The role of CD4_1hn+ helper T cells in immunity to acute friend retroviral infection [Elektronische Ressource] / submitted by Savita Nair

Savita Nair

Sujets

• Gesundheit

Informations

• Publié par: universitat_duisburg-essen
• Publié le: 01 janvier 2009
• Nombre de lectures: 30
• Langue: English
• Poids de l'ouvrage: 7 Mo

Extrait

+The role of CD4 helper T cells in immunity
to acute Friend retroviral infection


Inaugural Dissertation for the Degree of
Doctor of Natural Science
Dr. rer. nat.


A Thesis Presented to
The Faculty of Biology and Geography
University of Duisburg-Essen
Germany



submitted by
SAVITA NAIR

from Mumbai, India
February, 2009




Die der vorliegenden Arbeit zugrunde liegenden Experimente wurden am Institut für
Virologie der Universität Duisburg-Essen durchgeführt.

1. Gutachter: Prof. Dr. Ulf Dittmer
2. Gutachter: Prof. Dr. Helmut. Esche



Vorsitzender des Prüfungsausschusses: Prof. Dr. Perihan Nalbant

th
Tag der mündlichen Prüfung: 16 March 2009








































Dedicated to my Parents
















































The chess board is the world,
the pieces are the phenomena of the universe,
the rules of the game are what we call the laws of Nature.

A Liberal Education
Thomas Henry Huxley, 1868

Contents
TABLE OF CONTENTS

1. Introduction

1.1 History of retroviral research....................................................................... 1

1.2 Retroviruses................................................................................................. 2
1.2.1 Structure and genome of retroviruses......... 2
1.2.2 Taxonomy of retroviruses........................................................................... 3
1.2.3 Life cycle of retroviruses.................................................... 6
1.2.4 Genomic variation between complex versus simple retroviruses............... 9

1.3 Retrovirus and host interactions................................................................. 10
1.3.1 Retrovirus induced oncogenesis........................................ 10
1.3.2 Mechanism of oncogenesis........................................................................ 11
1.3.2.1 Nonacute transforming retroviruses: Insertional mutagenesis................... 11
1.3.2.2 Acute transforming retroviruses: Viral oncogenes..................................... 12
1.3.2.3 Trans-acting retroviruses: Viral oncogenes........................ 13

1.4 Infection and Immunity.............................................................................. 13
1.4.1 Innate immunity.......................................... 13
1.4.2 Adaptive immunity..................................................................................... 14
1.4.3 Helper T cells.............................................. 16
1.4.3.1 Helper T cells in virus infections ............................................................... 17
1.4.3.2 Helper T cells in HIV infections ....................................... 18
1.4.4 Regulatory T cells............................................................... 19
1.4.4.1 Regulatory T cells in virus infections......................................................... 20
1.4.4.2 Regulatory T cells in retrovirus infections.......................... 23

1.5 Friend virus complex to study immunity to retroviruses............................. 25
1.5.1 Friend virus-induced disease....................................................................... 26
1.5.1.1 Factors influencing susceptibility to FV-induced disease........................... 27
1.5.1.2 encing recovery from FV-induced splenomegaly.................... 28
1.5.2 Immunity to FV infection............................................................................. 29
1.5.2.1 Role of cytotoxic CD8+ T cells in FV immunity......................................... 29 Contents

1.5.2.2 Role of immunosuppressive Regulatory T cells in FV immunity................ 30
1.5.2.3 Role of B cells in FV immunity.................................................................... 31
1.5.2.4 Vaccine-induced protection against FV............................... 32
1.5.2.5 Role of helper T cells in FV immunity......................................................... 33


2. Aim and Scope of work............................................................................ 35

3. Materials..................................................................................................... 37

3.1 Laboratory animals..................................................................................... 37
3.1.1 Wild-type mice........................................... 37
3.1.2 Congenic mice............................................................................................ 37
3.1.3 Transgenic mice.......................................... 37

3.2 Virus........................................................................................................... 38
3.3 Cell line.............................. 38
3.4 Equipment and materials........................................................................... 39
3.5 Chemicals and media................................. 40
3.6 Antibiotics......................................................................... 41
3.7 Buffers and media…......................................................... 41
3.8 Antibodies......................................................................... 42
3.9 Fluorochromes....................................................43
3.10 Tetramers................................................................................................... 44


4. Methods..................................................................................................... 45

4.1 Animal trials............................................................................................... 45
4.2 Replication of the Friend virus in vivo....................................................... 45
4.3 In vivo depletion of lymphocyte subsets................................................... 45
4.3.1 Culturing of YTS191.1...................................................... 46
4.4 Measurement of Friend virus induced splenomegaly................................ 47
4.5 Preparation of single cell suspension of spleen……................................. 48 Contents

4.6 MACS technology for enrichment of CD4 T-cell subset.......................... 49
4.7 Cultivation and storage of Mus dunni cells................................................ 52
4.8 Estimation of viral loads by infectious center assay.................................. 52
4.9 Virus-neutralising antibody assay.............................................................. 54
4.10 Tetramers and Tetramer staining....................................... 54
4.11 Flow cytometry.......................................................................................... 56
4.11.1 Methodology of Flow cytometry....................................... 56
4.11.2 Staining of cells and FACS analyses......................................................... 60
4.11.3 Exclusion of dead cells in Flow cytometry....................... 60
4.12 Statistical analyses..................................................................................... 61


5. Results....................................................................................................... 62
5.1 Helper T cells are important in controlling viral replication
and preventing erythroleukemia during acute FV infections..................... 62
5.2 CD4 T cell help is needed for maintenance of FV- specific
+effector & memory CD8 T cell responses & for the induction
of neutralizing antibodies by B cells during acute FV infection................ 66
5.3 Resistance to FV-induced disease correlated with the
+magnitude of H-2A-restricted CD4 T cell responses................................ 70
+5.4 FV- specific CD4 T cells produced IFN- γ production and
reduced viral loads in FV-infected mice..................................................... 74
+5.5 Suppression of FV-specific CD4 T cell responses
by Regulatory T cells.................................................................................. 81

6. Discussion……………………………………………………………….. 87
7. Summary…….………….. 97
8. Bibliography…………………………………………………………….. 99
9. Appendix………………………………………………………………... 111
9.1 Abbreviations............................................................................................. 111
9.2 List of figures.....................................117
9.3 List of tables............................................................................................... 119