REGULATION OF T CELL

ACTIVATION BY FOXP3 AND

SIVA

Virginia K. Hench
Dissertation Defense
July 19
th
, 2010
TALK OUTLINE
Introduction and background:
Immune tolerance
T cell development in the thymus
T
regs
FoxP3
Biophysical interaction between FoxP3 and
Siva
Regulation of IL-2 by Siva
Regulation of T cell activation by FoxP3 and
Siva
IL-2 regulation
Apoptosis

Pathogen
Defense
Self
Tolerance
THE IMMUNE SYSTEM ~ A
STRUGGLE FOR
BALANCE
horror
autotoxicus

-Paul Ehrlich
Hematopoiesis generates
multiple blood cell lineages
Metcalf, D., STEM CELLS 2007;25: 390 2395
T cells develop in the thymus

HSC
Thymus T Cell Maturation
Stages
CD8
CD4
CD25
CD44
HSC
TECs
BM-derived APCs
Cortex
Medulla
CM Junction
TECs
Thymocytes
BM-derived APCs
Thymus T Cell Maturation
Stages
T
reg

DN1
DN2
DN3
DN4
Medulla
Cortex
CM Junction
CD8
CD4
CD25
CD44
HSC
DP
DP
SP4
SP8
SP4
SP8
SP8
Scanning Electron Microscopy
Image of cortical TECs and
thymocytes
van Ewijk, W. et al. Semin Immunol 11, 57-64
thymocyte
TEC
TECs
Thymocytes
BM-derived APCs
SP4
SP8
SP4
SP8
SP8
Positive and Negative
Selection
T
reg

Positive Selection &
Death by neglect
Negative Selection &
T
reg
selection
Apoptotic
thymocyte
DP
DP
Adapted from Berg, L. J. Nat Rev Immunol 7, 479-485
CD4
+

CD8
+

CD4
+
CD25
+

NKT

FoxP3
T Cell Subsets
Thymectomy in newborn mice
induces autoimmunity
Day 3, but NOT day 7, thymectomy leads to multiple
organ autoimmune diseases, which suggested the
presence of thymic-derived with suppressor cells
Reviewed by Sakaguchi in Eur. J. Immunology. 2007
T
regs
cells inhibit CD4
+
T cell-
mediated autoimmunity
Athymic Nude Mouse
CD4
+
CD25
-

T cells
WT mouse spleen
CD4
TCR
CD25
CD4
TCR
T
regs
protect
from multi-
organ
inflammation
Scaly skin
Lymphocyte infiltrates in the
skin and liver
Runting
Enlarged spleen, lymph
nodes, and liver
Ear thickening
Squinty eyes
Scurfy mouse phenotype:

Godfrey, V. L.,. Am J Pathol 138, 1379-1387 (1991).
IPEX patients and Scurfy mice have
autoimmune disease in multiple organs
IBD/Diarrhea
Eczema
Type I diabetes
Thyroiditis
Chronic wasting
Bleeding problems
Recurrent infections
IPEX Syndrome
And mutations in FoxP3
Foxp3 is enriched in T
regs
and FoxP3
converts nave CD4
+
cells into T
reg
-like
cells
CD4
TCR
CD25
FoxP3
CD4
TCR
FoxP3
Hori, Science 2003
FoxP3

positive T
regs
suppress immune
activation in vivo and in vitro
- Immune cell infiltration
leading to tissue
destruction in multiple
organs in vivo
- Nave T cell activation in
vitro(based on IL-2
production and
proliferation)
- APC function
CD4
TCR
CD25
FoxP3
Natural T
reg

T
regs
Inhibit:
FoxP3 confers cell intrinsic
phenotype
CD4
TCR
CD25
FoxP3
-Cytokines associated with T cell
activation are repressed, specifically
IL-2.

