Regulation of T cell Activation by FoxP3 and Siva

8/12/2019 Regulation of T cell Activation by FoxP3 and Siva

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REGULATIONOFT CELL

ACTIVATIONBYFOXP3ANDSIVA

Virginia K. Hench

Dissertation DefenseJuly 19th, 2010


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TALKOUTLINE

Introduction and background: Immune tolerance

T cell development in the thymus

Tregs FoxP3

Biophysical interaction between FoxP3 andSiva

Regulation of IL-2 by Siva

Regulation of T cell activation by FoxP3 andSiva

IL-2 regulation

Apoptosis


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PathogenDefense

Self

THE IMMUNE SYSTEM ~ ASTRUGGLE FORBALANCE


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horror

autotoxicus

-Paul Ehrlich


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Hematopoiesis generates

multiple blood cell lineages

Metcalf, D., STEM CELLS 2007;25: 3902395


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T cells develop in the thymus

HSC


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ymus e a ura onStages

CD8

CD4

CD25

CD44

HSC

TECs

BM-derived APCs

Cortex

Medulla

CM Junction


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TECs

Thymocytes

BM-derived APCs

ymus e a ura onStages

Treg

DN1

DN2

DN3

DN4

Medulla

Cortex

CM Juncti

CD8

CD4

CD25

CD44

HSC

DP

DP

SP4

SP8SP4

SP8

SP8


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Scanning Electron Microscopy

Image of cortical TECs and

thymocytes

van Ewijk, W.et al.Semin Immunol11, 57-64

thymocyte

TEC


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TECs ThymocytesBM-derived APCs

SP4

SP8SP4

SP8

SP8

os ve an ega veSelection

Treg

Positive Selection &

Death by neglect

Negative Selection &

Treg

selection

Apoptotic

thymocyte DP

DP


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Adapted from Berg, L. J. Nat Rev Immunol7, 479-485

CD4+

CD8+

CD4+CD25+

NKT

FoxP3

T Cell Subsets


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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.


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Tregscells inhibit CD4+T cell-

mediated autoimmunity

Athymic Nude Mouse

CD4+CD25-

T cells

WT mouse spleen CD4TCR

CD25

CD4TCR

Tregsprotect

from multi-

organ

inflammation


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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 Pathol138, 1379-1387 (19

IPEX patients and Scurfy mice have

autoimmune disease in multiple organs

IBD/DiarrheaEczema

Type I diabetes

Thyroiditis

Chronic wasting

Bleeding problems

Recurrent infections

IPEX Syndrome

And mutations in FoxP3

F 3 i i h d i T d F P3


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Foxp3is enriched in TregsandFoxP3

converts nave CD4+cells into Treg-like

cells

CD4

TCR

CD25

FoxP3

CD4

TCR

FoxP3

Hori, Science 200


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FoxP3positive Tregs suppress immune

activation in vivo and in vitro

- Immune cell infiltrationleading to tissue

destruction in multiple

organs in vivo

- Nave T cell activation in

vitro(based onIL-2production and

proliferation)

- APC function

CD4

TCR

CD25

FoxP3

Natural Treg

Tregs Inhibit:


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FoxP3 confers cell intrinsic

phenotype

CD4TCR

CD25

FoxP3

-Cytokines associated with T cell

activation are repressed, specifically

IL-2.

-Surface markers associated with Treg

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 Tregs


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Key points:

The thymus contributes to centraltolerance by positive and negative

selection the T cell repertoire

The thymus generates Tregswithsuppressor function

FoxP3 is required for Treg

development, function andmaintenance


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How does FoxP3 function atthe molecular level?

Antigen Presentation


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TCR complex

Ca2

+

Ca2+

Ca2+

T Cell

AP-1

Ca2

+Ca2+Ca2+

B

IB

BNFAT

DAG

IP3

PIP2

Ca2+ Ca2+

Ca2+

Ca2+

Ca2+

Ca2+

CalcineurinU

bU

bU

b

IKK

PKC

Antigen Presentation

CellPeptide-MHC & Co-receptors

Ub

NFAT

PMA

NFB

ERK/JNKactivation

MAPKinase

Ionomycin

IL-2 transcription

PKC


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FoxP3 is a transcription factor that

differentially enhances andrepresses geneexpression

IL-2 Promoter IL-2

CD25 Promoter CD25CTLA-4, GITR, HIV-LTR

FoxP3 inhibits the transcriptional activity of NFAT/AP1, Runx1, RORt, ROR

FoxP3 requires Eos to inhibit IL-2 gene transcription

ox s assoc a e w


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ox s assoc a e wchanges in chromatin

structure

Histone

Deacetylases (HDACs)

DNA Methyl

Transferases

Compacted Chromatin,

Transcriptionally Inactive

Open Chromatin,

Transcriptionally Active

Histone MethylTransferases (HMTs)

Methylated Histones

Methylated DNAHistone

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


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Organization of FoxP3 protein

N C

FKHL.ZipZn F

1 50 100 150 200 250 300 350 400 431

Transcriptional Repressor

Domains

RUNX bdHDAC7 / TIP60 bd

Eos bdROR

/T

bdNFAT bd

Proline rich region


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How does FoxP3 function atthe molecular level?


