Regulatory Lymphocytes of the Immune System. Dr. C. Piccirillo Canada Research Chair Department of Microbiology & Immunology McGill University MIMM-414A.

Regulatory Lymphocytes of the Immune System.

Dr. C. PiccirilloCanada Research Chair

Department of Microbiology & ImmunologyMcGill University

MIMM-414ALecture 2- Oct. 23, 2006

ActivatedEffector

T cell

APC

_

+ Peripheraldifferentiation

signals

Thymic CD4Thymic CD4++ T cell pool T cell pool

Peripherally-induced CD4+ Treg cells

( iTreg )

Autoimmunity Transplantation Tumor Immunity

Infectious disease

TCR

Piccirillo et al. Trends in Immunol. 2004.

Induced regulatory T cells

– CD4+•Th1 cell-mediated cross-regulation of Th2 cells (IL-4 <-> IFN)

•Th3 - Induced by oral tolerance–TGF-1 producing (IL-10) and TGF-1 dependent effect

•Tr1 - Induced by Ag + IL-10 and IL-10 dependent effect

• A. INDUCED - immunization, experimental manipulation…

– NK-T cells– CD8+ T cells T cells– DN T cells

A network of CD4+ regulatory T cellscontrol immune reponses.

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.

B. NATURALLY-OCCURRINGEndogenous in naïve repertoire: professional

Journal of Immunology 1995 155:1151

Lymph nodes from a 2 month old BALB/c

Normal Immune

responses

Tissue regeneration

Allergy

Tumor Immunity

Transplantation

Infectious disease

Autoimmunity

CD4+CD25+ Treg cells

MucosalImmunity

1 10 100 1000 100001

10

100

1000

100009

FL2-H: CD25 PE

FL

3-H

: C

D4

Tri

Colo

r

• Depletion• Day3 thymectomy• Genetic anomalies• Irradiation• Infections• Drugs

Dominant self-tolerance Unique lineage of CD25+ (IL-2R) T cells 1-10% of thymic or peripheral CD4+ T cells In resting, endogenous T cell repertoire Rodents, human and non-human primates

Naturally-occurring CD4+ regulatory T cells.

Gastritis Oophritis Orchitis Thyroiditis Pancreatitis ColitisAuto Abs

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

Reliable biomarkers for CD4+CD25+ regulatory T cells?

CD25

CTLA-4GITR

CD103 Galectin-1

Ly6

OX-40

4-1BB

Unidentified X,Y,Z ??

Neuropilin-1

CD4+CD25+

RegulatoryT cell

Piccirillo et al. Trends in Immunology July 2004

NONO marker after T cell activation.

OnlyOnly in the naïve T cell repertoire.

Need better marker.

MHC/peptide

TCR

Activated CD4+ T cell

RestingCD4+ T cell

T cell activation induces expression of functional T cell

surface molecules

TCR

APC

CD40L

CD25CD69CD44

CD25 is not a marker of Treg after T cell activation.Only in the naïve T cell repertoire.

Treg cells are partially-activated cells

Treg cells are anergic.

Hyporesponsive to TCR stimulation : restored by IL-2.

Do not produce IL-2, IL-4, IFN-, IL-10 (), TGF1 ()

Phenotypic and functional characteristics

CD4+CD25+ regulatory T cells suppress T cell functions. Consensus elements

IFN

- (

ng

/ml)

0

4

8

12

16

20

24

28

CD8+ CD8+/CD4+CD25-CD8+/CD4+CD25+

Cytokine Production

IFN-

CD8+ CD8+/- CD8+/+

24h

48h

CD8+

CD8+/-

CD8+/+

CP

M

# CD4+CD25- ( ) or CD4+CD25+ ( ) T cells

0

25000

50000

75000

100000

125000

0 10000 20000 30000 40000 50000

CD4+ or CD8+

T cells

Proliferation

• Requirement for TCR engagement• Antigen non-specific & MHC unrestricted

• Induce cell cycle arrest (G1-S transition)

CD4+CD25+ Immunoregulatory T Cells Suppress Polyclonal T Cell Activation In Vitro by Inhibiting Interleukin 2 Production Angela M. Thornton and Ethan M. Shevach J. Exp. Med. 1998 188: 287-296.

Cellular origin of CD4+ regulatory T cells

CD4+ Treg

EndogenousNaturally-occurring

Induced duringImmune response

?

