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"Towards a Cure”:HIV Reservoirs and Strategies to Control ThemIAS WORKSHOP, 16 & 17 JULY 2010
Purging the HIV-1 Reservoir Through the Disruption of the PD-1 Pathway
Sandrina DA FONSECA
Vaccine and Gene Therapy Institute – FloridaLaboratoire d’Immunologie - INSERM U743 - Université de Montréal
IntroductionIA
S H
IV R
ES
ER
VO
IRS
WO
RK
SH
OP,
16
& 1
7 J
ULY
20
10
, V
IEN
NA
- In viremic donors, HIV infected cells do generally survive for long enough to revert back to a resting state. However, a small fraction of these cells enter into latency and constitute a stable latent reservoir for the virus.
- Small pool of TCM (central memory) and TTM (transitional memory) latently infected CD4+T cells that persist in patients receiving HAART: main obstacle to HIV-1 eradication (N. Chomont et al, Nat. Med, 2009).
- Development of targeted strategies aimed at purging these reservoir cells needed.
- Mechanisms implicated in the establishment and persistence of the HIV reservoir: not fully understood.
- Factors that interfere with CD4+T cells activation and proliferation: major impact on the establishment and/or the maintenance of the HIV-1 reservoir ?
- HIV specific CD4+T cells are preferentially infected (DC. Douek et al, Nature, 2002) and express high levels of PD-1(CL. Day et al, Nature, 2006, L. Trautmann et al, Nat. Med, 2006). Can PD-1 play a role in the establishment of latency by repressing HIV transcription, leading to a small pool of quiescent, latently infected cells ?
Hypothesis:
Role for PD-1 in the establishment and/or maintenance of a cellular reservoir for HIV?
Part 1
A role for PD-1 in the ESTABLISHMENT of a reservoir
The establishment of a stable reservoir for HIV necessitates the establishment of viral latency = inhibition of viral production.
Does PD-1 triggering inhibit viral production in HIV infected primary CD4+T cells?
Triggering of the PD-1 pathway inhibits 95% of HIV-1 production in primary CD4+T cells
at day 3
IAS
HIV
RE
SE
RV
OIR
S W
OR
KS
HO
P, 1
6 &
17
JU
LY 2
01
0,
VIE
NN
A
CD3/CD28 + IgG2CD3/CD28 + PD-L1
NS
The negative signal conferred by the PD-1/PD-L1 interaction inhibits viral production in primary CD4+T cells.
- CD4+T cells from HIV infected viremic subjects purification- Stimulated with
CD3/CD28 in the presence or absence of PD-L1.
- Viral production measured by p24 ELISA.
p24
(pg/
ml)
Time (d) Time (d)
p24
(pg/
ml)
0 3 6 91
10
100
1000
10000
100000
1000000 Donor BDonor A
0 3 6 91
10
100
1000
10000
0 3 6 90.1
1
10
100
1000
0 3 6 91
10
100
1000
10000
100000
1000000
Donor C Donor D
Ultrasensitive assay to measure viral productionIA
S H
IV R
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VO
IRS
WO
RK
SH
OP,
16
& 1
7 J
ULY
20
10
, V
IEN
NA
1-5x106 CD4+T cells per well
PBMCs
HIV infected donor
Supernatant
Viral pellet
Viral RNA
Ultrasensitive RT-PCR
Centrifugation 17000 rpm, 30’ at 4°C
RNA extraction
DNase treatment
Blood : leukapheresis
Ficoll gradient
Stimulation CD3/CD28 +/- PD-L124 hours incubation at 37°C
Assay sensitivity : 1 RNA copy/mL
Triggering of the PD-1 pathway inhibits 60% of HIV-1 production in primary CD4+T cells
after 24h of stimulation
IAS
HIV
RE
SE
RV
OIR
S W
OR
KS
HO
P, 1
6 &
17
JU
LY 2
01
0,
VIE
NN
A
Very short term effect of PD-1 triggering on HIV viral production. PD-1 triggering may act directly on the LTR.
Non stimu-lated
CD3/CD28 + IgG2
CD3/CD28 + PD-L1
0
20000
40000
60000
80000
100000
120000
Non stimu-lated
CD3/CD28 + IgG2 beads
CD3/CD28 + PD-L1 beads
0
500
1000
1500
2000
2500
3000
3500
4000
0
2
4
6
8
10
12
Non s
timula
ted
CD3/CD28
+ Ig
G2
CD3/CD28
+ P
D-L1
0
2
4
6
8
10
12
Non s
timula
ted
CD3/CD28
+ Ig
G2
CD3/CD28
+ P
D-L1
0
2
4
6
8
10
12
Non s
timula
ted
CD3/CD28
+ Ig
G2
CD3/CD28
+ P
D-L1
0
2
4
6
8
10
12
Donor A Donor B Donor C
Donor D Donor E Donor F
HIV
RN
A c
opie
sH
IV R
NA
cop
ies
- HAART naïve chronically HIV-infected subjects → Total CD4+T cells negative selection- TCR triggering (CD3/CD28) with or without co-triggering of the PD-1 pathway with an Ig-PD-L1 chimera. - Viruses pellet + Viral RNA extraction and quantification by QRT-PCR
Triggering of the PD-1 pathway inhibits 45% of HIV-1 production in primary CD4+T cells
at day 3 in the presence of ARV
IAS
HIV
RE
SE
RV
OIR
S W
OR
KS
HO
P, 1
6 &
17
JU
LY 2
01
0,
VIE
NN
A
- The inhibition mediated by PD-L1 is still observed in the presence of ARV
- PD-1 directly impacts on viral production
- CD4+T cells from HIV infected viremic subjects purification- Stimulated with CD3/CD28 in the presence or
absence of PD-L1, and ARV- Viral production measured by p24 ELISA.
