The Modulation of Cell Wall-Associated Triton X-100 Soluble Protein (TSP) Antigens of Mycobacterium tuberculosis on Murine Dendritic Cells

Yung-Choon Yoo,Young-Joo Bae,Tae-Hyun Paik,Junglim Lee 한국실험동물학회 2008 Laboratory Animal Research Vol.24 No.4

Mycobacterium tuberculosis or its secreted protein antigens are known as a potent inducer of maturation process of dendritic cells, during which the expression of cell surface molecules and the capability of antigen presentation are increased, resulting in potent interaction with T cells to initiate the acquired immune response against tuberculosis. In this study, the cell wall-associated Triton X-100 soluble protein (TSP) antigens, TSP- BCG, TSP-H37Ra, TSP- H37Rv and TSP-K, were tested on murine bone marrowderived dendritic cells to determine the capability of their immunologic modulation. Only TSP-H37Rv stimulated murine dendritic cells showed moderate production of IL-6 and MIP-1α compared to those of LPS. TSP-H37Rv antigen did not induce the production of IL-12, TNF-α or IL-10. Other TSP antigenstimulated murine dendritic cells showed minimal or no induction of cytokines (IL-6, IL-12, TNF-α or IL-10) or chemokines (MIP-1α or RANTES). The murine dendritic cells showed the production of NO as well as the expression of iNOS mRNA after LPS stimulation, however, none of TSP antigen-stimulated murine dendritic cells induced either NO or iNOS mRNA expression. The allostimulation of T cells by murine dendritic cells, which were stimulated with each TSP antigens, showed insignificant increase in T cell proliferation activity in mixed lymphocyte reaction. These results suggested that cell wall-associated TSP antigen isolated from Mycobacterium tuberculosis H37Rv strain acts as a more potent stimulator on murine dendritic cells than any other TSP antigens, even the stimulation from TSP-H37Rv was not enough to activate the maturation of process of murine dendritic cells.

제대혈 CD34 양성세포를 이용한 수상돌기 세포의 배양

황보권,홍대식,김찬규,조성태,이규택,박성규,원종호,백승호,서원석,김숙자,박희숙 대한조혈모세포이식학회 1998 대한조혈모세포이식학회지 Vol.3 No.2

Background: Dendritic cells have a distinctive morphology and immunostimulatory potential. These cells are thought to be the major APC involved in triggering primary T-cell responses and express little linage specific surface markers. We tried to establish the method for the culture of dendritic cells with CD34^(+) cells from cord blood. Methods: Cord blood was collected during normal full-term delivery CD34^(+) cells were purified with Magnetic Separation Column, and cultured over 14 days in the presence of GM-CSF and TNF-α. Flow-cytometric analysis was conducted before and after culture. CFU-GM and CFU-DL culture was done with cells, harvested from 14 days of culture. Results: 1) After 14 days of culture with CD34^(+) cells (5×10^(5)), total cell number was expanded upto 3.2±3.35×10^(7), about 63 fold, and about 20% of them were adherent cells with dendritic morphology of numerals long and thin cytoplasmic projections. 2) Flow-cytometric analysis of surface marker expression were examined before and after culture for 14 days. The expression of CD34 was 69.1±13.01 (%), initially, and was decreased to 0.5±0.41 (%) at 14th day. The expression of HLA-DR, CD14 and CD1a were increased to 82.4±11.47 (%), 39.8±30.00 (%) and 15.7±7.73 (%) from 72.9±14.79 (%), 3.4±0.25 (%) and 1.5 ±0.64 (%). 3) The ratio of CFU-GM (94.5±86.97) and CFU-DL (8±8.49) colonies were about 10 to 1. Conclusion: CD34^(+) enriched cord blood can be expanded upto 63 folds with GM-CSF and TNF-α. This study established the method for the culture and identification of dendritic cells morphologically and immunophenotypically. Clinical application of dendritic cells as an adoptive immunotherapy and therapeutic vaccination in infectious or malignant disease needs further investigations about the right time of exposure to antigens, the dosage of cells, and the root of administration of the dendritic cells.

