The T-cell receptor and it's signaling in T-cell prolymphocytic leukemia:
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| 020 | |c Broschur | ||
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| 100 | 1 | |a Oberbeck, Sebastian Tobias Karl-Heinz |d 1984- |e Verfasser |0 (DE-588)1202484824 |4 aut | |
| 245 | 1 | 0 | |a The T-cell receptor and it's signaling in T-cell prolymphocytic leukemia |c vorgelegt von Sebastian Tobias Karl-Heinz Oberbeck |
| 264 | 1 | |a Köln |c 2019 | |
| 300 | |a XVII, 158 Seiten |b Illustrationen | ||
| 336 | |b txt |2 rdacontent | ||
| 337 | |b n |2 rdamedia | ||
| 338 | |b nc |2 rdacarrier | ||
| 502 | |b Dissertation |c Universität zu Köln |d 2019 | ||
| 650 | 0 | 7 | |a T-Zell-Leukämie |0 (DE-588)4509988-1 |2 gnd |9 rswk-swf |
| 650 | 0 | 7 | |a T-Lymphozyten-Rezeptor |0 (DE-588)4265934-6 |2 gnd |9 rswk-swf |
| 655 | 7 | |0 (DE-588)4113937-9 |a Hochschulschrift |2 gnd-content | |
| 689 | 0 | 0 | |a T-Zell-Leukämie |0 (DE-588)4509988-1 |D s |
| 689 | 0 | 1 | |a T-Lymphozyten-Rezeptor |0 (DE-588)4265934-6 |D s |
| 689 | 0 | |5 DE-604 | |
| 856 | 4 | 2 | |m B:DE-101 |q application/pdf |u https://d-nb.info/1208423703/04 |3 Inhaltsverzeichnis |
| 856 | 4 | 2 | |m DNB Datenaustausch |q application/pdf |u http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=032333053&sequence=000001&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA |3 Inhaltsverzeichnis |
| 943 | 1 | |a oai:aleph.bib-bvb.de:BVB01-032333053 | |
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| _version_ | 1811184397650493440 |
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| adam_text |
1
TABLE
OF
CONTENTS
ACKNOWLEDGEMENTS
II
ABSTRACT
AND
ZUSAMMENFASSUNG
IV
LIST
OF
ABBREVIATIONS
VIII
TABLE
OF
CONTENTS
XIII
1
INTRODUCTION
1
1.1
CLINICAL
CHARACTERISTICS
OF
T-PLL
AND
CURRENT
TREATMENT
OPTIONS
2
1.2
THE
MOLECULAR
MAKEUP
OF
T-PLL
IS
CHARACTERIZED
BY
INVERSIONS
/
TRANSLOCATIONS
OF
CHROMOSOME
14
LEADING
TO
ABERRANT
EXPRESSION
OF
TCL1A
4
1.3
ADDITIONAL
RECURRENT
GENETIC
ABERRATIONS
DOMINANTLY
AFFECT
THE
DNA
DAMAGE
RESPONSE
MEDIATOR
ATM
AND
PRO-SURVIVAL
SIGNALING
EFFECTORS
OF
THE
JAK/STAT
PATHWAY
7
1.4
T-PLL
CELL
PHENOTYPES
IN
THE
CONTEXT
OF
T-CELL
DEVELOPMENT
AND
DIFFERENTIATION
8
1.4.1
T-CELL
DEVELOPMENT
WITHIN
THE
THYMUS
8
1.4.2
GENOMIC
REARRANGEMENTS
OF
T-CELL
RECEPTOR
GENES
10
1.4.3
T-CELL
RECEPTOR
PROTEIN
STRUCTURE
12
1.4.4
T-CELL
EFFECTOR
FUNCTIONS
AND
IMMUNOLOGIC
MEMORY
13
1.4.5
ACTIVATION-INDUCED
CELL
DEATH
AND
CLONAL
COMPETITION
ARE
IMPORTANT
PROCESSES
OF
T-
CELL
HOMEOSTASIS
15
1.4.6
T-PLL
CELLS
APPEAR
AS
PREDOMINANTLY
TCR
POSITIVE
T-CELLS
OF
A
MATURE
POST-THYMIC
PHENOTYPE
16
1.5
THE
T-CELL
RECEPTOR
SIGNALING
PATHWAY
AS
POTENTIAL
ONCOGENIC
FORCE
IN
T-PLL.
18
1.5.1
PROXIMAL
TCR
ACTIVATION
18
1.5.2
IP3
AND
PIP2
MEDIATED
ACTIVATION
OF
AP-1
AND
AKT
20
1.5.3
PKC6
MEDIATED
ACTIVATION
OF
MAP
KINASES
AND
NF-
K
B
PATHWAYS
20
1.5.4
TCL1A
MODULATES
SURVIVAL
SIGNALS
DOWNSTREAM
OF
THE
TCR
21
1.6
OBJECTIVES
22
2
MATERIALS
AND
METHODS
24
2.1
MATERIALS
24
2.1.1
LABORATORY
INSTRUMENTS
24
2.1.2
DISPOSABLES
25
2.1.3
CELL
CULTURE
REAGENTS
26
2.1.4
CELL
LINES
27
2.1.5
MOUSE
MODELS
27
2.1.6
SOFTWARE
27
2.1.7
CHEMICALS
28
TABLE
OF
CONTENTS
2.1.8
BUFFERS
AND
SOLUTIONS
.
