2014 “Towards an HIV Cure” symposium
Melbourne
The Immune Checkpoints PD-1, LAG-3 and TIGIT
are Biomarkers of HIV Infected Cells During ART
and Identify Distinct Cellular Reservoirs
Remi Fromentin, Wendy Bakeman, Mariam B Lawani, Gabriela Khoury, Elizabeth Sinclair,
Frederick M. Hecht, Steven G. Deeks, Sharon R. Lewin, Jean-Pierre Routy, Rafick P. Sékaly,
Nicolas Chomont
Biomarkers of latently infected cells during ART
Identifying biomarkers of latently infected cells is of primary importance
to specifically target and eliminate the persistent reservoir
Approach: Combining the “Where” with the “How”
Where: HIV persists in discrete subsets of cells during ART
How: Mechanisms driving the establishment and persistence of the HIV
reservoir
Biomarkers of latently infected cells could be:
- surrogate markers of higher susceptibility to HIV infection
- markers of persistence providing selective advantages (latency maintenance,
immune escape, replenishment)
l
Why immune checkpoints (ICs) could be
biomarkers for HIV persistence during ART ?
b Co-inhibition of T cells following interaction with
counter-receptors on APCs
TReg cell
activation
MHC
class II
IDO
–
• ICs, negative regulators of T cell
activation, regulate T cell proliferation and
cytokine production.
TCR
LAG3
?
B7-2
CTLA4
B7-1
B7-H1
B7-DC
PD1
– B7-H1
B7-1
CD160
HVEM
BTLA
?
PD1H
Cell cycle
inhibition
Inhibition of
PD1H
Unknown
PD1H
receptor
Tolerance
Exhaustion
Apoptosis
Collagen
LAIR1
Unknown
PD1H
receptor
TIM1
?
– Galectin 9
TIM4
+ CD48
CD155
CD112
CD113
Unknown
TIM4
receptor
Immune dysregulations persist during
ART (residual immune activation,
incomplete CD4 T cell restoration, T cell
dysfunction).
(Hatano et al, JID 2012; Kelley et al, CID 2009)
2B4
TIGIT
Chen L, Nat Rev Imm. 2013
d Co-signalling interactions through multiple interfaces
ell
B7-H1
(Trautmann et al, NatMed 2006; Kaufmann et al, NatImm 2007;
Jones et al, JEM 2008, Peretz el al, PLoSPath 2012)
•
Unknown
TIM1
receptor
TIM3
?
• Several of these molecules are
associated with T cell dysfunction in
chronic HIV infection (PD-1, CTLA-4,
TIM-3, CD160).
Competition
Hypothesis
By inhibiting T cell activation, negative regulators (Immune Checkpoints, ICs)
may actively maintain viral latency and identify reservoir cells during ART.
A
B
ART
APC
ICs
ICs
+
-
Inhibitory signals
silencing HIV
Activated/Productively
infected CD4 T cell
ICs silence HIV
+
-
Persistent/Latently
infected CD4 T cell
ICs = selective advantage for
latently infected cells
Association between the expression of ICs and
virological markers of HIV persistence
CTLA-4
r=0.1747
p=0.2401
100
1000
10000
Int. HIV (cop/106 CD4 T cells)
2
1
0
10
100
1000
40
20
1000
10000
T cells)
1
100
1000
Int. HIV (cop/106 CD4
20
10
0
10
10000
10000
T cells)
1000
10000
2B4
r=0.2047
p=0.1676
10
80
8
6
4
2
0
10
100
Int. HIV (cop/106 CD4 T cells)
r=0.0615
p=0.6815
2
0
10
1000
30
CD160
CD160+ CD4 T cells (%)
TIM-3+ CD4 T cells (%)
60
100
Int. HIV (cop/106 CD4 T cells)
3
Int. HIV (cop/106 CD4
10
r=0.1940
p=0.1914
r=0.0167
p=0.9112
100
20
TIM-3
80
r=0.4453
p=0.0017
40
30
0
10
10000
Int. HIV (cop/106 CD4 T cells)
BTLA
0
10
r=0.3080
p=0.0352
TIGIT+ CD4 T cells (%)
20
TIGIT
40
3
LAG-3+ CD4 T cells (%)
CTLA-4+ CD4 T cells (%)
40
0
10
LAG-3
2B4+ CD4 T cells (%)
r=0.2788
p=0.0577
60
PD-1+ CD4 T cells (%)
Methods:
- Multiparametric flow
cytometry analysis of the
expression of 8 ICBs (PD1, LAG-3, TIGIT, CTLA-4,
BTLA, CD160, 2B4, TIM-3)
in PBMCs
- ultrasensitive
qPCRs to measure the
frequency of CD4 T cells
harboring virological
markers of HIV persistence
PD-1
BTLA+ CD4 T cells (%)
Study population:
48 HIV infected subjects
virally suppressed for at
least 3 years with
CD4>350 c/µL
100
1000
Int. HIV (cop/106 CD4
10000
T cells)
60
40
20
0
10
100
1000
10000
Int. HIV (cop/106 CD4 T cells)
The frequency of CD4 T cells expressing PD-1, LAG-3 and TIGIT are
positively correlated with the frequency of CD4 T cells harboring integrated
HIV DNA during ART.
