EBV/BLFZ1+

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EBV/BLFZ1+
(Multi) virus specific T cells
Spyridonidis Alexandros
SCAT Lab
Lab Meeting 03/22/2012
Cellular Immunotherapy for the treatment of Viral infection
and reactivation after Bone Marrow Transplantation
major cause of morbidity and mortality
CMV, EBV, AdV, BK, HHV-6, HHV-7, RSV
Depending on protocol, patient characteristics etc..
Obligatory for Haplo protocols
drug treatments
Efficacy
Partial effective (CMV, …..Adv?, RSV?)
Not effective (EBV, BK, HHV-6, HHV-7)
Side effects (cytopenias, renal insufficiency)
Expensive
Render viruses resistant
Why cellular immunotherapy
Central role of immune system
inverse relationship between viral antigenemia and circulating virus-reactive T
cells
40% CMV reactivation after autologous BMt but <5% disease
Efficacy
Clinical studies (Seattle, Baylor, Wurzburg, Leiden)
Adoptive cell therapy
IFN-g capture
Variables in generation of virus specific T cells
APCs (DCs)
Mo DCs vs Plasmacytoid DCs
TLR
Virus stimuli
peptide mix (CD4, CD8, polyclonal)
Other
Culture
Media
Cytokines, others
IL-7 (10 IU/ml), IL-15 (5 IU/ml), IL-2 (100 IU), IL-12
Wnt β- catenin inhibitor
T cell Donor
Initial donor
Patient (?)
Third party
Product
Potency markers
Viable
CD4 and CD8
Polyclonal? (minimize viral escape)
Tcm, IL-21R expression (for long term in vivo repopulation)
Efficacy
IFNγ –producing
Other tests (lysing ability etc)
Number
Probably few are enough. Efficacy has been shown with
IFNγ- capture assay: 1.2 x10e3 cells/ kg (AdV)
If a cellular product of 10x10e6 cells/kg contains 1% virus specific cells
= 1x10e5 cells / kg (virus specific)
Reproducible
Lack of alloreactivity
Purity?
GMP criteria / release
GMP materials
Sterility
Identity
Generation of human dendritic cells
elutriated monocytes
GM-CSF 800IU/mL
IL-4 1000IU/mL
Immature DCs
LPS 30ng/mL
IFN-γ 100IU/mL
mature DCs
Day 3-5
Day 0
10% FCS, NABS
Day7
Vivid
SSC
SSC
Vivid
 X Vivo serum
FSC
Immature DC
CD11b
Mature DC
HLA-DR
CD80
CD86
CD83
Generation of virus specific CTLs
Lymphos
Ellutriated
Monocytes
Immature
DCs
mature
DCs
(Re-expansion)
1st stim (x days)
Day 0
D3-5
2nd stim (x days)
D14?
D5-7
pulse
stimulate
Patient
Re-stimulate
Which virus (CMV, EBV, DC: lympho ratio
Yes or no
BK, ADV, … HHV-6?)
Cytokines (IL-7,
DC or monos
Which pep mix?
IL-15, IL-2, IL-4?)
Pulse?
Peptide dose
Days?
Days?
Wash vs not wash
competition? Which
Competition?
virus?
D21?
In vitro
evaluation
methodology
In vivo
evaluation
methodology
Important variable: Split the cells!!