29
2.1.9
DRUGS
.
30
2.1.10
ANTIBODIES
AND
CELL
ISOLATION
KITS
.
30
2.1.11
READY
TO
USE
KITS,
REAGENTS
AND
MARKERS
.
32
2.1.12
OLIGONUCLEOTIDES
.
34
2.2
METHODS
.
34
2.2.1
CELL
BIOLOGY
.
34
2.2.1.1
PRIMARY
HUMAN
MATERIAL
.
34
2.2.1.2
CELL
CULTURE
.
34
2.2.1.3
THAWING
AND
FREEZING
OF
CELL
LINES
AND
PRIMARY
T-PLL
CELLS
.
35
2.2.1.4
CELL
COUNTING
.
36
2.2.1.5
DENSITY
GRADIENT
CENTRIFUGATION
.
36
2.2.1.6
MAGNETIC
CELL
SORTING
AND
ENRICHMENT
.
36
2.2.1.7
FLOW
CYTOMETRY
.
37
2.2.1.7.1
PHENOTYPING
OF
T-PLL
SAMPLES
USING
FLOW
CYTOMETRY
.
37
2.2.1.7.2
FLOW
CYTOMETRIC
ANALYSIS
OF
CELL
LINES
AND
MURINE
SAMPLES
.
38
2.2.1.7.3
TCR
VP
SPECTRATYPING
IN
T-PLL
SAMPLES
.
38
2.2.1.7.4
TCR
VP
SPECTRATYPING
IN
MURINE
SAMPLES
.
38
2.2.1.7.5
DETECTION
OF
APOPTOSIS
USING
ANNEXIN
V
/
7AAD
STAINING
AND
FLOW
CYTOMETRY.
39
2.2.1.8
CELLTITER-GLO
LUMINESCENT
CELL
VIABILITY/TOXICITY
ASSAY
.
39
2.2.1.9
T-CELL
STIMULATION
USING
PLATE-BOUND
ANTI-CD3/CD28
ANTIBODIES
.40
2.2.1.10
ASSESSMENT
OF
BASAL
GLYCOLYSIS
AND
OXIDATIVE
PHOSPHORYLATION
.
41
2.2.2
PROTEIN
BIOCHEMISTRY
.
42
2.2.2.1
PREPARATION
OF
PROTEIN
LYSATES
.42
2.2.2
2
DETERMINATION
OF
PROTEIN
CONCENTRATION
.
42
2.2.2.3
IMMUNOBLOTTING
.43
2.2.2.3.1
SDS-PAGE
.43
2.2.2.3.2
WESTERN
BLOTTING
.43
2.2.2.3.3
PONCEAU
S
STAINING
.
43
2.2.2.3.4
IMMUNO
DETECTION
.44
2.2.2.3
5
DENSITOMETRIC
IMMUNOQUANTIFICATION
.
44
2.2.2.4
IL2
ELISA
.
44
2.2.2.5
SOLUBLE
FAS
ELISA
.45
2.2.2.6
MULTIPLEX
CYTOKINE
ARRAYS
.45
2.2.2.6.1
HUMAN
AND
MURINE
MULTIPLEX
CYTOKINE
ARRAYS
.45
2.2.2.6.2
HUMAN
PROCARTAPLEX
45-PLEX
CYTOKINE
ARRAY
.
46
2.2.3
MOLECULAR
BIOLOGY
.46
-XIV-
TABLE
OF
CONTENTS
2.2.3.1
ISOLATION
OF
NUCLEIC
ACIDS
.46
2.2.3.1.1
DNA
ISOLATION
.46
2.2.3.1.2
RNA
ISOLATION
.
47
2.2.3.1.2.1 RNEASY*
KIT
.
47
2.2.3.1.2.2
MIRVANA*
KIT
.47
2.2.3.2
DETERMINATION
OF
DNA/RNA
CONCENTRATION
.47
2.2.3
3
HUMAN
TCL1A
CDNA
DETECTION
IN
MURINE
SPLENOCYTES
BY
PCR
.
47
2.2.3.4
AGAROSE
GEL
ELECTROPHORESIS
.48
2.2.3.5
CDNA
SYNTHESIS
.
49
2.2.3.6
PRIMER
DESIGN
.
49
2.2.3.7
QUANTITATIVE
REAL-TIME
PCR
.
49
2.2.3.S
ARRAY
BASED
GENE
EXPRESSION
PROFILING
.
50
2.2
3.9
T-CELL
RECEPTOR
NGS
SEQUENCING
.
51
2.2.4
BIOINFORMATICS
AND
STATISTICS
.
51
2.2.4.1
BIOINFORMATIC
ANALYSIS
OF
GENE
EXPRESSION
PROFILING
.
51
2.2.4.2
T-CELL
RECEPTOR
NGS
BIOINFORMATIC
ANALYSIS
.
52
2.2
4.3
STATISTICAL
ANALYSIS
.
52
2.2.4.4
IL2
SECRETION
MODELING
BY
FITTING
KINETIC
SECRETION
MODELS
.
