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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
