BIOMARKERS OF T CELL RESPONSE IN CHRONIC INFECTIONS

Prof. Maria Nikolova, MD, DScNational Center of Infectious and Parasitic

Diseases, Bulgaria

SEEIS2013, 20 -23 September, Sofia, Bulgaria

Extracellular pathogens

В Ly

Infected cellphagocyte

Humoral immune response

MECHANISMS OF INFECTIOUS IMMUNITY

Intracellular bacteria

viruses

Th CD4+

аntibodies

phagocytosis

Specific cytotoxicityPer,GzB, IFNg

cytokines

Cellular immune response

Th CD4+

Tc CD8+

Mph

THE CENTRAL ROLE OF T CELL IMMUNE RESPONSE

Ab-mediated

Инфектирана клетка

cellular

В В LyLy

(cytolysis)IFNg, Per, GrB

CDCD88++ ТТ

CD4+CD4+ ТТ

inductorseffectors

regulators

A SIMPLIFED SCHEME OF T- CELL RESPONSE

naivenaiveT T LyLy

THYMUS

BLOOD SECONDARYLYMPHOID ORGANS

+ Аg

activated Tactivated T Ly Ly

+ АmemorymemoryTT Ly Ly

effectoreffectorT LyT Ly

activated Tactivated T Ly Ly

apoptosis

naive

MARKERS OF Т-CELL DIFFERENTIATION

Differentiation

memory effector

Function

Proliferative activityIFNγγγγ Gz Per

CD45RA+

CENTRAL MEMORY

EFFECTOR MEMORY

1

EFFECTOR TERMINAL EFFECTORS

EFFECTOR MEMORY

2

CD45RA-/CD45RO+CCR7+

CD28+

CD27+

CD45RA+

CD57+

spleen

LYMPHOCYTE DISTRIBUTION

Central lymphoid organs

bloodblood10 10 х101099

2%2%

70 х109

l.nodes190 х109

liver40х109

intestines50 х109

lungs30 х109

Secondary lymphoid organs

Parenchymal organs, tissues

BLOOD

Naïve poolhomeostasis

THYMUS

TN

SEC.LYMPHOID ORGANS

BLOOD IS A WINDOW TO IMMUNE SYSTEM

Proliferation

Differentiation

TNactivaion

TN

TE

TEM

TCMMemory pool homeostasis

TTE

Differentiation

Te

ApoptosisTe

TISSUES

TCM TEM

Proliferation

Th1 Th2

PROTECTIVE IMMUNE RESPONSE MEANS BALANCE

CD4 CD8

M E

CellularImmune response

HumoralImmune response

ACUTE INFECTION SCENARIOSA

NT

IGE

N L

OA

D

Complete elimination of pathogen: Influenza, RSV

1.Recovery = efficient immune response

AN

TIG

EN

LO

AD

2.Persistence of pathogen…..

CHRONIC INFECTION SCENARIOS

HIV+ART-,HBV,HCV

• Asymptomatic lifelong (CMV, EBV, МТВ)

• Asympomatic followed by clinical progression(HIV, Morbilli. Rubella: SSPE, JCV:PML )

• Acute reactivation(HSV, HZV)

AN

TIG

EN

LO

AD

CMV, EBV, HSV, VZV,HIV + АRТ-

(HSV, HZV)• Chronic course with relapses

and exacerbations (HBV, HCV).