52
2.2.5
ANIMALS
EXPERIMENTS
.
53
2.2.5.1
LABORATORY
MICE
.
53
2.2.5.1
.1
LCKP
R
-HTCL1A
T9
MOUSE
MODEL
.
53
2.2.5.1.2
CEA
TG
MOUSE
MODEL
.
53
2.2.5.1.3
CARP
EA
MOUSE
MODEL
.
54
2.2.5
2
PROCEDURES
IN
ANIMAL
EXPERIMENTS
.
54
2.2.5.2.1
BLOOD
COLLECTION
FROM
MICE
.
54
2.2.5.2.2
NARCOTIZATION
OF
MICE
BY
ISOFLURAN
INHALATION
.
54
2.2.5.2.3
SACRIFICATION
OF
MICE
.
55
2.2.5.2.4
ISOLATION
OF
SPLENOCYTES
FROM
MURINE
SPLEENS
.
55
2.2.5.2
5
ADOPTIVE
CELL
TRANSPLANT
EXPERIMENT
.
55
2.2.5.2.5.1
MONITORING
OF
CELL
REPOPULATION
AFTER
TRANSPLANTATION
BY
FLOW
CYTOMETRY
.
56
2.2.5.2.5
2
MAGNETIC
RESONANCE
IMAGING
AND
SPLEEN
VOLUMETRY
.
57
2.2.5.2
5.3
PREPARATION
OF
THE
INTESTINE
USING
THE
*
SWISS
ROLE
*
TECHNIQUE
.
57
2.2.6
HISTOLOGY
.
58
2.2.6.1
CRYOSECTIONING
.
58
2.2.6.2
HEMATOXYLIN
AND
EOSIN
STAINING
.
58
2.2.6.3
IMMUNOHISTOCHEMISTRY
.
58
2.2.6.4
PROCESSING
AND
STORAGE
OF
HISTOCHEMICAL
STAININGS
.
59
-XV-
TABLE
OF
CONTENTS
3
RESULTS
.
60
3.1
TCR
EXPRESSION
IN
T-PLL
SAMPLES
.
60
3.1.1
COMPREHENSIVE
CHARACTERIZATION
OF
T-PLL
CELL
PHENOTYPES
AND
EFFECTOR
FUNCTIONS.
60
3.1.2
THE
VAST
MAJORITY
OF
T-PLL
CELLS
RETAIN
SURFACE
TCR,
CD3,
AND
CD28
RECEPTOR
EXPRESSION
.
60
3.1.3
THE
CONSTITUTIONAL
TCR
PROFILE
OF
T-PLL
IS
DIVERSE
.
61
3.1.4
T-CELLS
OF
LEUKEMIC
LCK^-HTCLIA
TG
MICE
RETAIN
SURFACE
CD3
AND
CD28
EXPRESSION
AS
OBSERVED
IN
HUMAN
T-PLL
.
65
3.1.5
LEUKEMIC
LCK?
R
-HTCL1A
T9
MICE
DEVELOP
A
MONO
/
BI-CLONAL
T-PLL
LIKE
DISEASE
WITH
NO
PREVALENT
VP
CHAIN
USAGE
.
66
3.2
COMPREHENSIVE
CHARACTERIZATION
OF
T-CELL-SUBSET
SPECIFIC
PHENOTYPES
OF
T-PLL
CELLS
68
3.2.1
THE
MAJORITY
OF
T-PLL
CASES
RESEMBLES
A
PHENOTYPE
OF
MEMORY
TYPE
T-CELLS
.
68
3.2.2
T-PLL
CELLS
SHOW
A
GENE-EXPRESSION
SIGNATURE
THAT
IS
SIMILAR
TO
HEALTHY-DONOR
DERIVED
CENTRAL-MEMORY
T-CELLS
.
72
3.2.3
MURINE
T-PLL
CELLS
RESEMBLE
AN
EFFECTOR
MEMORY
PHENOTYPE
.
77
3.3
T-CELL
ACTIVATION
STATUS
AND
TCR
ACTIVATION-INDUCED
FUNCTIONAL
EFFECTS
IN
PRIMARY
HUMAN
AND
MURINE
T-PLL
CELLS
.
78
3.3.1
T-PLL
CELLS
SHOW
A
PHENOTYPE
OF
HIGH
ACTIVATION
ACCOMPANIED
BY
HETEROGENEOUS
EXPRESSION
OF
CYTOKINE/CHEMOKINE
RECEPTORS
.
78
3.3.2
THE
EXPRESSION
OF
IMMUNE
CHECKPOINT
REGULATORS
AND
CO-RECEPTORS
IS
DIMINISHED
IN
T-PLL
CELLS
.
81
3.3.3
SPLENOCYTES
OF
LEUKEMIC
LCK?
R
-HTCL1A
T9
MICE
SHOW
INCREASED
LEVELS
OF
PHOSPHORYLATED
ERK1/2
UPON
STIMULATION
COMPARED
TO
AGE-MATCHED
C57BL/6J
WILD
TYPE
ANIMALS
.
