"Towards a Cure”:
HIV Reservoirs and Strategies to Control Them
IAS 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 – Florida
Laboratoire d’Immunologie - INSERM U743 - Université de Montréal
IAS HIV RESERVOIRS WORKSHOP, 16 & 17 JULY 2010, VIENNA
Introduction
-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?
CD4+T cells at day 3
Donor A
1000000
p24 (pg/ml)
Donor B
1000000
100000
- 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.
100000
10000
10000
1000
1000
100
100
10
10
1
1
0
3
6
0
9
Donor C
10000
p24 (pg/ml)
IAS HIV RESERVOIRS WORKSHOP, 16 & 17 JULY 2010, VIENNA
Triggering of the PD-1 pathway inhibits 95% of HIV-1 production in primary
3
6
9
Donor D
1000
CD3/CD28 + IgG2
100
1000
CD3/CD28 + PD-L1
100
10
10
1
NS
0
1
3
6
9
0.1
0
3
6
Time (d)
9
Time (d)
The negative signal conferred by the PD-1/PD-L1 interaction inhibits viral
production in primary CD4+T cells.
IAS HIV RESERVOIRS WORKSHOP, 16 & 17 JULY 2010, VIENNA
Ultrasensitive assay to measure viral production
HIV infected donor
Blood : leukapheresis
PBMCs
Ficoll gradient
1-5x106 CD4+T cells per well
Stimulation CD3/CD28 +/- PD-L1
24 hours incubation at 37°C
Supernatant
Centrifugation
17000 rpm, 30’ at 4°C
Viral pellet
RNA extraction
Viral RNA
DNase treatment
Ultrasensitive RT-PCR
Assay sensitivity :
1 RNA copy/mL
CD4+T cells after 24h of stimulation
HIV RNA copies
120000
Donor A
Donor C
Donor B
4000
3000
3500
100000
3000
2500
80000
2500
2000
2000
1500
60000
1500
40000
1000
1000
500
500
20000
0
0
Non
stimulated
0
Non
CD3/CD28 + CD3/CD28 +
stimulated
IgG2
PD-L1
45000
HIV RNA copies
IAS HIV RESERVOIRS WORKSHOP, 16 & 17 JULY 2010, VIENNA
Triggering of the PD-1 pathway inhibits 60% of HIV-1 production in primary
Donor D
10000
40000
9000
35000
8000
30000
7000
Donor E
140000
Donor F
120000
100000
6000
25000
Non
CD3/CD28 CD3/CD28
stimulated
+ IgG2
+ PD-L1
CD3/CD28 + CD3/CD28 +
IgG2 beads PD-L1 beads
80000
5000
20000
15000
4000
60000
3000
40000
10000
2000
5000
1000
0
20000
0
Non
CD3/CD28 CD3/CD28
stimulated
+ IgG2
+ PD-L1
0
Non
CD3/CD28 CD3/CD28
stimulated
+ IgG2
+ PD-L1
Non
CD3/CD28 CD3/CD28
stimulated
+ IgG2
+ PD-L1
- 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
Very short term effect of PD-1 triggering on HIV viral production.
PD-1 triggering may act directly on the LTR.
CD4+T cells at day 3 in the presence of ARV
Donor A
200
180
p24 (pg/ml)
160
- 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.
140
120
100
80
60
40
20
0
0
3
6
9
Donor B
180
CD3/CD28 + IgG2
CD3/CD28 + PD-L1
160
NS
140
p24 (pg/ml)
IAS HIV RESERVOIRS WORKSHOP, 16 & 17 JULY 2010, VIENNA
Triggering of the PD-1 pathway inhibits 45% of HIV-1 production in primary
120
100
- The inhibition mediated by PD-L1 is still observed in the
presence of ARV
80
60
40
- PD-1 directly impacts on viral production
20
0
0
3
6
Time (d)
9
PD-1 High CD4+T cells
PD-1 Low CD4+T cells
400,0
1200,0
1000,0
p24 (pg/mL)
300,0
p24 (pg/mL)
IAS HIV RESERVOIRS WORKSHOP, 16 & 17 JULY 2010, VIENNA
Inhibition restricted to PD-1high cells
200,0
100,0
800,0
600,0
400,0
200,0
0,0
0,0
NS
CD3/CD28
+IgG2
CD3/CD28
+PD-L1
NS
CD3/CD28
+IgG2
CD3/CD28
+PD-L1
- 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).
Inhibition of viral production was exclusively observed in PD-1 high cells
indicating the specificity of this pathway
Part 2
A role for PD-1 in the MAINTENANCE of a reservoir
10000
1000
100
 = 0.45
p = 0.01
10
1
0
10
20
30
% PD-1+ CD4 T cells
PD-1 expression correlates with the
reservoir size.
Impact of PD-1 signaling on the HIV
reservoir?
HIV DNA copies per 106 cells
Integrated HIV DNA copies per
106 CD4 T cells
IAS HIV RESERVOIRS WORKSHOP, 16 & 17 JULY 2010, VIENNA
Sorted PD-1high cells preferentially harbor HIV-1 integrated DNA when
compared to their PD-1low counterparts
500
PD-1Hi CD4 T cells
400
PD-1Lo CD4 T cells
300
200
100
0
Cm
Tm
Em
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.
release of HIV-1 virions by CD4+T cells
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 600
p24 (pg/mL)
IAS HIV RESERVOIRS WORKSHOP, 16 & 17 JULY 2010, VIENNA
The disruption of the PD-1/PD-L1 interaction enhances the spontaneous
400
Mock
a-PD-1 blocking Ab
200
Isotype control
0
Donor A
Donor B
Donor C
HIV production is induced after blocking the PD-1 pathway suggesting a role for
PD-1 in the maintenance of HIV latency.
IAS HIV RESERVOIRS WORKSHOP, 16 & 17 JULY 2010, VIENNA
Conclusion
- 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?
"Towards a Cure”:
HIV Reservoirs and Strategies to Control Them
IAS WORKSHOP, 16 & 17 JULY 2010
Acknowledgments
Vaccine and Gene Therapy Institute, Florida
Université de Montréal, CHUM, INSERM U743
Nicolas Chomont
Rafick-Pierre Sékaly
Université de Montréal, CHUM, INSERM U743
Mohamed El Far
Royal Victoria Hospital, Mc Gill University
Mohamed-Rachid Boulassel
Jean-Pierre Routy