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