luis j. montaner, d.v.m., m.sc., d.phil. monday july 20, 2009 ias 2009, cape town
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Differential regulation of plasma TRAIL and IFN- a levels following therapy interruption during chronic HIV-1 infection Understanding the role of IFN- …. Luis J. Montaner, D.V.M., M.Sc., D.Phil. Monday July 20, 2009 IAS 2009, Cape Town. Innate Compartment & HIV Pathogenesis. NK-T Gd- T. - PowerPoint PPT PresentationTRANSCRIPT
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!