Isolation and characterization of antigen-specific T cell receptors:
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| 1. Verfasser: | |
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| Format: | Abschlussarbeit Buch |
| Sprache: | English |
| Veröffentlicht: |
Berlin
2015
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| Online-Zugang: | Inhaltsverzeichnis |
| Beschreibung: | 162 Seiten Illustrationen, Diagramme |
Internformat
MARC
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| 040 | |a DE-604 |b ger |e rda | ||
| 041 | 0 | |a eng | |
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| 100 | 1 | |a Lorenz, Felix |d 1982- |e Verfasser |0 (DE-588)108193848X |4 aut | |
| 245 | 1 | 0 | |a Isolation and characterization of antigen-specific T cell receptors |c von Mag. rer. nat. Felix Lorenz |
| 264 | 1 | |a Berlin |c 2015 | |
| 300 | |a 162 Seiten |b Illustrationen, Diagramme | ||
| 336 | |b txt |2 rdacontent | ||
| 337 | |b n |2 rdamedia | ||
| 338 | |b nc |2 rdacarrier | ||
| 502 | |b Dissertation |c Humboldt-Universität zu Berlin |d 2015 | ||
| 655 | 7 | |0 (DE-588)4113937-9 |a Hochschulschrift |2 gnd-content | |
| 776 | 0 | 8 | |i Reproduziert als |a Lorenz, Felix |t Isolation and characterization of antigen-specific T cell receptors |d Berlin : MIK-Center GmbH, 2015 |h 2 Mikrofiches |w (DE-604)BV043634230 |
| 856 | 4 | 2 | |m DNB Datenaustausch |q application/pdf |u http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=028752173&sequence=000001&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA |3 Inhaltsverzeichnis |
| 999 | |a oai:aleph.bib-bvb.de:BVB01-028752173 | ||
Datensatz im Suchindex
| _version_ | 1804175878541803520 |
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| adam_text | TABLE OF CONTENT
ZUSAMMENFASSUNG 9
1 SUMMARY 11
2 INTRODUCTION 13
2.1 TCELLS 13
2.1.1 CD8+ T CELLS PROVIDE CYTOTOXIC ADAPTIVE IMMUNITY 13
2.1.2 TCR SEQUENCES ARE UNIQUE SIGNATURES OF INDIVIDUAL T CELLS 13
2.1 *3 CD8+ T CELL SUBSETS 14
2.1.4 CD8+ T CELLS RECOGNIZE PEPTIDE-*** COMPLEXES 16
2.1.5 PEPTIDES ARE PROCESSED AND PRESENTED ON * * * COMPLEXES OF TARGET
CELLS 17
2.1.6 EFFECTOR FUNCTION OF T CELLS 20
2.2 IMMUNOTHERAPIES USING T CELLS 21
2.2.1 ADOPTIVE T CELL THERAPIES 21
2.2.1.1 TIL THERAPY 21
2.2.1.2 CAR-ENGINEERED T CELLS 21
2.2.1.3 TCR-ENGINEERED T CELLS 22
2.2.2 L_ U N E CHECKPOINT NHIBITO 24
2-3 ONCOGENIC VIRUSES 25
2.3.1 EBSTEIN-BARR VIRUS 25
2.3.2 KAPOSI SARCOMA VIRUS 27
2.3.3 POLYOMAVIRUS 27
2.3.4 HUMAN T CELL LYMPHOTROPIC VIRUSI 27
2.3.5 HUMAN PAPILLOMAVIRUS 27
2.3.5.1 USE-CYCLE OF HPV 28
