Critical Regulation of Thymic Epithelial Cell Function and Thymus Development by Transforming Growth Factor-Beta Signaling

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Critical Regulation of Thymic Epithelial Cell
Function and Thymus Development by Transforming
Growth Factor-Beta Signaling
Michael Blazar 2006
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Thymus
Blood vessels
Medulla
Cortex
Membrane
Hollander presentation
http//www.becomehealthynow.com/popups/thymus.htm
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Thymocytes and Thymic Epithelial Cells (TEC)
(Scanning Electron Microscopy)
Hollander presentation
Thymocyte
TEC
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Thymocyte Development
Thymic Epithelial Cells (TEC)
CD4-CD8-
Cortex
immature
Thymus
CD4CD8
intermediate
Corticomedullary Junction
Stem Cell
Medulla
CD4- CD8
CD4 CD8-
mature
mature
Hollander presentation
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Previous Studies

• Massagué et al. (1998, 2000)
• -Mutations in signaling of TGF-ß cause
  developmental disorders and cancer
• TGF-ß1 inhibits mitosis in epithelial cells
• TGF ß1-3 causes cell-cycle arrest in epithelial
  and hematopoietic cells

• Kim et al. (2005)
• TGF-ß stimulates formation of fibrous matrix
• TGF-ß impacts wound healing, the extracellular
  matrix, and immunosuppression

• Balciunaite et al. (2002)
• -Wnt glycoproteins stimulate in vivo growth of
  TEC in mice

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Thymocytes Produce Growth Factors to Stimulate TEC
T Cell
Thymocytes
TGF-ß Receptors (RI and RII)
Growth Factor
TEC
Hollander presentation
TEC
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Cre- and Cre Genotypes
Genotype Definition
Cre- lox/lox Cre (enzyme that cuts outs TGF-ßRII) 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
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• Goals of the First Objective

Effect of TGF-ßRII signaling
-Thymus structure
-Thymocyte development
-T-cell and B-cell populations in the spleen
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• Hypotheses

• Deletion of both TGF-ßRII alleles
• -abnormal thymus structure and development

• Deletion of one TGF-ßRII allele
• -normal thymus structure and development

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Methods
Fluorescent Activated Cell-Sorting (FACS) Staining
Immunofluorescence Staining
FACS Calibur Analysis
Confocal Microscopy Analysis
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Immunofluorescence Staining
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
Tears
Cysts
Abnormal blood vessels
Photos taken by author
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Results Percentages of thymocyte populations
Cre- lox/lox to lox/del p0.76
lox/del Cre- to Cre p0.27
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Results Percentages of thymocyte populations
Cre lox/lox to lox/del p0.46
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Results Percent of T-cells and B-cells in the
Spleen
Cre lox/lox to lox/del p0.34
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• Goals of the Second Objective

• Effect of adding TGF-ß1

-Cell growth of TEC cultures
-Programmed cell death of TEC cultures
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• Hypothesis
• TGF-ß will inhibit proliferation in TEC cultures
  proportional to expression level of TGF-ß
  receptors.

Hollander presentation
Line TGF-? Receptors Expression level 1.2
low 2.3 high C6 medium C9
medium
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Methods
Measure programmed cell death after 24 and 48
hours in culture with/without TGF-ß
Measure cell growth after 24 and 48 hours in
culture with/without TGF-ß
FACS Calibur Analysis
FACS Calibur Analysis
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Results Percent cell growth with/without TGF-ß
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Results Percent programmed cell death
with/without TGF-ß
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Results Percent programmed cell death
with/without TGF-ß
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Conclusions
First
Objective

• 1. One TGF-ß Receptor II allele was required for
  normal thymus structure

2. TGF-ß Receptor II did not affect thymocyte
development and T-cell and B-cell
populations in the spleen
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Conclusions
Second Objective

1. TGF-ß addition did not affect cell growth in
the TEC culture with the lowest receptor
expression level of TGF-ß
2. TGF-ß addition caused a consistent increase in
cell growth in the TEC culture with the highest
receptor expression level of TGF-ß
3. The effect of TGF-ß on cell growth in TEC
cultures was lower after 48 hours of culture as
compared to 24 hours
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Conclusions
Second Objective
4. TGF-ß addition increased programmed cell
death proportional to the receptor expression
level of TGF-ß
5.
When baseline programmed cell death was large
(gt10), addition of TGF-ß reduced cell death
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Future Studies
1. Ideal time of incubation with TGF-ß in intact
and damaged TEC
2. Effects of TGF-ß on cell growth and death of
injured TEC in vivo
3. Optimal TGF-ß 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
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Acknowledgements

• Dr. Georg A. Hollander
• Mathias Hauri
• Jason Gill
• Annick Peter
• Ms. Fruen
• Advanced Science Research team

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Critical Regulation of Thymic Epithelial Cell
Function and Thymus Development by Transforming
Growth Factor-Beta Signaling
Michael Blazar 2005/06