갑상선암 환자의 말초혈액 백혈구로부터 수지상세포의 유도분화

이대희 고신대학교(의대) 고신대학교 의과대학 학술지 2006 고신대학교 의과대학 학술지 Vol.21 No.1

Background : Recent studies suggest that immunization with autologous dendritic cells(DCs) result in protective immunity and rejection of established tumors in various human malignancies, It has been reported that a dense infiltration of dendritic cells correlates with a favorable prognosis in several types of cancer. The purpose of this study is to determine whether DCs are generated from peripheral blood monocytes by using cytokines such as Fit-3 ligand, granulocyte macrophage-colony stimulating factor (GM-CSF), IL-4, and TNF- a,and whether cytotoxic T cells activated against the medullary thyroid carcinoma(MTC) tissues by the DCs. Methods : Peripheral blood was obtained from 2 patients with MTC. DCs were established from mononuclear leukocytes by culturing in the presence of Flt-3 ligand,GM-CSF, I卜4, and TNF- a for 14 days. At day 14,the differentiated DCs was analyzed morphologically. The immunophenotypic features of DCs such as CDla,CD83, and CD86 were analyzed by Immunofluorelescence microscopy. At day 15, DCs were incubated with thyroid cancer tissues and normal thyroid tissues for 7 additional days,respectively. Results : DCs were generated from the peripheral blood mononuclear cells. The generated cells showed the typical morphology of DCs. Activated cytotoxic T lymphocytes (CTLs) were observed. DCs attached to the thyroid cancer tissues and the CTLs were attached to the MTC tissues on scanning electron microscope. Conclusion : We could differentiate DCs from the peripheral blood mononuclear cells,And the DCs activate the CTLs which able to attack the MTC tissues. These results suggest that DCs can be used as adjuvants for immunotherapy of MTC. And this study represent the basis for the develop of new therapeutic strategies not only in MTC but also in the other malignancies. Background : Recent studies suggest that immunization with autologous dendritic cells(DCs) result in protective immunity and rejection of established tumors in various human malignancies, It has been reported that a dense infiltration of dendritic cells correlates with a favorable prognosis in several types of cancer. The purpose of this study is to determine whether DCs are generated from peripheral blood monocytes by using cytokines such as Fit-3 ligand, granulocyte macrophage-colony stimulating factor (GM-CSF), IL-4, and TNF- a,and whether cytotoxic T cells activated against the medullary thyroid carcinoma(MTC) tissues by the DCs. Methods : Peripheral blood was obtained from 2 patients with MTC. DCs were established from mononuclear leukocytes by culturing in the presence of Flt-3 ligand,GM-CSF, I卜4, and TNF- a for 14 days. At day 14,the differentiated DCs was analyzed morphologically. The immunophenotypic features of DCs such as CDla,CD83, and CD86 were analyzed by Immunofluorelescence microscopy. At day 15, DCs were incubated with thyroid cancer tissues and normal thyroid tissues for 7 additional days,respectively. Results : DCs were generated from the peripheral blood mononuclear cells. The generated cells showed the typical morphology of DCs. Activated cytotoxic T lymphocytes (CTLs) were observed. DCs attached to the thyroid cancer tissues and the CTLs were attached to the MTC tissues on scanning electron microscope. Conclusion : We could differentiate DCs from the peripheral blood mononuclear cells,And the DCs activate the CTLs which able to attack the MTC tissues. These results suggest that DCs can be used as adjuvants for immunotherapy of MTC. And this study represent the basis for the develop of new therapeutic strategies not only in MTC but also in the other malignancies.