2.3.5.2 HPV CAUSES CANCER 29
2.3.53 PRIMARY TREATMENTS OF HPV-POSITIVE CANCERS 29
2.3.5.4 PROPHYLACTIC VACCINATION 30
2.3.5.5 IMMUNOTHERAPEUTIC TREATMENTS 31
2.36 CYTOMEGALOVIRUS 31
2.4 ISOLATION OF ANTIGEN-SPECIFIC TCRS 32
2.4.1 SOURCES OF ANTIGEN-SPECIFIC TCRS 33
2.4.1.1 TCR ISOLATION FROM PBMCS AND TILS 33
2.4.1 *2 ALLO-RESTRICTED T CELLS 33
2.4.1.3 TCR STATION ^OM HLATRANSGENIC MICE 33
2.4.2 CURRENT METHODS TO DETECT AND SOLATE ANTIGEN-SPECIFIC T CELLS 35
2.4.2.1 ANTIGEN-SPECIFIC STIMULATION 35
2.4.2.2
***
MULTIMERS
36
2.4.2.3 T CELL ACTIVATION MARKERS 36
2.4.2.4 CYTOKINE CAPTURE 36
2.4.2.5 REQUIREMENTS FOR NOVEL STRATEGIES TO ISOLATE ANTIGEN-SPECIFIC
TCRS 37
3 AIM 39
4 RESULTS 41
4.1 * * *
CELL LIBRARY
41
4.1.1 GENERATION OF A VECTOR LIBRARY CONTAINING * * * ALLELE GENES 41
4.1.2 GENERATION OF *** CELL LIBRARY 42
4.1.3
ANTIGEN EXPRESSION IN *562 CELLS OF THE
***
CELL LIBRARY
43
4.1.4 THE * * * CELL LIBRARY INDUCES ANTIGEN-SPECIFIC T CELL RESPONSES
44
4.2 CODON OPTIMIZATJON OF HPV16 *7 INDUCES T CEL* RESPONSE TO A CRYPTIC
EPJTOPE* 47
4.2.1 CODON OPTIMIZATION ELEVATES *7 EXPRESSION LEVELS 47
4.2.2 ENRICHMENT OF ANTIGEN-SPECIFIC T CELLS THROUGH STIMULATION WITH
ANTIGEN-
EXPRESSING DCS 47
4.2.3 GENERATION OF T CELL CLONES AND TESTING FOR ANTIGEN SPECIFICITY 49
4.2.4 ISOLATION
*
OPTIMIZATION AND RETROVIRAL EXPRESSION OF ANTIGEN-SPECIFIC TCRS 49
4.2.5 TCR-TRANSDUCED T CELLS ARE RESTRICTED TO HLA-B*27:05 AND RECOGNIZE
E7C0
ANTIGEN BUT NOT E7WT 51
4.2.6 ISOLATED TCRS RECOGNIZE A **-NUCLEOTIDE MICROGENE ^AGMENT OF E7C0
52
4.2.7 IN SILICO PREDICTION OF EPITOPES FROM THE ANTIGENIC REGION OF E7CO
53
4.2.8 EPITOPE MAPPING IDENTIFIES A CRYPTIC EPITOPE FROM THE +3 ARF OF
E7C0 AS TARGET
OF TCR-TRANSDUCED T CELLS 55
4.2.9 REDESIGNING THE +3 ARF PREVENTS E7C0 ^OM RECOGNITION BY
TCR-TRANSDUCED T
CELLS 56
4.3 PLATFORM TO DETECT AND ISOLATE V
**
S-SPECFCTCRS 59
4.3.1 VIRUS ANTIGEN-SPECIFIC STIMULATION OF PBMCS 59
4.3.2 SCREENING FOR VIRUS-SPECIFIC T CELLS 60
4.3.3 SORTING OF VIRUS-SPECIFIC T CELLS TO ANALYZE THE TCR REPERTOIRE 62
4.3.4 TCR REPERTOIRE ANALYSIS USING NEXT GENERATION SEQUENCING 64
4.3.5 FUNCTIONAL ANALYSIS OF TRA AND TRB CHAIN COMBINATIONS 66
4.3.6 OPTIMIZATION AND FUNCTIONAL ANALYSIS OF HPV- AND CMV-SPECIFIC TCRS
68
4.4 IDENTIFICATION OF THE ANTIGENIC PEPTIDE RECOGNIZED BY THE
E5-SPECFC TCR 70
4.4.1 EPITOPE MAPPING OF THE HPV16E5-SPECIFIC TCR 70
4.4.2 TCR-TRANSDUCED T CELLS RECOGNIZE ANTIGEN-EXPRESSING CELL LINES 73
D IS C U S S IO N 77
5.1 THE *** * * LIBRARY 79
5.1.1 PROFESSIONAL AND ARTIFICIAL APCS 79
5.1.1.1 DCS 79
5.1.1.2 AUTOLOGOUS TUMOR CELLS 79
