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