2014 “Towards an HIV Cure” symposium
Melbourne
Following in vitro culture with myeloid dendritic
cells, negative regulators of T cell activation are
expressed preferentially on latently infected CD4+
T cells
Vanessa A. Evans, Renée M. van der Sluis, Nitasha A. Kumar, Rafick-Pierre
Sekaly, Remi Fromentin, Nicolas Chomont, Paul U. Cameron, Sharon R.
Lewin
Infection of resting CD4+ T cells:
a role for cell-cell interactions
Resting
CD4+ T cell
Unactivated
resting cells
Ex vivo tissue blocks
Eckstein et al. J Virology 2001
Endothelial
Cells
Shen et al. J Virology 2013
Dendritic Cells
Evans et al. PLoS Pathogens 2013
Myeloid DC induce latency in resting
memory CD4+ T cells
L a te n t In fe c tio n
P r o d u c tiv e In fe c tio n
**
1000
**
c e lls
1000
**
ns
4
c e lls / 1 0
100
100
10
+
10
EGFP
E G F P + c e lls / 1 0
4
c e lls
**
<1
A lo n e
+pDC
+m DC
1
A lo n e
+pD C
+mD C
hi
T o ta l C e lls
S o rNon-proliferating
te d e F lu o r 6 7 0 E G F P
++
CD4
CD4 T
T ccells
e lls
-

Not mediated by soluble factors: CCL19, CCL21, CXCL10, IL-10, IL-6

Close DC-T cell proximity required for induction of latency
Evans et al. PLoS Pathogens 2013
Negative regulators and latency

Negative regulators dampen the immune
response and can be found on exhausted T cells
Ahmed et al. J Immunol 2010; Day et al. Nature 2006

Latently infected T cells express negative
regulators of T cell activation eg. PD-1, Tim-3
and TIGIT
Chomont et al. Nat Med 2010; Fromentin et al. CROI 2014

Blocking PD-L1/PD-1 in vivo restores the SIVspecific cellular and humoral immune responses,
improves viral control and reduces immune
activation
Velu et al. Nature 2009; Dyavar Shetty et al. J Clin Invest 2012;
Finnefrock et al. J Immunol 2009
Hypothesis
Expression of negative regulators
during DC-T cell interactions may
actively suppress viral replication and
maintain latency
Resting CD4+ T cells
+ mouse anti-human
CD8, CD11b, CD14, CD16,
Resting CD4+ T cells
>98%
CD4
PBMC
Magnetic Bead
Depletion
CD69
CD19, CD69, HLA-DR
CD25 HLA-DR
CD3
+ mouse anti-human
CD3, CD11b, CD19
Bulk DC
Plasmacytoid DC
CD123
PBMC
Magnetic Bead
Depletion
HLA-DR
Dendritic Cells
Myeloid DC
Lineage Cocktail
CD11c
Resting
CD4+ T cells
eFluor
670
DC added
1:10
AND
1 day
R5-EGFP-HIV-1 (2h pulse)
Non-proliferating eFluor670hi
Day 5 p.i.
Not productively infected EGFP-
eFluor670hiEGFPCD4+ T cells
+ integrase inhibitor L8
eFluor670
aCD3/aCD28
Activation
3 Days
>99%
EGFP
Productive infection
Latent infection
Expression of negative regulators following
DC-T cell co-culture
eFluor-resting
CD4+ T cells
OR
+ mDC
Baseline PD-1/Tim-3/CTLA-4
1 day
R5-EGFP-HIV-1 (2h pulse)
Phenotyping on days 1, 2, 3, 4, 5 post-infection
CD3
SSC-A
FSC-A
SSC-H
eFluorhiEGFP-
T cells
eFluor
Single cells
SSC-W
Viable
HLA-DR
PD-1, Tim-3
and CTLA-4
EGFP
Expression of negative regulators following
DC-T cell co-culture
% Positive cells
50
PD-1
CD4+T cells
T cells(+ mDC)
40
30
Mean fold change 32
Tim-3
15
Mean fold change 24
10
20
5
10
0
0
n=3
BL
1
2
3
4
5
20
CTLA-4
15
No change
10
5
0
BL
1
2
3
BL
1
2
3
Days post-infection
Days post-infection
% Positive cells
20
4
Days post infection
5
4
5
Differential expression of negative regulator
ligands on mDC and pDC
% positive DC
100
75
mDC
pDC
50
25
0
Ligand
Neg Reg
PDL1
PDL2
PD-1
Gal9
CD80
Tim3
Leitner PLoS Pathog 2013
CD86
CTLA-4
Are PD-1hi cells enriched for latency?
PD-1 hi cells
eFluorhiEGFPAnti-PD-1
aCD3/28 +L8
PD-1 lo/- cells
Latent
infection
eFluor670
CD4+ T cells
EGFP
HIV latency is enriched in PD-1hi cells
Latent Infection
EGFP+ cells/104 cells
1000
p=0.04
FC range 1.9-7.7
Mean FC = 4.1
100
+1uM L8
10
PD-1hi
PD-1lo
Sorted memory
eFluorhiEGFP- CD4+ T cells
HIV latency is enriched in Tim3hi cells
F C r a n g e 2 .2 - 6 .4
M e a n F C = 4 .3
1000
4
c e lls /1 0
+
100
EGFP
Tim-3 lo/- cells
L a t e n t In f e c tio n
Anti-Tim-3
c e lls
p = 0 .0 4
Tim-3 hi cells
eFluorhiEGFPCD4+ T cells
+1uM L8
10
T im 3
hi
T im 3
lo
S o rte d m e m o ry
e F lu o r
hi
-
EGFP CD4
+
T c e lls
Summary
 Myeloid DC facilitate establishment of HIV latency in resting
memory CD4+ T cells via direct infection
 Negative regulators PD-1 and Tim-3 but not CTLA-4 are upregulated on resting CD4+ T cells upon co-culture with mDC
 Negative regulator ligands are differentially expressed by
mDC and pDC
 mDC-induced HIV latency is enriched in PD-1hi and Tim-3hi
cells
Blocking interactions between negative regulators
and their ligands
EGFP+ productively
infected CD4+ T cells
PD-1/-L1
blockade
PD-1
PD-L1
Cell death
 virus production
 immune clearance
Latent Infection
in sorted
eFluorhiEGFPCD4+ T cells
Implications
 mDC may facilitate ongoing latent infection of resting CD4+
T cells leading to replenishment of the reservoir
 Disrupting the function of PD-1 and/or Tim-3 could
potentially be exploited to inhibit replenishment of the
reservoir and/or reverse latency
Acknowledgements
Monash University
–
–
–
–
–
–
Sharon Lewin
Paul Cameron
Renée van der Sluis
Nitasha Kumar
Suha Saleh
Candida da Fonseca
AMREP Flow Cytometry
–
–
–
–
Geza Paukovicks
Michael Thompson
Jeanne Le Masurier
Phil Donaldson
VGTI Florida
– Rafick Sekaly
– Nicolas Chomont
– Remi Fromentin
University of Melbourne
– Damian Purcell