-Surface markers associated with T
reg

function and survival are upregulated
(CD25 = IL-2 receptor, GITR, and
CTLA-4)
- Gene array analysis has identified
hundreds of genes that are
differentially regulated in T
regs

Key points:
The thymus contributes to central
tolerance by positive and negative
selection the T cell repertoire
The thymus generates T
regs
with
suppressor function
FoxP3 is required for T
reg

development, function and
maintenance


How does FoxP3 function at
the molecular level?
TCR complex
Ca
2
+

Ca
2+

Ca
2+

T Cell
AP-1
Ca
2
+

Ca
2+

Ca
2+

B
IB
B NFAT
DAG
IP
3

PIP
2

Ca
2+

Ca
2+

Ca
2+

Ca
2+

Ca
2
+

Ca
2+

Calcineurin
U
b
U
b
U
b
IKK
PKC
Antigen Presentation
Cell
Peptide-MHC & Co-receptors
Ub
NFAT
PMA
NFB
ERK/JNK
activation
MAP
Kinase
Ionomycin
IL-2 transcription
PKC
FoxP3 is a transcription factor that
differentially enhances and
represses gene expression
IL-2 Promoter IL-2
CD25 Promoter
CD25
CTLA-4, GITR, HIV-LTR
FoxP3 inhibits the transcriptional activity of NFAT/AP1, Runx1, RORt, ROR
FoxP3 requires Eos to inhibit IL-2 gene transcription
FoxP3 is associated with
changes in chromatin
structure
Histone
Deacetylases (HDACs)
DNA Methyl
Transferases
Compacted Chromatin,
Transcriptionally Inactive

Open Chromatin,
Transcriptionally Active

Histone Methyl
Transferases (HMTs)
Methylated Histones
Methylated DNA
Histone
Acetylytransferases (HATs)
Acetylated Histones
`
FoxP3 interacts with:
HDAC7,
HDAC9,
TIP60 (a HAT)
FoxP3 correlates with :
acetylation at IL-2 promoter
acetylation at CD25, GITR,
CTLA-4, & HIV LTR
Organization of FoxP3 protein
N C
FKH L.Zip Zn F
1 50 100 150 200 250 300 350 400 431
Transcriptional Repressor
Domains
RUNX bd
HDAC7 / TIP60 bd
Eos bd
ROR
/T
bd
NFAT bd
Proline rich region
How does FoxP3 function at
the molecular level?
What other proteins bind to FoxP3?
Use yeast two-hybrid screen
Yeast-two-hybrid screen for FoxP3 binding partners
Bait
GAL4 DNA BD/Foxp3
Trp
AD/cDNA from
human thymus
library
Leu
Reporter Genes
Prey
FoxP3
GAL4
DNA B.D.
AD
Library
encoded
protein
His, Ade, Gal
Reporter Genes
Yeast-two-hybrid screen criteria
FoxP3
GAL4
DNA B.D.
AD
Library
encoded
protein

Triple Drop-out: His- Trp- Leu-
- Stringency was increased with
3AT(Inhibitor of His)

Quadruple Drop-out: His- Trp- Leu- Ade-


-Galactosidase Activity: X-Gal
Select for Growth
Screen Relative
Colony Size
Screen Relative
Color Intensity
His, Ade, Gal
FoxP3 binding partners identified
by yeast two-hybrid screen
Courtesy W. Ince and R. Hales
Siva was named after the Hindu God of
destruction based on its ability to induce cell
death
Siva is expressed in:
Hematopoietic cells,
neurons, and myocardial
cells

Siva binds to the
cytoplasmic tail of CD27

Siva binds and inhibits
BCL-2 and BCL-XL

Siva represses NFB

Tumor suppressors, p53
and E2F1, activate Siva



Runx
APOPTOSIS
TCR
complex
APOPTOSIS
SIVA-1
B Box
DDHR
Zinc Finger
SAH
The Siva N-terminus is required for nuclear
translocation
The SAH domain induces apoptosis in
breast cancer cells
The DDHR domain did not induce
apoptosis in T cells
The C-terminus is enriched with cysteine
residues
Both the N- and C-terminus are sufficient to
induce apoptosis in a HPB-T cell line
Siva-2 lacks exon 2, which encodes most of
the SAH and DDHR domains
Absent from SIVA-2
C N
Project Goals:
Characterize the FoxP3 and Siva
interaction.