5.1.1.3 LCLS 80
5.1.1.4 ARTIFICIAL APCS 80
5.1.1.5 ACELLULAR APCS 81
5.1.2 *562 CELLS AS ARTIFICIAL APC SCAFFOLD 82
5.1.3 CONCLUSION AND OUTLOOK 83
5.2 TCRS RECOGNIZE A CRYPTIC EPITOPE FROM CODON-OPTIMIZED HPV16 *7 85
5.2.1 IMMUNOTHERAPIES TARGETING HPV ONCOGENES *6 AND *7 85
5.2.2 CODON OPTIMIZATION OF HPV16 *7 86
5.2.3 ALTERNATIVE SOURCES OF EPITOPES 86
5.2.4 CODON OPTIMIZATION ALGORITHMS 88
5.2.5 CLINICAL APPLICATIONS OF CODON-OPTIMIZED GENE SEQUENCES 89
5.2.6 CONCLUSION AND OUTLOOK 89
5-3 ISOLATION OF VIRUS-SPECIFIC TCRS 91
5.3.1 THE E5-SPECIFIC TCR TARGETS A NOVEL EPITOPE RESTRICTED TO
HLA-B*15:01 91
5.3.2 TARGETING *5 IN HPVISPOSITIVE CANCERS 92
5.3.3 THE CMV-SPECIFIC TCR TARGETS AN IMMUNODOMINANT PEPTIDE-MHC 94
5.3.4 TREATMENT STRATEGIES FOR CMV DISEASE USING T CELLS 95
5.3.5 ESTABLISHMENT OF A PLATFORM TO ISOLATE ANTIGEN-SPECIFIC TCRS 96
5.3.5.1 T CELLS HAVE FREE CHOICE OF PEPTIDE-*** COMBINATION 96
5.3.5.2 T CELLS ARE SELECTED UPON RECOGNITION OF ENDOGENOUSLY PROCESSED
EPITOPES 97
5.3.5.3 T CELLS ARE SELECTED UPON FUNCTIONAL ANTIGEN RECOGNITION 98
5.3-5.4 SHORT-TERM IN VITRO CELL CULTURE IMPROVES THE ANALYSIS OF T CELL
RESPONSES 99
5.3.5.5 TCR REPERTOIRE ANALYSIS ENABLES RECONSTITUTION OF FUNCTIONAL
TCRS 99
5.3.6 CONCLUSION AND OUTLOOK 101
6 MATERIALS AND METHODS 102
6.1 MOLECULAR BIOLOGY 102
6.1.1 RNA EXTRACTION AND CDNA GENERATION 102
6.1.2 ISOLATION OF GENOMIC DNA 103
6.1.3 POLYMERASE CHAIN REACTION (PCR) 103
6.1.4 OVERLAP EXTENSION PCR 104
6.1 *5 RESTRICTION DIGEST, KLENOW FILL-UP OF AND DEPHOSPHORYLATION *F
PLASMID DNA 106
6.1.6 DNA FRAGMENT PURIFICATION 107
6.1.7 PLASMID LIGATION AND BACTERIAL TRANSFORMATION 108
6.1.8 DNA ISOLATION AND PREPARATION 108
6.1 *9 SITE DIRECTED MUTAGENESIS 110
6.1.10 TCR REPERTOIRE PCR PANELS 111
6.1.11 RAPID AMPLIFICATION OF CDNA ENDS (RACE) AND BLUNT END CLONING OF
PCR
PRODUCTS 112
6.1.12 NEXT GENERATION SEQUENCING ^ * 5
6.1.13 QUANTITATIVE REAL-TIME PCR 115
6.1.14 GENERATION OF IVTRNA 116
6.2 IN SILICO ANALYSIS 117
6.2.1 DATA ANSYSIS SOFTWARE 117
6.2.2 EPITOPE PREDICTION PROGRAMS 118
6.2.3 TCR GENE SEQUENCE ANALYSIS 118
6.2.4 OPTIMIZATION OF GENE SEQUENCES AND TCR CASSETTES 118
6.3 CELL CULTURE 119
6.3.1 ISOLATION OF HUMAN PBMCS 119
6.3.2 GENERATION OF DCS 119
6.3.3 TRANSFECTION OF TARGET CELLS WITH ANTIGEN VTRNA 120
6.3.4 ISOLATION OF CD8+T CELLS 120
6.3.5 ANTIGEN-SPECIFIC STIMULATION OF CD8+ T CELLS AND PBMCS 120
6.3.6 T CELL CLONE CULTURE 121
6.3.7
***
CELL LIBRARY-BASED SCREENING 122
6.3.8
***
CELL LIBRARY-BASED SORTING 122
6.3.9 GENERATION OF RETROVIRAL VECTOR PARTICLES 123
