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

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A

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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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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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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

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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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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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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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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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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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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)

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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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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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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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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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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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