Melbourne Induction and Clearance of Latent HIV Infection an ExVivo Assessment of Immune Effectors using Cells from ARTtreated Patients DM Margolis 1 C Garrido 1 JM Sung 1 S Lam 2 ID: 227143
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Slide1
2014 “Towards an HIV Cure” symposiumMelbourne
Induction and Clearance of Latent HIV Infection:
an Ex-Vivo Assessment of Immune Effectors using Cells from ART-treated Patients
DM Margolis
1
, C Garrido
1
, JM Sung
1
, S Lam
2
, R Bateson
1
, B Allard
1
, N Dahl
1
, CR Cruz
2
, P Castillo-Caro
3
, MT Ngo
3
, J Kuruc
1
, A Crooks
1
, CM Rooney
3
, CM Bollard
2
, and NM Archin
1
1
University of North Carolina Chapel Hill, NC;
2
Children’s National Medical Center, Washington, DC;
3
Baylor College of Medicine, Houston, Texas Slide2
Model systems to assess clearancePrimary cell systems
Cytotoxic T cellsNK cellsADCC antibodies
ImmunotoxinsSelective apoptosis inducersSlide3
Why Ex-vivo Expanded CTLsBypasses impaired immune response
Precisely controlled quantity & timing of administrationCTLs can persist
Safe and effective for treatment of viral infections in oncology patients
AM Leen, et al. Nat Med. 2006
Bollard, et al. Blood. 2007Slide4
Mature DC
+ gag/ pol/ nef
++
HIV-CTLs
T cell
Immature
DC
PBMC
freeze
PHAblast
IL-7 IL-15 IL-2
IL-12
IL-15
CD80/86
4-1BBL
CD32
+ K562
ART to prevent
In vitro spread
Cath Bollard, DC Children’s
Clio Rooney, Baylor and PACT
Ex vivo
Expansion of HIV-specific T cells
HXTCsSlide5
HXTCs are a Mixture of Phenotypes
82% CD8+ T cells
19% CD4+ T cells
80% EM T cells
13% CM T cells
IFN
γ
Release
to Cognate PeptidesSlide6
HXTCs Inhibit Productively Infected Cells
CD8 deplete patient PBMCs
Activate 2-3 days
Superinfect w/ virus via spinoculation
+ autologous unexpanded CD8 or expanded HIV-CTLs (or no effector control)
Co-culture x 7 days
Media change q3-4 days
Supernatant harvested for p24 ELISA
Yang, et al. JID 2012
Freel, et al. J Virol. 2012
wash
Plate in triplicate
HIV-CTLsSlide7
HXTCs Inhibit Autologous Reservoir Virus
E:T
E:T
E:T
E:T
HIV p24 (% of no effector at day 7)Slide8
Ex-Vivo Latency Clearance Assay:
A modified quantitative viral outgrowth Assay
Resting CD4
Negative selection
CD8, CD14, CD16, CD19,
CD56, glycophorin A
CD8
HXTCs
Induction
PHA/IL2
or
VOR
Plate: 0.5-1x10
6
/well, 12 wells group
PBMCs
No Effectors
or
or
Add CTLsCo Cx 24HAdd AlloFeedersCoCxx2Measure p24atDay 15washwashMedia changes q3-4 daysCx with ARVs24HCTL Expansion CD8 negative selectionSlide9
HXTCs Clear Infected Cells Emerging from Latency
PHA Stimulation VOR Induction
No Effectors
CD8
HXTC
0
1
2
3
4
5
6
7
8
#wells positive
(out of 12 total)
425
532
250
0
20
40
60
80
100
120
% viral recovery
Et 1:10
Et 1:10
p <0.03 Wilcoxon signed rank
p <0.02 t testSlide10
VOR does not Impair CD8 Antiviral Activity at Physiogically Relevant Exposures
HIV p24 (
ng/ml)
Viral inhibition
assay at E:T
ratio 1:1
hours
B.
Control
24
48
72
0
20
40
60
80
100
No CD8 Control
335nM
500nM
1000nM
No VOR
Patient 532Slide11
Why NKs to target residual HIVCrucial innate immune effectors against viral infections
Do not recognize specific antigens nor require prior antigen sensitizationFunction is balanced by inhibitory and activating receptorsKill cells by release of granzymes and perforins, which causes apoptosis
NK cells may help control HIV-1 infectionMemory NK cells may also provide a more effective antiviral responseNK function may be augmented by clinically applicable cytokinesSlide12
VIRAL INHIBITION ASSAY
PBMCs
Negative isolation
NK cells
CD4
+
T cells
PHA 24h
Super-infection (
autologous reservoir
virus)
HIV-CD4
+
cells
+/- effectors
Unstimulated NKs
IL-2 /IL-15
stimulated NKs
HIV-p24
7 daysNegative isolationNo effectors≠ E:T ratiosSlide13
VIRAL INHIBITION ASSAY
1:1 1:10 1:100
1:1 1:10 1:100
1:1 1:10 1:100
NK cells
CD4+
Targets
only
IL2-stimulated NK cells
IL15-stimulated NK cells
†
†
†
†
†
†
†: p <0.01Slide14
LATENCY CLEARANCE ASSAY
PBMCs
Negative isolation
NK cells
Resting CD4+T cells
ARV 24h
Stimulation
CD4+T cells
+/- effectors
Add feeders
Unstimulated NKs
IL-2 stimulated NKs
Measure number of positive wells for p24 at day 15
24h
24h
Negative isolation
No effectors
Compare number of positive wells with/without effectorsSlide15
LATENCY CLEARANCE ASSAY
PHA reactivation
VOR reactivation
VOR
Positive trends but more assays neededSlide16
IMPACT OF VOR ON NK FUNCTION
NKs were treated with or without 335 nM VOR overnight
For degranulation cells 4 hours in the presence of K562 cell targets
VOR did not impair NK cytotoxicity or antiviral activity
Viral inhbition assays
Degranulation
VOR
Slide17
SummaryPolyclonal HXTCs with activity against multiple peptides can be generated in clinically relevant numbers
HXTCs and cytokine-treated NK cells show enhanced antiviral activity Using both lab strain JR-CSF virus and autologous reservoir virus
NK cells and HXTCs reduce viral recovery from resting CD4 cells reactivated with PHA/IL-2, demonstrating a potential to clear latent HIV infection ex-vivoPreliminary results also show reduction in viral recovery from resting CD4 cells reactivated with VOR, and no adverse effect on function at relevant VOR exposuresSlide18
Special thanks to the
HIV+ volunteers
Juila Sung MD
Carolina Garrido Pavon, PhD
Natalia Soriano, PhD
Nancie Archin, PhD
Noelle Dahl
Rosalie Bateson
Brigette Allard
Joann Kuruc, MSN RN
Amanda Crooks
Cynthia Gay, MD, MPH
Children’s National Medical Center
Catherine Bollard, MD
Sharon Lam