• Malignant proliferation (EBV, HTLV-1 HPV, HBV, HCV, HHV-8)

AN

TIG

EN

LO

AD

NN

АА

+antigen

PATHOLOGY OF T-CELL RESPONSE IN CHRONIC INFECTION

Imbalanced immune response characterized by inefficient functional differentiation of effectors or continuous differentiation of effectors at the expense of memory, leading to clonal exhaustion;

EECMCM

CHRONIC INFECTIONS: HIV, HBV, HCV, MTB, CFS

Treg

clonal exhaustion; accompanied by a persistently high level of Treg with inhibitory function

CHRONIC INFECTION

IMMUNE DEFICIENCY

PATHOGENESIS OF CHRONIC INFECTIONS

INDIVIDUAL FACTORS: genetic, immune history:

cytokine background

ANTIGEN LOAD

IMMUNE ACTIVATION

CHRONIC IMMUNE ACTIVATION

DYSBALANCEat the subset level

DYSBALANCEDExpression of co-

regulatory molecules

IMMUNE EXHAUSTION

THE BIOMARKERS CONCEPT

Phenotypic, functional and kinetic characteristics defining the effectiveimmune response = CORRELATES OF PROTECTIVE IMMUNITY

WHAT DOES IT MEAN?WHAT DOES IT MEAN?

WHY DO WE NEED THEM?WHY DO WE NEED THEM?

Diagnostic evaluation Prognosis & monitoringPrognosis & monitoringTreatment strategies

HOW ARE THEY DETERMINED?HOW ARE THEY DETERMINED?

FLOW CYTOMETRY IN CHRONIC INFECTIONS:evolution of technology&methods

Increasing number of Fl channels: 3 - 8

Single-platformenumeration

Quantitative analysis of antigen

expressionIntracellular

cytokine expression

МНС-tetrameresProliferation index by vital stains

(CFSE)

MULTIPARAMETER ANALYSIS:Antigen-specific response at the subset level (balance, functions)

cytokine expression

BIOMARKERS OF T CELL RESPONSE: THE EVOLUTIONBIOMARKERS OF T CELL RESPONSE: THE EVOLUTIONOF CONCEPTOF CONCEPT

1. Memory is a “must” (IL-7R, IL-2, CD27, prolifera tion) Wherry EJ, et al. Nat Immunol. 2003;4:225-234.

2. Different phenotypes of antigen-specific cells corr espond to different antigen burdens (Tussey LG et al. Antigen J Infect Dis. 2003;187:364-374.)

3. The functional signature of protective T -cell immunity: poly -3. The functional signature of protective T -cell immunity: poly -and multifunctional lymphocytes (Harari A et al. Immunol Rev. 2006 Jun;211:236-54.)

4. Co-stimulatory & co-inhibitory receptors ( CD28/ C TLA-4,CD160/2B4; PD1/PD-L1, LAG3/Tim) Rey J, et al. Eur J Immunol. 2006;.Petrovas, JEM, 2006; Elahi, Nat Med, 2011; Nikolova et al., 2010)

5. The regulatory T cells: a friend or foe (Weiss L. et al, Blood 2004; Kinter A. et al, J. Exp. Med 2004 , Nikolova М. et al., Blood, 2009)

EE

+RSV

PATHOLGY OF T-CELL RESPONSE

+CMV

NN

АА

+HIVNN

АА

NN

АА

ART

EECMCM

TETE

Phenotype of antigen-specific cells depends on anti gen burden

(after Tussey LG et al. Antigen J Infect Dis. 2003;187:364-374.)

EE CMCM

BIOMARKERS OF T CELL RESPONSE: THE EVOLUTIONOF CONCEPT

1. Memory is a “must” (IL-7R, IL-2, CD27, prolifera tion) Wherry EJ, et al. Nat Immunol. 2003;4:225-234.

2. Different phenotypes of antigen-specific cells corr espond to different antigen burdens (Tussey LG et al. Antigen J Infect Dis. 2003;187:364-374.)

3. The functional signature of protective T -cell immunity: poly -3. The functional signature of protective T -cell immunity: poly -and multifunctional lymphocytes (Harari A et al. Immunol Rev. 2006 Jun;211:236-54.)

