The importance of CD8_1hn+ T-cells and antigen-presenting cells in the immune reaction of primary inflammatory versus degenerative diseases [Elektronische Ressource] / vorgelegt von Nicholas Schwab

Nicholas Breton

Sujets

• Gesundheit

Informations

• Publié par: julius-maximilians-universitat_wurzburg
• Publié le: 01 janvier 2009
• Nombre de lectures: 33
• Langue: Deutsch
• Poids de l'ouvrage: 5 Mo

Extrait

+The importance of CD8 T cells and
antigen-presenting cells in the immune reaction
of primary inflammatory versus degenerative
diseases




Dissertation zur Erreichung des
naturwissenschaftlichen Doktorgrades
der Bayerischen Julius-Maximilians-Universität Würzburg





vorgelegt von
Nicholas Schwab
aus Würzburg






Würzburg 2009




















Eingereicht am:

Mitglieder der Promotionskommission:
Vorsitzender: Prof. Dr. M. J. Müller
Gutachter: Prof. Dr. H. Wiendl
Gutachter: Prof. Dr. E. Buchner

Tag des Promotionskolloquiums:

Doktorurkunde ausgehändigt am:






Erklärung:



Hiermit erkläre ich ehrenwörtlich, die vorliegende Arbeit selbständig angefertigt
und keine anderen als die angegebenen Quellen und Hilfsmittel verwendet zu
haben.

Diese Arbeit hat weder in gleicher noch in ähnlicher Form in einem anderen
Prüfungsverfahren vorgelegen.

Ich habe in keinem früheren Verfahren einen akademischen Titel erworben oder
zu erwerben versucht.





...............................................................................................
Nicholas Schwab
Würzburg, April 2009




Für meine Eltern.















I could not prove the Years had feet —
Yet confident they run
Am I, from symptoms that are past
And Series that are done —

I find my feet have further Goals —
I smile upon the Aims
That felt so ample — Yesterday —
Today's — have vaster claims —

I do not doubt the self I was
Was competent to me —
But something awkward in the fit —
Proves that — outgrown — I see —


Emily Dickinson
#563


Table of contents


1. Summary ........................................................................................ 7

2. Zusammenfassung........................................................................ 9

3. Introduction................. 11

3.1. Overview of the immune system.......11

3.2. T-cell development...........................................................11

3.3. The T-cell receptor.............................................................13

3.3.1. V(D)J recombination of the T-cell receptor...............................................13

3.3.2. The CDR3 of the TCR..................................14

3.3.3. P- and N-nucleotides................................................................15

3.4. T-cell activation and proliferation.....................................15

3.5. PD-1...................................................................................16

3.6. Proteolipid protein............................................................17

3.7. Multiple sclerosis and gliopathy-induced inflammation..17

3.8. The PLP-tg mouse as a model for gliopathy-induced inflammation..................18

3.9. The CNS - a site of immunological privilege?.....................19

+ +3.10. CD8 T cells and CD8 T-cell-mediated cytotoxicity .......................................20

3.11. Rasmussen encephalitis..................................................21

3.12. Myositis ...........................................22

3.13. Antigen-presenting cells.................................................22

3.13.1. Dendritic cells...........................................................................................................22

3.13.2. Macrophages..............24

3.14. Goal of this study.............................................................26

4. Materials....................................................... 27

4.1. Buffers and media27

4.2. Antibodies.........................................................................28

4.3. Cytokines...........28

4.4. Primers...............29

4.4.1. Spectratyping primers (human samples).....................................................29

4.4.2. Spectratyping primers (murine samples)....................................................30

4.5. Mice ...................................................................................32

4.5.1. Proteolipid transgenic (PLP-tg) mice ............................................................32

4.5.2. PD-1 deficient mice..................................................................32

4.5.3. Double transgenic mice.........................32

4.5.4. Bone marrow chimerization...............................................................................................................33

4.6. Reagents............................................33

4.7. Consumables.....34

4.8. Equipment.........................................................................34

4.9. Software............35

5. Methods ....................................................... 36

5.1. Isolation of peripheral blood mononuclear cells (PBMC)...................................36

5.2. Isolation of splenocytes.....................................................................................36

5.3. Lymphocyte purification from murine CNS36

5.4. Magnetic activated cell separation (MACS®)36

5.5. Fluorescence staining for flow cytometry..........................37

5.6. Cell culture.........................................................................................................37

5.7. Assessment of cell proliferation by CFSE labeling............38

5.8. Macrophages phagocytosis assay.....38

5.9. RNA isolation.....39

5.9.1. RNA isolation from tissue ......................................................................................................................39

5.9.2. RNA isolation from cells.........................39

5.9.2.1. TriZol®...........................................................39

5.9.2.2. Column-based spin kit..........................................................................................................................39

5.10. cDNA synthesis40

5.10.1. Unspecific.....................................................40

5.10.2. TCR specific (human samples)..........................................................................................................40

5.11. CDR3 spectratyping PCRs...............40

5.11.1. Human system...........................................40

5.11.2. Murine system...........................................................................................................41

5.12. Fragment analysis...........................41

5.13. Data normalization and analysis.....................................41

5.14. Statistics ..........................................................................42

6. Results and Discussion 43

7. Final assessment.......................................................................... 44

8. Abbreviations.............. 47

9. Acknowledgements.... 50

10. References .................................................................................. 51

11. Curriculum Vitae........ 60

12. Publications............... 61

12.1. Peer-reviewed publications............................................................................61

12.2. Poster presentations.......................62

12.3. Oral presentations...........................................................................................62

13. Appendix.................... 64

13.1. Manuscripts for the cumulative dissertation...................64

7

1. Summary
The bidirectional influence of parenchymal cells and cells of the immune system,
+especially of antigen-presenting and CD8 T cells, in situations of putative auto-
immune pathogenicity and degeneration was the main topic of this thesis.
In the first part, the influence of human muscle cells on antigen-presenting cells was
investigated. In inflammatory myopathies prominent infiltrates of immune cells
containing T cells and antigen-presenting cells like macrophages and dendritic cells are
present. The hypothesis was that human myoblasts have an inhibiting influence on
these antigen-presenting cells under homeostatic conditions. A dysfunction or
impairment under inflammatory circumstances might contribute to the development
of myopathic conditions. The surface analysis of dendritic cells cocultured with
myoblasts showed that immature dendritic cells could be driven into a reversible semi-
mature state with significantly elevated levels of CD80. These dendritic cells were
additionally characterized by their inhibiting function on T-cell proliferation. It was also
shown that the lysates of healthy myoblasts could strongly enhance the phagocytic
ability of macrophages, which could help with muscle regeneration and which might
be disturbed in myositis patients.
+The second part of this thesis was about the clonal specificity of CD8 T cells in a
mouse model with genetically induced over-expression of PLP in oligodendrocytes.
Here, we could