Identification of preferentially targeted tumor-associated antigens in melanoma patients via mRNA stimulation of CD8+ blood lymphocytes [Elektronische Ressource] / Emmanuelle Wesarg (born Vaniet)

Sujets

• Biologie
• Gesundheit

Informations

• Publié par: johannes_gutenberg-universitat_mainz
• Publié le: 01 janvier 2009
• Nombre de lectures: 15
• Langue: English
• Poids de l'ouvrage: 2 Mo

Extrait

Identification of preferentially targeted tumor-associated
antigens in melanoma patients via mRNA stimulation of
CD8+ blood lymphocytes
DISSERTATION
For achievement of the academic degree
DOCTOR OF PHILOSOPHY (PHD)
At the Faculty of Medicine
And the Faculty of Biology
Johannes-Gutenberg University of Mainz
Emmanuelle WESARG (born VANIET)
ndBorn on 22 of October 1975 in Boulogne-Sur-Mer (FRANCE)
Mainz, 2008thDay of the oral examination: 4 of November 2008„Ich erkläre, dass ich die vorgelegte Thesis selbständig, ohne unerlaubte fremde Hilfe und
nur mit den Hilfen angefertigt habe, die ich in der Thesis angegeben habe. Alle Textstellen,
die wörtlich oder sinngemäß aus veröffentlichten oder nicht veröffentlichten Schriften
entnommen sind, und alle Angaben, die auf mündlichen Auskünften beruhen, sind als solche
kenntlich gemacht. Bei den von mir durchgeführten Untersuchungen habe ich die
Grundsätze guter wissenschaftlicher Praxis, wie sie in der Satzung der Johannes Gutenberg
Universität Mainz zur Sicherung guter wissenschaftlicher Praxis niedergelegt sind,
eingehalten.“SUMMARY I
SUMMARY
Until now, therapeutic vaccination of cancer patients has mainly relied on rather few T cell
epitopes processed from structurally normal shared tumor antigens and presented by
frequent HLA alleles. So far the design of these studies has not addressed the individuality of
tumor-host interactions, which are not only determined by the antigenic tumor phenotype or
the natural HLA polymorphism, but also by the individual T cell repertoire. The procedure
described herein was developed to identify the preferential targets of the individual repertoire
from a panel of known shared tumor-associated antigens.
Lymphocytes were isolated from the peripheral blood of cancer patients or healthy donors
and stimulated twice with autologous mRNA-transfected FastDC (Dauer et al., J Immunol.
170:4069, 2003). FastDC were generated from blood monocytes and separately transfected
via lipofection with in vitro transcribed mRNAs encoding the panel antigens. Responder
lymphocytes were tested on day 12 in a 20-hour IFN-? ELISPOT assay for recognition of
293T cells co-transfected pairwise with plasmids encoding the stimulation antigens and the
respective individual’s HLA class I alleles. In a first step, stimulation parameters were
optimized for the detection of anti-HCMV pp65 responses. A maximum amplification of pp65-
specific CD8+ T cell responses was obtained at a rather low IL-2 concentration (25 IU/ml)
and at a minimum APC-to-effector ratio of 1:10. Addition of IL-4, IL-7 or IL-15 did not
substantially improve the stimulatory potential.
The test was applied to the human melanoma models D05 and MZ2, in both of which
multiple T cell-defined antigens had previously been identified by expression screening.
Blood lymphocytes were stimulated in parallel with autologous tumor cells and with mRNA-
transfected FastDC. In D05, T cell reactivities against three out of eleven epitopes induced
by stimulation with tumor cells were also found after stimulation with mRNA-transfected
FastDC. Two further T cell target epitopes were identified with mRNA but not with tumor cell
stimulation. In MZ2, T cell responses against five distinct epitopes were detected on day 12
after stimulation with mRNA transfectants. The same responses were detectable after
stimulation with tumor cells only on day 32. mRNA stimulations against 21 tumor-associated
antigens in addition to HCMV pp65 were performed in four healthy individuals. In all cases,
CD8+ T cells against HCMV pp65 could be expanded. Among tumor-associated antigens,
only reactivity Melan-A/MART-1 in association with HLA-A*0201 was detectable in
one of the donors.
The vaccination of patients with targets a priori known to be recognized by their T cell
repertoire may help to improve the outcome of therapeutic vaccination.TABLE OF CONTENTS II
TABLE OF CONTENTS
SUMMARY.................................................................................................................................I
TABLE OF CONTENTS ...............................................................................................................II
ABBREVIATIONS...................... 1
1 INTRODUCTION................................................................................................................ 3
1.1 Melanoma............. 3
1.2 Antigens recognized on melanoma by autologous T cells ............................... 4