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What other proteins bind to FoxP3?

Use yeast two-hybrid screen

t t h b id f F P3 bi di t


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east-two-hybrid screen for FoxP3 binding partner

Bait

GAL4 DNA BD/Foxp3

Trp

AD/cDNA from

human thymus

library

Leu

Reporter Genes

Prey

FoxP3

GAL4

DNA B.D.

ADLibrary

encoded

protein

His, Ade, Gal


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FoxP3 binding partners identified

by yeast two-hybrid screen

Courtesy W. Ince and R. Hales

Siva was named after the Hindu God of


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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 myocardialcells

Siva binds to thecytoplasmic tail of CD27

Siva binds and inhibitsBCL-2 and BCL-XL

Siva represses NFB

Tumor suppressors, p53

and E2F1, activate Siva


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Runx

APOPTOSIS

TCR

complex

APOPTOSIS

SIVA-1


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SIVA 1

B Box

DDHR Zinc Finger

SAH

The Siva N-terminus is required for nucleartranslocation

The SAH domain induces apoptosis inbreast cancer cells

The DDHR domain did not induceapoptosis 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

CN


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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?

o mmunoprec p a on


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My

c Myc

o- mmunoprec p a onProtocol

1. Transfect 293T cells with plasmids

expressing EGFP/Siva andMyc/FoxP3 or vector.

SIV

A

2. Lyse cells to release proteins

FoxP3My

cSIVA

EGFP

3. Add Anti-Myc monoclonal

Antibody (Incubate overnight)

4. Add sepharose beads,

Incubate

FoxP3My

c

5. Wash beads

6. Run precipitated proteins on SDS-gel.

F P3 i i h b h Si i f


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FoxP3 interacts with both Siva isoforms

Si i f ti ll d i


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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 Biol10, R130 (2009

Lattin, J. E.et al. Immunome Res4, 5 (200


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Siva and Foxp3 co-localize in the

nucleus of co-transfected U2OS cells

Siva and FoxP3 both display nuclear staining

Courtesy S.Mackey-Cushm

Wh t i f F P3 bi d t Si ?


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Siva-binding activity is

within a central portion of

the FoxP3 protein (AAs

106-332)

What region of FoxP3 binds to Siva?

What region of Siva binds to FoxP3


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The Siva C-terminus is sufficient to bind FoxP3

What region of Siva binds to FoxP3

?


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DOSIVAANDFOXP3 FUNCTIONALLY

INTERACT?

SIVAANDFOXP3 PHYSICALLYINTERACT


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Runx

IL-2

SIVA

?

What is Sivas effect on IL-2


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Experimental outline to test the effect of


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Siva overexpression on endogenous IL-

2

VSV-gHSPG

Gag/Pol

293T cells

Spinoculate

TransducedJurkat T cells

pHSPG-Siva-1

Retrovirus

Activate with PMA/Ion

for 18 hours.

Collect SNs.

Evaluate

endogenous IL-2 by

ELISA

Calcium phosphate

Si i


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Siva overexpression represses

endogenous IL-2

0

500

1000

1500

2000

2500

vector Siva-1

E6.1 pHSPG

IL-2(pg/m

L)


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Siva knockdown on endogenous IL-2

VSV-gpLKO

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

.

f S


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Knockdown of endogenous Siva

enhances endogenous IL-2

Experimental outline to test

Sivas effect on


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pGL-IL-2

Luciferase

pHSP

G-Siva

pLKO-

shSIVA

OR

Experimental outline to test Siva s effect onIL-2 promoter activity

Minimal IL-2 promoter

Measure luciferase

activity (RLUs) on

luminometer

pGL-IL2 Luc

Luciferase reporter


for 8 hours

Culture transfected Jurkat

cells for ~16 hours

Collect lysates

Si i


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Siva overexpression represses

IL-2promoter activity

Si k kd h IL 2


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Siva knockdown enhances IL-2

promoter activity


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Conclusions:- Siva represses IL-2gene expression.- Siva repression of IL-2is at the

promoter level.


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Runx

IL-2 promoter

IL-2

SIVA

?

How does Siva affect FoxP3srepressive effect on IL-2?

Outline for experiment to evaluate Sivas effect on


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Outline for experiment to evaluate Siva seffect on

FoxP3sability to repress endogenous IL-2

Perform two rounds ofspinoculation

1. pHSPG-Siva

2. pHSPG-FoxP3


for 18 hours.

Evaluate

endogenous IL-2 by

Collect SNs.