CD3/CD28 + IgG2CD3/CD28 + PD-L1
NS
Donor A
Donor B0 3 6 9
0
20
40
60
80
100
120
140
160
180
200
0 3 6 90
20
40
60
80
100
120
140
160
180
p24
(pg/
ml)
p24
(pg/
ml)
Time (d)
Inhibition restricted to PD-1high cellsIA
S H
IV R
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VO
IRS
WO
RK
SH
OP,
16
& 1
7 J
ULY
20
10
, V
IEN
NA
Inhibition of viral production was exclusively observed in PD-1 high cells indicating the specificity of this pathway
p24
(pg/
mL)
p24
(pg/
mL)
0,0
100,0
200,0
300,0
400,0
0,0
200,0
400,0
600,0
800,0
1000,0
1200,0
PD-1 High CD4+T cells PD-1 Low CD4+T cells
CD3/CD28+PD-L1
CD3/CD28+IgG2
NS CD3/CD28+PD-L1
CD3/CD28+IgG2
NS
- Total CD4+T cells form viremic HIV infected patients isolation- PD-1high and PD-1low subsets highly purified by cell sorting- Stimulation with different combination of triggering antibodies (CD3/CD28 with PD-L1 or the
corresponding IgG isotype).
Part 2
A role for PD-1 in the MAINTENANCE of a reservoir
Sorted PD-1high cells preferentially harbor HIV-1 integrated DNA when compared to their
PD-1low counterparts
IAS
HIV
RE
SE
RV
OIR
S W
OR
KS
HO
P, 1
6 &
17
JU
LY 2
01
0,
VIE
NN
A
PD-1 expression correlates with the reservoir size. Impact of PD-1 signaling on the HIV reservoir?
Inte
gra
ted
HIV
DN
A c
op
ies
pe
r 1
06
CD
4 T
ce
lls
% PD-1+ CD4 T cells 0 10 20 30
1
10
100
1000
10000
= 0.45p = 0.01
0
100
200
300
400
500
Cm Tm Em
PD-1Hi CD4 T cells
PD-1Lo CD4 T cells
HIV
DN
A c
opie
s pe
r 10
6 c
ells
Sorted PD-1 high cells are enriched in total and integrated HIV DNA compared to sorted PD-1 low cells : PD-1 high cells constitute a preferential reservoir for the virus.
The disruption of the PD-1/PD-L1 interaction enhances the spontaneous release of HIV-1
virions by CD4+T cells
IAS
HIV
RE
SE
RV
OIR
S W
OR
KS
HO
P, 1
6 &
17
JU
LY 2
01
0,
VIE
NN
A
HIV production is induced after blocking the PD-1 pathway suggesting a role for PD-1 in the maintenance of HIV latency.
Donor A Donor B Donor C
Mock
a-PD-1 blocking Ab
Isotype control200
400
600
0
p24
(pg/
mL)
If PD-1 triggering inhibits viral production, blocking the PD-1/PD-L1 interaction should increase viral production.
- CD4+T cells isolated from HIV infected subjects - CD4+T cells incubated with an anti-PD-1 antibody that prevents the interaction between PD-1
and its ligands
- PD-1+ Central and Transitional memory CD4+T cells are enriched for HIV integrated DNA
- PD-1 receptor may be used as a specific marker to target HIV-1 reservoir cells
- PD-1 receptor triggering inhibits viral production. Role in the establishment of a reservoir?
- Blocking PD-1/PD-L1 interaction induces viral production. Role in the maintenance of a reservoir?
Can we purge the HIV reservoir by disrupting the PD-1 negative pathway in HAART individuals?
ConclusionIA
S H
IV R
ES
ER
VO
IRS
WO
RK
SH
OP,
16
& 1
7 J
ULY
20
10
, V
IEN
NA
"Towards a Cure”:HIV Reservoirs and Strategies to Control ThemIAS WORKSHOP, 16 & 17 JULY 2010
Acknowledgments
Vaccine and Gene Therapy Institute, FloridaUniversité de Montréal, CHUM, INSERM U743Nicolas ChomontRafick-Pierre Sékaly
Université de Montréal, CHUM, INSERM U743Mohamed El Far
Royal Victoria Hospital, Mc Gill UniversityMohamed-Rachid BoulasselJean-Pierre Routy