rafael correa rocha day 3...3rd international conference and exhibition on...

3rd International Conference and Exhibition on

ClinicalClinical & & CellularCellular

ImmunologyImmunologySeptember 29 - October 01, 2014

Baltimore, USA

HIV infection of Human Treg cells downregulates

Foxp3 expression and produces a loss of the

suppressive capacity of these cells

Rafael Correa RochaRafael Correa Rocha

Laboratory of Immune-regulationInstitute of Health Research “Gregorio Marañón” (IISGM). Madrid (Spain)

RegulatoryRegulatory TT cellscells (Treg(Treg)) �������� SubpopulationSubpopulation ofof CDCD44++ TT

cellscells withwith aa suppressivesuppressive activityactivity

�� TregTreg areare identifiedidentified asas CDCD44+CD+CD2525+Foxp+Foxp33++ cellscells

�� TregTreg areare consideredconsidered aa crucialcrucial componentcomponent ofof immuneimmune systemsystem forfor

preservingpreserving peripheralperipheral tolerancetolerance andand thethe correctcorrect immuneimmune homeostasishomeostasis

�� TheThe potentpotent suppressorsuppressor functionfunction ofof TregsTregs mightmight presentpresent aa seriousserious obstacleobstacle

toto establishingestablishing robustrobust protectiveprotective immunityimmunity towardtoward pathogenspathogens..

�� TregsTregs playplay anan essentialessential rolerole inin controllingcontrolling immuneimmune responseresponse--mediatedmediated

inflammationinflammation..

�� StudiesStudies suggestsuggest thatthat byby limitinglimiting latelate immuneimmune responsesresponses toto anan infectiousinfectious

agent,agent, TregsTregs minimizeminimize associatedassociated tissuetissue damagedamage butbut alsoalso diminishingdiminishing

pathogenpathogen clearanceclearance..

Thus, with several scenarios proposed, the role for Thus, with several scenarios proposed, the role for

TregsTregs during HIV infection remains unclear.during HIV infection remains unclear.

HIVHIV

HIVHIV

HIVHIV

HIVHIV

HIVHIV

ObjectivesObjectives

�� ToTo investigateinvestigate whetherwhether TregTreg cellscells fromfrom healthyhealthy donordonor areare infectedinfected inin vitrovitro

PrecedentsPrecedents

�� TregTreg areare recruitedrecruited andand expandedexpanded inin infectionsinfections toto controlcontrol thethe immuneimmune

hyperactivationhyperactivation.. DoesDoes notnot workwork inin HIVHIV--infectedinfected patientspatients

�� TregTreg cellscells expressexpress CDCD44,, CCRCCR55 andand CXCRCXCR44 (viral(viral entry)entry) andand couldcould bebe susceptiblesusceptible ofof

beingbeing infectedinfected byby HIVHIV

�� TheThe effecteffect ofof HIVHIV infectioninfection inin thethe phenotypephenotype andand functionfunction ofof TregTreg waswas unknownunknown..

�� ToTo investigateinvestigate whetherwhether TregTreg cellscells fromfrom healthyhealthy donordonor areare infectedinfected inin vitrovitro

byby HIVHIV..

�� InIn thatthat case,case, toto studystudy thethe effectseffects ofof HIVHIV infectioninfection onon thethe phenotypephenotype andand

functionfunction ofof TregTreg

�� ToTo investigateinvestigate thethe rolerole ofof TregTreg cellscells inin HIVHIV--infectedinfected patientspatients

PBMC

sorter

Treg

HIV

infection

Phenotype

Cytometry: Foxp3; CD4

Treg suppressive function

Gene Expression

Q-PCR: Foxp3; DNMTs

Blood from 15 healthy volunteers Blood from 15 healthy volunteers

Age: 25Age: 25--40 years40 years

Purity of isolated Treg > 95 %Purity of isolated Treg > 95 %

Sorter

Non-Infected HIV 0.1

76.73% 58.57%

HIV infection2 hours

Treg culture3 � 7 days

Wash virus

Day 3

0

20

40

60

80

100

120

140

160

180

p24

gag

(n

g/m

L)

p24 (HIV)76.73% 58.57%

Foxp3

CD

25

HIV infects and replicate in Treg cellsHIV infects and replicate in Treg cells

HIVHIV--infection decrease Foxp3 expressioninfection decrease Foxp3 expression

0,2

0,4

0,6

0,8

1

1,2

**

**

**

*

N

.