Figure 1. TR cells may arise from relatively high-avidity interactions with self-peptide–MHC complexes, just below the threshold for negative selection (green area). This narrow avidity selection window ensures that Tr cells will constitute only a small fraction of the mature T cell pool and have a greater sensitivity to self-peptide–MHC than potentially pathogenic autoreactive T cells.

TReg cells are a normal product of thymic selection.

Thymic development and TCR specificity: Unknown

Diverse T cell repertoire

Self-specific

Cross-reactive to foreign?

Altered negative selection?

Unique signals for CD4+CD25+ Treg cell homeostasis.

MHC II engagement in periphery Foxp3

Requirement for co-stimulation: CD40/CD40L B7/CD28

IL-2 production IL-2-/- mice IL-2R signaling : IL-2R, STAT5, IL-2c Functional correlate: survival, mechanism…? Other signals: TGF-1

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

Hypothetical pathway to suppression.

Ag/TCR

IL-2 transcription

IL-2 protein

IL-2 driven expansion

T cell differentiation & effector function

Self-reactiveTeff cell

nTreg

CD4+CD25+

√

T cellEffector

CD4+

CD25+

Pathways to T cell suppression

Antigen Presenting CellCo-stimulation

Adhesion

XA

B

• CD4+CD25+ cells modulate co-stimulation?• MHC class I/II, B7.1/2, CD40 expression is unaffected.• Suppression is still operative with fixed LPS blasts. • Not overcome with numbers of APCs.

CD4+CD25+ mediated suppression is contact-dependent.

Requirement for APC?

Piccirillo et al. J.Immunology 167:1137-1140.

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

• Cytokine independent

• Suppress IL-2 mRNA in T cells.

• Suppression of effector functions

• proliferation• inflammatory cytokines• differentiation

• Effector molecules are unknown.

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) ?

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

EffectorT cell

CD4+

CD25+

Cytokines ?

In vitro and in vivo role of cytokines in CD4+CD25+ regulatory T cell-mediated suppression?

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

•Suppression is cytokine independent• 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 TGF-1?

Suppressor T cell-derived IL-10 ?

Initial studies showed that anti-IL-10 or anti-TGF-B1abrogated Treg-mediated suppression of disease.

Nakamura et al JEM 2001Membrane-bound TGFSimon Read et al. JEM 2000.

Requirement for Transforming Growth Factor Transforming Growth Factor 1 1

(TGF-(TGF-1) 1) ?

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?

100806040200

TGF-b KO CD25pos

WT CD25neg + TGF-b KO CD25pos

WT CD25neg + WT CD25pos

WT CD25neg

No cells

Severe colitis (3.5 - 4.0)Moderate colitis (2.0 - 3.0)Mild colitis (1.0 - 1.5)No colitis (0 - 0.5)

Incidence of colitis (%)

Colon grades (HEL129 + HEL141)

n=8

n=7

n=8

n=8

n=9

CD25– CD25+

cells cells

— — WT —

WT WT

WT TGF-1-/-

— TGF-1-/-

A

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

Incidence of colitis (%)

B

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

CD25– CD25+ cells cells

— —

WT —

WT WT

WT TGF-1-/-

— TGF-1-/-

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?

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 !

Diversity of modes of action

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

• IBD is a bacterially-driven disease, not gastritis.• Lessons from germ-free mice.

• Genetic background

• Possible subsets of CD4+CD25+ Treg:– Cytokine versus Contact– Adaptable to inflammatory milieu.– Induction of other Treg cells.

Re-evaluation of CTLA-4 autoimmune phenotype

• Could CTLA-4 deficiency result in defective Treg function and thus autoimmunity ? (rather than role of CTLA-4 on T cell autonomous regulation)

• CTLA-4 phenotype rescued by complementing CTLA-4-/- BM with wild type BM (mixed BM chimera)

• CTLA-4 deficient bone marrow can make CD25+CD4+ (function vs development)

CTLA-4 is expressed constitutively on CD4+CD25+CD45RBlow cells.

Read et al. J. Exp. Med. July 2000

CD4+CD25+ (CD45low) inhibit IBD Anti–CTLA-4 treatment abrogates the Treg function.

Open: control Ig Closed: CTLA-4.

Adoptive transfer of CD4 T cell into scid mice.

MHC:peptide

NaiveCD4+ or CD8+

T cells

Possible Mechanisms of Action of CD4+ Regulatory T Cells

TCR

APC

CD25

Activated effector T cells

CD25

Naturally-occurring Treg

Induced Treg

IL-10TGF-1

IL-10TGF-1

Cellularcontact

A BA+B

Tolerance Autoimmunity

Transplantation Tumor Immunity

Infectious disease