84
3.3.4
BASAL
AND
TCR
ACTIVATION
INDUCED
NAIVE/MEMORY-,
ACTIVATION-MARKER
AND
CYTO-
/CHEMOKINE
RECEPTOR
EXPRESSION
IS
SIMILAR
COMPARING
HEALTHY-DONOR
DERIVED
CENTRAL
MEMORY
T-CELLS
AND
T-PLL
CELLS
.
86
3.3.5
T-PLL
CELLS
SHOW
INCREASED
SECRETION
OF
CYTOKINES
AND
ENHANCED
METABOLIC
ACTIVITY
UPON
ANTI-CD3/CD28
CROSS-LINKING
.
89
3.3.6
BASAL
AND
TCR-ACTIVATION
INDUCED
METABOLIC
ACTIVITY
IS
ENHANCED
IN
T-PLL
CELLS.
92
3.3.7 GENE
EXPRESSION
PROFILES
OF
PRIMARY
HUMAN
T-PLL
CELLS
AND
MURINE
T-PLL
LIKE
T-CELL
EXPANSIONS
(LCKP
R
-HTCL1A
T9
MICE)
REFLECT
GLOBAL
TCR
SIGNALING
DEREGULATIONS
ON
TRANSCRIPT
LEVEL
.
94
3.4
APOPTOSIS
AND
ACTIVATION
INDUCED
CELL
DEATH
IN
T-PLL
.
98
-XVI-
TABLE
OF
CONTENTS
3.4.1
CD95
/
CD95L
MEDIATED
ACTIVATION-INDUCED-CELL-DEATH
(AICD)
IS
IMPAIRED
IN
T-PLL
CELLS
98
3.4.2
IMPAIRED
APOPTOSIS
INDUCTION
IS
REFLECTED
IN
A
GLOBAL
DEREGULATION
OF
GENES
ENCODING
FOR
PRO-
/
ANTI-APOPTOTIC
FACTORS
99
3.4.3
CD95
RECEPTOR
SURFACE
EXPRESSION
IS
HETEROGENEOUS
IN
T-PLL
101
3.4.4
APOPTOTIC
RESPONSE
OF
T-PLL
CELLS
TO
AGONISTIC
ANTI-CD95
ANTIBODY
TREATMENT
IS
DIMINISHED
102
3.4.5
TCL1A
EXPRESSION
EXHIBITS
A
PROTECTIVE-EFFECT
AGAINST
CD95
MEDIATED
APOPTOSIS
104
3.4.6
LEVELS
OF
SOLUBLE
CD95
RECEPTOR
ARE
ELEVATED
IN
THE
PLASMA
OF
T-PLL
PATIENTS.
105
3.5
TCL1A
IS
A
PRO-TUMORIGENIC
AMPLIFIER
OF
TCP
SIGNALING
106
3.5.1
ECTOPIC
TCL1A
EXPRESSION
IN
T-CELL
LINES
INCREASES
PHOSPHORYLATION
OF
ERK1/2
UPON
TCP
STIMULATION
106
3.5.2
TCL1A
EXHIBITS
A
CONCENTRATION
DEPENDENT
EFFECT
ON
TCP
SIGNALING
AS
SHOWN
BY
THE
ACTIVATION
OF
PHOSPHOKINASES
AND
DOWNSTREAM
EFFECTOR
FUNCTIONS
109
3.5.3
A
MULTI-DIMENSIONAL-TITRATION-APPROACH
REVEALS
CONCENTRATION
DEPENDENT
EFFECTS
OF
TCL1A
PREFERENTIALLY
ON
LOW
LEVEL
TCP
ACTIVATION
AS
DETECTED
BY
IL2
SECRETION
LEVELS.
.110
3.5.4
INDUCED
SURFACE
EXPRESSION
OF
THE
T-CELL
ACTIVATION
MARKERS
CD25,
CD38,
AND
CD69
UPON
TCP
STIMULATION
IS
ENHANCED
BY
ECTOPIC
TCL1A
EXPRESSION
IN
JURKAT
AND
IHH-TCL1A
CELLS
113
3.5.5
TCL1A
AND
TCP
ACTIVATION
COOPERATE
TOWARDS
AN
ACCELERATED
T-CELL
EXPANSION
IN
THE
CONTEXT
OF
CHRONIC
STIMULATION
IN
A
MURINE
MODEL
115
4
DISCUSSION
123
5
CONCLUSION
AND
OUTLOOK
135
6
REFERENCES
136
7
CURRICULUM
VITAE
153
8
EIDESSTATTLICHE
ERKLARUNG
157
-XVII- |
| adam_txt |
1
TABLE
OF
CONTENTS
ACKNOWLEDGEMENTS
II
ABSTRACT
AND
ZUSAMMENFASSUNG
IV
LIST
OF
ABBREVIATIONS
VIII
TABLE
OF
CONTENTS
XIII
1
INTRODUCTION
1
1.1
CLINICAL
CHARACTERISTICS
OF
T-PLL
AND
CURRENT
TREATMENT
OPTIONS
2
1.2
THE
MOLECULAR
MAKEUP
OF
T-PLL
IS
CHARACTERIZED
BY
INVERSIONS
/
TRANSLOCATIONS
OF
CHROMOSOME
14
LEADING
TO
ABERRANT
EXPRESSION
OF
TCL1A
4
1.3
ADDITIONAL
RECURRENT
GENETIC
ABERRATIONS
DOMINANTLY
AFFECT
THE
DNA
DAMAGE
RESPONSE
MEDIATOR
ATM
AND
PRO-SURVIVAL
SIGNALING
EFFECTORS
OF
THE
JAK/STAT
PATHWAY
7
1.4
T-PLL
CELL
PHENOTYPES
IN
THE
CONTEXT
OF
T-CELL
DEVELOPMENT
AND
DIFFERENTIATION
8
1.4.1
T-CELL
DEVELOPMENT
WITHIN
THE
THYMUS
8
1.4.2
GENOMIC
REARRANGEMENTS
OF
T-CELL
RECEPTOR
GENES
10
1.4.3
T-CELL
RECEPTOR
PROTEIN
STRUCTURE
12
1.4.4
T-CELL
EFFECTOR
FUNCTIONS
AND
IMMUNOLOGIC
MEMORY
13
1.4.5
ACTIVATION-INDUCED
CELL
DEATH
AND
CLONAL
COMPETITION
ARE
IMPORTANT
PROCESSES
OF
T-
CELL
HOMEOSTASIS
15
1.4.6
T-PLL
CELLS
APPEAR
AS
PREDOMINANTLY
TCR
POSITIVE
T-CELLS
OF
A
MATURE
POST-THYMIC
PHENOTYPE
16
1.5
THE
T-CELL
RECEPTOR
SIGNALING
PATHWAY
AS
POTENTIAL
ONCOGENIC
FORCE
IN
T-PLL.
