Myeloid dendritic cells induce HIV-1 latency in non-proliferating CD4+ T cellsIAS WORKSHOP, 16 & 17 JULY 2010
Resting CD4+ T cells: a cellular reservoir of
latent HIV-1 Memory – considered major reservoir
Observed early in infectionLong half life ~ 6 - 40 monthsResistant to
AntiretroviralsHost immune recognition
Finzi et al., Science 1997; 278:1295; Wong et al., Science 1997; 278:1291; Chun et al., PNAS 1997; 94:13193; Siliciano et al., Nat Med 2003; 9:727; Ramratnam et al., JAIDS 2004; 35:33
IAS
HIV
RE
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RV
OIR
S W
OR
KS
HO
P,
16
& 1
7 J
UL
Y 2
01
0,
VIE
NN
A
IAS
HIV
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RV
OIR
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P,
16
& 1
7 J
UL
Y 2
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0,
VIE
NN
A Resting CD4+ T cell latency
Resting CD4+ T cell
Blood
Lymphoid blocks
Possible role for cellular secretions and/or cell-cell interactions within lymphoid tissues
Pre-integration
Post-integration
OR
Eckstein et al., Immunity 2001, 15: 671; Kreisberg et al., J Exp Med 2006, 203:865; Saleh et al., Blood 2007, 110:416
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Y 2
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A High frequency of integrated HIV-1 with low level of productive infection in resting
cells incubated with CCL19
Saleh et al., Blood 2007; 110:4161
100
1000
RT C
PM
/ul
0 1 2 3 4 5 6 7
Days after infection
Unconditioned
PHA/IL-2
CCL19
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A
Hypothesis
Establishment of latency occurs as a result of signalling by DC during the recirculation of CD4+ T cells through lymphoid tissue
DC are found within T-zones of lymphoid
tissues
IAS
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A In vitro model
R5 EGFP-HIV-124 hrsSNARF
- 1 0
Resting CD4+ T cells +/- SEB 10ng/mL
DC added 1:10
Resting CD4+ T cells
+ DC
+/- SEB 10ng/mL
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& 1
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A R5 EGFP-HIV-1 (2h pulse)
Non-proliferating SNARFhiNot productively infected EGFP-
EGFP
SN
AR
F
PHA
5 Days
SNARFhiEGFP-
CD4+ T cells
Amplification in PBMC
SN
AR
F
EGFP
5 Days
Detection of replication competent latent infection
PBMC
EGFP+ feeder PBMC a surrogate for latently infected cells
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A
SNARFhi EGFP-
CD4+ T cells 0 5Days post sort
Unstimulated
PHA-PBMC
DC induce latent infection of SNARFhiEGFP- CD4+ T
cells
Unstimulated
Amplification of HIV-1 from latently NOT productively infected cells
SEB Stimulated
p<0.05
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A Activation state of the SNARFhi EGFP- CD4+ T cells
Unstimulated
Sorted SNARFhiEGFP- cells were non-proliferating CD4+ T cells
Pe
rce
nt
po
sit
ive
ce
lls CD69HLA-DRKi67
SEB Stimulated
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A
24 hrs
EGFP HIV-1
Resting CD4+ T
DC
DC
Soluble factors play a role in DC-induced latency
Is DC-T cell contact required?
EG
FP
+ c
ells
/10
4 ce
lls
(L
aten
tly
infe
cted
cel
ls)
p=0.03
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ABlocking CD18
inhibits DC-T cell adhesion
10
100
1000Aloneanti-IgGanti-CD18
<1
CD4+T(post DC)
CD4+T
EG
FP
+ c
ells
/10
4 c
ells
(Lat
entl
y in
fect
ed c
ells
)
Cell-cell contact AND soluble factors are required for maximal DC-induced latency
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OIR
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& 1
7 J
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A
10
100
1000
<1
p=0.03
p=0.01
DC +/- HIV-1
+ + +
NO DC
CD4+T + HIV-1
+ + -
+
Membrane + +--
EG
FP
+ c
ells
/10
4 c
ells
(Lat
entl
y in
fect
ed c
ells
)24 hrs
Resting CD4+ T
DC
EGFP HIV-1
Latency inducing soluble factors are secreted by DC as a result of HIV-1 stimulation
Do DC condition resting CD4+ T cells towards latency without HIV-1
exposure?