4. Co-stimulatory & co-inhibitory receptors ( CD28/ C TLA-4,CD160/2B4; PD1/PD-L1, LAG3/Tim) Rey J, et al. Eur J Immunol. 2006;.Petrovas, JEM, 2006; Elahi, Nat Med, 2011; Nikolova et al., 2010)

5. The regulatory T cells: a friend or foe (Weiss L. et al, Blood 2004; Kinter A. et al, J. Exp. Med 2004 , Nikolova М. et al., Blood, 2009)

BIOMARKERS OF T CELL RESPONSE: OUR BIOMARKERS OF T CELL RESPONSE: OUR EXPERIENCEEXPERIENCE

Can we do it at the level of the global T cell population?

Detecting and measuring immune activation

T-subset differentiation and balance associated with immune activation(antigen load )

Phenotypic & functional parameters of protective response

Levels of regulation and possibilities for restoration of Т-cellimmunity

“Models” of infection with different characteristics of antigen load : HIV+ART-, of HIV+ART+, EBV-H, HBV-H, CMV, FOI, A-M TB, L-MTB,

QUANTITATIVE EXPRESSION OF CD38: MARKER OF IMMUNE ACTIVATION

eNADеАТF

cADFRadenosine

ADFCD39

CD73 CD38

CD38 ABC on CD8 T

� correlates with viral replication� prognostic significance in НІV

CD38 ABC on CD4 T ? CD38 in other infections?

ADFR

cADFRadenosine

**

***

*

*

a

QUANTITATIVE EXPRESSION OF CD38

FOI

CD38 ABC on CD8 T cells differentiate

between EBV-hepatitis and NAFLD

ns

***

*

CD

38 A

BC

CD8 T

1.M. Nikolova, et al. Scand. J, Gastroeneterol, 2007

FOI = focal odontogenic infection

FOI

QUANTITATIVE EXPRESSION OF CD38

***

* **

б

***

**

* ****

а

CD38 ABC on CD4 T is an early indicator of FOI

б

FOI FOI-T

FOI

FOI -T= focal odontogenic infection treated

PROPORTIONS OF PERIPHERAL BLOOD Т-SUBSETS

TE

CMEM

CD

45R

A

N

“Models” CD8

N М ТЕ

IM ns ** ns

LTB ns ns ns

TB ns ns ns

CMV *** ** ns

HIV-S *** *** ns

HIV-ART *** *** ns

“Models” CD8

CM EM

IM * ns

CMV ns **HIV-S *** ***

CCR7HIV-L *** *** ns

EBV *** *** ns

HBV *** *** ns

HIV-S *** ***HIV-ART *** ***

HIV-L *** ***EBV ns ***HBV ns ***

CD27

CD

45R

A

THE MEMORY/ EFFECTOR Т CELL RATIO...

CCR7

CD

45R

A

ТЕ N

EM CM

ТЕ Н

E М

CD45RA/CD27CD27

Р<0.01

CD45RA/CD27

Р>0.05

CD45RA/CCR7

**

***

***

CD45RA/CD27

CD8 T subset proportions in active and latent MTB infection

A higher memory/ effector Т cell ratio...

….is associated with protective immune response in chronic infection

*** MTB infection

RHC L-MTB A-MTB MTB (-)

M. Nikolova, et al.Diagn Microbiol Infect Dis, 2012

FUNCTIONAL SIGNATURES OF T CELLS IN CHRONIC INFECTION

**

МТВ-specific CD8

H А-MTB L-MTB RHC H A-MTB L-MTB RHC

МТВ-specific CD4

МТВ: IFNγγγγ+CD8 correlatewith protection inrecent infection

Extreme immune activation results in reduced funct ion of CD8 T cells

0.0

0.2

0.4

0.6

0.8

1.0 **

HIV+

ART-

HIV+

ART+

EBV-HK

****

Gz+Per+/Gz+Per-(mature/immature effectors)

FUNCTIONAL SIGNATURES OF T CELLS IN CHRONIC INFECTION

120

140

160

180

25

30

35ns

**ns

1,2

1,4

1,6

1,8

CD

4

RRP (HPV) is characterized with decreased FN γ / IL-4 and IFNγ/IL-10 ratios and increased share of Th17+CD4 T, restored after 20 mo of immune modulation