18
1.5.1
PROXIMAL
TCR
ACTIVATION
18
1.5.2
IP3
AND
PIP2
MEDIATED
ACTIVATION
OF
AP-1
AND
AKT
20
1.5.3
PKC6
MEDIATED
ACTIVATION
OF
MAP
KINASES
AND
NF-
K
B
PATHWAYS
20
1.5.4
TCL1A
MODULATES
SURVIVAL
SIGNALS
DOWNSTREAM
OF
THE
TCR
21
1.6
OBJECTIVES
22
2
MATERIALS
AND
METHODS
24
2.1
MATERIALS
24
2.1.1
LABORATORY
INSTRUMENTS
24
2.1.2
DISPOSABLES
25
2.1.3
CELL
CULTURE
REAGENTS
26
2.1.4
CELL
LINES
27
2.1.5
MOUSE
MODELS
27
2.1.6
SOFTWARE
27
2.1.7
CHEMICALS
28
TABLE
OF
CONTENTS
2.1.8
BUFFERS
AND
SOLUTIONS
.
29
2.1.9
DRUGS
.
30
2.1.10
ANTIBODIES
AND
CELL
ISOLATION
KITS
.
30
2.1.11
READY
TO
USE
KITS,
REAGENTS
AND
MARKERS
.
32
2.1.12
OLIGONUCLEOTIDES
.
34
2.2
METHODS
.
34
2.2.1
CELL
BIOLOGY
.
34
2.2.1.1
PRIMARY
HUMAN
MATERIAL
.
34
2.2.1.2
CELL
CULTURE
.
34
2.2.1.3
THAWING
AND
FREEZING
OF
CELL
LINES
AND
PRIMARY
T-PLL
CELLS
.
35
2.2.1.4
CELL
COUNTING
.
36
2.2.1.5
DENSITY
GRADIENT
CENTRIFUGATION
.
36
2.2.1.6
MAGNETIC
CELL
SORTING
AND
ENRICHMENT
.
36
2.2.1.7
FLOW
CYTOMETRY
.
37
2.2.1.7.1
PHENOTYPING
OF
T-PLL
SAMPLES
USING
FLOW
CYTOMETRY
.
37
2.2.1.7.2
FLOW
CYTOMETRIC
ANALYSIS
OF
CELL
LINES
AND
MURINE
SAMPLES
.
38
2.2.1.7.3
TCR
VP
SPECTRATYPING
IN
T-PLL
SAMPLES
.
38
2.2.1.7.4
TCR
VP
SPECTRATYPING
IN
MURINE
SAMPLES
.
38
2.2.1.7.5
DETECTION
OF
APOPTOSIS
USING
ANNEXIN
V
/
7AAD
STAINING
AND
FLOW
CYTOMETRY.
39
2.2.1.8
CELLTITER-GLO
LUMINESCENT
CELL
VIABILITY/TOXICITY
ASSAY
.
39
2.2.1.9
T-CELL
STIMULATION
USING
PLATE-BOUND
ANTI-CD3/CD28
ANTIBODIES
.40
2.2.1.10
ASSESSMENT
OF
BASAL
GLYCOLYSIS
AND
OXIDATIVE
PHOSPHORYLATION
.
41
2.2.2
PROTEIN
BIOCHEMISTRY
.
42
2.2.2.1
PREPARATION
OF
PROTEIN
LYSATES
.42
2.2.2
2
DETERMINATION
OF
PROTEIN
CONCENTRATION
.
42
2.2.2.3
IMMUNOBLOTTING
.43
2.2.2.3.1
SDS-PAGE
.43
2.2.2.3.2
WESTERN
BLOTTING
.43
2.2.2.3.3
PONCEAU
S
STAINING
.
