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Regulatory Lymphocytes of the Immune System. Dr. C. Piccirillo Canada Research Chair Department of Microbiology & Immunology McGill University MIMM-414A Lecture 2- Oct. 23, 2006 Induced regulatory T cells Thymic CD4 + T cell pool Peripherally-induced CD4 + Treg cells ( iTreg ) TCR

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slide1

Regulatory Lymphocytes of the Immune System.

Dr. C. Piccirillo

Canada Research Chair

Department of Microbiology & Immunology

McGill University

MIMM-414A

Lecture 2- Oct. 23, 2006

slide2

Induced regulatory T cells

Thymic CD4+ T cell pool

Peripherally-induced

CD4+ Treg cells

( iTreg )

TCR

+ Peripheral

differentiation

signals

APC

_

Activated

Effector

T cell

Autoimmunity

Transplantation

Tumor Immunity

Infectious disease

Piccirillo et al. Trends in Immunol. 2004.

slide3

A network of CD4+ regulatory T cells

control immune reponses.

  • A. INDUCED- immunization, experimental manipulation…
    • NK-T cells
    • CD8+ T cells
    •  T cells
    • DN T cells
  • CD4+
    • Th1cell-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
slide4

B. NATURALLY-OCCURRING

Endogenous in naïve repertoire: professional

Thymic CD4+ T cell pool

Thymically-derived

naturally-occurring

CD4+CD25+ Treg cells

(nTreg )

Peripherally-induced

CD4+ Treg cells

( iTreg )

Foxp3+

GITR+

CTLA-4+

CD25+

TCR

TCR

+ Peripheral

differentiation

signals

CD25

GITR

CTLA-4

Foxp3

APC

_

_

Activated

Effector

T cell

Autoimmunity

Transplantation

Tumor Immunity

Infectious disease

Piccirillo et al. Trends in Immunol. 2004.

slide5

Journal of Immunology 1995 155:1151

Lymph nodes from a

2 month old BALB/c

slide6

Naturally-occurring CD4+ regulatory T cells.

Autoimmunity

  • 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

Allergy

Infectious

disease

CD4+CD25+

Treg cells

Tissue

regeneration

Mucosal

Immunity

Transplantation

  • Gastritis
  • Oophritis
  • Orchitis
  • Thyroiditis
  • Pancreatitis
  • Colitis
  • Auto Abs
  • Depletion
  • Day3 thymectomy
  • Genetic anomalies
  • Irradiation
  • Infections
  • Drugs

Normal

Immune

responses

Tumor

Immunity

reliable biomarkers for cd4 cd25 regulatory t cells
Reliable biomarkers for CD4+CD25+ regulatory T cells?

CD25

GITR

CTLA-4

CD4+CD25+

Regulatory

T cell

NO marker after T cell activation.

Only in the naïve T cell repertoire.

Need better marker.

Galectin-1

CD103

Ly6

4-1BB

Neuropilin-1

Unidentified

X,Y,Z ??

OX-40

Piccirillo et al. Trends in Immunology July 2004

slide9

T cell activation induces expression of functional T cell surface molecules

Activated

CD4+ T cell

MHC/peptide

CD40L

TCR

TCR

CD25

Resting

CD4+ T cell

APC

CD69

CD44

CD25 is not a marker of Treg after T cell activation.

Only in the naïve T cell repertoire.

phenotypic and functional characteristics
Phenotypic and functional characteristics

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 (), TGF1 ()
cd4 cd25 regulatory t cells suppress t cell functions consensus elements
CD4+CD25+ regulatory T cells suppress T cell functions.Consensus elements

CD8+

24h

Proliferation

Cytokine Production

CD8+/-

CD8+/+

CD4+ or CD8+

T cells

CPM

48h

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

  • Requirement for TCR engagement
  • Antigen non-specific & MHC unrestricted
  • Induce cell cycle arrest (G1-S transition)

(ng/ml)

IFN-

g

IFN-

CD8+/-

CD8+/+

CD8+

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.

slide12

Cellular origin of CD4+ regulatory T cells

CD4+ Treg

?

Endogenous

Naturally-occurring

Induced during

Immune response

slide13

TReg cells are a normal product of thymic selection.

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.

  • 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
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-2Ra, STAT5, IL-2gc
        • Functional correlate: survival, mechanism…?
        • Other signals: TGF-b1
foxp3 transcription factor
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
Fontenot et al.

More selective and faithful

marker than CD25

hypothetical pathway to suppression
Hypothetical pathway to suppression.

Self-reactive

Teff cell

Ag/TCR

nTreg

IL-2 transcription

CD4+CD25+

IL-2 protein

IL-2 driven

expansion

T cell differentiation &

effector function

slide18

CD4+CD25+ mediated suppression

is contact-dependent.

Requirement for APC?

CD4+

CD25+

T cell

Effector

B

Pathways to

T cell suppression

A

X

Antigen Presenting Cell

Co-stimulation

Adhesion

  • 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.

