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2014 “Towards an HIV Cure” symposiumMelbourne
Following in vitro culture with myeloid dendritic cells, negative regulators of T cell activation are expressed preferentially on latently infected CD4+ T cellsVanessa 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
Unactivated resting cells
Resting CD4+ T cell
Dendritic Cells
Evans et al. PLoS Pathogens 2013
Endothelial Cells
Shen et al. J Virology 2013
Ex vivo tissue blocksEckstein et al. J Virology 2001
Myeloid DC induce latency in resting memory CD4+ T cells
Productive Infection
Alone +pDC +mDC
10
100
1000
<1
****
ns
Total Cells
EG
FP
+ c
ells
/ 104
ce
lls
EG
FP
+ c
ells
/ 1
04 c
ellsAlone +pDC +mDC
1
10
100
1000
Sorted eFluor670hiEGFP-
CD4+ T cells
Latent Infection
****
Not mediated by soluble factors: CCL19, CCL21, CXCL10, IL-10, IL-6
Close DC-T cell proximity required for induction of latency
Non-proliferating CD4+ T cells
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 SIV-specific 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
CD11cC
D12
3
Plasmacytoid DC
Myeloid DC
PBMC
Resting CD4+ T cells
CD69 CD25 HLA-DR
+ mouse anti-human CD8, CD11b, CD14, CD16, CD19, CD69,
HLA-DR
Magnetic Bead Depletion
CD
4
CD3
>98%
Resting CD4+ T cells
Bulk DCPBMC
+ mouse anti-human CD3, CD11b, CD19
Magnetic Bead Depletion
Lineage Cocktail
HL
A-D
RDendritic Cells
Resting CD4+ T cells
eFluor670
DC added 1:10AND
R5-EGFP-HIV-1 (2h pulse)
1 day
Productive infection Latent infection
>99%
aCD3/aCD28
3 Days
Activation
+ integrase inhibitor L8
Non-proliferating eFluor670hi Not productively infected EGFP-
EGFP
eFlu
or6
70
eFluor670hiEGFP-
CD4+ T cells
Day 5 p.i.
Expression of negative regulators following DC-T cell co-culture
eFluor-resting CD4+ T cells
+ mDC
OR
R5-EGFP-HIV-1 (2h pulse)
1 day
Baseline PD-1/Tim-3/CTLA-4
Phenotyping on days 1, 2, 3, 4, 5 post-infection
Viable Single cells T cells eFluorhiEGFP-
PD-1, Tim-3 and CTLA-4
EGFP
eFlu
or
HLA-DR
CD
3
SSC-H
SS
C-W
FSC-A
SS
C-A
Days post-infection
% P
osi
tive
cel
lsExpression of negative regulators following DC-T cell co-culture
0
5
10
15
20
BL 1 2 3 4 5
0
10
20
30
40
50
BL 1 2 3 4
CD4+T cellsT cells + mDC
5
0
5
10
15
20
BL 1 2 3 4 5
PD-1 Tim-3
CTLA-4
% P
osi
tive
cel
ls
Days post infection
Mean fold change 24
No change
n=3
Days post-infection
Mean fold change 32
( )
0
25
50
75
100
% p
osi
tiv
e D
C
PDL1 PDL2 Gal9 CD80 CD86
mDC
pDC
PD-1 CTLA-4Tim3Neg Reg
Ligand
Differential expression of negative regulator ligands on mDC and pDC
Leitner PLoS Pathog 2013
Are PD-1hi cells enriched for latency?
eFluorhiEGFP-
CD4+ T cellsAnti-PD-1
Latent infection
PD-1 lo/- cells
PD-1 hi cells
EGFP
eFlu
or6
70
aCD3/28 +L8
HIV latency is enriched in PD-1hi cells
PD-1hi PD-1lo
10
100
1000
Lat
ent
Infe
ctio
n
EG
FP
+ c
ells
/104
cel
ls
Sorted memory
eFluorhiEGFP- CD4+ T cells
+1uM L8
p=0.04
Mean FC = 4.1
FC range 1.9-7.7
HIV latency is enriched in Tim3hi cells
Tim3hi Tim3lo
10
100
1000
Lat
ent
Infe
ctio
n
EG
FP
+ c
ells
/104
cel
ls
+1uM L8
Sorted memory
eFluorhiEGFP- CD4+ T cells
FC range 2.2-6.4
Mean FC = 4.3
p=0.04eFluorhiEGFP-
CD4+ T cells
Anti-Tim-3
Tim-3 lo/- cells
Tim-3 hi cells
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 up-regulated 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
PD-L1PD-1
PD-1/-L1blockade
Cell death virus production immune clearance
Blocking interactions between negative regulators and their ligands
EGFP+ productively infected CD4+ T cells
Latent Infection in sorted eFluorhiEGFP- CD4+ 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