu // Experimental Oncology. — 2007. — Т. 29, № 2. — С. 137-143. — Бібліогр.: 49 назв. — англ. 1812-9269 http://dspace.nbuv.gov.ua/handle/123456789/138573 en Experimental Oncology Інститут експериментальної патології, онкології і радіобіології ім. Р.Є. Кавецького НАН України

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