No split
CD3
Split
CD14-CD19-Vivid
FACS analysis
CD14-CD19-Vivid
CD4 V500
SCS
FCS
CD3 efluo 610
CD3, CD4, CD8, (CD27 CD45RO)
IFN-g
TNFa
IL-2
CD154
CD107
FCS
FCS
CD8 H7-APC
uDC/SEB
CD4
Donor 32102, CMV+, 1st stim
CD8+ cells
uDC/-
CD8
EBV/LMP1- EBV/BLFZ1-
BK/LT
-
BK/ST-
ADV-
MIX-
CMV+
EBV/LMP1+ EBV/BLFZ1+
BK/LT+
BK/ST+
ADV+
MIX+
SS
CMV-
IFN-g(PE)
uDC/SEB
CD4
Donor 32102, CMV+, 1st stim
CD4+ cells
uDC/-
CD8
EBV/LMP1- EBV/BLFZ1-
BK/LT
-
BK/ST-
ADV-
MIX-
CMV+
EBV/LMP1+ EBV/BLFZ1+
BK/LT+
BK/ST+
ADV+
MIX+
SS
CMV-
IFN-g (PE)
uDC/SEB
CD4
Donor 32102, CMV+, 2nd stim
CD8+ cells
uDC/-
CD8
EBV/LMP1- EBV/BLFZ1-
BK/LT
-
BK/ST-
ADV-
MIX-
CMV+
EBV/LMP1+ EBV/BLFZ1+
BK/LT+
BK/ST+
ADV+
MIX+
SS
CMV-
IFNg (PE)
Donor 32102 (CMV pos)
10
6
4
% cells
8
2
0
CD4
CD8
uDC/SEB
CD4
Donor 100108, CMV+, 1st stim
CD4+ cells
uDC/-
CD8
EBV/LMP2-
EBV/BLFZ1-
BK/LT-
BK/VP1-
CMV+
EBV/LMP2+
EBV/BLFZ1+
BK/LT+
BK/VP1+
MIX-
SS
CMV-
IFN-g (PE)
MIX+
Donor 61710 (CMV pos)
3
% cells
2.5
2
1.5
1
0.5
0
CD4
CD8
CD4+
2 stim
CD8+
CD4+
1 stim
CD8+
Mutlivirus CTLs from different donors
IFN-g (PE)
The role of CMV in
CMV seropos patients
Donor 100108, CMV+, 2nd stim
CD4+ cells
SEB
CMV-
CMV+
2nd stim
Irradiated monos
+/- CMV pulsed
unpulsed
EBV/LMP2-
EBV/BLFZ1-
EBV/LMP2+ EBV/BLFZ1+
BK/LT-
BK/LT+
BK/VP1-
BK/VP1+
MIX-
MIX+
MIX+
(CMV CTLs)
Donor 100108, CMV+, 2nd stim
CD8+ cells
SEB
CMV-
CMV+
2nd stim
Irradiated monos
+/- CMV pulsed
unpulsed
EBV/LMP2-
EBV/BLFZ1-
EBV/LMP2+ EBV/BLFZ1+
BK/LT-
BK/LT+
BK/VP1-
BK/VP1+
MIX-
MIX+
MIX+
(CMV CTLs)
Donor 60607, CMV+, 2nd stim
CD8+ cells
CMVpp65 -
SS
CMVpp65 +
IFN-g (PE)
CMV/IE1 -
CMV/IE1 +
Which peptides?
1 or 2 stimulations?
Variability
The tough case
Subject ID
CMV seronegative donors
One stimulation
CMV
Exp Number
101310
pos
14
1st and 2nd stim
101410
pos
14
1st and 2nd stim
50107
101706
11310
90110
12810
62311
31307
60607
100108
80510
32111
111010
60110
6123
32102
61710
321012
32111
31711
53111
53111
101911
xxx
xxx
neg
neg
neg
neg
neg
neg
neg
pos
pos
neg
pos
pos
pos
pos
pos
pos
pos
pos
neg
pos
pos
xxx
xxx
xxx
14
14
13
13
13
13
12
12
11
11
10
10
9
9
8
8
7
7
6
5
4
3
2
1
1st and 2nd stim
to analyze
1st and 2nd stim
2nd stim
2nd stim
2nd stim
2nd stim
withdrawn
Exp #13
Donor 11310, CMV1st stim
Donor 11310, CMV-, 1st stim
CD4+ cells
uDC/-
CMVpp65-
CMV-IE1-
EBV-EBNA1-
EBV/BLFZ1-
BK-LT-
ADV -
MIX-
CMVpp65+
CMV-IE1+
EBV-EBNA1+
EBV/BLFZ1+
BK-LT+
ADV +
MIX+
SS
uDC/SEB
IFN-g (PE)
Donor 11310, CMV-, 1st stim
CD4+ cells
uDC/SEB
uDC/-
CMV-IE1-
EBV-EBNA1-
EBV/BLFZ1-
BK-LT-
ADV -
MIX-
CMVpp65+
CMV-IE1+
EBV-EBNA1+
EBV/BLFZ1+
