Differential regulation of plasma TRAIL

and IFN- levels following therapy

interruption during chronic HIV-1 infection

Understanding the role of IFN-Understanding the role of IFN-….….

Luis J. Montaner, D.V.M., M.Sc., D.Phil.

Monday July 20, 2009

IAS 2009, Cape Town

NK-TT

Innate Compartment & HIV Pathogenesis

Steady-state HIV-1 replication decreases the functionality of the

innate effector compartment and PDC-mediated responses (cross-

sectional studies to uninfected subjects).

Immune reconstitution of innate effector cell function is possible

following antiretroviral therapy yet degree of recovery varies

relative to pre-therapy immune status.

PDC, NK response & HIV-infected Subjects

Journal of Immunology 168:4796, 2002Journal of Immunology 168:5764, 2002Journal of Infectious Diseases 188: 873, 2003Journal of Infectious Diseases 191:1451, 2005 AIDS. 2007 Jan 30;21(3):293-305.Journal of Immunology 179:2642, 2007.

Working Questions (#1)

• Does plasma Interferon- levels increase in parallel

to increases in sTRAIL levels after therapy

interruption in chronic infection?

• Are circulating PDC activated by viremia and is their

state associated with enhanced or decreased potential

for Interferon- secretion?

PDC, TRAIL, Interferon-alpha & HIV Viremia

Baseline(<50)

Week 2(50-19,700)

Week 4(50-517,000)

Week 6(824-493,000)

010002000300040005000600070008000

p=0.0005

p=0.0003p=0.002

0

25

50

75

100

Baseline<50)

Week 2(50-19,700)

Week 4(50-517,000)

Week 6(824-493,000)

ns

IFN

- (

pg

/ml)

sTR

AIL

(p

g/m

l)

Controls (N=31)

All HIV+ (N=69)

Viremic(N=37)

Aviremic (N=32)

0

1200

2400

3600

4800

6000

p=0.0024p=0.0023p<0.0001

p=0.011

IFN

- (

pg

/ ml)

0

100

200

300

400

ns

Controls (N=31)

All HIV+ (N=69)

Viremic(N=37)

Aviremic (N=32)

p=0.0016

Higher plasma TRAIL but not IFN- after viral rebound during ART interruption

IFN-

CYTOPLASM

NUCLEUS

p-IRF-7 p-IRF-3 p-IRF-5

Dimer Dimer

IRF-7 IRF-3 IRF-5

p-IRF-7 p-IRF-3 p-IRF-3 p-IRF-3 p-IRF-5P-IRF-7 p-IRF-7 p-IRF-3 p-IRF-5 p-IRF-5

PromotersType 1 IFNs

IFNAR-1/2

p-STAT1p-STAT1IRF-9

STAT1 IRF9 STAT2

JAK/STAT pathway

PromotersIFN-stimulated genes:

PKR, OAS, ISG, MX, IRF-7

ISGF3

Autocrine IFN amplification loop

Evidence for IFN amplification loop?

PDC Protein Expression of mTRAIL, IRF-7

Controls(N=8)

HIV+ (N=11)

0102030

40506070

0.0035

% P

DC

exp

res s

ing

mT

RA

IL

1

Control HIV+ (VL=10,807))

IRF-7-PE

Ce

ll C

ou

nt

GM

F/IR

F-7

0.002

0.02.55.07.5

10.012.515.017.520.0

Controls HIV+(N=10 (N=11)

Working Questions #1

• Do Interferon- plasma levels increase in parallel to increases

in sTRAIL levels after therapy interruption in chronic infection?

ANSWER: Evidence for sTRAIL increase but not soluble IFN-

in asymptomatic subjects upon ART interruption.

• Are circulating PDC as target cells activated by viremia in

association with Interferon- secretion?

ANSWER: PDC upregulates mTRAIL upon viremia in

cojunction with decreased IRF-7 levels consistent with

decreased IFN- secretion potential.

Section Conclusions

Working Question (#2)

• Do HIV-induced TRAIL expressing PDCs (or

Interferon- mediate cytotoxic responses against

autologous HIV-infected CD4 T-cell targets?

• Does HIV-1 infection of CD4 T-cells (in vivo or in

vitro) result in increased DR5 as a ligand for TRAIL?

PDC, Interferon-alpha, DR5 & CD4 T Cell Loss

Herbeuval et al. PNAS 103:7000, 2006; 104:17453, 2007Boasso, et al. Blood 109:3357, 2006.

Cytotoxicity against Autologous HIV-1-Infected CD4Cytotoxicity against Autologous HIV-1-Infected CD4++ T Cells T Cells

Day 1Day 1

PBMCsPBMCs

PHA/IL-2PHA/IL-2

Day 3Day 3

CD4CD4++ T Cells T Cells

Day 4Day 4

SpinfectionSpinfection

IL-2IL-2

4 hr.4 hr.