관해된 급성 골수성 백혈병 환자의 말초 혈액 단핵구로 부터수지상 세포 분화 유도 및 자가 T 림프구 활성화 유도

김양수 고신대학교(의대) 고신대학교 의과대학 학술지 2003 고신대학교 의과대학 학술지 Vol.18 No.1

Background and purposes Evidences for the expression of immunogenecity of AML blast cells are supported by graft versus leukemic reaction, the effect of donor lymphocyte transfusion and in vitro studies with cytotoxic T cell. Dendritic cells (DCs) are important antigen presenting cells in the development of antileukemic T cell cytotoxic responses. The purpose of this study was to analyze the immunophenotype of dendritic cells cultured from peripheral blood mononuclear cells of AML patients in complete remission and to evaluate whether this immunomodulated DC evoked cytotoxic T cells are capable to kill the leukemic blast or not. Methods Peripheral blood was obtained from 6 patients of AML in complete remission and 2 healthy controls(HC). Dendritic cell were established from monocyte by culturing in the presence of GM-CSF, Flt-3 ligand, TNF-α, and IL-4 for 8 days. At day 8, cell number was counted and expression of surface marker typical for DCs was analyzed morphologically. The immunophenotypic feature of DCs such as CD1a, CD83, and CD86 was anlayzed by flow cytometry. Thereafter in same day, day 8, DCs were incubated with lysate of leukemic blast and cultured with autologous lymphocyte, because DCs were potent stimulators in an allogenic mixed lymphocyte reaction(MLR). Cytotoxicity against autologouse blast cells was measured by PKH67 assay. Results After 8 days of culture, cells from all 8 samples exhibited morphological and immunophenotypic features of DCs including expression of CD1a, CD83, and CD86. There was no difference in expression of surface marker by DC from AML patients and healthy control. Of 6 samples, 2 were analyzed as an effective stimulator in the autologous MLR and induced antileukemic cytotoxicity. At an effector: target -ratio of over 10: 1(and 20: 1) lymphocyte from two patients stimulated with DC pulsed with blast lysates killed about 40-80 % of autologouse blast. Conclusion Dendritic cell can be generated in AML patients especially during hematopoietic regeneration following chemotherapy. This study indicated that dendritic cells with enhanced antigenecity can be generated from mononuclear cell of AML in CR, that these cells can effectively prime autologous cytotoxic T cell in vitro, and that they may be used as potential vaccines in the immunotherapy or therapeutic strategy for minimal residual disease of AML

관해된 급성 골수성 백혈병 환자의 말초 혈액 단핵구로 부터 수지상 세포 분화 유도 및 자가 T 림프구 활성화 유도

김양수 고신대학교 의학부 2003 高神大學校 醫學部 論文集 Vol.18 No.1

Background and purposes: Evidences for the expression of immunogenecity of AML blast cells are supported by graft versus leukemic reaction, the effect of donor lymphocyte transfusion and in vitro studies with cytotoxic T cell. Dendritic cells (DCs) are important antigen presenting cells in the development of antileukemic T cell cytotoxic reponses. The purpose of this study was to analyze the immunophenotype of dendritic cells cultured from peripheral blood mononuclear cells of AML patients in complete remission and to evaluate whether this immunomodulated DC evoked cytotoxic T cells are capable to kill the leukemic blast or not. Methods: Peripheral blood was obtained from 6 patients of AML in complete remission and 2 healthy controls(HC). Dendritic cell were established from monocyte by culturing in the presence of GM-CSF, Flt-3 ligand. TNF-a, and IL-4 for 8 days. At day 8, cell number was counted and expression of surface marker typical for DCs was analyzed morphologically. The immunophenotypic feature of DCs such as CD1a. CD83, and CD86 was anlayzed by flow cytometry. Thereafter in same day, day 8, DCs were incubated with lysate of leukemic blast and cultured with autologous lymphocyte, because DCs were potent stimulators in an allogenic mixed lymphocyte reaction(MLR). Cytotoxicity against autologouse blast cells was measured by PKH67 assay. Results: After 8 days of culture, cells from all 8 samples exhibited morphological and immunophenotypic features of DCs including expression of CDla, CD83, and CD86. There was no difference in expression of surface marker by DC from AML patients and healthy control. Of 6 samples, 2 were analyzed as an effective stimulator in the autologous MLR and induced antileukemic cytotoxicity. At an effector: target -ration of over 10: 1(and 20: 1) lymphoyte from two patients stimulated with DC pulsed with blast lysates killed about 40-80 % of autologouse blast. Conclusion: Dendritic cell can be generated in AML patients especially during hematopoietic regeneration following chemotherapy. This study indicated that dendritic cells with enhanced antigenecity can be generated from monouclear cell of AML in CR, that these cells can effectively prime autologous cytotoxic T cell in vitro, and that they may be used as potential vaccines in the immunotherapy or therapeutic strategy for minimal residual disease of AML