BK-LT+
ADV +
MIX+
SS
CMVpp65-
IL-2 (PE-efluo710)
Donor 11310, CMV-, 1st stim
CD8+ cells
uDC/SEB
uDC/-
CMV-IE1-
EBV-EBNA1-
EBV/BLFZ1-
BK-LT-
ADV -
MIX-
CMVpp65+
CMV-IE1+
EBV-EBNA1+
EBV/BLFZ1+
BK-LT+
ADV +
MIX+
SS
CMVpp65-
IFN-g (PE)
Donor 11310, CMV-, 1st stim
CD8+ cells
uDC/SEB
uDC/-
CMV-IE1-
EBV-EBNA1-
EBV/BLFZ1-
BK-LT-
ADV -
MIX-
CMVpp65+
CMV-IE1+
EBV-EBNA1+
EBV/BLFZ1+
BK-LT+
ADV +
MIX+
SS
CMVpp65-
TNFa (PE-Cy7)
Donor 11310, CMV-, 1st stim
CD8+ cells
uDC/SEB
uDC/-
CMV-IE1-
EBV-EBNA1-
EBV/BLFZ1-
BK-LT-
ADV -
MIX-
CMVpp65+
CMV-IE1+
EBV-EBNA1+
EBV/BLFZ1+
BK-LT+
ADV +
MIX+
SS
CMVpp65-
IL-2 (PE-efluo710)
Donor 11310, CMV-, 1st stim
CD4+
CD8+
CMVpp65+
EBV/
BLFZ1+
IFNg(PE)
CD45 RO (APC)
SS
MIX+
SS
CD45 RO (APC)
ADV +
CD27 (FITC)
IFNg(PE)
CD27 (FITC)
Exp. #14, Stim #2, Donor 101310
CMV pp65
CMV IE-1
EBV BLZF1 EBV EBNA1 BKV ST-1
Adv. Hex5
Mix-CMV
Mix +CMV
Adv. Hex5
Mix-CMV
Mix +CMV
SSC-A
CD4+
IFN-γ
CMV pp65
CMV IE-1
EBV BLZF1 EBV EBNA1 BKV ST-1
SSC-A
CD8+
IFN-γ
Exp. #14, Stim #2, Donor 101310
CMV pp65
CMV IE-1
EBV BLZF1 EBV EBNA1 BKV ST-1
Adv. Hex5
Mix-CMV
Mix +CMV
SSC-A
CD4+
TNF-α
CMV pp65
CMV IE-1
EBV BLZF1 EBV EBNA1 BKV ST-1
SSC-A
CD8+
TNF-α
Adv. Hex5
Mix-CMV
Mix +CMV
Exp #13
Donor 012180, CMV1st stim
Donor 012180, CMV-, 1st stim
CD4+ cells
uDC/-
CMVpp65-
CMV-IE1-
EBV-EBNA1-
EBV/BLFZ1-
BK-LT-
ADV -
MIX-
CMVpp65+
CMV-IE1+
EBV-EBNA1+
EBV/BLFZ1+
BK-LT+
ADV +
MIX+
SS
uDC/SEB
IFN-g (PE)
Donor 012180, CMV-, 1st stim
CD4+ cells
uDC/SEB
uDC/-
CMV-IE1-
EBV-EBNA1-
EBV/BLFZ1-
BK-LT-
ADV -
MIX-
CMVpp65+
CMV-IE1+
EBV-EBNA1+
EBV/BLFZ1+
BK-LT+
ADV +
MIX+
SS
CMVpp65-
IL-2 (PE-efluo710)
Donor 012180, CMV-, 1st stim
CD8+ cells
uDC/SEB
uDC/-
CMV-IE1-
EBV-EBNA1-
EBV/BLFZ1-
BK-LT-
ADV -
MIX-
CMVpp65+
CMV-IE1+
EBV-EBNA1+
EBV/BLFZ1+
BK-LT+
ADV +
MIX+
SS
CMVpp65-
IFN-g (PE)
Donor 012180, CMV-, 1st stim
CD8+ cells
uDC/SEB
uDC/-
CMV-IE1-
EBV-EBNA1-
EBV/BLFZ1-
BK-LT-
CMVpp65+
CMV-IE1+
EBV-EBNA1+
EBV/BLFZ1+
BK-LT+
ADV -
MIX-
SS
CMVpp65-
TNFa (PE-Cy7)
ADV +
MIX+
Donor 012180, CMV-, 1st stim
CD8+ cells
uDC/SEB
uDC/-
CMV-IE1-
EBV-EBNA1-
EBV/BLFZ1-
BK-LT-
ADV -
MIX-
CMVpp65+
CMV-IE1+
EBV-EBNA1+
EBV/BLFZ1+
BK-LT+
ADV +
MIX+
SS
CMVpp65-
IL-2 (PE-efluo710)
Exp #13
Donor 062311, CMV1st stim
Donor 062311, CMV-, 1st stim
CD4+ cells
uDC/-
CMVpp65-
CMV-IE1-
EBV-EBNA1-
EBV/BLFZ1-
BK-LT-
ADV -
MIX-
CMVpp65+
CMV-IE1+
EBV-EBNA1+
EBV/BLFZ1+
BK-LT+
ADV +
MIX+
SS
uDC/SEB
IFN-g (PE)
Donor 062311, CMV-, 1st stim
CD4+ cells
uDC/SEB
uDC/-
CMV-IE1-
EBV-EBNA1-
EBV/BLFZ1-
BK-LT-
ADV -
MIX-
CMVpp65+
CMV-IE1+
EBV-EBNA1+
EBV/BLFZ1+
BK-LT+
ADV +
MIX+
SS
