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HIV infection of Human Treg cells downregulates Foxp3 expression and produces a loss of the
suppressive capacity of these cells
3rd International Conference and Exhibition on
Clinical & Cellular
ImmunologySeptember 29 - October 01, 2014
Baltimore, USA
Rafael Correa Rocha
Laboratory of Immune-regulationInstitute of Health Research “Gregorio Marañón” (IISGM). Madrid (Spain)
Regulatory T cells (Treg) Subpopulation of CD4+ T cells with a suppressive activity
Treg are identified as CD4+CD25+Foxp3+ cells
Treg are considered a crucial component of immune system for preserving peripheral tolerance and the correct immune homeostasis
Treg cells
The potent suppressor function of Tregs might present a serious obstacle to establishing robust protective immunity toward pathogens.
Tregs play an essential role in controlling immune response-mediated inflammation.
Studies suggest that by limiting late immune responses to an infectious agent, Tregs minimize associated tissue damage but also diminishing pathogen clearance.
Thus, with several scenarios proposed, the role for Tregs during HIV infection remains unclear.
Treg cells in
infections
Treg
B cell
Y
YY
Y
Plasma B cell
T CD4
T CD8
HIV
Y
Y
Y
Y HIV
Linf.T
InflamationActivation
Tissue damag
eCelular death
Inf. HIV
HIV
?Treg-HIV
Objectives
To investigate whether Treg cells from healthy donor are infected in vitro by HIV.
In that case, to study the effects of HIV infection on the phenotype and function of Treg
To investigate the role of Treg cells in HIV-infected patients
Precedents
Treg are recruited and expanded in infections to control the immune hyperactivation. Does not work in HIV-infected patients
Treg cells express CD4, CCR5 and CXCR4 (viral entry) and could be susceptible of being infected by HIV
The effect of HIV infection in the phenotype and function of Treg was unknown.
Treg-HIV
PBMC
sorter
Treg
HIV infection
PhenotypeCytometry: Foxp3; CD4
Treg suppressive function
Gene ExpressionQ-PCR: Foxp3; DNMTs
Blood from 15 healthy volunteers
Age: 25-40 years
Purity of isolated Treg > 95 %
Sorter
Methods
HIV infection of Treg
NI
HIV 0
.01
HIV 0
.05
HIV 0
.1
0
20000
40000
60000
80000
100000
120000
140000
160000
p24 (HIV)
Non-Infected HIV 0.1
76.73% 58.57%
Foxp3
CD
25
HIV infects and replicate in Treg cells
HIV-infection decrease Foxp3 expression
HIV infection2 hours
Treg culture3 7 days
Wash virus
Day 3
0
0.2
0.4
0.6
0.8
1
1.2
NI HIV R5 HIV X4 HIV X4+AZT
HIV X4+T20
Fo
xp3
Exp
ress
ion
**
HIV infection of Treg
HIV effect on Foxp3 expression is dose-dependent ….
but is not observed with R5-tropic viruses
0
0,2
0,4
0,6
0,8
1
1,2
Dia 3 Dia 5 Dia 7
Expr
esió
n de
Fox
p3
NI VIH 0,01 VIH 0,1
**
****
*
% d
e ex
pres
ión
de F
oxp3
N.D
Day 3 Day 5 Day 7
HIV 0.01 HIV 0.1
Norm
alis
ed F
oxp
3 e
xpre
ssio
n
Epigenetic Control
CD4 DNMT
Foxp3 expression
HIV infection of Treg cells modifies the methylation pattern of Foxp3 gene ?
Enhancer 5' flanking STAT 50
0.5
1
1.5
2
2.5
3
3.5
4
4.5
5
1.00 1.00 1.001.11
3.79
4.43
Methylation increase
NI
HIV
Treg NI Treg HIV 0.01
Treg HIV 0.1
CD4 NI0
2
4
6
8
10
Dnmt3b
*
*
Mechanism Foxp3
Treg NI Treg HIV 0.01
Treg HIV 0.1
CD4 NI0
0.2
0.4
0.6
0.8
1
1.2
1.4
1.6
Dnmt1
Treg NI Treg HIV 0.01
Treg HIV 0.1
CD4 NI0
0.2
0.4
0.6
0.8
1
1.2
1.4
1.6
Dnmt3a
“de novo” methylation
binding site in Foxp3 gene
Increased DNMT3b expression and subsequent methylation would be responsible of HIV-mediated decrease in Foxp3
0
10
20
30
40
50
60
70
% o
f T
cel
l pro
life
rati
on
Treg NI
mean NI
Treg HIV 0.1
mean HIV 0.1
mean Teff-NT
mean Teff-act
Ratio Treg:Teff
1:10.2:10.1:1
Teff-
act.