Modulation of dendritic cell function by Trichomonas vaginalis-derived secretory products

( Min Ji Song ),( Jong Joo Lee ),( Young Hee Nam ),( Tae Gyun Kim ),( Youn Wook Chung ),( Mik Young Kim ),( Ye Eun Choi ),( Myeong Heon Shin ),( Hyoung Pyo Kim ) 생화학분자생물학회(구 한국생화학분자생물학회) 2015 BMB Reports Vol.48 No.2

Trichomoniasis caused by the parasitic protozoan Trichomonas vaginalis is the most common sexually transmitted disease in the world. Dendritic cells are antigen presenting cells that initiate immune responses by directing the activation and differentiation of naive T cells. In this study, we analyzed the effect of Trichomonas vaginalis-derived Secretory Products on the differentiation and function of dendritic cells. Differentiation of bone marrow-derived dendritic cells in the presence of T. vaginalis-derived Secretory Products resulted in inhibition of lipopolysaccharide-induced maturation of dendritic cells, down-regulation of IL-12, and up-regulation of IL-10. The protein components of T. vaginalis-derived Secretory Products were shown to be responsible for altered function of bone marrow- derived dendritic cells. Chromatin immunoprecipitation assay demonstrated that IL-12 expression was regulated at the chromatin level in T. vaginalis-derived Secretory Productstreated dendritic cells. Our results demonstrated that T. vaginalis- derived Secretory Products modulate the maturation and cytokine production of dendritic cells leading to immune tolerance. [BMB Reports 2015; 48(2): 103-108]

결핵성 경부 림프절염에서 수지상돌기세포의 침윤과 임상양상의 연관성

임성용 ( Seoung Young Lim ),신종욱 ( Jong Wook Shin ),김재열 ( Jae Yoel Kim ),박인원 ( In Whn Park ),김미경 ( Mi Kyung Kim ),최병휘 ( Byoung Whui Choi ),정재우 ( Jae Woo Jung ),이영우 ( Young Woo Lee ),최재철 ( Jae Cheol Choi ) 대한결핵 및 호흡기학회 2006 Tuberculosis and Respiratory Diseases Vol.60 No.5

