Regulatory Lymphocytes of the Immune System.

II

Dr. C. PiccirilloCanada Research Chair

Department of Microbiology & ImmunologyMcGill University

MIMM-414ALecture 3- Oct. 25, 2006

CD25GITRCTLA-4Foxp3

ActivatedEffector

T cell

APC

__

TCR

+ Peripheraldifferentiation

signals

Thymic CD4Thymic CD4++ T cell pool T cell pool

Thymically-derived naturally-occurring

CD4+CD25+ Treg cells (nTreg )

Peripherally-induced CD4+ Treg cells

( iTreg )

Autoimmunity Transplantation Tumor Immunity

Infectious disease

TCR

Foxp3+

GITR+

CTLA-4+

CD25+

Piccirillo et al. Trends in Immunol. 2004.

NATURALLY-OCCURRING versus INDUCEDTreg cells in the immune system.

IL-10,TGF-1iDCVitDDexamethasone

CD4CD4++CD25CD25++ nTreg cells nTreg cellsMasterswitch of peripheral toleranceMasterswitch of peripheral tolerance

Self antigens Non-self antigensPathogens

TumorsAllergens

Grafts

FoxP3 transcription factor FoxP3 spontaneous mutations induces autoimmunity:

-IPEX in humans: Immunodysregulation, polyendocrinopathy, enteropathy,X-linked syndrome

-Scurfy in mice.

FoxP3-/- develop spontaneous autoimmunity- defective Treg cells

FoxP3 is preferentially expressed in CD4+CD25+ T cells

FoxP3 Tg have cellular frequency of CD4+CD25+ Treg cells.

FoxP3 Tg mice x CTLA-4-/- = resolved/delayed autoimmunity

FoxP3 retroviral transduction in non-regulatory CD4+CD25- T cells induces regulatory potential.

- Phenotypically and functionally similar to naturally occuring lineage.

Genes induced by FoxP3 remain unknown.

Fontenot et al.

More selective and faithful marker than CD25

nTregTeff cell

Antigen Presenting Cell

CD4+CD25+

CD4+ Teff

APC

CD4+

CD25+

• Requires TCR engagement

• Antigen non-specific

• Cell-cell contact dependent• Co-stimulation/APC independent• T-T suppressor synapse

• Suppress IL-2 mRNA in T cells.

• Suppression of effector functions

• proliferation• inflammatory cytokines• differentiation

• Effector molecules are unknown.

•Suppressive cytokines?

Cellular and molecular requirements of CD4+CD25+ nTreg cell suppressor function.

Suppressor Synapse

Mechanism of CD4+CD25+ regulatory T cell function ?

Contribution of Transforming Growth Factor 1 (TGF-1) ?

J.Exp. Med. 196:237-250.

EffectorT cell

CD4+

CD25+

Cytokines ?

Role of cytokines in CD4+CD25+ Treg cell-mediated suppression?

• IL-4, IL-10• Immunosuppressive effects on APC and T cells

•Suppression is cytokine independent in vitro• Cytokine neutralization

• Absence of cytokines in suppressor supernatants

• Cytokine-deficient Treg cells

• Transwell chamber experiments

CD4+CD45Rbhigh

CD4+CD45Rbhigh

CD4+CD45Rblow

ColitisNo colitis

(CD25+ subset)

SCID

CD4+CD25+ Treg cells control bacterial-driven intestinal inflammation.

Bacterially-driven, Th1 cell-mediated

Inflammatory bowel disease(IBD)Colitis

T cell infiltration of colon ->weight loss

Suppressor T cell-derived IL-10 needed.

Suppressor T cell-derived TGF-1?

Initial studies showed that anti-IL-10 or anti-TGF-1abrogated Treg-mediated suppression of disease.

Nakamura et al JEM 2001Membrane-bound TGF ?

Powrie et al. JEM 1994Simon Read et al. JEM 2000.

Requirement for TGF-TGF-1 1 ?

Potent immunosuppressive cytokine on various immune cell subsetsSuppression of T, B and DC responses: proliferation, cytokine, MHC/Ag

presentation and co-stimulation.Role is best exemplified in TGF-1 knock-out mice which die of a fulminant,

multi-organ, lymphoproliferative disease.

Piccirillo et al. J.Exp. Med. 196:237-250.

nTreg

TGF-TGF-11

RII

Smad3

TGF-R

DNRIITg

Smad3-/-

X

XTGF-TGF-11-/--/-

Y

Y

CD4+CD25- and CD4+CD25+ T cells produce TGF-1?

CD4+CD25-

CD4+CD25-

WT CD4+CD25+

CD4+CD25-

TGF-1-/-CD4+CD25+

No colitis

?

B6RAG-/-

3-7 day oldneonates

WT B6/Sv129 WT B6/Sv129TGF-1-/-

CD4+CD25+ Treg cell-mediated control of mucosal inflammation.