43
2.2.2.3.4
IMMUNO
DETECTION
.44
2.2.2.3
5
DENSITOMETRIC
IMMUNOQUANTIFICATION
.
44
2.2.2.4
IL2
ELISA
.
44
2.2.2.5
SOLUBLE
FAS
ELISA
.45
2.2.2.6
MULTIPLEX
CYTOKINE
ARRAYS
.45
2.2.2.6.1
HUMAN
AND
MURINE
MULTIPLEX
CYTOKINE
ARRAYS
.45
2.2.2.6.2
HUMAN
PROCARTAPLEX
45-PLEX
CYTOKINE
ARRAY
.
46
2.2.3
MOLECULAR
BIOLOGY
.46
-XIV-
TABLE
OF
CONTENTS
2.2.3.1
ISOLATION
OF
NUCLEIC
ACIDS
.46
2.2.3.1.1
DNA
ISOLATION
.46
2.2.3.1.2
RNA
ISOLATION
.
47
2.2.3.1.2.1 RNEASY*
KIT
.
47
2.2.3.1.2.2
MIRVANA*
KIT
.47
2.2.3.2
DETERMINATION
OF
DNA/RNA
CONCENTRATION
.47
2.2.3
3
HUMAN
TCL1A
CDNA
DETECTION
IN
MURINE
SPLENOCYTES
BY
PCR
.
47
2.2.3.4
AGAROSE
GEL
ELECTROPHORESIS
.48
2.2.3.5
CDNA
SYNTHESIS
.
49
2.2.3.6
PRIMER
DESIGN
.
49
2.2.3.7
QUANTITATIVE
REAL-TIME
PCR
.
49
2.2.3.S
ARRAY
BASED
GENE
EXPRESSION
PROFILING
.
50
2.2
3.9
T-CELL
RECEPTOR
NGS
SEQUENCING
.
51
2.2.4
BIOINFORMATICS
AND
STATISTICS
.
51
2.2.4.1
BIOINFORMATIC
ANALYSIS
OF
GENE
EXPRESSION
PROFILING
.
51
2.2.4.2
T-CELL
RECEPTOR
NGS
BIOINFORMATIC
ANALYSIS
.
52
2.2
4.3
STATISTICAL
ANALYSIS
.
52
2.2.4.4
IL2
SECRETION
MODELING
BY
FITTING
KINETIC
SECRETION
MODELS
.
52
2.2.5
ANIMALS
EXPERIMENTS
.
53
2.2.5.1
LABORATORY
MICE
.
53
2.2.5.1
.1
LCKP
R
-HTCL1A
T9
MOUSE
MODEL
.
53
2.2.5.1.2
CEA
TG
MOUSE
MODEL
.
53
2.2.5.1.3
CARP
EA
MOUSE
MODEL
.
54
2.2.5
2
PROCEDURES
IN
ANIMAL
EXPERIMENTS
.
54
2.2.5.2.1
BLOOD
COLLECTION
FROM
MICE
.
54
2.2.5.2.2
NARCOTIZATION
OF
MICE
BY
ISOFLURAN
INHALATION
.
54
2.2.5.2.3
SACRIFICATION
OF
MICE
.
55
2.2.5.2.4
ISOLATION
OF
SPLENOCYTES
FROM
MURINE
SPLEENS
.
55
2.2.5.2
5
ADOPTIVE
CELL
TRANSPLANT
EXPERIMENT
.
55
2.2.5.2.5.1
MONITORING
OF
CELL
REPOPULATION
AFTER
TRANSPLANTATION
BY
FLOW
CYTOMETRY
.
56
2.2.5.2.5
2
MAGNETIC
RESONANCE
IMAGING
AND
SPLEEN
VOLUMETRY
.
57
2.2.5.2
5.3
PREPARATION
OF
THE
INTESTINE
USING
THE
*
SWISS
ROLE
*
TECHNIQUE
.
57
2.2.6
HISTOLOGY
.
58
2.2.6.1
CRYOSECTIONING
.
58
2.2.6.2
HEMATOXYLIN
AND
EOSIN
STAINING
.
58
2.2.6.3
IMMUNOHISTOCHEMISTRY
.
58
2.2.6.4
PROCESSING
AND
STORAGE
OF
HISTOCHEMICAL
STAININGS
.
59
-XV-
TABLE
OF
CONTENTS
3
RESULTS
.
60
3.1
TCR
EXPRESSION
IN
T-PLL
SAMPLES
.
60
3.1.1
COMPREHENSIVE
CHARACTERIZATION
OF
T-PLL
CELL
PHENOTYPES
AND
EFFECTOR
FUNCTIONS.
60
3.1.2
THE
VAST
MAJORITY
OF
T-PLL
CELLS
RETAIN
SURFACE
TCR,
CD3,
AND
CD28
RECEPTOR
EXPRESSION
.
60
3.1.3
THE
CONSTITUTIONAL
TCR
PROFILE
OF
T-PLL
IS
DIVERSE
.
61
3.1.4
T-CELLS
OF
LEUKEMIC
LCK^-HTCLIA
TG
MICE
RETAIN
SURFACE
CD3
AND
CD28
EXPRESSION
AS
OBSERVED
IN
HUMAN
T-PLL
.
