Tumor immune suppression mechanism of tumor-derived osteopontin and enhanced antitumor immunotherapeutic effect of B cell-based vaccine transduced with modified adenoviral vector : 암 유래 osteopontin의 암 면역억제 작용기전 및 변형 adenovirus를 이용한 B 세포백신의 향상된 항암효과 연구

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dc.description학위논문 (박사)-- 서울대학교 대학원 : 약학과, 2014. 2. 강창율.-
dc.description.abstractBreaking the tumor-derived immunosuppressive environment and initiating strong immune responses against tumor antigens are both necessary for eliminate established tumors. Therefore, we investigated mechanisms of tumor-derived immunosuppression and attempted to develop more effective B cell based vaccine. These two different strategies were described in different sections respectively part I and part II.
Tumor cells secrete soluble factors to suppress host immunity against tumor for improvement of their survival. Expansion of myeloid derived suppressor cells (MDSCs) is one of host immune suppression mechanism derived by tumors. Extramedullary myelopoiesis has been known as a common phenomenon in the tumor bearing hosts, which cause abnormal myeloid cell expansion in the peripheral lymphoid organ and tumor site. Although it is assumed that inflammatory mediators contribute to tumor-induced extramedullary myelopoiesis, responsible factor and its contribution remains still elusive. Using cytokine array, we found that osteopontin was increased in both spleen lysates and sera in the tumor bearing mice. Based on this, we investigated whether tumor-derived osteopontin affect extramedullary myelopoiesis and MDSC accumulation in the tumor environment. Knock down and neutralizing of osteopontin in the tumor cells showed reduced tumor growth, splenomegaly and extramedullary myelopoiesis in the tumor bearing mice. In the primary cultured splenocytes, osteopontin increased the proliferation of lineageneg/CD127-/CD117+/Sca1- heterogeneous myeloid progenitor populations (LK cells). Moreover, osteopontin increased migration of bone marrow LK cells. These results suggest that osteopontin induced increased extramedullary myelopoiesis and subsequently accumulated immunosuppressive MDSCs. Taken together, these findings suggested that osteopontin could become therapeutic target to overcome tumor-derived immunosuppressive environment.
For successful clinical tumor immunotherapy outcomes, strong immune responses against tumor antigens must be generated. Cell based vaccines compromise one strategy with which to induce appropriate strong immune responses. Previously we established a natural killer T cell (NKT) ligand loaded and Her2/neu introduced modified adenoviral vector (Ad-k35HM) transduced-B cell based anticancer cellular vaccine. An Ad-k35HM transduced B cell vaccine elicited strong antigen specific cellular and humoral immune responses, subsequently showed inhibition of the in vivo growth of established tumors in a mouse model. In this study, we additionally investigated diverse characteristics of Ad-k35HM introduced B cell-based vaccine. Furthermore, the vaccine elicited HLA-A2 epitope specific cytotoxic T cell responses in B6.Cg (CB)-Tg (HLA-A/H2-D) 2Enge/Jat mice (AAD mice). These findings indicated that the Ad-k35 could be appropriate for the preclinical and clinical development of B cell-based anticancer immunotherapies.
dc.description.tableofcontentsTable of Contents

Abstract --------------------------------------------------------------------------------------i
Table of Contents --------------------------------------------------------------------------iv
List of Figures ------------------------------------------------------------------------------vii
Abbreviations -------------------------------------------------------------------------------ix

Part I. Tumor-derived osteopontin act as an immune suppressor factor through accumulating myeloid-derived suppressor cells (MDSC)

I. 1. Introduction-----------------------------------------------------------------------------2
I. 2. Materials and Methods --------------------------------------------------------------5
Cell culture and lentiviral vector shRNA transfection
FACS staining and ELISA
Quantitative real-time PCR
Bromodeoxyuridine (BrdU) assay
Neutralizing experiment
Cytokine array and western blot
I. 3. Results ---------------------------------------------------------------------------------12
Extramedullary myelopoiesis was increased in tumor bearing mice
Osteopontin increased in the tumor environment
Extramedullary myelopoiesis was induced by tumor-derived osteopontin
Neutralizing of osteopontin ameliorate splenomegaly and accumulation of myeloid progenitor cells
Tumor-derived osteopontin increased proliferation of myeloid progenitor cells
Osteopontin increased migration of bone marrow myeloid progenitor cells
Osteopontin aggravated tumor immunosuppressive environment
I. 4. Discussion------------------------------------------------------------------------------21

Part II. Enhanced antitumor immunotherapeutic effect of B cell-based vaccine transduced with modified adenoviral vector containing type 35 fiber structures

II. 1. Introduction--------------------------------------------------------------------------41
II. 2 Materials and methods -------------------------------------------------------------44
Regent and antibodies
Cell lines and culture
Adenoviral vectors
B cell isolation and B cell-based vaccine preparation
Quantitative real-time PCR
In vivo NKT activation and in vivo transferred B cell analysis
Evaluation of HLA-A2 epitope specific cytotoxic T cell response
II. 3. Results--------------------------------------------------------------------------------49
Expression kinetics of Her2/neu following adenoviral vector transduction
Expressions of various co-stimulatory molecules on adenoviral vector-transduced human and murine B cells
Soluble NKT ligand, KBC009 loaded- B cells directly activate NKT cells in vivo
dc.description.tableofcontentsNKT cells subsequently stimulate transferred B cells and activate them to become immunogenic APCs
Induction of a human HLA-A2 epitope-restricted CD8+ T cell immune response by Ad-k35HM transduced B cell-based vaccine
II. 4. Discussion----------------------------------------------------------------------------54

국문 초록------------------------------------------------------------------------------------75
감사의 글------------------------------------------------------------------------------------77
dc.format.extent2280627 bytes-
dc.publisher서울대학교 대학원-
dc.subjectMyeloid derived suppressor cells (MDSC)-
dc.subjectExtramedullary myelopoiesis-
dc.subjectMyeloid progenitor cells (lineageneg/CD127-/CD117+/Sca1--
dc.subjectLK cells). B cell-based vaccine-
dc.subjectAAD mice-
dc.titleTumor immune suppression mechanism of tumor-derived osteopontin and enhanced antitumor immunotherapeutic effect of B cell-based vaccine transduced with modified adenoviral vector-
dc.title.alternative암 유래 osteopontin의 암 면역억제 작용기전 및 변형 adenovirus를 이용한 B 세포백신의 향상된 항암효과 연구-
dc.citation.pagesx, 76-
dc.contributor.affiliation약학대학 약학과-
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College of Pharmacy (약학대학)Dept. of Pharmacy (약학과)Theses (Ph.D. / Sc.D._약학과)
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