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:// 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 Stem Cell 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 presentation TEC

GenotypeDefinition Cre- lox/loxCre (enzyme that cuts outs TGF-βRII) absent Cre- lox/delCre absent One allele deleted in all cells Cre+ lox/loxCre present Both alleles deleted in Cre+ cells Cre+ lox/delCre 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 lox/del: Cre- to Cre+ p=0.27 Cre-: lox/lox to lox/del p=0.76

Results: Percentages of thymocyte populations Cre+: lox/lox to lox/del p=0.46

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 LineTGF-  Receptors’ Expression level 1.2 low 2.3 high C6medium C9medium 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

Results: Percent programmed cell death with/without TGF-β

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