D

No

rmal

ise

d F

ox

p3

ex

pre

ssio

n

0

0.2

0.4

0.6

0.8

1

1.2

NI HIV R5 HIV X4 HIV X4+AZT

HIV X4+T20

Fo

xp

3 E

xp

res

sio

n

**

0

0.2

0.4

0.6

0.8

1

1.2

NI HIV R5 HIV X4 HIV X4+AZT

HIV X4+T20

Fo

xp

3 E

xp

res

sio

n

**

HIV effect on Foxp3 expression is HIV effect on Foxp3 expression is

dosedose--dependent …. dependent ….

but is not observed with R5but is not observed with R5--tropic tropic

virusesviruses

0

0,2

Dia 3 Dia 5 Dia 7

NI VIH 0,01 VIH 0,1

D

Day 3 Day 5 Day 7

HIV 0.01 HIV 0.1

No

rmal

ise

d F

ox

p3

ex

pre

ssio

n

HIV infection of Treg cells HIV infection of Treg cells

modifies the methylation modifies the methylation

pattern of Foxp3 gene ?pattern of Foxp3 gene ?

1.00 1.00 1.001.11

3.79

4.43

1

2

3

4

5

6

7

8

Methylation increase

NI

HIV

Increased DNMT3b expression and Increased DNMT3b expression and

subsequent methylation would be subsequent methylation would be

responsible of HIVresponsible of HIV--mediated decrease mediated decrease

in Foxp3in Foxp3

1.00 1.00 1.001.11

0

Enhancer 5' flanking STAT 5

0

2

4

6

8

10

12

14

Treg NI Treg HIV

0.01

Treg HIV

0.1

CD4 NI

Dnmt3b

*

*

0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

1.8

Treg NI Treg HIV 0.01

Treg HIV 0.1

CD4 NI

Dnmt1

0

0.5

1

1.5

2

Treg NI Treg HIV 0.01

Treg HIV 0.1

CD4 NI

Dnmt3a

� “de novo” methylation� binding site in Foxp3 gene

10

20

30

40

50

60

70

% o

f T

cell

pro

life

rati

on

Treg NI

mean NI

Treg HIV 0.1

mean HIV 0.1

mean Teff-NT

mean Teff-act

10

20

30

40

50

60

70

% o

f T

cell

pro

life

rati

on

Treg NI

mean NI

Treg HIV 0.1

mean HIV 0.1

mean Teff-NT

mean Teff-act

20

25

30

35

40

Pe

rcen

t su

pp

res

sio

n o

f a

cti

va

tio

n m

ark

ers

in

PB

MC

s

PBMC +Treg NI

PBMC +Treg HIV

PBMC +Treg HIV +AZT

20

25

30

35

40

Pe

rcen

t su

pp

res

sio

n o

f a

cti

va

tio

n m

ark

ers

in

PB

MC

s

PBMC +Treg NI

PBMC +Treg HIV

PBMC +Treg HIV +AZT

* *

0

10

0

10

Ratio Ratio Treg:TeffTreg:Teff

1:11:10.2:10.2:10.1:10.1:1

aCD3 APC

0

5

10

15

CD69 CD154

Pe

rcen

t su

pp

res

sio

n o

f a

cti

va

tio

n m

ark

ers

in

PB

MC

s

0

5

10

15

CD69 CD154

Pe

rcen

t su

pp

res

sio

n o

f a

cti

va

tio

n m

ark

ers

in

PB

MC

s

* *

aCD3

�� HIVHIV--infectedinfected TregTreg lossloss thethe FoxpFoxp33 expressionexpression andand decreasedecrease itsits suppresivesuppresive capacitycapacity

�� TheThe impairmentimpairment inin TregTreg populationpopulation andand thethe lossloss ofof itsits suppresivesuppresive functionfunction couldcould bebe

relatedrelated withwith thethe presencepresence ofof thethe immuneimmune hyperactivationhyperactivation inin HIVHIV--infectedinfected

patients,patients, whichwhich hashas beenbeen correlatedcorrelated withwith thethe progressionprogression ofof thethe diseasedisease

Pion et al. AIDS. (2013). 27:2019-29

�� 14 non14 non--infected Controlsinfected Controls

�� 20 HIV20 HIV--infected patients with infected patients with

undetectable VLundetectable VL

�� 15 HIV15 HIV--infected patients with infected patients with

VL >5,000 copiesVL >5,000 copies20,0

30,0

40,0

50,0

60,0

70,0

80,0

d F

oxp

3+C

D25+

(cé

l/µ

L)

***

***

abso

lute

cou

nts

(ce

lls/u

L)

� HIV-infected patients has decreased number of Treg cells

� Which is the effect of VL in Treg counts ?