0

500

1000

1500

2000

2500

PG Siva-1

IL-2(pg

/mL)

PG

2000

2608

1200

2705

PG Vector (IL-2 pg/ml)

FoxP3 (IL-2 pg/ml)

= FoxP3s IL-2repressive activity

Calculating FoxP3s IL-2 repressive activity:

How does Siva overexpression affect FoxP3s


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How does Siva overexpression affect FoxP3 s

repressive effect on endogenous IL-2?

Endogenous IL-2

0

12

3

4

5

6

7

8

9

PG Siva-1

Foxp3'sRepressiveA

ctivity

FoxP3-mediated IL-2Repression

0

500

1000

1500

2000

2500

PG Siva-1

IL-2(pg/mL)

PG

Foxp3

Transduction efficiency

based on GFP expression

99% 99%

99% 99%


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How does Siva overexpression affect FoxP3s


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At lower doses of FoxP3, Sivareduced FoxP3s

repressive effect on IL-2 promoter activity

How does Siva overexpression affect FoxP3 s

repressive effect on IL-2 promoter activity?

FoxP3-mediated IL-2 RepressionIL-2 Promoter Activity

0

50000

100000

150000

200000

250000

300000

350000

400000

1.13 2.25 4.5PG Foxp3 (ug)

IL-2RLUs

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

Foxp3'sRepress

iveActivity

Foxp3 (ug)

PG

Siva (4.5 ug)


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Mini-summary:

Siva overexpression tends to

reduce FoxP3s repressive effect

on IL-2

Does knockdown of endogenous

Siva expression affect FoxP3srepressive effect on IL-2?

How does Siva knockdown affect FoxP3s repressive


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Sivaknockdown does not effect

FoxP3s repressive effect on

endogenous IL-2

How does Siva knockdown affect FoxP3 srepressive

effect on endogenous IL-2?

Endogenous IL-2

FoxP3-mediated IL-2 Repression

0

2

4

6

8

10

12

14

shEGFP shSIVA

Foxp3'sRepressiveActivity

00.5

11.5

22.5

33.5

44.5

shEGFP shSIVA

R

elativeSiva/18S PG

Foxp3

Efficiency of Siva Knockdown

by Realtime PCR

0

2000

4000

6000

8000

10000

12000

shEGFP shSIVA

IL-2(pg/m

l)

PG

Foxp3

How does Siva knockdown affect


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How does Siva knockdown affect

FoxP3srepressive effect on IL-2

promoter activity?

0

2000

4000

6000

8000

1000012000

14000

16000

18000

20000

shNS shSIVA

IL-2RLUs

PG

Foxp3

0

1

2

3

4

5

6

shNS shSIVA

Foxp3'sRepressi

veActivity

FoxP3-mediated IL-2 Repressio

Siva knockdown enhances FoxP3srepressive effect on IL-2 promoteractivity

IL-2 Promoter Activity

*


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Mini-summary:

Siva knockdown enhanced FoxP3s

repressive effect at the IL-2

promoter.


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Siva modifies FoxP3s repressive

effect on IL-2 promoter activity

FoxP3

SIV

A

IL-2 promoter activity

FoxP3SIV

A

IL-2 promoter activity

SIV

A

SIV

A

SIVA

SIV

A

SIV

A


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Ionomycin


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TCR

complex

Ca2

+

Ca2+

Ca2+

T Cell

AP-1

Ca2

+Ca2+Ca2+

IB

NFAT

Ca2+ Ca2+

Ca2+

Ca2+

Ca2

+

Ca2+

NFAT

PMA

NFB

MAPKKK

Ionomycin

PKC


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PMA

Ionomycin


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p50

IB

p65

NFB

SIV

A

PMA

Ionomycin


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p50

IB

p65

NFB

SIV

A

NFAT AP-1

SIVA

?

How does FoxP3 effect Sivas pro


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APOPTOSIS

FoxP3?

SIV

A

How does FoxP3 effect Siva s pro-

apoptotic activity?

Siva and FoxP3 promote apoptosis in


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Siva and FoxP3 promote apoptosis in

Jurkat cells in the absence of stimulation

0

2

4

6

8

10

12

PG Siva

%7A

ADPositiveCell

s

PG

Foxp3

*

Effects of Siva and FoxP3 in


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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

%

7AADPositiveC

ells

PG

Foxp3

*

*

*


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Summary of apoptosis data

Siva and FoxP3 both promoteapoptosis 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


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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 affectsFoxP3s repressive effect on IL-2promoter activity


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Future Directions

Mapping studies: Narrow the region of Siva binding activity

within FoxP3

Functional studies Evaluate Sivas IL-2 repressive activity in

primary cells


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Is Siva required for thymocyteselection?

-DP thymocytes express high levels ofSiva

-Hypothesis: Siva-mediated apoptosiscontributes to thymocyte selection

The Su Lab Committee Members


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Thank You!

The Su Lab Committee Members

Lishan Su

All lab members past andpresent

Jeff Frelinger

Jon Serody Roland Tisch

Jenny Ting

Karen McKinnon

Regulation of T cell Activation by FoxP3 and Siva

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