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Anti-Nucleic Acid Antibody-Mediated Cross Presentation of An Antigen and Its CD8+ T Cell Activation 항-핵산 항체의 교차항원제시 및 항원 특이 T 세포의 활성화 기전연구 Pham, Dinh-Chuong 권, 명희 대학원 의생명과학과 200624567 Pham, Dinh-Chuong Cross-presentation Anti-nucleic acid antibody Cell-penetrating antibody OVA peptide Internalization 항 핵산 항체 교차항원제시 T 세포 활성 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. 수지상 세포에 의한 교차항원제시 (cross-presentation)는 종양에 대한 세포독성 CD8+ T 림프구 반응 유도에 중요하다. 본 연구는 수지상 세포에서 3D8 scFv 항체의 교차항원제시 능력 및 기전, 교차항원제시에 의한 항원 특이적 T 세포 활성화 분석에 관한 것이다. 이를 위하여 CD8+ T 세포의 모델에 피톱으로서 잘 알려진 닭 오발부민 단백질 유래의 펩티드 SIINFEKL (OVA257-264)를 3D8 scFv 항체에 연결시킨 3D8-OVA250-264 융합단백질을 박테리아 배양으로부터 정제하여 본 연구에 사용하였다. DC2.4 수지상 세포 (H-2Kb)에 3D8-OVA250-264 단백질을 처리했을 때, 3D8-OVA250-264이 DC2.4 세포 내로 유입된 후, 세포질에 위치한 프로테아좀에 의해 항원처리(antigen processing) 되어, OVA257-264 에 특이적인 CD8+ T 세포가 활성화됨을 확인하였다. 생체 내 수지상 세포에서 3D8-OVA250-264 단백질의 교차항원제시 능력을 확인하기 위하여, 3D8-OVA250-264 단백질을 C57BL/6 생쥐 (H-2Kb)에 접종한 후, 유출 림프절 (draining lymph nodes)에 존재하는 CD11c+수지상 세포의 표면분자를 분석하였다. 그 결과, CD11c+수지상 세포의 성숙이 유도 (B7.1+/MHC classIIhigh 세포 검출) 되었을뿐만 아니라, 이들 성숙된 수지상 세포에 의한 OVA 항원의 교차항원제시를 통하여, OVA 특이 CD8+ T 세포가 활성화됨을 확인하였다. 또한 OVA 에피톱을 발현하는 종양이 자라고 있는 생쥐에, 어쥬번트 추가없이, 3D8-OVA250-264을 단독 접종했을 때 종양성장이 현저히 억제되었다. 본 연구결과는 항-핵산 항체의 교차항원제시 능력을 최초로 보여주는 것이며, 교차항원제시 능력이 있는 항체를 활용하는 새로운 항암, 항바이러스 백신 개발 전략을 제공한다. 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 Doctor 2013-12-12T05:24:51Z 2013-12-12T05:24:51Z 2013 2013 Thesis Theses http://repository.ajou.ac.kr/handle/201003/8582 http://dcoll.ajou.ac.kr:9080/dcollection/jsp/common/DcLoOrgPer.jsp?sItemId=000000013450 000000013450 en application/pdf