Teff-
NT
TregLinf. TCD4
aCD3 APC
0
5
10
15
20
25
30
35
40
CD69 CD154
Pe
rce
nt
su
pp
res
sio
n o
f a
cti
vati
on
ma
rke
rs in
PB
MC
s
PBMC +Treg NI
PBMC +Treg HIV
PBMC +Treg HIV +AZT
* *
Linf. TCD4
aCD3
Treg suppressive function
HIV-infected Treg loss the Foxp3 expression and decrease its suppresive capacity
The impairment in Treg population and the loss of its suppresive function could be related with the presence of the immune hyperactivation in HIV-infected patients, which has been correlated with the progression of the disease
Pion et al. AIDS. (2013). 27:2019-29
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 ?
HIV-infected patients
(Data not published)
14 non-infected Controls
20 HIV-infected patients with undetectable VL
15 HIV-infected patients with VL >5,000 copies
0,0
1,0
2,0
3,0
4,0
5,0
6,0
7,0
8,0
9,0
10,0
Controles Sin CV CV
% d
e F
oxp
3+CD25
+ (c
él/µ
L)
0,0
10,0
20,0
30,0
40,0
50,0
60,0
70,0
80,0
Controles Sin CV CV
Nº A
bsd
Foxp
3+CD25
+ (c
él/µ
L)
B C**
***
***
CVi CViNon-HIV Controls
HIV u.d. VL
HIV high VL
Treg a
bso
lute
counts
(ce
lls/u
L)
HIV-infected patients
(Data not published)
CVi CV
r= 0,745r= —0,429
Nº Abs de Treg Foxp3+ (cél/ µL)
% de
T C
D4
acti
vada
s (c
él/µ
L)
u.d. VL high VL
Treg counts (cel/uL)Act
ivate
d C
D4+
T-c
ells
(ce
l/uL)
p=0.013p=0.289
Nº Abs de Treg Foxp3+ (cél/ µL)
Carg
a vi
ral (c
opia
s/m
L)
Individuos VIH+r= —0,610
A HIV-infected Patients
Treg counts (cel/uL)
Vir
al Lo
ad (
cp/m
L) p=0.004
The balance between Treg and effector T-cells is broken in HIV-infected patients
Control
% I
L2-
pro
duci
ng T
ce
lls
High VL ud VL
% Treg cells
A)
R² = 0.99910
1
2
3
0 0.5 1 1.5
R² = 0.82470
1
2
3
4
0 1 2 3 4
R² = 0.03270
5
10
15
20
25
30
0 0.5 1 1.5
Mechanism of Treg/ T-effector imbalancein HIV-infected patients
CD25
Foxp3
CD
25
ud VL High VL
B) D)
ud VL
high V
L0
1
2
3
4
CD
25
MF
I
p=0.031
C) ud VL High VL
CD25
A)
NI Tre
g
HIV T
reg
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
CD25
Foxp3
CD
25
ud VL High VL
B) D)
ud VL
high V
L0
1
2
3
4
CD
25
MF
I
p=0.031
C) ud VL High VL
p=0.031
D)
0
10
20
30
40
50
60
70
80
90
NI Treg HIV Treg
% p
STA
T5
p=0.016
NI Tre
g
HIV T
reg
0
2
4
6
8
pS
TA
T5
MF
I
p=0.008E)
C)
pSTAT5
NI Treg HIV Treg
pSTAT5 MFI:NI Treg= 6.14HIV Treg= 1.89
Méndez-Lagares et al. J Acquir Immune Defic Syndr (2014). 65:278–282
HIV infection decreases IL2-Rc expression in Treg, diminishing the IL-2 signal that maintain the balance between effector and Treg cells
HIV-infected patients
Treg Linf. TCD4
0
10
20
30
40
50
60
CD69 CD154
% d
e ex
pres
ión
CMSP Estim
Treg control
Treg CV
PBMC alone
+ Treg non-HIV control
+ Treg HIV
0
10
20
30
40
50
60
CD69 CD154
% de expresión
CMSP Estim
Treg control
Treg CV
% o
f act
ivati
on m
ark
er
(Data not published)
The suppressive capacity of Treg cells is impaired in HIV-infected patients
The impairment of Treg suppressive function could be responsible of immune hyperactivation in HIV-infected patients, which is related with the progression of the disease.
Preserving or boosting Treg population could avoid the deterioration of immune system and to improve the immune homeostasis in these patients.
Treg-HIV
Laboratory of Immune-regulation. IiSGM, Madrid (SPAIN)
Didiana JaramilloJacobo López-AbenteRafael Correa-Rocha*
Hospital Virgen del Rocío. IBIS, Sevilla (Spain)
Laboratory of Molecular Immunobiology. IiSGM
Gema Méndez-LagaresManuel LealYolanda Pacheco
Marjorie PionMarta Martínez-BonetAlberto MartínezMª Angeles Muñoz-Fernández
*: rafael.correa@iisgm.com
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