Mechanisms of rejection of high grade B cell lymphoma in mice [Elektronische Ressource] / Madhusudhanan Sukumar

Madhusudhanan Sukumar

Sujets

• Biologie

Informations

• Publié par: ludwig-maximilians-universitat_munchen
• Publié le: 01 janvier 2008
• Nombre de lectures: 36
• Langue: Deutsch
• Poids de l'ouvrage: 1 Mo

Extrait

MECHANISMS OF REJECTION OF HIGH GRADE B CELL
LYMPHOMA IN MICE














A Thesis Submitted for the Degree of Doctor of Natural Sciences
at the Faculty of Biology,
Ludwig-Maximilians-Universität München









Madhusudhanan Sukumar
München, 2008






Conducted at the Helmholtz Zentrum München,
German Research Center for Environmental Health
Institute of Clinical Molecular Biology and Tumor Genetics



ERKLÄRUNG




Hiermit versichere ich, Madhusudhanan Sukumar, ehrenwörtlich, dass meine
Dissertation selbstständig und ohne unerlaubte Hilfsmittel angefertigt worden ist.
Die vorliegende Dissertation wurde weder ganz, noch teilweise bei einer anderen
Prüfungskommission eingereicht. Ich habe noch zu keinem früheren Zeitpunkt
versucht eine Dissertation einzureichen oder an einer Doktorprüfung teilzunehmen.


Munchen, 30.09.08
Madhusudhanan Sukumar















2
















First Examiner: Prof. Dr. Elisabeth Weiss

Additional Examiners: Dr. rer. nat. habil. Christine Falk

Prof. Dr. Heinrich Jung

Prof. Dr. John Parsch



Date of the oral examination: 25.09.2008
3Table of Contents
Table of Contents ....................................................................................................................... 4
A. Synopsis................................................................................................................................. 7
B. List of Abbreviations ..................................................................................... 9
1. Introduction ................................................................................................... 11
1.1 Lymphomas of B cell origin................................................................... 11
1.2 Burkitt’s Lymphoma ........................................................................................ 12
1.2.1 Translocations .................................................................................. 13
1.2.2 EBV and Burkitt’s lymphoma................................................................... 13
1.2.3 c-myc function in normal cell growth and cancer development ............................. 14
1.2.4 Models for studying Burkitt’s lymphoma ............................................................... 14
1.3 Immune response to tumors ........................................................................................... 15
1.3.1 Cancer immunoediting .................................................................... 15
1.3.2 Elimination phase............................................................................ 15
1.3.3 Equilibrium phase ..................................................................................... 18
1.3.4 Escape phase ........................................................................................................... 19
1.4 Foreign antigen-mediated tumor rejection ...................................................... 20
1.5 Role of interferons in cancer surveillance.............................................. 22
1.5.1 IFN- γ in tumor immunity .......................................................................... 23
1.5.2 Stat1 as tumor promoter for leukemia development ....................... 25
1.6 Immunosuppressive strategies of tumor cells .................................................. 26
1.6.1 Abnormalities in antigen presentation machinery................................................... 26
1.6.2 Secretion of immunosuppressive factors................................................................. 26
1.6.3 Tumor-induced antigen-specific CD8+ T cell tolerance... 26
1.6.4 MYC overexpression imposes non-immunogenic phenotype on human B cells.... 27
1.7 Aim of the Study ............................................................................................................ 29
2. Materials and Methods ................................. 30
Materials............................................................................................................................... 30
2.1 Surgical related reagents and equipment........................................................................ 30
2.2 Mammalian cell lines ...................................................................................... 30
2.3 Oligonucleotides..................................................................................... 30
2.4 Plasmids ......................................................................................................................... 30
2.5 Antibodies ....................................................................... 30
42.6 Reagents, media and apparatus ...................................................................................... 31
2.6.1 Molecular biology ........................................................................... 31
2.6.2 Cell and tissue culture ...................................................... 31
2.7 Mice................................................................................................................................ 33
Mice lines used in this study ........................ 33
Methods................................................................................................................................ 34
2.8 Establishment of cell lines.............................................................................................. 34
2.9 Cloning of chicken ovalbumin ................................................. 35
2.10 Retroviral production and transduction................................................. 36
2.11 Western blotting ................................................................................... 37
2.12 Tumor transfer and monitoring ............................................... 38
2.13 Immunization protocol and transfer of naïve and activated OT-I CD8+ T cells ......... 38
2.14 SIINFEKL pentamer staining....................................................................................... 38
2.15 Flow cytometric analysis........................................................ 39
2.16 CFSE staining.............................................................. 39
2.16 LDH cytotoxicity assay......................................................................... 39
3. Results .......................................................................................................... 41
3.1 Determination of lethal dose for lymphoma transfer ..................................................... 41
3.2 Introduction of foreign antigens and establishment of stable cell lines of λ-hu-c-myc
lymphoma cells .................................................................................................................... 42
3.3 Introduction of OVA into 291 lymphoma cells leads to delay in tumor formation ....... 43
3.4 Loss of antigen in outgrowing lymphomas .................................................................... 47
3.5 Detection of SIINFEKL specific T cells in splenocytes of mice that have rejected
lymphoma cells expressing ovalbumin and GFP ................................................................. 48
3.6 Stat1 and IFN- γ deficient mice fail to reject OVA-GFP cells........................................ 49
3.7 Organ infiltration by 291-OVA-GFP cells is higher in Stat1 and IFN- γ deficient mice 51
-/- -/-3.8 Expression of antigen is preserved in IFN- γ and Stat1 mice .................................... 52
3.9 IFN- γ-dependent induction of MHC class I and MHC class II........................ 53
-/-3.10 OT-I T cells fail to reject 291-OVA-GFP lymphomas in Stat1 recipients ................ 55
-/-3.11 Primed OT-I T cells persist in tumor bearing Stat1 animals ...................... 56
3.12 Generation of IFN unresponsive lymphoma cells........................................................ 57
-/-3.13 Stat1 lymphomas are unresponsive to IFN- γ............................................... 58
3.14 Stat1 deficient lymphoma is rejected upon transfer into immunocompetent mice ...... 59
-/- -/-3.15 IFN- γ and Stat1 recipients fail to reject Stat1 deficient lymphomas....................... 61
53.16 Lymphoma cells are lysed by NK cells in vitro ........................................................... 62
4. Discussion ............................................................................................................................ 64
4.1 MYC overexpression in human B cells imposes a non-immunogenic phenotype in vitro
.............................................................................................................................................. 64
4.2 Mouse model to evaluate the immune response against a B cell lymphoma expressing a
foreign antigen in vivo ...................................................................................