T-Cell development

T Cell Cell in the body

TCR MHC

CD4/CD8

DP in the thymus: peptide is ‘self’

SP in the periphery: peptide is ‘altered self’

In vitro stimulation

Use plate-bound antibodies to

crosslink TCR and CD28

Mature SP cell

Immature DP cell

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-XTM

Bcl-2 family members in development

DP SPDN

Bcl-2hi

Bcl-XLlo

Bcl-2lo

Bcl-XLhi

Bcl-2hi

Bcl-XLlo

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.

Initial Protocol Revised Protocol

RT-PCR

Bcl-2

Bcl-XL

Data from RT-PCR

Actin Actin

Bax

Bak

SP

SP

T/2

8

DP

DP

T/2

8

SP

SP

T/2

8

DP

DP

T/2

8

Intracellular Staining

Bcl-2 expression in DP thymocytes

Modification of Bcl-XL

Inactive bcl-XL

Mitochondrion

Pro-death

bcl-XL

Mitochondrion

Phosphorylation? Cleavage?

P

What’s Good About Bad?

bcl-XL

Mitochondrion

bcl-XL

Mitochondrion

bad

Active bcl-XL Inactive bcl-XL

bad

14-3-3

Death signal

P

Isoforms of Bcl-XL and Bad

bad

bcl-XL

Inte

stin

e

Kid

ney

Hea

rt

Liv

er

Lym

ph N

ode

Bra

in

SP T

-cel

ls

Lun

g

Thy

mus

26 kD

30 kD

Bcl-XL and Bad in T-Cells

bad

bcl-XL

DP

4 hr

DP

4 hr

TC

R

DP

4 hr

T/2

DP

4 hr

T/2

8

DP

8 hr

T/2

8

DP

8 hr

T/2

DP

8 hr

TC

R

DP

8 hr

Fres

h D

P

Liv

er

Fres

h D

P

Fres

h no

rmal

SP

SP 4

hr

T/2

8 (d

ex)

SP 4

hr

TC

R (

dex)

SP 4

hr

- (d

ex)

Fres

h SP

(dex

)

Lym

ph n

ode

bad

Fres

h D

P

Pann

ed S

P

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.

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.

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