Does Siva affect FoxP3s repressive
effect on IL-2?

Does FoxP3 affect the pro-apoptotic
effect of Siva?
My
c
My
c
Co-Immunoprecipitation
Protocol
1. Transfect 293T cells with plasmids
expressing EGFP/Siva and
Myc/FoxP3 or vector.
SIV
A
2. Lyse cells to release proteins
FoxP3
My
c
SIV
A
EGFP
3. Add Anti-Myc monoclonal
Antibody (Incubate overnight)
4. Add sepharose beads,
Incubate
FoxP3
My
c
5. Wash beads
6. Run precipitated proteins on SDS-gel.
FoxP3 interacts with both Siva isoforms
Siva is preferentially expressed in
CD4
+
CD25
+
cells
Siva in Human Primary
Cells by RT-PCR
Siva in Mouse Primary
Cells by Gene Array
Wu, C. et al.. Genome Biol 10, R130 (2009).
Lattin, J. E. et al. Immunome Res 4, 5 (2008)
Siva and Foxp3 co-localize in the
nucleus of co-transfected U2OS cells
Siva and FoxP3 both display nuclear staining
Courtesy S.Mackey-Cushman
Siva-binding activity is
within a central portion of
the FoxP3 protein (AAs
106-332)
What region of FoxP3 binds to Siva?
The Siva C-terminus is sufficient to bind FoxP3
What region of Siva binds to FoxP3
?
DO SIVA AND FOXP3 FUNCTIONALLY
INTERACT?
SIVA AND FOXP3 PHYSICALLY
INTERACT
Runx
IL-2
SIVA
?
What is Sivas effect on IL-2?
Investigating IL-2 regulation
Two methods were used to study IL-2
regulation.
Endogenous IL-2 in Jurkats transduced
with RV and/or LV to modulate Siva and
FoxP3 expression levels.

IL-2 promoter-driven luciferase activity in
Jurkats transfected with plasmids
expressing the reporter, FoxP3, Siva,
and/or shSIVA.
pHSPG-Siva-1 plasmid expresses Siva in 293T
cells
N
o
t

T
r
a
n
s
f
e
c
t
e
d

WB: Rabbit
-SIVA
Sc 175-FL
293T Cells
52
38
31
WB: -Actin
EGFP/
Siva-1
Siva-1
Transfected
with:
pHSPG-Siva-1
LTR
LTR
PGK
Promo
Psi
Siva-1
EGFP
CMV
prom.
Experimental outline to test the effect of
Siva overexpression on endogenous IL-
2
VSV-g
HSPG
Gag/Pol
293T cells
Spinoculate
Transduced
Jurkat T cells
pHSPG-Siva-1
Retrovirus
Activate with PMA/Ion
for 18 hours.

Collect SNs.

Evaluate
endogenous IL-2 by
ELISA
Calcium phosphate
Siva overexpression represses
endogenous IL-2
0
500
1000
1500
2000
2500
vector Siva-1
E6.1 pHSPG
I
L
-
2

(
p
g
/
m
L
)

Experimental outline to test the effect of
Siva knockdown on endogenous IL-2
VSV-g
pLKO
NRF
293T cells
Spinoculate
Select transduced
Jurkat T cells in
puromycin for a week
pLKO
Lentivirus(expres
sing shSIVA or
shEGFP) Activate with PMA/Ion
for ~18 hours