1.2.1 Mutated antigens............................................................................................ 5
1.2.2 Shared ............................. 5
1.2.2.1 Shared tumor-specific antigens................................. 5
1.2.2.2 Differentiation antigens.............................................. 6
1.2.2.3 Overexpressed............................................ 6
1.3 Processing and presentation of tumor antigens............... 7
1.3.1 Antigens presented by MHC class I molecules 7
1.3.2by MHC class II .............................................. 8
1.3.3 Antigen recognition by T cells......................................... 9
1.3.3.1 HLA polymorphism.................... 9
1.3.3.2 T cell repertoire ......................................................................................... 9
1.3.3.3 Mechanisms of tolerance......... 10
1.4 The tumor-immune escape................ 10
1.4.1 Antigen and HLA loss................................................................................... 10
1.4.2 Tumor immune barriers................ 11
1.4.2.1 Immune suppressive effects directly induced by the tumor...................... 11
1.4.2.2 Indirect immune suppressive effects........................................................ 12
1.5 Antigen-specific immunotherapy of melanoma............... 12
1.5.1 Cellular vaccines .......................................................... 12
1.5.2 Vaccination with defined antigens ................................................................ 13
1.6 Methods to quantify antigen-specific T cell responses... 15
1.6.1 Fluorescent HLA/peptide tetramers.............................. 15
1.6.2 Enzyme Linked Immunospot (ELISPOT) assay............................................ 16
1.6.3 Intracellular cytokine assay .......................................... 17
1.6.4 Cytokine secretion assay.............. 17TABLE OF CONTENTS III
1.7 The rationale and requirements of a procedure identifying the preferential
targets of the individual T cell repertoire..................................................................... 17
1.7.1 Short-term in vitro stimulation....... 18
1.7.2 The stimulation procedure............ 18
1.7.3 Antigen format.............................................................................................. 19
1.7.4Presenting Cells (APC).................................... 19
1.7.5 Readout assay............................. 20
2 MATERIAL AND METHODS.............................................................................................. 21
2.1 Material ............................................................................................................... 21
2.1.1 Laboratory instruments................. 21
2.1.2 Chemicals .................................................................................................... 22
2.1.3 Buffers and solutions.................................................................................... 23
2.1.4 Material for bacterial culture and molecular biology...... 25
2.1.4.1 Plasmids.................................................................................................. 25
2.1.4.2 Escherichia coli strains............ 26
2.1.4.3 Substances for bacterial culture............................... 26
2.1.4.4 Media for bacterial culture and conservation............................................ 26
2.1.4.5 Enzymes ................................................................. 27
2.1.4.6 Molecular markers................... 27
2.1.4.7 Kits.......................................... 28
2.1.5 Material for cell culture and cellular assays.................. 28
2.1.5.1 Cell lines.................................................................. 28
2.1.5.2 Healthy donors........................ 30
2.1.5.3 Substances for cell culture....................................... 30
2.1.5.4 Media for cell culture ............................................... 31
2.1.5.5 Cytokines ................................................................ 32
2.1.5.6 Antibodies............................... 32
2.1.5.7 Synthetic peptides ................................................................................... 33
2.2 Methods .............................................. 34
2.2.1 Molecular biology......................... 34
2.2.1.1 Amplification and isolation of plasmid DNA.............................................. 34
2.2.1.2 Generation of in vitro transcribed (IVT)-mRNA........ 35
2.2.1.3 Polyadenylation of IVT-mRNA ................................................................. 37
2.2.1.4 Spectrophotometric quantification of nucleic acids... 39
2.2.1.5 Electrophoretic visualization of nucleic acids........... 40
2.2.1.6 HLA RT-PCR........................................................................................... 41TABLE OF CONTE