PD-1 identifies TCM and TTM CD4 T cells enriched in
integrated HIV DNA
B
PD-1 expression in CD4 T cell subsets
TCM
80
TTM
p=0.019
40
20
p=0.330
10000
Integrated HIV DNA
(cop/106 cells)
60
TEM
p=0.004
10000
Integrated HIV DNA
(cop/106 cells)
PD-1+ CD4 T cells (%)
100
1000
100
10
1
10000
Integrated HIV DNA
(cop/106 cells)
A
1000
100
10
1
PD-1+
PD-1-
1000
100
10
1
PD-1+
PD-1-
PD-1+
0
TN
T CM
T TM
T EM
T TD
Differentiation
The frequency of cells harboring integrated HIV DNA is significantly
higher in PD-1 expressing TCM and TTM when compared to their PD-1
negative counterparts.
PD-1-
LAG-3 identifies TCM and TTM CD4 T cells enriched
in integrated HIV DNA
A
B
LAG-3 expression in CD4 T cell subsets
TCM
TTM
1000
100
10000
Integrated HIV DNA
(cop/106 cells)
Integrated HIV DNA
(cop/106 cells)
20
p=0.156
10000
10000
40
TEM
p=0.047
p=0.031
60
Integrated HIV DNA
(cop/106 cells)
LAG-3+ CD4 T cells (%)
80
1000
100
10
10
LAG-3+ LAG-3-
1000
100
10
LAG-3+ LAG-3-
0
TN
T CM
TTM
T EM
TTD
Differentiation
The frequency of cells harboring integrated HIV DNA is significantly
higher in LAG-3 expressing TCM and TTM when compared to their
LAG-3 negative counterparts.
LAG-3+ LAG-3-
TIGIT identifies TEM CD4 T cells enriched in
integrated HIV DNA
A
B
TIGIT expression in CD4 T cell subsets
TCM
TTM
p=0.496
20
10000
10000
Integrated HIV DNA
(cop/106 cells)
40
p=0.027
p=0.496
10000
60
TEM
1000
100
Integrated HIV DNA
(cop/106 cells)
80
Integrated HIV DNA
(cop/106 cells)
TIGIT+ CD4 T cells (%)
100
1000
100
10
10
TIGIT+ TIGIT-
1000
100
10
TIGIT+ TIGIT-
0
TN
T CM
T TM
T EM
T TD
Differentiation
The frequency of cells harboring integrated HIV DNA is significantly
higher in TIGIT expressing TEM when compared to their TIGIT
negative counterparts.
TIGIT+ TIGIT-
Can we further enrich in the reservoir by combining
multiple ICs?
A
B
LAG3+/PD1+/TIGIT+
LAG3+/PD1+
LAG3+/TIGIT+
PD1+/TIGIT+
LAG3+
PD1+
Fold over frequency
of infected CD4 T cells
12
CD4
9
Triple Negative
Single Positive
6
Double Positive
Triple Positive
3
TIGIT+
LAG3-/PD1-/TIGIT-
0
Means +/-SD from 5 subjects
Memory CD4 T cells expressing multiple ICs are highly enriched for
integrated HIV DNA
Is the virus carried by latently infected cells
expressing ICs functional ?
A Principle of “Tat/Rev Induced Limiting Dilution Assay” (TILDA)
PMA/Ionomycin
(12h)
Nested RT-PCR
For msHIV RNA
(24+40 cycles)
18000 cells/well
9000 cells/well
3000 cells/well
1000 cells/well
Maximum
likelihood
method
Frequency of cells
with inducible
msHIV RNA
Sorted CD4+ T cells
B
TILDA (cells expressing
ms HIV RNA/106 cells)
103
102
101
100
mLPT+ mLPT-
Memory CD4 T cells expressing LAG-3 and/or PD-1 and/or TIGIT are
highly enriched for inducible HIV latently infected cells.
Conclusions
1.
CM
TM
EM
Biomarkers
PD-1
LAG-3
TIGIT
2.
Enrichment for HIV infected cells
Altogether, our data suggest that blocking ICs may reactivate HIV from
latency and paves the way for the development of novel strategies to
cure HIV infection.
Acknowledgements
• VGTI Florida
Wendy Bakeman
Amanda McNulty
Mariam B. Lawani
Rafick-Pierre Sekaly
Nicolas Chomont
• Burnet Institute
Gabriela Khoury
Sharon R. Lewin
• UCSF
Steven Deeks
Hiroyu Hatano
Rick Hecht
Rebecca Hoh
Elisabeth Sinclair
Lorrie Epling
• Merck
Daria Hazuda
Mike Miller
Richard J.O. Barnard
Dan Gorman
• McGill University
Jean-Pierre Routy
The study participants