� Treg decrease is related with immune hyperactivation ?

(Data not published)

VL >5,000 copiesVL >5,000 copies

0,0

10,0

20,0

Nº

Ab

sd

Fo

xp

3+C

D25+

(

Non-HIVControls

HIV u.d. VL

HIV high VL

Tre

g a

bso

lute

Carg

a v

iral

(cop

ias/

mL)

Individuos VIH+

r= —0,610

HIV-infected Patients

Vir

al L

oad

(cp

/mL) p=0.004

(Data not published)

CVi CV

r= 0,745r= —0,429

Nº Abs de Treg Foxp3+ (cél/µL)

u.d. VL high VL

Treg counts (cel/uL)

Act

ivat

ed

CD

4+

T-c

ells

(ce

l/uL)

p=0.013p=0.289

Nº Abs de Treg Foxp3+ (cél/µL)Treg counts (cel/uL)

Control

pro

du

cin

g T

ce

lls

High VL ud VLA)

2

3

2

3

4

15

20

25

30

TheThe balancebalance betweenbetween TregTreg andand

effectoreffector TT--cellscells isis brokenbroken inin HIVHIV--

infectedinfected patientspatients

% I

L2

-pro

du

cin

g T

% Treg cells

R² = 0.99910

1

0 0.5 1 1.5

R² = 0.82470

1

2

0 1 2 3 4

R² = 0.03270

5

10

15

0 0.5 1 1.5

Foxp3

CD

25

ud VL High VL

C)ud VL High VL

Foxp3

CD

25

ud VL High VL

C)ud VL High VL ud V

L

high V

L

0

1

2

3

4

CD

25

MF

I

ud VL

high V

L

0

1

2

3

4

CD

25

MF

I

p=0.031

CD25CD25

CD25

A)

NI T

reg

HIV

Tre

g

0

50

100

150

CD

25

MF

I

B)p=0.028

CD25

A)

NI T

reg

HIV

Tre

g

0

50

100

150

CD

25

MF

I

B)p=0.028

� Treg from HIV-infected patients show a deficient expression of IL2-Rc (CD25)

� In vitro experiments confirms that CD25 downregulation is due to the direct HV

infection

0

10

20

30

40

50

60

70

80

90

% p

STA

T5

p=0.016

0

10

20

30

40

50

60

70

80

90

% p

STA

T5

p=0.016

0NI Treg HIV Treg

NI T

reg

HIV

Tre

g

0

2

4

6

8

pS

TA

T5

MF

I

p=0.008

0NI Treg HIV Treg

NI T

reg

HIV

Tre

g

0

2

4

6

8

pS

TA

T5

MF

I

p=0.008

Méndez-Lagares et al. J Acquir Immune Defic Syndr (2014). 65:278–282

HIVHIV infectioninfection decreasesdecreases ILIL22--RcRc expressionexpression inin

Treg,Treg, diminishingdiminishing thethe ILIL--22 signalsignal thatthat maintainmaintain thethe

balancebalance betweenbetween effectoreffector andand TregTreg cellscells

50

60

PBMC alone

+ Treg non-HIV control

mar

ker

0

10

20

30

40

CD69 CD154

% d

e e

xpre

sión + Treg HIV

% o

f ac

tiva

tio

nm

arke

r

(Data not published)

TheThe suppressivesuppressive capacitycapacity ofof

TregTreg cellscells isis impairedimpaired inin HIVHIV--

infectedinfected patientspatients

�� TheThe impairmentimpairment ofof TregTreg suppressivesuppressive functionfunction couldcould bebe responsibleresponsible ofof immuneimmune hyperactivationhyperactivation

inin HIVHIV--infectedinfected patients,patients, whichwhich isis relatedrelated withwith thethe progressionprogression ofof thethe diseasedisease..

�� PreservingPreserving oror boostingboosting TregTreg populationpopulation couldcould avoidavoid thethe deteriorationdeterioration ofof immuneimmune systemsystem andand

toto improveimprove thethe immuneimmune homeostasishomeostasis inin thesethese patientspatients..

Laboratory of Immune-regulation. IiSGM, Madrid (SPAIN)

Didiana JaramilloJacobo López-AbenteRafael Correa-Rocha*

Laboratory of Molecular Immunobiology.

MarjorieMarjorie PionPionMarta Martínez-BonetAlberto Martínez

Hospital Virgen del Rocío. IBIS, Sevilla (Spain)

Gema Méndez-LagaresManuel LealYolanda Pacheco

Alberto MartínezMª Angeles Muñoz-Fernández

*: rafael.correa@iisgm.com