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OIR
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7 J
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Y 2
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A Do pDC or mDC induce latency?
Myeloid DC induce HIV-1 latency in non-proliferating CD4+ T cells
Days-1 0
CD4+ T ± HIV-1 EGFP
5
+ PHA-PBMCSNARFhiEGFP-
CD4+ T cells
SNARF-resting CD4+ T cells
+ pDC + mDC
5 Days
Unstimulated
CD4+ T CD4+T CD4+T (post pDC) (post mDC)
EG
FP
+ c
ells
/104
cells
(L
aten
tly
infe
cted
cel
ls) p=0.02
p=0.01
SEB Stimulated
CD4+ T CD4+T CD4+T (post pDC) (post mDC)
p=0.02
p=0.02
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RV
OIR
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HO
P,
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& 1
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Y 2
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VIE
NN
AmDC induce post-
integration latency in non-proliferating CD4+ T
cellsSNARFhiEGFP-
CD4+ T cells
Integrated HIV-1 DNA
(Alu-LTR nested PCR)
Unstimulated
Alu
-LT
R c
op
ies
/106
cells
CD4+ T pDC: CD4+T mDC: CD4+T
SEB Stimulated
CD4+ T pDC: CD4+T mDC: CD4+TCD4+ T CD4+T CD4+T (post pDC) (post mDC)
CD4+ T CD4+T CD4+T (post pDC) (post mDC)
IAS
HIV
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RV
OIR
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HO
P,
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& 1
7 J
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Y 2
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A Summary
mDC induce post-integration latency
Mediated by DC-T cell contact Soluble factors (HIV-1 exposure) Partial activation?
Enhanced by SEB
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OIR
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& 1
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Y 2
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A
Days post infection-1 0
± DC HIV-1 EGFP MOI 1
5
Non-proliferating (SNARFhi) Not productively infected (EGFP-)
Lysed for microarrays
MOCK
Gene profiles of DC-induced latently infected CD4+ T
cells
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A
MockLatent p<0.05
Non-proliferating CD4+ T cells co-cultured with DC
+- 0
461 Genes
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A65 genes involved in cell
cycle 28 genes
Arrest in cell division (24) Delay in cell division (9) Arrest in cell cycle progression (13) Arrest G0/G1 phase transition (2)
GADD45A – growth arrest
IFI16 – growth arrest
11 Upregulated
CHKA – G0/G1 transition
CDC20/AURKB – mitosis
IL-16/BIRC5 – cell cycle
17 Downregulated
Latently infected non-proliferating CD4+ T cells
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OIR
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& 1
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VIE
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A
TRIM22 OAS1
TXNRD1
Upregulation
Genes that inhibit HIV-1
IKBE ITGAL
PRKCATNFRSF14
Downregulation
Genes involved in NFkB activation
Latently infected CD4+ T cells
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OIR
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HO
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& 1
7 J
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Y 2
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VIE
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A
Suboptimal activation – ↑CD69 Enhanced integration
Productive infection prevented: Blocks in HIV-1 transcription Blocks in cell proliferation
mDC-induced post-integration latency in non-proliferating
CD4+ T cells
In vitro model to study the establishment and/or reactivation of latency in primary resting CD4+ T cells
A mechanism for the establishment of latency in vivo within lymphoid tissues
Acknowledgements MONASH UNIVERSITY
Sharon LewinPaul CameronSuha SalehGeza Paukovics
UNIVERSITY OF MONTREAL
Rafick-Pierre SekalyElias HaddadNadia KettafJean-Philipe Goulet
UNIVERSITY OF MELBOURNE
Damian Purcell
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OIR
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A