Mouse model of Inflammatory bowel disease (IBD)

Colitis• T cell infiltration of colon• Th1 response to gut bacteria• Weight loss

Colitis

Kullberg M., and C.A. Piccirillo Euro. J. Immunol. 2005

Is nTreg cell functionTGF- dependent in vivo?

CD25– CD25+

cells cells

— — WT —

WT WT

WT TGF-1-/-

— TGF-1-/-

80

90

100

110

0 10 20 30 40 50 60 70

Days post cells

Bo

dy

We

igh

t (%

of

da

y 4

we

igh

t)B

od

y w

eig

ht

(% o

f d

ay 4

wei

gh

t)

Days post cells

TGF-1-/- CD4+CD25+ nTreg cells suppress IBD.

TGF-1-/- CD4+CD25+ nTreg cells suppresscolonic inflammation.

AG

rad

e o

f in

flam

mat

ion

B

IFN

- /

G3P

DH

mR

NA

rat

io

CD25– cells — WT WT WT —

CD25+ cells — — WT TGF-1-/- TGF-1-/-

CD25– cells — WT WT WT —

CD25+ cells — — WT TGF-1-/- TGF-1-/-

C.

A.

D.

E.

B.A. B. C. D. E.

CD4+CD25-

WT CD4+CD25-

CD4+CD25+

Smad 3-/- CD4+CD25-

CD4+CD25+

Colitis

No colitis

?

B6RAG-/-

4-6 weeks old

WT B6/Sv129WT B6/Sv129

Smad3 -/-

FACS sort

CD4+CD25+-mediated regulation of Smad3-deficient effector T cells in vivo.

0

2

4

6

8

10

Gra

de

of

infl

am

ma

tio

n

CD25- cells – WT WT WT Smad3-/- Smad3-/- Smad3-/-

CD25+ cells – – WT Smad3-/- – WT Smad3-/-

A

B

Bo

dy

we

igh

t (%

of

da

y 4

we

igh

t)G

rad

e o

f in

fla

mm

ati

on

CD25– CD25+

— — WT Smad3-/- Smad3-/- WT Smad3-/- Smad3-/- WT WT Smad3-/- — WT —

Days post cells

Smad3-/- effector T cells are highly susceptible to suppression mediated by CD4+CD25+ T cells in vivo.

Powrie group observesabrogation of protection withTGFR-/- Effector T cells

Why?

CD45RBLow

CD4+CD25-

CD45RBHigh

+ TGF-1

- TGF-1

1:4 1:2 1:10

20

40

60

80% S

up

pre

ssio

n

Suppressor: Effector Cell

TGF-TGF-11

iTreg nTreg

Regulation of immune responses via Foxp3

induction

CD4+Foxp3+

IL-10IL-10++CD4+Foxp3Foxp3++

CD25-Rblow

Any role for TGF-1 in Treg responses?

Tissue-specific CD4+CD25+ mediated disease protection in the absence of IL-10.

CD4+CD25-

CD4+CD25-

CD4+CD25+ CD4+CD25-

IL-10-/- CD4+CD25+

GastritisIBD

No Gastritis

NoIBD

Nude

No GastritisIBD develops !IBD develops !

• Context-dependent regulation in vivo.– Tissue-specific differentiation of Treg?– Any role for bacteria?

• IBD is a bacterially-driven disease, not gastritis.

• Lessons from germ-free mice.

• Genetic background

• Subsets of CD4+CD25+ Treg?– Cytokine versus Contact– Adaptable to inflammatory milieu.– Induction of other Treg cells.

Control of immune responses by CD4+CD25+ regulatory T cells.

CD4+

CD8+

T cells

CD4+CD25+

RegulatoryT cells

Infectious disease

Immunity to intracellular pathogens ??

Belkaid/Piccirillo et al. Nature 420:502-7, 2002

100

103

104

105

106

0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

1.8

0 5 10 15 20P

aras

ite

nu

mb

er

Lesio

n s

ize (mm

)

Weeks post-infection

Chronic phase : Transmits back to vector Resistant to re-infection. Life-long immunity : concomitant immunity

Site of immune pressure : IL-10IFN-IL-10-/- or anti-IL-10R -> Sterile cure.

Sile

nt

Acute

Chronic

Role for nTreg cells?

Weeks post-infection4 80

Th2 Non-healing

Les

ion

siz

e /

par

asit

e #

BALB/c

Susceptibility and resistance to Leishmania major infection

Healing Th1

B6

CD4+CD25+ CD4+CD25-

Chronic Dermal site

Cell Sorting

CD4+ nTreg cells accumulate in sites of chronic infection.

L.major infected DC

CytokineProduction

0

200

400

600

800

1000

1200

I-10

0

5000

1 104

1.5 10 4

IFN-

pg/

ml

CD4+CD25-

++++

CD4+CD25+

+

+

+

+

DCs

Infected DCs

45-60%

CD

25

CD4

8 months

CTLA-4 +Foxp3+GITR+CD45Rblow

CD4+CD25+ T cells from chronic sites are regulatory.