CMVpp65-
TNF-a (PE-Cy7)
Donor 062311, CMV-, 1st stim
CD8+ cells
uDC/SEB
uDC/-
CMV-IE1-
EBV-EBNA1-
EBV/BLFZ1-
BK-LT-
ADV -
MIX-
CMVpp65+
CMV-IE1+
EBV-EBNA1+
EBV/BLFZ1+
BK-LT+
ADV +
MIX+
SS
CMVpp65-
IFN-g (PE)
Donor 062311, CMV-, 1st stim
CD8+ cells
uDC/SEB
uDC/-
CMV-IE1-
EBV-EBNA1-
EBV/BLFZ1-
BK-LT-
ADV -
MIX-
CMVpp65+
CMV-IE1+
EBV-EBNA1+
EBV/BLFZ1+
BK-LT+
ADV +
MIX+
SS
CMVpp65-
TNFa (PE-Cy7)
Exp #13
Donor 090110, CMV1st stim
Donor 090110, CMV-, 1st stim
CD4+ cells
uDC/-
CMVpp65-
CMV-IE1-
EBV-EBNA1-
EBV/BLFZ1-
BK-LT-
ADV -
MIX-
CMVpp65+
CMV-IE1+
EBV-EBNA1+
EBV/BLFZ1+
BK-LT+
ADV +
MIX+
SS
uDC/SEB
IFN-g (PE)
Donor 090110, CMV-, 1st stim
CD8+ cells
uDC/SEB
uDC/-
CMV-IE1-
EBV-EBNA1-
EBV/BLFZ1-
BK-LT-
ADV -
MIX-
CMVpp65+
CMV-IE1+
EBV-EBNA1+
EBV/BLFZ1+
BK-LT+
ADV +
MIX+
SS
CMVpp65-
IFN-g (PE)
Donor 090110, CMV-, 1st stim
CD8+ cells
uDC/SEB
uDC/-
CMV-IE1-
EBV-EBNA1-
EBV/BLFZ1-
BK-LT-
ADV -
MIX-
CMVpp65+
CMV-IE1+
EBV-EBNA1+
EBV/BLFZ1+
BK-LT+
ADV +
MIX+
SS
CMVpp65-
TNFa (PE-Cy7)
Reproducibility
Do virus reactive cells proliferate in clusters?
Ellutriated
monocytes
GM-CSF L
IL-4
Day 0
Im DCs
LPS
IFN-γ
Day 4
mature DCs
Pulse with CMV mix
+Lymphos
IL-7 / IL-15
CTL
Separate
Clusters vs
single cells
Restimulate
Day12
Day6
32 μm filter
Day16 Day19
Single cell fraction
Unmanipulated well
clusters
Exp#4
ICC
stain
Donor 080510, CMV-, 2nd stim
CD8+ cells
Unmanipulated well
clusters
Single cell fraction
EBV/BLFZ1-
EBV/BLFZ1-
EBV/BLFZ1-
EBV/BLFZ1+
EBV/BLFZ1+
EBV/BLFZ1+
Reproducibility
Hypothesis:
Virus specific T cells contact rapidly with DCs presenting viral peptides
These contacts last > 4 hours (in contrast to transient non specific contacts 15 min- < 2h) and are
stable (> streptavidin biotin force)
Isolated high specific cells will expand more efficiently in vitro in the absence of “other” cells
DC-T cluster selection
A)
B)
Step A:
6 h incubation of
magnetic bead- loaded DCs with T cells
(magnets, shaker)
Step B:
Negative selection
Legends
Tube,
placed in shaker
Magnetic Dendritic Cell,
Field
Peptide pulsed
CD1a
Magnetic Bead
Ag specific
T cell
C)
T cells
Step C:
Positive selection
Donor 60607, CMV+, 2nd stim
CD8+ cells
CMV/IE1 -
SS
CMV/IE1 -
Magnetic selection
CMV/IE1 +
IFN-g (PE)
CMV/IE1 +
Next steps
Scale up, GMP production
Test alloreactivity
Evaluate methods of reproducibility, eg through in vitro selection
Evaluate whether donor markers can predict results of in vitro
expansion (preevaluation of virus cells in donors?)
Phase I/II clinical trial
ΕΥΧΑΡΙΣΤΩ

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