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Anti-Nucleic Acid Antibody-Mediated Cross Presentation of An Antigen and Its CD8+ T Cell Activation
| DC Field | Value | Language |
|---|---|---|
| dc.contributor.advisor | 권명희 | - |
| dc.contributor.author | Pham, Dinh-Chuong | - |
| dc.date.accessioned | 2019-10-21T07:19:42Z | - |
| dc.date.available | 2019-10-21T07:19:42Z | - |
| dc.date.issued | 2013-02 | - |
| dc.identifier.other | 13450 | - |
| dc.identifier.uri | https://dspace.ajou.ac.kr/handle/2018.oak/18174 | - |
| dc.description | 학위논문(박사)아주대학교 일반대학원 :의생명학과,2013. 2 | - |
| dc.description.tableofcontents | TABLE OF CONTENTS ABSTRACT i TABLE OF CONTENTS iii LIST OF FIGURES vi LIST OF TABLES vii I. INTRODUCTION 1 A. Canonical pathway of antigen presentation 2 B. Cross-presentation pathway 4 C. Chicken ovalbumin as the model antigen 18 D. 3D8 single chain variable fragment 19 E. Purposes of this research 21 II. MATERIALS AND METHODS 22 A. Cells 22 B. Production of 3D8-OVA fusion proteins 22 C. Isolation of CD11c+ dendritic cells from lymph nodes 23 D. DNA binding assay by Enzyme-linked immunosorbent assay (ELISA) 25 E. DNA/RNA hydrolyzing assay by Agarose gel electrophoresis 25 F. Cross-presentation assay 26 G. Confocal microscopy 27 H. Flow cytometry 28 I. MTT assay 29 J. Cytosolic fractionation 30 K. Western blotting 30 L. Immunization of mice with 3D8-OVA250-264 31 M. 51Cr release assay 32 N. Tumor growth 33 III. RESULTS 34 A. Bacterial expression, purification, and biochemical properties of fusion protein 34 B. Involvement of cell surface HSPGs in the internalization of 3D8-OVA proteins 37 C. 3D8-OVA fusion proteins have little effect on the viability of DC2.4 dendritic cells 40 D. Cytosolic translocation and proteasome-involved degradation of 3D8-OVA fusion protein in DC2.4 dendritic cells 42 E. 3D8-OVA proteins are cross-presented by dendritic cells 47 F. Cross-presentation of 3D8-OVA proteins by DC2.4 dendritic cells occurs via a proteasome-dependent pathway 52 G. 3D8-OVA250-264 induces the maturation of CD11c+ dendritic cells 56 H. Generation of CTL by 3D8-OVA250-264 61 I. Tumor challenge experiment 63 IV. DISCUSSION 66 V. CONCLUSION 71 REFERENCES 72 국문요약 91 | - |
| dc.language.iso | eng | - |
| dc.publisher | The Graduate School, Ajou University | - |
| dc.rights | 아주대학교 논문은 저작권에 의해 보호받습니다. | - |
| dc.title | Anti-Nucleic Acid Antibody-Mediated Cross Presentation of An Antigen and Its CD8+ T Cell Activation | - |
| dc.title.alternative | 추옹 팜 딘 | - |
| dc.type | Thesis | - |
| dc.contributor.affiliation | 아주대학교 일반대학원 | - |
| dc.contributor.alternativeName | 추옹 팜 딘 | - |
| dc.contributor.department | 일반대학원 의생명과학과 | - |
| dc.date.awarded | 2013. 2 | - |
| dc.description.degree | Master | - |
| dc.identifier.localId | 570801 | - |
| dc.identifier.url | http://dcoll.ajou.ac.kr:9080/dcollection/jsp/common/DcLoOrgPer.jsp?sItemId=000000013450 | - |
| dc.description.alternativeAbstract | Cross-presentation is important for initiating cytotoxic T lymphocyte (CTL) responses against tumors. Delivery of exogenous antigens to the cross-presentation pathway in dendritic cells using a number of different carriers has been attempted to further understand the mechanisms underlying cross-presentation and to develop therapeutic tumor vaccines. The present study reports a new antigenic carrier molecule: a single chain variable region fragment (scFv) of a nucleic acid-hydrolyzing antibody, 3D8. As the CD8-T cell epitopes, chicken ovalbumin amino acid 257-264 (OVA257-264-SIINFEKL) and 250-264 (OVA250-264-SGLEQLESIINFEKL) were used for 3D8 scFv fusion partners. Purified 3D8 scFv-OVA fusion proteins with ~95% purity retained DNA-binding, DNA-hydrolyzing, cell-penetrating, and cytotoxic activity comparable to 3D8 scFv. Although 3D8-OVA proteins induced B16 and MO5 melanoma cancer cell death up to 80%, they just had little effect on the viability of DC2.4 dendritic cells. Both 3D8-OVA257-264 and 3D8-OVA250-264 showed OVA-specific CD8 T cell stimulatory effect but the activity of 3D8-OVA250-264 was higher than that of 3D8-OVA257-264. Furthermore, 3D8-OVA250-264 stimulated OVA-specific CD8 T cells through cross-presentation by dendritic cells via a proteasome dependent, brefeldin- and cycloheximide-sensitive, chloroquine- and primaquine-insensitive pathway, resulting in loading of the CTL epitope onto H-2Kb. In vivo cross-presentation and cross-priming was efficient, even without adjuvant; injection of mice with 3D8 scFv-OVA250-264 induced cross-presentation of the CTL epitope by draining lymph node CD11c+ B7.1+ MHC class IIhigh dendritic cells, elicited a CTL response, and suppressed the growth of tumors expressing the OVA epitope. This is the first report of an anti-nucleic acid antibody used to deliver exogenous antigen to the cross-presentation pathway and inhibit in vivo tumor growth. | - |
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