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Critical Regulation of Thymic Epithelial Cell Function and Thymus Development by Transforming Growth Factor-Beta Signaling
Michael Blazar 2006
Thymus
Blood vessels
Membrane
Medulla
Hollander presentationhttp://www.becomehealthynow.com/popups/thymus.htm
Cortex
Thymocytes and Thymic Epithelial Cells (TEC)
(Scanning Electron Microscopy)
ThymocyteTEC
Hollander presentation
Thymus
Thymocyte Development
CD4-CD8-
CD4+CD8+
CD4- CD8+
CD4+ CD8-
Cortex
Corticomedullary Junction
Medulla
Thymic Epithelial Cells (TEC)
immature
intermediate
mature mature
StemCell
Hollander presentation
Previous Studies
•Kim et al. (2005)
-TGF-β stimulates formation of fibrous matrix
-TGF-β impacts wound healing, the extracellular matrix, and immunosuppression
•Massagué et al. (1998, 2000)
-Mutations in signaling of TGF-β cause developmental disorders and cancer
-TGF-β1 inhibits mitosis in epithelial cells
-TGF β1-3 causes cell-cycle arrest in epithelial and hematopoietic cells
•Balciunaite et al. (2002)
-Wnt glycoproteins stimulate in vivo growth of TEC in mice
T Cell
Thymocytes
Growth Factor
TEC
TGF-β Receptors (RI and RII)
Thymocytes Produce Growth Factors to Stimulate TEC
Hollander presentationTEC
Genotype Definition
Cre- lox/lox •Cre (enzyme that cuts outs TGF-βRII) absent
Cre- lox/del •Cre absent
•One allele deleted in all cells
Cre+ lox/lox •Cre present
•Both alleles deleted in Cre+ cells
Cre+ lox/del •Cre present
•One allele deleted in all cells
•Both alleles deleted in Cre+ cells
Cre- and Cre+ Genotypes
Goals of the First Objective
-Thymus structure
-Thymocyte development
-T-cell and B-cell populations in the spleen
Effect of TGF-βRII signaling
Hypotheses
•Deletion of both TGF-βRII alleles -abnormal thymus structure and development
•Deletion of one TGF-βRII allele -normal thymus structure and development
Methods
Fluorescent Activated Cell-Sorting (FACS) Staining
FACS Calibur Analysis
Immunofluorescence Staining
Confocal Microscopy Analysis
Immunofluorescence Staining
Tears Cysts Abnormal blood vessels
Cre- lox/lox Cre- lox/lox Cre- lox/lox Cre- lox/lox Cre- lox/del
Cre+ lox/lox Cre+ lox/lox Cre+ lox/lox Cre+ lox/lox Cre+ lox/del
Photos taken by author
Results: Percentages of thymocyte populations
5.55 4.80 6.1311.82
6.50
87.43 88.26
82.05
90.47
3.04
6.937.03
0
10
20
30
40
50
60
70
80
90
100
Cre- lox/lox Cre- lox/del Cre+ lox/lox Cre+ lox/del
Genotype
% Thymocytes
Double Negative
Single Positive
Double Positive
lox/del: Cre- to Cre+ p=0.27
Cre-: lox/lox to lox/del p=0.76
0.62 1.04 0.99
8.32 9.32 7.76
92.8789.88 90.50
0
10
20
30
40
50
60
70
80
90
100
Cre- lox/lox Cre+ lox/lox Cre+ lox/del
Genotype
% Thymocytes
Double Negative
Single Positive
Double Positive
Results: Percentages of thymocyte populations
Cre+: lox/lox to lox/del p=0.46
10.85 10.85 9.32
27.47 28.3630.23
44.6547.25
49.73
0
10
20
30
40
50
60
Cre- lox/lox Cre+ lox/lox Cre+ lox/del
Genotype
% Cells that express marker
CD8
CD4
CD19
Results: Percent of T-cells and B-cells in the Spleen
Cre+: lox/lox to lox/del p=0.34
Goals of the Second Objective
•Effect of adding TGF-β1
-Cell growth of TEC cultures
-Programmed cell death of TEC cultures
TEC cultures
Line TGF- Receptors’ Expression level1.2 low 2.3 highC6 mediumC9 medium
Hollander presentation
Hypothesis
•TGF-β will inhibit proliferation in TEC cultures proportional to expression level of TGF-β receptors.
Methods
Measure programmed cell death after 24 and 48 hours in culture
with/without TGF-β
FACS Calibur Analysis
Measure cell growth after 24 and 48 hours in culture
with/without TGF-β
FACS Calibur Analysis
Results: Percent cell growth with/without TGF-β
34.1
42.8
56.1
51.4
17.3
33.2
50.148.1
55.053.9
48.9
53.6
0
10
20
30
40
50
60
1.2: 24h 2.3: 24h C9: 24h 1.2: 48h 2.3: 48h C9: 48h
TEC culture; Timepoint
% Cells undergoing proliferation
without TGF-beta
with TGF-beta
Results: Percent programmed cell death with/without TGF-β
10.4
5.4
8.88.3
6.3
8.0
13.1
10.2
4.03.3
4.0
3.1
0
2
4
6
8
10
12
14
1.2: 24h 2.3: 24h C9: 24h 1.2:48h 2.3: 48h C9: 48h
TEC culture; Timepoint
% Cells undergoing apoptosis
without TGF-beta
with TGF-beta
Results: Percent programmed cell death with/without TGF-β
2.8 3.1 2.7
3.8
5.3
3.2
0
2
4
6
8
10
12
14
1.2 2.3 C6
TEC culture
% Cells undergoing apoptosis
without TGF-beta
with TGF-beta
Conclusions
First Objective
1. One TGF-β Receptor II allele was required for normal thymus structure
2. TGF-β Receptor II did not affect thymocyte development and T-cell and B-cell populations in the spleen
Conclusions
Second Objective
1. TGF-β addition did not affect cell growth in the TEC culture with the lowest receptor expression level of TGF-β
2. TGF-β addition caused a consistent increase in cell growth in the TEC culture with the highest receptor expression level of TGF-β
3. The effect of TGF-β on cell growth in TEC cultures was lower after 48 hours of culture as compared to 24 hours
5. When baseline programmed cell death was large (>10%), addition of TGF-β reduced cell death
Conclusions
Second Objective
4. TGF-β addition increased programmed cell death proportional to the receptor expression level of TGF-β
Future Studies
1. Ideal time of incubation with TGF-β in intact and damaged TEC
2. Effects of TGF-β on cell growth and death of injured TEC in vivo
3. Optimal TGF-β signaling to inhibit cell growth in vivo before chemotherapy and radiation therapy
4. Prevention and repair of injured TEC to aid immune system deficiency and recovery during chemotherapy and radiation therapy
Acknowledgements
•Dr. Georg A. Hollander
•Mathias Hauri
•Jason Gill
•Annick Peter
•Ms. Fruen
•Advanced Science Research team
Critical Regulation of Thymic Epithelial Cell Function and Thymus Development by Transforming Growth Factor-Beta Signaling
Michael Blazar 2005/06