Treg Alterations Lead To Systemic And Treg Alterations Lead To Systemic And Local Immune Deregulation In Local Immune Deregulation In

Idiopathic Pulmonary Fibrosis And Idiopathic Pulmonary Fibrosis And Collagen Vascular Disease-Associated Collagen Vascular Disease-Associated

Interstitial PneumoniaInterstitial Pneumonia

Nakou ENakou E11, Tzouvelekis A, Tzouvelekis A11, Bouchliou I, Bouchliou I22, , Kouliatsis GKouliatsis G11, Froudarakis M, Froudarakis M11, Steiropoulos P, Steiropoulos P11, ,

Kotsianidis IKotsianidis I22 and Bouros and Bouros DD11

11Dep of Pneumonology and Dep of Pneumonology and 22Dep of Haematology, Dep of Haematology, Democritus Thrace University Hospital Democritus Thrace University Hospital

Alexandroupolis, GreeceAlexandroupolis, Greece

CD4CD4++CD25CD25++ T regulatory cells T regulatory cells

Essential for the maintenance of Essential for the maintenance of immunological toleranceimmunological tolerance

Prevent of autoimmunity by active Prevent of autoimmunity by active suppression suppression

High expression of a-chain of the IL-2 High expression of a-chain of the IL-2 receptor (CD25)receptor (CD25)

Foxp3 is essential for their development Foxp3 is essential for their development and functionand function

Suppression requires cell contactSuppression requires cell contact through through CTLA-4 moleculeCTLA-4 molecule

Treg

Foxp3

GITRCD4 CD25

CTLA-4

BackgroundBackground

ILDs are a heterogeneous group of lung diseases.

Imbalance of Th1/Th2 cytokine profile

TGF-β: pro-fibrotic effect IFN-γ: anti-fibrotic effect Krein et al,Chest ,(2002). Strieter et al, Am J Respir Crit Care Med(2004). Burdick et al. Am J Respir

Crit Care Med,(2005).

Local and systemic immune activation remitting after steroids in IPF/UIP Homolka, J. et al. Respiration (2003)

Oligoclonal expansion of T cells Shimizudani, N. et al Clin Exp Immunol (2002) Yurovsky, V.V et al.Hum Immunol (1996)

Evidence for immune system involvement in the Evidence for immune system involvement in the pathogenesis of ILDspathogenesis of ILDs

Tregs are the main regulatory subset responsible for the are the main regulatory subset responsible for the control of control of immunologic tolerance immunologic tolerance

Systemic and/or local, qualitative and/or quantitative Treg alterations are involved in the pathophysiology of ILDs

Hypothesis - ObjectiveHypothesis - Objective

Experimental DesignExperimental DesignPatients & controls:

Two groups of diseases (CVD-IP n=10, IPF n=5)

24 Normal donors (ND only PB) 24 Normal donors (ND only PB)

6 non-ILDs 6 non-ILDs (as BAL controls) Phenotypic analysis:: 4-colour flow cytometry in PB and BAL (CD4, CD25, CD152)

Modified MACS-isolation of CD4+CD25High Tregs

CFSE-based proliferation assays MACS-isolated PB and BAL CD4+CD25+ or – cells after polyclonal stimulation

IPF: 22%IPF: 22%±2.9%±2.9% CVD-IP: 18.6%±3%CVD-IP: 18.6%±3% Non-ILD: 7%±1.3% Non-ILD: 7%±1.3%

Treg frequency in BAL and PBTreg frequency in BAL and PB

Treg percentage in BAL and PB

0

5

10

15

20

25

NON-I LD CVD-I P I PF ND

% o

f C

D4

+ c

ell

s

PB

BAL

P<0.03 P=0.01

Treg frequency in BALTreg frequency in BAL

CD4

CD

2530% 28%

5.6%

IPF

NonILD

CVD-IP

TheThe absolute numberabsolute number of of CD4CD4++CD25CD25hi hi TregsTregs in the peripheryin the periphery is the same in all groupsis the same in all groups

Absolute number of Tregs in PBAbsolute number of Tregs in PB

0

2

4

6

8

10

12

14

non-ILDs CVD-IP IPF ND

Tre

gs

pe

r m

m3

non-ILDs CVD-IP IPF ND

TheThe percentage ofpercentage of CTLA-4CTLA-4 inin CD4CD4++CD25CD25hihi Tregs Tregs of BALof BAL andand PBPB is the same in all groups is the same in all groups Significant difference between BAL and PB in CVD-IP andSignificant difference between BAL and PB in CVD-IP and IPF IPF ((*p<0.05).*p<0.05).

CTLA-4 expression in Treg

0

20

40

60

80

100

NON-I LD CVD-I P I PF

% o

f C

D4

+C

D2

5+

cell

s

PB

BAL

** **

CTLA-4 expression in TregsCTLA-4 expression in Tregs

Tregs inTregs in PBPB ofof IPF and CVD-IP patients are IPF and CVD-IP patients are 2 2 times lesstimes less suppressive in comparison with ND and non-ILDs suppressive in comparison with ND and non-ILDs

(Τ(Τreg:Teff = 1:2, reg:Teff = 1:2, *p<0.05*p<0.05, , nn=5=5))..

CFSE-based proliferation assays in PBCFSE-based proliferation assays in PB

0%

20%

40%

60%

80%

100%

ND non-ILDs

CVD-IP IPF 0-1

% o

f p

rolife

rati

on

** **

CFSE-based proliferation assays in PBCFSE-based proliferation assays in PB

CFSE-based proliferation assays in BALCFSE-based proliferation assays in BAL

Tregs in BAL of IPF and CVD-IP patients Tregs in BAL of IPF and CVD-IP patients exerted exerted comparable suppressioncomparable suppression with the non-ILD BAL with the non-ILD BAL Treg Treg (Τ(Τreg:Teff = 1:2, n=5)reg:Teff = 1:2, n=5)

0%

25%

50%

75%

100%

non-ILDs CVD-IP IPF 0-1

%of

pro

lifer

atio

n

Conclusions Conclusions

In both IPF and CVD-IP Tregs exert impaired In both IPF and CVD-IP Tregs exert impaired suppression in the suppression in the peripheral blood blood, allowing , allowing the onset of autoimmunitythe onset of autoimmunity

In contrast, TregsTregs are expanded and also functionally normal in the lung of the above diseases, potentially resulting in aberrant oversuppression of the local immune response

Our results indicate systemic and local immune indicate systemic and local immune system involvement in the pathophysiology of ILDssystem involvement in the pathophysiology of ILDs