Collect SNs

Evaluate
endogenous IL-2 by
ELISA
Calcium phosphate
.
Knockdown of endogenous Siva
enhances endogenous IL-2
pGL-IL-2
Luciferase
pHSP
G-Siva
pLKO-
shSIV
A
OR
Experimental outline to test Sivas effect on
IL-2 promoter activity
Minimal IL-2 promoter
Measure luciferase
activity (RLUs) on
luminometer
pGL-IL2 Luc
Luciferase reporter
Activate with PMA/Ion
for 8 hours
Culture transfected Jurkat
cells for ~16 hours
Collect lysates
Siva overexpression represses
IL-2 promoter activity
Siva knockdown enhances IL-2
promoter activity
Conclusions:
- Siva represses IL-2 gene expression.
- Siva repression of IL-2 is at the
promoter level.
Runx
IL-2 promoter
IL-2
SIVA
?
How does Siva affect FoxP3s
repressive effect on IL-2?
Outline for experiment to evaluate Sivas effect on
FoxP3s ability to repress endogenous IL-2
Perform two rounds of
spinoculation
1. pHSPG-Siva
2. pHSPG-FoxP3
Activate with PMA/Ion
for 18 hours.
Evaluate
endogenous IL-2 by
ELISA
Collect SNs.
0
500
1000
1500
2000
2500
PG Siva-1
I
L
-
2

(
p
g
/
m
L
)

PG
2000
260
8
1200
270
5
PG Vector (IL-2 pg/ml)
FoxP3 (IL-2 pg/ml)

= FoxP3s IL-2 repressive activity
Calculating FoxP3s IL-2 repressive activity:
How does Siva overexpression affect FoxP3s
repressive effect on endogenous IL-2?
Endogenous IL-2
0
1
2
3
4
5
6
7
8
9
PG Siva-1
F
o
x
p
3
'
s

R
e
p
r
e
s
s
i
v
e

A
c
t
i
v
i
t
y

FoxP3-mediated IL-2
Repression
0
500
1000
1500
2000
2500
PG Siva-1
I
L
-
2

(
p
g
/
m
L
)

PG
Foxp3
Transduction efficiency
based on GFP expression
99% 99%
99% 99%
Experimental outline to test Sivas effect on
FoxP3s IL-2 promoter repressive activity
Minimal IL-2 promoter
pGL-IL2 Luc
Luciferase reporter
Measure luciferase
activity (RLUs) in
activated,
transfected Jurkat
cells
PG Vector (IL-2 RLUs)
FoxP3 (IL-2 RLUs)

= FoxP3s IL-2 promoter
repressive activity
Calculating FoxP3s IL-2
promoter repressive
activity:
Calculate FoxP3 repressive
activity in the presence and
absence of Siva
Fixed
amount:
Titrate:
At lower doses of FoxP3, Siva reduced FoxP3s
repressive effect on IL-2 promoter activity
How does Siva overexpression affect FoxP3s
repressive effect on IL-2 promoter activity?
FoxP3-mediated IL-2 Repression IL-2 Promoter Activity
0
50000
100000
150000
200000
250000
300000
350000
400000
1.13 2.25 4.5
PG Foxp3 (ug)
I
L
-
2

R
L
U
s

Vector
Siva (4.5 ug)
*
*
0
0.5
1
1.5
2
2.5
3
3.5
4
4.5
1.13 2.25 4.5
F
o
x
p
3
'
s

R
e
p
r
e
s
s
i
v
e

A
c
t
i
v
i
t
y

Foxp3 (ug)
PG
Siva (4.5 ug)
Mini-summary:
Siva overexpression tends to
reduce FoxP3s repressive effect
on IL-2
Does knockdown of endogenous
Siva expression affect FoxP3s
repressive effect on IL-2?
Siva knockdown does not effect
FoxP3s repressive effect on
endogenous IL-2
How does Siva knockdown affect FoxP3s repressive
effect on endogenous IL-2?
Endogenous IL-2
FoxP3-mediated IL-2 Repression
0
2
4
6
8
10
12
14
shEGFP shSIVA
F
o
x
p
3
'
s

R
e
p
r
e
s
s
i
v
e

A
c
t
i
v
i
t
y

0
0.5
1
1.5
2
2.5
3
3.5
4
4.5
shEGFP shSIVA
R
e
l
a
t
i
v
e

S
i
v
a
/
1
8
S

PG
Foxp3
Efficiency of Siva Knockdown
by Realtime PCR
0
2000
4000
6000
8000
10000
12000
shEGFP shSIVA
I
L
-
2

(
p
g
/
m
l
)

PG
Foxp3
How does Siva knockdown affect
FoxP3s repressive effect on IL-2
promoter activity?
0
2000
4000
6000
8000
10000
12000
14000
16000
18000
20000
shNS shSIVA
I
L
-
2

