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T-Cell development

T-Cell development. CD4/CD8. Cell in the body. T Cell. TCR MHC. DP in the thymus: peptide is ‘self’ SP in the periphery: peptide is ‘altered self’. In vitro stimulation. Mature SP cell. Use plate-bound antibodies to crosslink TCR and CD28. Immature DP cell. The bcl-2 family members.

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T-Cell development

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  1. T-Cell development CD4/CD8 Cell in the body T Cell TCR MHC DP in the thymus: peptide is ‘self’ SP in the periphery: peptide is ‘altered self’

  2. In vitro stimulation Mature SP cell Use plate-bound antibodies to crosslink TCR and CD28 Immature DP cell

  3. The bcl-2 family members • Killer: • bax • bak • bad • bid • bik • bim • bok • bcl-XS • Mcl • Hrk • Protective: • bcl-2 • bcl-XL • bcl-w • bag • bcl-X • Al • bcl-XTM

  4. Bcl-2 family members in development DN DP SP Bcl-2hi Bcl-XLlo Bcl-2lo Bcl-XLhi Bcl-2hi Bcl-XLlo

  5. Why are DPs so sensitive? Expression of a pro-apoptotic family member: bax, bad, bak? Goal: Determine what renders DPs sensitive to apoptosis; gain insight into selection.

  6. RT-PCR Initial Protocol Revised Protocol

  7. Data from RT-PCR Actin Actin Bax Bcl-XL Bcl-2 Bak DP T/28 DP T/28 SP T/28 SP T/28 DP DP SP SP

  8. Intracellular Staining Bcl-2 expression in DP thymocytes

  9. Modification of Bcl-XL P Pro-death bcl-XL Inactive bcl-XL Mitochondrion Mitochondrion Cleavage? Phosphorylation?

  10. What’s Good About Bad? 14-3-3 P bad bad Death signal bcl-XL bcl-XL Mitochondrion Mitochondrion Active bcl-XL Inactive bcl-XL

  11. Isoforms of Bcl-XL and Bad bad 26 kD 30 kD bcl-XL Lymph Node SP T-cells Intestine Thymus Kidney Lung Liver Heart Brain

  12. Bcl-XL and Bad in T-Cells bad bcl-XL DP 8 hr TCR DP 4 hr TCR DP 8 hr T/28 DP 4 hr T/28 DP 8 hr T/2 DP 4 hr T/2 Fresh DP DP 8 hr DP 4 hr Liver bad SP 4 hr TCR (dex) SP 4 hr T/28 (dex) Fresh normal SP SP 4 hr - (dex) Fresh SP(dex) Lymph node Panned SP Fresh DP Fresh DP

  13. Conclusions: • RT-PCR can serve as a semi-quantitative method to assay gene expression. • Bcl-2 may be upregulated in DPs in response to TCR stimulation. • Differential expression of bak and bax does not appear to be responsible for the sensitivity of DPs to apoptosis. • Bcl-XL isoforms appear not to be a factor in this sensitivity. • The phosphorylation state of bad may be a factor in this sensitivity, and may play a role in selection.

  14. Future Directions: • Is the RNA data accurate? • Confirm the bcl-2, bak and bax data at the protein level, using flow cytometry or Western blotting. • Are isoforms of bcl-XL truly unimportant? • Blot for bcl-XL in TCR/CD28 stimulated SPs to compare isoform content to DPs. • Use another antibody to bcl-XL. • Is bad important in T-cell development? • Determine if the doublet of bad truly represents phosphorylation by immunoprecipitation and phosphatase treatment. • FACS sort SPs to confirm the expression of the higher isoform of bad in a pure population. • Immunoprecipitate bad in DPs and SPs to determine if it is associated with bcl-XL or 14-3-3. • Construct a bad knockout or a knock-in of a serine mutant of bad to determine if bad plays a role in selection.

  15. Acknowledgements: Jennifer Punt Harjeet van der Keyl, Bruce Freedman, and Mary Taneko Dave Allman Carolyn Harding and Brian Bean Everyone in the lab: Rachel, Mariam, Jon, Jesse, Princess, Sarah The bio department The mice

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