5151CrCr

CD107aCD107aTargets Targets FicollFicoll PurifyPurify

Day 7Day 7

IL-2IL-2

Day 7Day 7

Effector:Effector:PBMCsPBMCs NKsNKs

++

Wilcoxon Matched Pairs Test Non-parametric, n=9

PBMC 100:1PBMC 100:1

PBMCPBMC+ HIV CD4+ HIV CD4

PBMCPBMC+ uCD4+ uCD4

p=0.0039p=0.0039

Per

cen

tage

Cyt

otox

icit

yP

erce

nta

ge C

ytot

oxic

ity

0

10

20

30

40

PBMC 100:1PBMC 100:1

Per

cen

tage

Cyt

otox

icit

yP

erce

nta

ge C

ytot

oxic

ity

PBMCPBMC+ HIV CD4+ HIV CD4

PBMCPBMC+ uCD4+ uCD4

uCD4

HIV+ CD4

0

10

20

30

40

50

aCD4-HIV stimulation can Augment NK-mediated Lysis of Autologous Infected CD4 T-cells

Ig anti-TRAIL

0102030405060

% s

pe

cif

i c l

ys

is

HIV-infected SupT1

Uninfected primary aCD4

HIV-infected primary aCD4

HIV-infected SupT1

% s

pec

ific

lysi

s

Activated PDC Activated NK

0

20

40

60

Activated PDC do not Lyse aHIV-infected CD4+ T cells

% D

R5+

CD

4+T

Controls HIV+(N=10) (N=16)

0

1

2

3

4

10 0 10 1 10 2 10 3 10 4

1.7%

DR

5

10 10 10 10 10 0 1 2 3 4

0.58%

CD3+CD4+ T cells

Control HIV+

DR

5

0.076% 0.02%

0.013%

Uninfected

p24 Ag

NL4-3-infected

0.058% 0.22%

53%

10 0 101 102 103 104100 101 102 1030

500

1000

1500

100 101 102 103 10

DR5-PE

Sup-T1 Uninfected HIV-IIIB

0 101 102 103

HIV-SHIP

Cel

l C

ou

nt

No evidence for DR5 expression on HIV-infected CD4+ T cells

In Vivo-derived In Vitro-derived

Working Question (#2)

• Do HIV-activated PDCs (via TLR interactions) or Interferon-

mediate PDC-mediated TRAIL-dependent cytotoxic responses

against autologous CD4 T-cell HIV-infected targets?

ANSWER: PDC show no TRAIL-mediated activity against

autologous infected CD4 cells but instead show activity to

mediate NK cytotoxic responses upon HIV-infected cell

activation.

• Do infected CD4 T-cells express DR5 as a ligand for TRAIL?

ANSWER: Evidence for lack of surface DR5 expression in

infected CD4 T-cells (in vivo or following in vitro PDC

activation) in contrast to HIV-infected transformed T cell lines.

PDC, Interferon-alpha & CD4 Cell Loss

Using Innate Activation as Antiviral Strategy after ART?

Discussion question (#3)

Can we directly test (proof-of-concept) the effect of

innate activation and/or viral control via IFN- following

immune reconstitution?

Suppressive Anti-retroviral Therapy

T-cell activation

Serum HIV RNA

CD4+ T cells

Viremia early benefit delayed benefit

pDC, IFN-

NK cells

NK cytotoxicityNo study to date has investigated the effects of

Interferon-alpha against HIV-1 if the immune system

is fully recovered following ART.

Test of IFN-alpha monotherapy after ART

ConclusionsActivation of PDC in chronic HIV Infection unlikely

to be a major factor in progressive CD4 T cell depletion via direct cytotoxic activity.

Functional PDC/NK axis may represent a major innate antiviral response mechanism acutely lost upon acute infection in progressive disease.

Remains to be determined how suppressive is IFN- in the presence of a fully functional immune reconstituted subject.

Livio Azzoni

Craig Carty

Jihed Chehimi

Jennifer Dubin

Betsy Gekonge

Agnes Mackiewicz

Emmanouil Papasavvas

Maria Picone

Max Pistilli

Griffin Reynolds

Andrea Raymond

Brian Ross

Costin Tomescu

Kavita Vinekar

Acknowledgments

Acknowledgments

University of Pennsylvania Ronald CollmanJay Kostman Robert DomsRobert GrossLan ZouCFAR Cores

BD BioSciencesSkip MainoMaria Suni

Jonathan Lax Immune Disorder ClinicCele GalloKaram MounzerJane Shull & Clinical Research Staff

The Wistar InstituteL. Showe & LabH. ErtlJeffrey Faust Phlebotomy Unit

U. MassAndrea Foulkes

Gladstone Inst.Robert GrantMichael McCuneDouglas NixonGabriel Ortiz

Children’s HospitalOf PhiladelphiaAlma NowmosRick M RutsteinCarol Vincent

DAIDS, NIHLarry FoxDaniella LivnatCarolyn Williams

Multicenter AIDS Cohort Study

Women’s Interagency HIV Study

NIAID, NIHThe Philadelphia FoundationCommonwealth of Pennsylvania

THANK YOU!