HIV infection of Human Treg cells downregulates Foxp3 expression and produces a loss of the...

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 HIV 0.1

CD4 NI0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

Dnmt1


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)



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


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


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

*: [email protected]

HIV infection of Human Treg cells downregulates Foxp3 expression and produces a loss of the...

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