65
3.1.5
LEUKEMIC
LCK?
R
-HTCL1A
T9
MICE
DEVELOP
A
MONO
/
BI-CLONAL
T-PLL
LIKE
DISEASE
WITH
NO
PREVALENT
VP
CHAIN
USAGE
.
66
3.2
COMPREHENSIVE
CHARACTERIZATION
OF
T-CELL-SUBSET
SPECIFIC
PHENOTYPES
OF
T-PLL
CELLS
68
3.2.1
THE
MAJORITY
OF
T-PLL
CASES
RESEMBLES
A
PHENOTYPE
OF
MEMORY
TYPE
T-CELLS
.
68
3.2.2
T-PLL
CELLS
SHOW
A
GENE-EXPRESSION
SIGNATURE
THAT
IS
SIMILAR
TO
HEALTHY-DONOR
DERIVED
CENTRAL-MEMORY
T-CELLS
.
72
3.2.3
MURINE
T-PLL
CELLS
RESEMBLE
AN
EFFECTOR
MEMORY
PHENOTYPE
.
77
3.3
T-CELL
ACTIVATION
STATUS
AND
TCR
ACTIVATION-INDUCED
FUNCTIONAL
EFFECTS
IN
PRIMARY
HUMAN
AND
MURINE
T-PLL
CELLS
.
78
3.3.1
T-PLL
CELLS
SHOW
A
PHENOTYPE
OF
HIGH
ACTIVATION
ACCOMPANIED
BY
HETEROGENEOUS
EXPRESSION
OF
CYTOKINE/CHEMOKINE
RECEPTORS
.
78
3.3.2
THE
EXPRESSION
OF
IMMUNE
CHECKPOINT
REGULATORS
AND
CO-RECEPTORS
IS
DIMINISHED
IN
T-PLL
CELLS
.
81
3.3.3
SPLENOCYTES
OF
LEUKEMIC
LCK?
R
-HTCL1A
T9
MICE
SHOW
INCREASED
LEVELS
OF
PHOSPHORYLATED
ERK1/2
UPON
STIMULATION
COMPARED
TO
AGE-MATCHED
C57BL/6J
WILD
TYPE
ANIMALS
.
84
3.3.4
BASAL
AND
TCR
ACTIVATION
INDUCED
NAIVE/MEMORY-,
ACTIVATION-MARKER
AND
CYTO-
/CHEMOKINE
RECEPTOR
EXPRESSION
IS
SIMILAR
COMPARING
HEALTHY-DONOR
DERIVED
CENTRAL
MEMORY
T-CELLS
AND
T-PLL
CELLS
.
86
3.3.5
T-PLL
CELLS
SHOW
INCREASED
SECRETION
OF
CYTOKINES
AND
ENHANCED
METABOLIC
ACTIVITY
UPON
ANTI-CD3/CD28
CROSS-LINKING
.
89
3.3.6
BASAL
AND
TCR-ACTIVATION
INDUCED
METABOLIC
ACTIVITY
IS
ENHANCED
IN
T-PLL
CELLS.
92
3.3.7 GENE
EXPRESSION
PROFILES
OF
PRIMARY
HUMAN
T-PLL
CELLS
AND
MURINE
T-PLL
LIKE
T-CELL
EXPANSIONS
(LCKP
R
-HTCL1A
T9
MICE)
REFLECT
GLOBAL
TCR
SIGNALING
DEREGULATIONS
ON
TRANSCRIPT
LEVEL
.
94
3.4
APOPTOSIS
AND
ACTIVATION
INDUCED
CELL
DEATH
IN
T-PLL
.
98
-XVI-
TABLE
OF
CONTENTS
3.4.1
CD95
/
CD95L
MEDIATED
ACTIVATION-INDUCED-CELL-DEATH
(AICD)
IS
IMPAIRED
IN
T-PLL
CELLS
98
3.4.2
IMPAIRED
APOPTOSIS
INDUCTION
IS
REFLECTED
IN
A
GLOBAL
DEREGULATION
OF
GENES
ENCODING
FOR
PRO-
/
ANTI-APOPTOTIC
FACTORS
99
3.4.3
CD95
RECEPTOR
SURFACE
EXPRESSION
IS
HETEROGENEOUS
IN
T-PLL
101
3.4.4
APOPTOTIC
RESPONSE
OF
T-PLL
CELLS
TO
AGONISTIC
ANTI-CD95
ANTIBODY
TREATMENT
IS
DIMINISHED
102
3.4.5
TCL1A
EXPRESSION
EXHIBITS
A
PROTECTIVE-EFFECT
AGAINST
CD95
MEDIATED
APOPTOSIS
104
3.4.6
LEVELS
OF
SOLUBLE
CD95
RECEPTOR
ARE
ELEVATED
IN
THE
PLASMA
OF
T-PLL
PATIENTS.
