Characterization of the cytokine profile in adults with latent and active tuberculosis from a high endemic country [Elektronische Ressource] : and on the role of the cytotoxic protein granulysin in childhood tuberculosis / Henrik Müller. Gutachter: Stefan Kaufmann ; Hans-Dieter Volk ; Ulrich Steinhoff

Henrik Müller

Sujets

• Gesundheit

Informations

• Publié par: humboldt-universitat_zu_berlin
• Publié le: 01 janvier 2011
• Nombre de lectures: 29
• Langue: Deutsch
• Poids de l'ouvrage: 4 Mo

Extrait

Characterization of the cytokine profile in adults with latent and active tuberculosis
from a high endemic country
And
On the role of the cytotoxic protein granulysin in childhood tuberculosis

Dissertation
zur Erlangung des akademischen Grades
des Doktors doctor rerum naturalium
(Dr. rer. nat.)
im Fach Biologie
eingereicht an der

Mathematisch-Naturwissenschaftlichen Fakultät I
der Humboldt-Universität zu Berlin

vorgelegt von
Diplom-Biologe
Henrik Müller


Präsident der Humboldt-Universität zu Berlin
Prof. Dr. Dr. h.c. Christoph Markschies

Dekan der Mathematischen-Naturwissenschaftlichen Fakultät I
Prof. Dr. Andreas Herrmann
Gutachter/-innen:
1. Prof. Dr. Dr. h.c. Stefan Kaufmann.
2. Prof. Dr. med. Hans-Dieter Volk
3. Prof. Dr. Ulrich Steinhoff

Tag der mündlichen Prüfung: 16.11.2010















„Der Mensch hat dreierlei Wege, klug zu
handeln: Erstens durch Nachdenken, das ist der
Edelste, zweitens durch Nachahmen, das ist der
Leichteste, und drittens durch Erfahrung, das ist
der Bitterste.“
Konfuzius



III



Table of contents

Summary ................................................................................................................................. IX
1 Introduction ....................................................................................................................... 1
1.1 Immune system .............................................................................................................. 1
1.1.1 Innate immune system .......................................................................................... 1
1.1.2 Adaptive immune system ..................................................................................... 3
1.2 T cell activation and co-stimulation ............................................................................. 5
1.3 e, effector and memory T cells 6
1.3.1 Naive T cells ......................................................................................................... 6
1.3.2 Effector T cells ..................................................................................................... 6
1.3.3 Memory T cells .................................................................................................... 6
+1.4 CD4 T cell populations ................................................................................................ 9
1.4.1 T-helper cells type 1 (T 1) lineage ...................................................................... 9 H
1.4.2 T-helper cells type 2 (T 2) lineage .................................................................... 10 H
1.4.3 T-helper cells type 17 (T 17) lineage ................................................................ 11 H
1.4.4 Natural and adaptive regulatory T cells ............................................................. 12
1.5 Cytotoxic T lymphocytes ............................................................................................ 14
1.5.1 The Fas-Fas ligand linkage-mediated pathway .................................................. 14
1.5.2 Granule-dependent exocytosis pathway 14
1.6 M. tuberculosis ............................................................................................................. 17
1.6.1 Epidemiology and disease .................................................................................. 17
1.6.2 Latency versus active disease ............................................................................. 18
1.6.3 Diagnosis of M. tuberculosis .............................................................................. 19
1.6.4 Vaccines and biomarkers ................................................................................... 20
1.6.5 Childhood tuberculosis ....................................................................................... 21
1.7 Adaptive immune response to M. tuberculosis-infection ......................................... 23
1.7.1 T-cell cytokines in response to M. tuberculosis ................................................. 23
+1.7.2 CD4 T cell subsets involved in the immune response to M. tuberculosis ........ 24
1.8 Aims of the study ......................................................................................................... 26
V

2 Methods ............................................................................................................................ 27
2.1 Study population of adults from high endemic country .......................................... 27
2.2 Study population for childhood tuberculosis ............................................................ 27
2.3 Cell culture handling ................................................................................................... 28
2.4 Isolation of PBMC from human blood ...................................................................... 28
2.5 Intracellular staining after short-term stimulation assay ....................................... 28
2.6 Intracellular CD107a/b staining ................................................................................ 29
2.7 7d in vitro restimulation assay .................................................................................... 29
®2.8 Magnetic cell separation (MACS ) ............................................................................ 30
2.8.1 Isolation of monocytes ....................................................................................... 30
2.9 Proliferation assays ..................................................................................................... 30
2.10 Generation of granulysin expressing T cell lines ...................................................... 31
2.11 Quantitative real-time polymerase chain reaction ................................................... 31
2.12 Assessment of cytolytic activity of T cell lines in monocytes ................................... 31
2.13 Assessment of cytolytic activity of T cell lines in macrophages .............................. 32
3 Results .............................................................................................................................. 33
3.1 Characterization of the cytokine profile in adults with latent and active
tuberculosis from a high endemic country ...................................................... 33
3.1.1 Study design ....................................................................................................... 33
3.1.2 Cytokine response in latent and active TB patients under therapy .................... 33
+3.1.3 Characterization of multi-functional CD4 T cells from patients
undergoing therapy and in comparison to LTBI ................................................ 36
+3.1.4 Analysis of CD4 memory T cells from patients undergoing treatment
and in comparison to LTBI ................................................................................ 39
+3.1.5 Characterization of multi-functional memory CD4 T cells from
patients undergoing therapy and in comparison to LTBI .................................. 41
3.1.6 Co-stimulation with IL-7 induces higher cytokine expression and
leads to increased multi-functionality ................................................................ 43
VI
3.1.7 Impact of T 2 and T 17 on cytokine response during treatment and in H H
comparison to LTBI ........................................................................................... 44
+3.1.8 Concluding remarks on the cytokine profile of CD4 T cells from
TB patients and LTBI ........................................................................................ 45
3.2 The role of the cytotoxic protein granulysin in childhood tuberculosis ................. 47
3.2.1 Granulysin expression is upregulated in latently infected children and
children with active TB after antigen stimulation ............................................. 47
3.2.2 Granulysin expressing cells show central and effector
memory-like phenotype ..................................................................................... 51
3.2.3 Granulysin expression is highly inducible in patients that
completed therapy .............................................................................................. 52
+3.2.4 Phenotyping of antigen-specific granulysin expressing CD4 T cells ............... 54
3.2.5 Granulysin expressing T cell lines show non-polarized phenotype ................... 57
3.2.6 Antigen-specific cytotoxic T cell lines expressing granulysin
reduce viability of M. tuberculosis in infected target cells ................................ 63
3.2.7 Concluding remarks on granulysin expression in childhood tuberculosis ......... 64
4 Discussion ......................................................................................................................... 65
4.1.1 Differences in T 1 cytokine expression between LTBI and TB patients .......... 65 H
4.1.2 Effects of antimicrobial treatment on T 1 cytokine expression ......................... 67 H
+4.1.3 Dynamic changes in CD4 T cell cytokine profile under
anti-tuberculosis drug treatment ..