infection of resting cd4 + t-cells
DESCRIPTION
Myeloid dendritic cells and HIV latency in resting T-cells Nitasha A Kumar, Vanessa A Evans, Suha Saleh, Candida de Fonseca Pereira, Paula Ellenberg, Paul U Cameron, Sharon R Lewin. International AIDS Conference, Washington DC, July 2012 . Infection of resting CD4 + T-cells. - PowerPoint PPT PresentationTRANSCRIPT
Myeloid dendritic cells and HIV latency in resting T-cells
Nitasha A Kumar, Vanessa A Evans, Suha Saleh, Candida de Fonseca Pereira, Paula Ellenberg, Paul U Cameron, Sharon R Lewin.
International AIDS Conference, Washington DC, July 2012
Ex vivo tissue blocks
Eckstein et al, Immunity 2001; 15: 671; Kreisberg et al., J Exp Med 2006; 203:865;
Infection of resting CD4+ T-cells
Unactivated resting cellsResting CD4+ T-cell
Swiggard et al., J Virol 2005; 79(22):14179-88; Lassen et al., Plos One 2012; 7(1):e30176
chemokinesIn v
itro
Saleh et al., Blood 2007;110:416; Cameron et al., Proc Natl Acad Sci 2010;107(39):16934-9
CCR7 (CCL19/21)CXCR3 (CXCL9/10)CCR6 (CCL20)
Ex vivo tissue blocks
Eckstein et al, Immunity 2001; 15: 671; Kreisberg et al., J Exp Med 2006; 203:865;
Infection of resting CD4+ T-cells
Unactivated resting cellsResting CD4+ T-cell
Swiggard et al., J Virol 2005; 79(22):14179-88; Lassen et al., Plos One 2012; 7(1):e30176
Saleh et al., Blood 2007;110:416; Cameron et al., Proc Natl Acad Sci 2010;107(39):16934-9
In v
itro
Dendritic cells
Resting CD4+ T-cells
SNARF or
Efluor
DC added 1:10AND
PBMC + Mouse-anti-human CD3, CD11b,
CD19
CD11c
CD
123
Myeloid DC
Plasmacytoid DC
HLA
-DR
Lineage cocktail
Bulk DC
PHA
5 Days
Amplification in PBMC
PBMC
Non-proliferating SNARFhiNot productively infected EGFP-
EGFP
SNA
RF
5 days
SNARFhiEGFP-
CD4+ T-cells
Resting CD4+ T cells
SNARF or
Efluor
DC added 1:10AND
Productive infection
SNA
RF
EGFP
Latent infection
R5-EGFP-HIV-1 (2h pulse)
1 day
DC induce latent infection in non-proliferating (SNARFhi) cells
SNARFhiEGFP-
CD4+ T cells
LatentEG
FP+
cells
/104 c
ells
Total Cells
Productive
Alone +DC
10
100
1000
<1
*
Alone +DC
10
100
1000
<1
*
n=5
SNARFhiEGFP- T-cells express CD69
Alone +DC
0
2
4
6
8
10
Ki67
CD69HLA-DR
*
% P
ositi
ve c
ells
ActivationMarkers
SNARFhiEGFP-
CD4+ T cells
n=4
Alone +DC
0
10
20
Ki67
CD69HLA-DR
*
0
10
20* *
% P
ositi
ve c
ells
- SEB+ SEB
ActivationMarkers
SNARFhiEGFP-
CD4+ T cells
n=4
mDC specifically mediate HIV latency
Days-1 0 5
+pDC
Sorted SNARFhiEGFP - CD4+ T cells
Integrated HIV DNA
copies/ million cells
Latent Infection Integrated HIV DNAProductive Infection
Total Cells
EGFP+ cells/ 10
4 cells
EGFP+ cells/ 10
4 cells
-SEB
Alone +pDC +mDC
10
100
1000
<1
NS****
Alone +pDC +mDC
10
100
1000
<1
******
Alone +pDC +mDC102
103
104
105
n=10 n=10 n=3
Productive
+mDC
+
+
Alu-LTR PCR
Sorted SNARFhiEGFP - CD4+ T cells
Integrated HIV DNA
copies/ m
illion cells
Latent Infection Integrated HIV DNAProductive Infection
Total Cells
EGFP+ cells/ 10
4 cells
EGFP+ cells/ 10
4 cells
-SEB
Alone +pDC +mDC
10
100
1000
<1
NS****
Alone +pDC +mDC
10
100
1000
<1
******
Alone +pDC +mDC102
103
104
105
n=10 n=10 n=3
Sorted SNARFhiEGFP - CD4+ T cells