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

slide19

Mechanism of CD4+CD25+ regulatory T cell function ?

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

  • 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.

nTreg

Teff cell

Antigen Presenting Cell

CD4+CD25+

Suppressor Synapse

APC

CD4+

CD25+

CD4+

Teff

slide20

In vitro and in vivo role of cytokines in

CD4+CD25+ regulatory T cell-mediated suppression?

CD4+

CD25+

Effector

T cell

Cytokines ?

  • 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

Contribution of

Transforming Growth Factor 1 (TGF-1) ?

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

slide21

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

CD4+CD45Rbhigh

CD4+CD45Rblow

(CD25+ subset)

Bacterially-driven,

Th1 cell-mediated

Inflammatory bowel disease

(IBD)

Colitis

T cell infiltration of colon ->weight loss

CD4+CD45Rbhigh

SCID

Colitis

No colitis

Initial studies showed that

anti-IL-10 or anti-TGF-B1

abrogated Treg-mediated

suppression of disease.

Suppressor T cell-derived TGF-1?

Suppressor T cell-derived IL-10 ?

Nakamura et al JEM 2001

Membrane-bound TGF

Simon Read et al. JEM 2000.

slide22

RII

  • Requirement for
  • Transforming Growth Factor 1

(TGF-1) ?

Y

TGF-R

TGF-1

nTreg

Y

X

DNRIITg

Smad3-/-

TGF-1-/-

X

Smad3

CD4+CD25- and CD4+CD25+

T cells produce TGF-b1?

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

slide23

Is nTreg cell function

TGF- dependent in vivo?

CD4+CD25+ Treg cell-mediated control of mucosal inflammation.Mouse model of Inflammatory bowel disease (IBD)

WT B6/Sv129

WT B6/Sv129

TGF-b1-/-

3-7 day old

neonates

Colitis

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

CD4+CD25-

WT CD4+CD25+

CD4+CD25-

TGF-b1-/-CD4+CD25+

CD4+CD25-

B6

RAG-/-

?

Colitis

No

colitis

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

tgf 1 cd4 cd25 ntreg cells suppress ibd
TGF-1-/- CD4+CD25+ nTreg cells suppress IBD.

A

CD25–CD25+

cellscells

— —

WT —

WT WT

WT TGF-b1-/-

— TGF-b1-/-

Body weight (% of day 4 weight)

Days post cells

CD25– CD25+

cells cells

— —

WT —

WT WT

WT TGF-b1-/-

— TGF-b1-/-

B

Incidence of colitis (%)

tgf 1 cd4 cd25 ntreg cells suppress colonic inflammation
TGF-1-/- CD4+CD25+ nTreg cells suppresscolonic inflammation.

B.

A.

A

A.

B.

C.

D.

E.

Grade of inflammation

C.

D.

CD25– cells — WT WT WT —

CD25+ cells — — WT TGF-b1-/- TGF-b1-/-

B

E.

IFN-g / G3PDH mRNA ratio

CD25– cells — WT WT WT —

CD25+ cells — — WT TGF-b1-/- TGF-b1-/-

slide26

CD4+CD25+-mediated regulation of

Smad3-deficient effector T cells in vivo.

WT B6/Sv129

Smad3 -/-

WT B6/Sv129

4-6 weeks old

FACS sort

WT CD4+CD25-

CD4+CD25+

Smad 3-/- CD4+CD25-

CD4+CD25+

CD4+CD25-

B6

RAG-/-

Colitis

?

No

colitis

slide27

Smad3-/- effector T cells are highly susceptible to

suppression mediated by CD4+CD25+ T cells in vivo.

A

CD25– CD25+

— —

WT Smad3-/-

Smad3-/- WT

Smad3-/- Smad3-/-

WT WT

Smad3-/- —

WT —

Body weight (% of day 4 weight)

B

Days post cells

Powrie group observes

abrogation of protection with

TGFR-/- Effector T cells

Why?

Grade of inflammation

tissue specific cd4 cd25 mediated disease protection in the absence of il 10
Tissue-specific CD4+CD25+ mediated disease protection in the absence of IL-10.

CD4+CD25-

CD4+CD25+

CD4+CD25-

IL-10-/- CD4+CD25+

CD4+CD25-

Nude

Gastritis

IBD

No Gastritis

IBD develops !

No

Gastritis

No

IBD

diversity of modes of action
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
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 t reg function
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.

slide32

Possible Mechanisms of Action

of CD4+ Regulatory T Cells

Naturally-occurring Treg

CD25

IL-10

TGF-1

Cellular

contact

A

A+B

B

MHC:

peptide

Activated

effector T cells

TCR

Naive

CD4+ or CD8+

T cells

APC

Tolerance

Autoimmunity

Transplantation

Tumor Immunity

Infectious disease

IL-10

TGF-1

Induced Treg

CD25