0

10000

20000

30000

40000

50000

60000

CP

M

0 10000200003000040000500000

50000

100000

150000

200000

CP

M

-IL-2 +IL-2

CD4+CD25+CD4+CD25-

CD4+CD25+

CD4+CD25-

0

3

6

9

12

15

IFN

- (

ng

/ml)

CD25- CD25-/CD25+CD25-/CD25+

Anti-IL-10R# CD4+CD25±

- -/+ -/+

IL10R

L.major Infected

macrophages

CD4+CD25+

CD4+CD25-

L. majorIntradermally

C57BL/6RAG-/-

1/10

Chronic site

CD4+CD25+T cells control immunity to pathogens. L

y5.

1

CD25

50%12 % 65 %

9 WksChronic

5 WksAcute

3 WksSilent

0

2

4

6

8

10

12

0 2 4 6 8 10 12

CD25-/CD25+

CD25-

RAG -/-

1. Rapid nTreg accumulation2. Prevent effector T cell functions.3. Promote susceptibility to infection4. Remain in chronic site5. Favor persistence of pathogen

IL-10+ Treg / IFN+ Teff cells

Model of cutaneous L.major infection.

Pa

ras

ite

nu

mb

er

/ea

rCD4+CD25+T cells from chronic sites prevent

anti-parasite effector T cell function.

CD4

IFN-

1

10

100

1000

104

105

106

107

RAG-/- CD25+ CD25- CD25-/CD25+

10/1

CD25-/CD25+

CD25-

CD25+

Lesi

on s

ize

Weeks post infection

IL-10 dependent and independent modes of disease control by nTreg cells.

-/+ WT

-/+ IL-10-/-0

0.5

1

1.5

2

0 2 4 6 8 10 12 14 16

CD25-

CD25-

RAG-/-

109

103

108

107

106

105

104

102

10

Par

asite

num

ber

CD25-CD25+

WT

CD25-CD25+IL-10-/-

3 103 103 10

Parasite persistence is required for immunity to re-infection

Implications :

•Parasite • Long-term maintenance of

infectious reservoirs.

•Host • Role of parasite

persistence in immunity ?

Homing of Treg cells to the infected sites

Preferential tropism for Treg cells to infected sites?

Chemokine-mediated selective recruitment of CD4+CD25+T cells ?

Journal of Experimental Medicine Oct. 2006

CCR5 gene expression

A.

B.

CCR5

Resting Activated

CCR5 is required for CD4+CD25+ nTreg cell chemo-attraction but not suppressive activity in vitro.

Gated on Foxp3+ cells

C.

0

40000

80000

120000

160000

CP

M

0 10000 20000 30000 40000 50000

# CD4+CD25+ T cells

CCR5-/-

WTD.

0

10

20

30

40

50

% M

igra

ting

CD

4+ T

ce

lls

CD4+Foxp3- CD4+Foxp3+

RANTES

MIP-1

MIP-1

CCR5-/- mice are resistant to L.major infection.

CCR5-/- CD4+CD25+ nTreg cells fail to promote parasite persistence.

Weeks post infection

0

0.5

1

1.5

0 1 2 3 4 5 6 7 8

WT

CD4+CD25+

102

103

104

105

+ +

Par

asit

e n

um

ber

7 weeks

Les

ion

siz

e (m

m)

WT CD4+CD25+

CCR5-/- CD4+CD25+

WT CD4+CD25-

+

-

-

+

CCR5-/-

CD4+CD25+101

+

--

0

10

20

30

40

50

60

70 WT

CCR5-/-

WT

CCR5-/-

WT

CCR5-/-

1.5 3 5.5 10

ND

% C

D4+

CD

25+

T c

ells

Weeks post-infection

ND

Spleen

Lymphnode

Skin38% 4%

CD

4+C

D2

5+ L

y5

.1+

3 weeks3 weeks

CD

4+C

D2

5+ L

y5

.2+

CD4+ effector T cells

WT CCR5-/-

CCR5 dependent homing of CD4+CD25+ Treg cells in sites of infection.

Summary• nTreg cells home preferentially to sites of

inflammation:

– nTreg cells express CCR5 and responds to its ligands.

– CCR5-mediated signals may drive the early recruitment of nTreg cells in sites of infection.

– CCR5 mediated homing into sites of pathogen infection regulates pathogen persistence.

– Pathogen persistence may itself provide a major benefit to the

host by maintaining life long immunity to re-infection.

– Blockade of CCR5 chemotaxis may hinder nTreg/Teff balance and provoke anti-pathogen immune responses.

– Mechanism of immune evasion ?

– Other receptors: CCR4 and CCR6 ( tumors and CNS homing)

CD4+ nTreg cell function in health and disease Diversification versus adaptability model

CD4+CD25+

nTreg

Self Autoimmunity

Foreign Pathogens

Tumors Grafts

Loss of tolerance

Increased immunity

Subset Diversification

Adaptability

Current Drug Targets 2006.

Genetic determinantsInnate signals

Adaptive signals

Teff

-