**

0

20

40

60

80

100

IFNγ/IL-40

5

10

15

20

IFNγ/IL-10

0,0

0,2

0,4

0,6

0,8

1,0

CD4+CD69+ IL17+

% o

fC

D4

E. Vetskova, et al.. Folia Medica , 2013

0 9 20 K 0 9 20 K

� Expression of PD-1 ( Programmed D eath Rreceptor -1) and its ligand PD-L1 is induced on activated T cells

� Increased PD-1 expression is detected on virus-spec ific CD8 and CD4 T in chronic progressive infections: LCMV, CMV, HCV, VIH

MARKERS OF REPLICATIVE SENESCENCE and EXHAUSTION

PD1PD1

T in chronic progressive infections: LCMV, CMV, HCV, VIH(Day, Nature,2006; Petrovas, J Exp Мed, 2006) that is not reversed after antigen withdrawal (Utzschneider DT et al.,Nat. Medecine, 2013)

� Blocking PD-1 / PD-L1 pathway restores T cell response (Trautmann

L,2006) Palmer BE , J. Immunology, 2013) and decreases immune activation and microbial translocation in SIV (R. Amara et al., J.Clin. Invest. 2012; )

� PD-1 /PD-L1 pathway : a target to restore protecti ve immune response? (Sakthivel P et al, Rev Recent Clin Trials, 2012)

Is characterized by

� decreased proliferation of memory at the expense o f effector cells

� low efficiency of effector T cells

� “exhausted” phenotype of effectors (PD1/PD-L1. LAG-3, GTLA-4)

T CELL RESPONSE IN CHRONIC INFECTIONS

� high level of regulatory FoxP3+CD4+Т(Treg) with inhibitoryfunction

• differentiate in thymus to maintain immune tolerance• are induced in the course of antigen-specific response, and exert non-specific inhibition on CD4 and CD8 Т

• have homeostatic function : promote the formation of memory in case of strong immune activation

% p

rolif

erat

ion

inhi

bitio

nа

M Е

HOMEOSTATIC EFFECTS OF Treg

*

M Е

% A

nnex

in V

+ *

M/Е

аCD3 aCD3+Treg

*

+ Treg

Treg inhibit the proliferation of effectors

Treg inhibit the apoptosis of memory cells

Treg inhibit the differentiation of memory into eff ector cells

Тreg favor the formation of immune memory and prevent clonal exhaustion

(Nikolova М. , Lelievre JD, Bensussan A., Y. Levy , Blood, 2009)

w/o Treg

+ Treg

are associated with PD1/PD-L1 pathway

р<0.01

ns

CD8 Tр<0.01 ns

HOMEOSTATIC EFFECTS OF Treg

PD-1 PD-L1 CD25-CD4 T

PD-L1 PD-1

Treg

Treg express lower levels of PD-L1, and reduce its expression on activated CD8 and CD4 T

� What is the role of Treg in chronic infection?

Treg IN CHRONIC HIV INFECTION…

Inhibit the proliferation …. ….and differentiation of effector T cells,%

CF

SE

low

клетки

*** **

Е ТЕ

0

25

50

75

100

% от C

D8

T

* *

N M Е ТЕЕ ТЕ N M Е ТЕ

As well as IFN γ γ γ γ expression, but …

%in

hibi

tion

HIV+/аCD3 HIV-/aCD3

*** ***

CEF Gag

PD1 / PD-L1 expression on Gag-specific CD8 T is no longer regulated by Treg

4000

5000

6000

3000

4000

5000

6000

***

**ns

ns

MF

I

MF

I

GAG-specific CD8 CEF-specific CD8

Treg IN CHRONIC HIV INFECTION…

In chronic infection Тreg employ different signaling mechanisms

0

1000

2000

3000

0

1000

2000

3000

PD1PD-L1

**

PD1 PD-L1

MF

I

MF

I

CD39: A BIOMARKER OF Тreg IN CHRONIC HIV INFECTION

3500

* *

eNADеАТF

ADFR

cADFRadenosine

ADFCD39

CD73CD38

0

500

1000

1500

2000

2500

3000

CD

39 M

FI /

Tre

g

c-ART+HIV+

HIV-c-ART-HIV+

* *

CD39 expression on Treg increases

T Ly are more sensitive to adenosine (increased A2AR expresion)