연구배경 : 결핵성 경부 림프절염은 우리나라에서 폐결핵만큼 빈도가 높은 질환이다. 이 질병에서 수지 상돌기세포는 초기의 항원 제시역할을 하고 있다. 그러나 림프절염의 임상 양상과 관련된 항원제시세포의 역할은 아직 명확하게 밝혀져 있지 않은 상태이다. 경부 림프절의 수지상 돌기세포의 침윤과 임상양상과의 연관성을 알아보기 위해 본 연구를 시행하였다. 방법 : 환자들의 입원기록 및 방사선사진을 바탕으로 후향적으로 고찰하였다. 72례의 조직표본을 대상으로 항산균도말염색을 다시 시행하였고, 수지상돌기세포의 단클론항체로 S-100b를 이용하여 면역조직 화학염색을 시행한 후, 각각 결핵성 육아종안의 수지 상돌기세포의 수를 세어 비교분석하였다. 결과 : 결핵성 경부 림프절염이 있는 환자들의 30%가 폐결핵의 과거력이 있거나 현재 폐결핵을 앓고 있는 상태이었고 21%의 환자에서 항산균도말염색 양성을 보였다. 이들 한 육아종안에 침윤된 수지상돌기세포의 수는 평균 113.0±7.0개이었다. 육아종내 수지상돌기세포의 침윤수가 증가됨에 따라 발열과 기침의 빈도는 감소하였고 항산균도말염색상에서 결핵균의 수가 더 감소하는 결과를 보였으며, 다중로짓회귀분석을 보면, 수지상돌기세포의 침윤은 특징적으로 발열에 기여하여하는 것으로 나타났다. 결론 : 수지상돌기세포가 결핵성 경부 림프절염에서 발열과 기침 등의 전신증상을 줄이고, 결핵균의 침윤정도를 감소시키는 것으로 확인되었고, 이는 수지상돌기세포가 Mycobacterium tuberculosis의 감염을 조절하고 이와 함께 면역반응도 조절하여, 결핵성 경부 림프절염에서의 임상양상을 결정하는 것으로 생각된다. Background : Cervical tuberculous lymphadenopathy is a very common disease with a similar incidence to pulmonary tuberculosis. Dendritic cells play a role of initial antigen presentation of this illness. Nevertheless, the precise role of these antigen-presenting cells according to the clinical features in unclear. The aim of this study was to determine the clinical implication of dendritic cell infiltration in the cervical lymph nodes. Methods : A review of the clinical characteristics was carried out retrospectively based on the clinical records and radiography. Immunohistochemical staining was performed on the available histology specimens of 72 cases using the S-100b polyclonal antibody for dendritic cells. The number of dendritic cells with tuberculous granuloma were determined. A X2 test, unpaired T test and multiple logistic regression analysis were performed. Results : Thirty percent of subjects had previous or concurrent pulmonary TB. Twenty one percent of cases showed a positive reaction on the AFB stain. Within a granuloma, the number of infiltrated dendritic cells was 113.0±7.0. The incidence of fever and cough decreased with increasing infiltration of dendritic cells Multivariate regression analysis showed that the infiltration of dendritic cells could significantly contribute to fever. Conclusion : Overall, dendritic cells can control a Mycobacterium tuberculosis infection and modulate the immune response, as well as resolve the clinical manifestations of TB lymphadenopathy. (Tuberc Respir Dis 2006; 60: 523-531)

Optimization of the concentration of autologous serum for generation of leukemic dendritic cells from acute myeloid leukemic cells for clinical immunotherapy

Choi, Bo-Hwa,Kang, Hyun-Kyu,Park, Jung-Sun,Kim, Sang-Ki,Pham, Than-Nhan Nguyen,Zhu, Xiao-Wei,Cho, Duck,Nam, Jong-Hee,Chung, Ik-Joo,Kim, Young-Jin,Rhee, Joon-Haeng,Kim, Hyeoung-Joon,Lee, Je-Jung Wiley-Liss 2006 JOURNAL OF CLINICAL APHERESIS Vol.21 No.4

<P>Clinical application of immunotherapy for acute myeloid leukemia (AML) requires the efficient induction of dendritic cells (DCs) from AML blast cells using in vitro culture. We examined the effect of autologous serum on the properties of leukemic DCs derived from leukemic cells of AML patients by culture in AIM-V medium with GM-CSF, IL-4, TNF-α, and 0, 2, 5, or 10% human autologous serum. The expressions of CD80, CD83, CD86, and HLA-DR were upregulated under all culture conditions; however, 10% autologous serum induced the highest expression levels of several molecules. The capacity of leukemic DCs to stimulate allogeneic T cells increased with increasing serum concentration. Stimulation of autologous CD3+ T cells with leukemic DCs grown in the presence of various concentrations of autologous serum resulted in induction of more IFN-γ-secreting cells than was the case for unprimed CD3+ T cells. Leukemic DCs cultured with 10% autologous serum induced the highest numbers of IFN-γ-secreting cells and CD8+CD56+ T cells from autologous T cells. These results suggest that culture of AML blast cells in the presence of autologous serum could be used to generate leukemic DCs for immunotherapy against AML. The highest serum concentration appeared optimal for generating the most potent leukemic DCs. J. Clin. Apheresis. © 2006 Wiley-Liss, Inc.</P>