6.3.10 RAPID EXPANSION AND RETROVIRAL TRANSDUCTION OF PBMCS 124
6.3.11 CELL LINES 125
6.3.12 RET
*
OVI
*
AL TRANSDUCTION OF CELL LINES 125
6.3.13 CELL CULTURE AND CRYO-PRESERVATION 126
6.4 FUNCTIONAL ASSAYS 126
6.4.1 FLOW CYTOMETRY 126
6.4.2 FLUORESCENT-ACTIVATED CELL SORTING (FACS) 127
6.4.3 MAGNETIC-ACTIVATED CELL SORTING (MACS) 127
6.4.4 ENZYME-LINKED IMMUNOSORBANT ASSAY (EUSA) 128
7 REFERENCES 129
8 APPENDIX 153
ABBREVIATIONS 153
ANTIBODIES 155
PRIMER LISTS 156
ACKNOWLEDGEMENTS 159
PUBLICATIONS 160
PUBLICATIONS 160
PATENTS AND FUNDINGS 160
CURRICULUM VITAE 161
PERSONAL DATA 161
EDUCATION 161
SELBSTSTAENDIGKEITSERKLAERUNG 162
|
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| spelling | Lorenz, Felix 1982- Verfasser (DE-588)108193848X aut Isolation and characterization of antigen-specific T cell receptors von Mag. rer. nat. Felix Lorenz Berlin 2015 162 Seiten Illustrationen, Diagramme txt rdacontent n rdamedia nc rdacarrier Dissertation Humboldt-Universität zu Berlin 2015 (DE-588)4113937-9 Hochschulschrift gnd-content Reproduziert als Lorenz, Felix Isolation and characterization of antigen-specific T cell receptors Berlin : MIK-Center GmbH, 2015 2 Mikrofiches (DE-604)BV043634230 DNB Datenaustausch application/pdf http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=028752173&sequence=000001&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA Inhaltsverzeichnis |
| spellingShingle | Lorenz, Felix 1982- Isolation and characterization of antigen-specific T cell receptors |
| subject_GND | (DE-588)4113937-9 |
| title | Isolation and characterization of antigen-specific T cell receptors |
| title_auth | Isolation and characterization of antigen-specific T cell receptors |
| title_exact_search | Isolation and characterization of antigen-specific T cell receptors |
| title_full | Isolation and characterization of antigen-specific T cell receptors von Mag. rer. nat. Felix Lorenz |
| title_fullStr | Isolation and characterization of antigen-specific T cell receptors von Mag. rer. nat. Felix Lorenz |
| title_full_unstemmed | Isolation and characterization of antigen-specific T cell receptors von Mag. rer. nat. Felix Lorenz |
| title_short | Isolation and characterization of antigen-specific T cell receptors |
| title_sort | isolation and characterization of antigen specific t cell receptors |
| topic_facet | Hochschulschrift |
| url | http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=028752173&sequence=000001&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA |
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