Blocking CD39 on Treg restores CD8 Tcell function

Nikolova M, Carriere M, Jenabian MA, et al. PLoS Pathog, 2011

CD39: A BIOMARKER OF Тreg IN CHRONIC HIV INFECTION

c-ART-HIV+ c-ART+HIV+Р<0.01, R=-0.6

Р<0.01, R=-0.8

CD39 /Treg inversely correlate with CD4 T АС

independently of ART

Nikolova M, Carriere M, Jenabian MA, et al. PLoS Pathog, 2011

GRIVcohort

LTNP K

rela

ted

The allel rs11188513-C is associated with low CD39, slower progression and better prognosis

of HIV infection

CD39: A BIOMARKER OF Тreg IN CHRONIC HIV INFECTION

ACScohort

MACS cohort

Pro

port

ion

of A

IDS

-rel

ated

Dea

th fr

ee

Pro

port

ion

of A

IDS

free

Time after seroconversion (yrs)

CCCT

TT

Nikolova M, Carriere M, Jenabian MA, et al. PLoS Pathog, 2011

0

2500

5000

7500

10000M

FI

CD

39***

Treg/CD39+Is a specific characteristic of T cell

response in progressive chronic infections

CD39: A BIOMARKER OF Тreg IN CHRONIC INFECTION

0

в HBV HC

% CD39+Treg

ALA

T (

IU/L

)

MFI CD39/Treg

Log1

0 H

BV

DN

A c

opie

s/m

l

R= 0.66P=0.03

R=0.77P<0.01

IU/L

ASAT ALAT HBV VL

R=0.67P=0.05

M. Nikolova , et al. Probl. Infect. Parasit. Dis. 2011

� PROTECTIVE IMMUNE RESPONSE = BALANCE� CHRONIC IMMUNE ACTIVATION RESULTS IN PHENOTYPIC ANDFUNCTIONAL CHANGES DETECTABLE IN PERIPHERAL BLOOD

� increased number of CD38 molecules on CD8 & CD4 T� skewed proportions of T cell subsets (CD28/CD27/CD57)� decreased M/E T cell ratio� decreased/skewed functionality

BIOMARKERS OF T CELL RESPONSE IN CHRONIC INFECTIONS :

WHAT to RETAIN?WHAT to RETAIN?

� decreased/skewed functionality� increased expression of inhibitory and apoptosis-reducin greceptors

� THESE EFFECTS ARE ASSOCIATED WITH INCREASED LEVEL OFTreg

�Treg homeostatic functions and mechanisms change in chroni cinfection� Differentiated approaches for restoration of T cell respon seshould be employed depending on the characteristics of infe ctionand antigen load

CHU Henri Mondor, APHP, UPEC, Inserm U841, Créteil, France

Dr. Matthieu CarrièreDr. Mohammad-Ali Jenabian

MewishYounasDr. Christine LacabaratzPr. Jean-Daniel Lelièvre

Pr. Yves Lévy

ACKNOWLEDGMENTSACKNOWLEDGMENTS

PHC « RILA »

NRLI, National Center of Infectious Parasitic Diseases, Sofia

M. Muhtarova, PhDD. Stankulova, PhD

A. MihovaPr. H. Taskov, DSc

E. Vetskova

DIAID, Hospital of Infectious Diseases, Sofia

I. Elenkov, MDN. Yancheva, MDM. Yankova, MD

T. Tomov, MDPr. T. Tchervenjakova