Inte
grat
ed H
IV D
NA
copi
es/ m
illio
n ce
lls
Latent Infection Integrated HIV DNAProductive Infection
Total Cells
EGFP
+ ce
lls/ 1
04 c
ells
EGFP
+ ce
lls/ 1
04 c
ells
-SEB
Alone +pDC +mDC
10
100
1000
<1
NS****
Alone +pDC +mDC
10
100
1000
<1
******
Alone +pDC +mDC10 2
10 3
10 4
10 5
n=10 n=10 n=3
5 days
Latent
Sorted SNARFhiEGFP - CD4+ T cells
Integrated HIV DNA
copies/ m
illion cells
Latent Infection Integrated HIV DNAProductive Infection
Total Cells
EGFP+ cells/ 10
4 cells
EGFP+ cells/ 10
4 cells
-SEB
Alone +pDC +mDC
10
100
1000
<1
NS****
Alone +pDC +mDC
10
100
1000
<1
******
Alone +pDC +mDC102
103
104
105
n=10 n=10 n=3
PHA
SNARFhiEGFP-
CD4+ T-cells
PBMC
SNARFhiEGFP-
CD4+ T cells
% P
D-1
-pos
itive
cel
ls
Alone +mDC0
2
4
6p=0.007
n=6
SNARFhiEGFP- T-cells express PD1
Chomont et al., Nature Med 2010
Establishment of latency not related to degree of productive infection
Productive Latent
EGFP
+ cel
ls/ 1
04 cel
ls
10
100
1000
<1
Alone +DC
Total Cells
n=2
10
100
1000
<1
Alone+imMDDC+matMDDC+mDC
Alone +DC
Sorted SNARFhiEGFP-
CD4+ T cells
MDDC = monocyte derived DCs
Myeloid DC induce latency in memory not naïve CD4+ T-cells
Naive/Memory CD4+ TLa
tent
Infe
ctio
nEG
FP+
cells
/ 104 c
ells
Sorted SNARFhiEGFP-
CD4+ T cells
10
100
1000
<1
Alone +mDC
NaiveMemory*
*
n=5
Cytokines/chemokines in mDC-T-cell co-cultures
Days
-1 0 5
+pDC
+mDC
+
+Bead arrays
Neutralization of DC-derived chemokines does not inhibit latency
Neutralisation of select cytokines does not inhibit latency
Late
nt In
fect
ion
EGFP
+ cel
ls /1
04 cel
ls
10
100
Alone +mDC
<1n=3
NS
Late
nt In
fect
ion
EGFP
+ cel
ls /1
04 cel
ls
10
100
<1
Alone +mDCn=5
NS
anti-IL-10R anti-IL-6
Sorted eFluorhiEGFP- CD4+ T cells
24 hrs
EGFP HIV-1
DC
DC
DC-T cell proximity required for DC-induced latency
CD4+ T
DC
CD4+ T CD4+ T
DC
Late
nt In
fect
ion
EGFP
+ cel
ls/1
04 cel
ls10
100
1000
<1
Alone+mDC
* *
DC-T-cell culture supernatant doesn’t facilitate latency
Late
nt In
fect
ion
EGFP+ c
ells
/10
4 cel
ls10
100
<1
ii iiii
CD4+ T
DC
CD4+ T CD4+ T
Supernatant
Alone+mDC
*
n=3
CD4+ T
DC
CD4+ T
n=3
CD4+ T
Supernatant
Conclusions
Myeloid DC facilitate establishment of HIV latency in resting memory CD4+ T-cells via direct infection.
Close proximity (± cell contact) of mDC and T-cells required
Role for negative regulators in the establishment/maintenance of DC-induced latency
Implications
Efficient infection of resting T-cells in close contact with mDC and HIV may explain the rapid early establishment of the latent reservoir.
If infectious virus persists in tissues such as LN in patients on cART, mDC may facilitate ongoing infection of resting T-cells leading to replenishment of the reservoir.
Acknowledgements
Department of Infectious Diseases, Monash University– Sharon Lewin – Paul Cameron– Vanessa Evans– Suha Saleh– Paula Ellenberg– Candida de Fonseca Pereira– Ajantha Solomon
University of Melbourne– Damian Purcell
Flow Cytometry Facility, Monash University– Geza Paukovics– Michael Thomson– Jeanne Le Masurier
Vaccine Gene Therapy Institute– Rafick Sekaly– Nicholas Chomont