위암 환자의 말초혈액 조혈모세포로부터 수지상세포로의 분화 유도

최지연,이대희,정현기,조성래 고신대학교(의대) 고신대학교 의과대학 학술지 2002 고신대학교 의과대학 학술지 Vol.17 No.1

Background : Throughout the body, Dendritic Cells (DCs) capture the antigens, migrate to draining lymphoid organs and mature to present the processed antigens to naive T cells to generate the effector cytotoxic T cells (CTLs) or helper T cells. The granulocyte-macrophage colony stimulating factor (GM-CSF) and tumor necrosis factor-a (TNF-a) cooperate in vitro generation of DCs from Peripheral Blood Stem Cells (PBSCs). Interleukin-4 (IL-4) is an another important factor in promoting DCs outgrowth and expression of CD1a with costimulatory molecules and in blocking monocytic differentiation. The aim of this study was induction of differentiation and maturation of dendritic cells from PBSCs in patient with stomach cancer. Methods : The CD34+ PBSCs were obtained from the patients with stomach cancer and divided the samples into 6 groups. Flt-3 Ligand (FL) (F group), GM-CSF (G group), IL-4 (I group), TNF-a (T group), GM-CSF and IL-4 (GI group), FL, GM-CSF, IL-4 and TNF-a (FGIT group) were added to X-VIVO culturing media. The cells were cultured for 2 weeks, then, examined the morphology of and functions the cells with phase contrast and fluorescence microscopes. Results : The generated cells with FL, GM-CSF, IL-4 and TNF-a showed typical morphology of DCs including multiple dendrites and profuse cytoplasm. The cells stained positively with CD1a, CD83 and CD86. FL, GM-CSF, IL-4 and TNF-a. Conclusion : Large quantities of mature DCs from PBSCs in a patient with stomach cancer was possible using FL, GM-CSF, IL-4 and TNF-a. Further studies are needed with various types of diseases and culture environments.

Ursolic acid isolated from Uncaria rhynchophylla activates human dendritic cells via TLR2 and/or TLR4 and induces the production of IFN-γ by CD4+ naive T cells

Jung, T.Y.,Pham, T.N.N.,Umeyama, A.,Shoji, N.,Hashimoto, T.,Lee, J.J.,Takei, M. North-Holland ; Elsevier Science Ltd 2010 european journal of pharmacology Vol.643 No.2

Ursolic acid is triterpene isolated from Uncaria rhynchophylla and is a pharmacologically active substance. The induction of dendritic cell maturation is critical for the induction of Ag-specific T-lymphocyte response and may be essential for the development of human vaccine relying on T cell immunity. In this study, we investigated that the effect of Ursolic acid on the phenotypic and functional maturation of human monocyte-derived dendritic cells in vitro. Dendritic cells harvested on day 8 were examined using functional assay. The expression levels of CD1a, CD80, CD83, CD86, HLA-DR and CCR7 on Ursolic acid-primed dendritic cells was slightly enhanced. Ursolic acid dose-dependently enhanced the T cell stimulatory capacity in an allogeneic mixed lymphocyte reaction, as measured by T cell proliferation. The production of IL-12p70 induced by Ursolic acid-primed dendritic cells was inhibited by the anti-Toll-like receptor-2 (TLR2) mAb and anti-TLR4 mAb. Moreover, Ursolic acid-primed dendritic cells expressed levels of mRNA coding for both TLR2 and TLR4. The majority of cells produced considerable interferon-gamma (IFN-γ), but also small amounts of interleukin (IL-4)-4. Ursolic acid-primed dendritic cells have an intermediate migratory capacity towards CCL19 and CCL21. These results suggest that Ursolic acid modulates human dendritic cells function in a fashion that favors Th1 polarization via the activation of IL-12p70 dependent on TLR2 and/or TLR4, and may be used on dendritic cells-based vaccines for cancer immunotherapy.