105
3.5
TCL1A
IS
A
PRO-TUMORIGENIC
AMPLIFIER
OF
TCP
SIGNALING
106
3.5.1
ECTOPIC
TCL1A
EXPRESSION
IN
T-CELL
LINES
INCREASES
PHOSPHORYLATION
OF
ERK1/2
UPON
TCP
STIMULATION
106
3.5.2
TCL1A
EXHIBITS
A
CONCENTRATION
DEPENDENT
EFFECT
ON
TCP
SIGNALING
AS
SHOWN
BY
THE
ACTIVATION
OF
PHOSPHOKINASES
AND
DOWNSTREAM
EFFECTOR
FUNCTIONS
109
3.5.3
A
MULTI-DIMENSIONAL-TITRATION-APPROACH
REVEALS
CONCENTRATION
DEPENDENT
EFFECTS
OF
TCL1A
PREFERENTIALLY
ON
LOW
LEVEL
TCP
ACTIVATION
AS
DETECTED
BY
IL2
SECRETION
LEVELS.
.110
3.5.4
INDUCED
SURFACE
EXPRESSION
OF
THE
T-CELL
ACTIVATION
MARKERS
CD25,
CD38,
AND
CD69
UPON
TCP
STIMULATION
IS
ENHANCED
BY
ECTOPIC
TCL1A
EXPRESSION
IN
JURKAT
AND
IHH-TCL1A
CELLS
113
3.5.5
TCL1A
AND
TCP
ACTIVATION
COOPERATE
TOWARDS
AN
ACCELERATED
T-CELL
EXPANSION
IN
THE
CONTEXT
OF
CHRONIC
STIMULATION
IN
A
MURINE
MODEL
115
4
DISCUSSION
123
5
CONCLUSION
AND
OUTLOOK
135
6
REFERENCES
136
7
CURRICULUM
VITAE
153
8
EIDESSTATTLICHE
ERKLARUNG
157
-XVII- |
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| author | Oberbeck, Sebastian Tobias Karl-Heinz 1984- |
| author_GND | (DE-588)1202484824 |
| author_facet | Oberbeck, Sebastian Tobias Karl-Heinz 1984- |
| author_role | aut |
| author_sort | Oberbeck, Sebastian Tobias Karl-Heinz 1984- |
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| building | Verbundindex |
| bvnumber | BV046923908 |
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| discipline_str_mv | Medizin |
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| index_date | 2024-07-03T15:32:06Z |
| indexdate | 2024-09-25T16:00:42Z |
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| spelling | Oberbeck, Sebastian Tobias Karl-Heinz 1984- Verfasser (DE-588)1202484824 aut The T-cell receptor and it's signaling in T-cell prolymphocytic leukemia vorgelegt von Sebastian Tobias Karl-Heinz Oberbeck Köln 2019 XVII, 158 Seiten Illustrationen txt rdacontent n rdamedia nc rdacarrier Dissertation Universität zu Köln 2019 T-Zell-Leukämie (DE-588)4509988-1 gnd rswk-swf T-Lymphozyten-Rezeptor (DE-588)4265934-6 gnd rswk-swf (DE-588)4113937-9 Hochschulschrift gnd-content T-Zell-Leukämie (DE-588)4509988-1 s T-Lymphozyten-Rezeptor (DE-588)4265934-6 s DE-604 B:DE-101 application/pdf https://d-nb.info/1208423703/04 Inhaltsverzeichnis DNB Datenaustausch application/pdf http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=032333053&sequence=000001&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA Inhaltsverzeichnis |
| spellingShingle | Oberbeck, Sebastian Tobias Karl-Heinz 1984- The T-cell receptor and it's signaling in T-cell prolymphocytic leukemia T-Zell-Leukämie (DE-588)4509988-1 gnd T-Lymphozyten-Rezeptor (DE-588)4265934-6 gnd |
| subject_GND | (DE-588)4509988-1 (DE-588)4265934-6 (DE-588)4113937-9 |
| title | The T-cell receptor and it's signaling in T-cell prolymphocytic leukemia |
| title_auth | The T-cell receptor and it's signaling in T-cell prolymphocytic leukemia |
| title_exact_search | The T-cell receptor and it's signaling in T-cell prolymphocytic leukemia |
| title_exact_search_txtP | The T-cell receptor and it's signaling in T-cell prolymphocytic leukemia |
| title_full | The T-cell receptor and it's signaling in T-cell prolymphocytic leukemia vorgelegt von Sebastian Tobias Karl-Heinz Oberbeck |
| title_fullStr | The T-cell receptor and it's signaling in T-cell prolymphocytic leukemia vorgelegt von Sebastian Tobias Karl-Heinz Oberbeck |
| title_full_unstemmed | The T-cell receptor and it's signaling in T-cell prolymphocytic leukemia vorgelegt von Sebastian Tobias Karl-Heinz Oberbeck |
| title_short | The T-cell receptor and it's signaling in T-cell prolymphocytic leukemia |
| title_sort | the t cell receptor and it s signaling in t cell prolymphocytic leukemia |
| topic | T-Zell-Leukämie (DE-588)4509988-1 gnd T-Lymphozyten-Rezeptor (DE-588)4265934-6 gnd |
| topic_facet | T-Zell-Leukämie T-Lymphozyten-Rezeptor Hochschulschrift |
| url | https://d-nb.info/1208423703/04 http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=032333053&sequence=000001&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA |
| work_keys_str_mv | AT oberbecksebastiantobiaskarlheinz thetcellreceptoranditssignalingintcellprolymphocyticleukemia |
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Inhaltsverzeichnis