R
L
U
s

PG
Foxp3
0
1
2
3
4
5
6
shNS shSIVA
F
o
x
p
3
'
s

R
e
p
r
e
s
s
i
v
e

A
c
t
i
v
i
t
y

FoxP3-mediated IL-2 Repression
Siva knockdown enhances FoxP3s
repressive effect on IL-2 promoter
activity
IL-2 Promoter Activity
*
Mini-summary:
Siva knockdown enhanced FoxP3s
repressive effect at the IL-2
promoter.
Siva modifies FoxP3s repressive
effect on IL-2 promoter activity
FoxP3
SIV
A
IL-2 promoter activity
FoxP3
SIV
A
IL-2 promoter activity
SIV
A
SIV
A
SIV
A
SIV
A
SIV
A
WHAT OTHER MOLECULAR
MECHANISMS MIGHT BE
INVOLVED?
TCR
complex
Ca
2
+

Ca
2+

Ca
2+

T Cell
AP-1
Ca
2
+

Ca
2+

Ca
2+

IB
NFAT
Ca
2+

Ca
2+

Ca
2+

Ca
2+

Ca
2
+

Ca
2+

NFAT
PMA
NFB
MAPKKK
Ionomycin
PKC
Transcriptional activity of three IL-2 regulatory
elements in response to FoxP3 and Siva.
MHC MHC
Luc.
MHC
Luc.
TRE TRE TRE Luc.
IL-2
NFAT
RE
IL-2
NFAT
RE
IL-2
NFAT
RE
0
10000
20000
30000
40000
50000
60000
70000
80000
PG SIVA
R
L
U
s

PG Foxp3
0
10000
20000
30000
40000
PG SIVA
R
L
U
s

PG Foxp3
NFkB Reporter NFAT Reporter
0
5000
10000
15000
20000
25000
30000
PG SIVA
R
L
U
s

PG Foxp3
AP-1 Reporter
p50
IB
p65
PMA
NFB
Ionomycin
SIV
A
p50
IB
p65
PMA
NFB
Ionomycin
SIV
A
NFAT AP-1
SIVA
?
APOPTOSIS
FoxP3
?
SIV
A
How does FoxP3 effect Sivas pro-
apoptotic activity?
Siva and FoxP3 promote apoptosis in
Jurkat cells in the absence of stimulation
0
2
4
6
8
10
12
PG Siva
%
7
A
A
D

P
o
s
i
t
i
v
e

C
e
l
l
s

PG
Foxp3
*
Effects of Siva and FoxP3 in
stimulated cells on apoptosis
0
5
10
15
20
25
30
35
PG Siva PG Siva
No Activation PMA/Ion
%
7
A
A
D

P
o
s
i
t
i
v
e

C
e
l
l
s

PG
Foxp3
*
*
*
Summary of apoptosis data
Siva and FoxP3 both promote
apoptosis in un-stimulated cells
Sivas pro-apoptotic activity is inhibited
in PMA/Ion stimulated cells
FoxP3 protects from apoptosis in
response to PMA and Ionomycin
treatment


Summary of Results
FoxP3 and Siva biophysically interact
The FoxP3 binding domain has been
mapped to Sivas C-terminus.
A central portion of FoxP3 binds Siva.
Siva represses IL-2 gene expression
Siva repression of IL-2 is probably
mediated by NFB
Modulation of Siva expression affects
FoxP3s repressive effect on IL-2
promoter activity


Future Directions
Mapping studies:
Narrow the region of Siva binding activity
within FoxP3
Functional studies
Evaluate Sivas IL-2 repressive activity in
primary cells

Is Siva required for thymocyte
selection?

-DP thymocytes express high levels of
Siva

-Hypothesis: Siva-mediated apoptosis
contributes to thymocyte selection
Thank You!
The Su Lab Committee Members
Lishan Su
All lab members past and
present
Jeff Frelinger
Jon Serody
Roland Tisch
Jenny Ting
Karen McKinnon