Terapia cellulare con
Linfociti T Regolatori
Marco Romano, PhD student
Dipartimento di Medicina Specialistica, Diagnostica e Sperimentale (DIMES)
Università di Bologna
Ospedale Sant’Orsola – Malpighi
Via Massarenti, 9
40138 Bologna
GRAFT REJECTION
(A) Alloantigen presentation via the direct, semi-direct and indirect pathways following organ transplantation,
and (B) the relative intensity of each antigen-presentation pathway during the post-transplantation (post-Tx)
period. Sagoo et al 2010.
Chronic GRAFT VERSUS HOST DISEASE
phatogenesis
Bruce R. Blazar et al Nature Reviews Immunology 12, 443-458 June 2012
Common Immunosuppressive Drugs
Treg Discovery
FOXP3+ regulatory T cells in the human immune system S. Sakaguchi Nature Reviews Immunology 10, 490-
500 (July 2010)
“How regulatory T cells work”. Vignali D., Lauren W. Collison and Creg J. Workman – Nat Rev Immunol 2008
How Regulatory T cells work
Donor Tregs
In vivo expansion
Ex-vivo
expansion
Infusion
Infusion
Clinical strategy
Infusion of freshly
isolated cells
Background
• First-in-man clinical results of the treatment of patients with graft
versus host disease with human ex vivo expanded
CD4+CD25+CD127- T regulatory cells. Trzonkowski P. et al Clin Immunol. 2009 Oct;133(1):22-6.
• Infusion of ex vivo expanded T regulatory cells in adults transplanted
with umbilical cord blood: safety profile and detection kinetics. Brunstein CG et al. Blood. 2011 Jan 20;117(3):1061-70.
• Donor Regulatory T Cells Infusion in Patients With Chronic Graft-
versus-host Disease
ClinicalTrials.gov Identifier: NCT01903473.
Treg will be administered fresh. A dose of 0.5 x106 Treg/kg should be ideally administered.
PI: Baron F. University Hospital of Liege
Background
• Trial of Regulatory T-cells Plus Low-Dose Interleukin-2 for
Steroid-Refractory Chronic Graft-versus-Host-Disease. ClinicalTrials.gov Identifier: NCT01937468 . PI: John Koreth, MD, Dana-Farber
Cancer Institute
• Therapy of type 1 diabetes with CD4+CD25highCD127- regulatory T cells prolongs survival of pancreatic islets — Results of one year follow-up. N. Marek-Trzonkowska. Clinical Immunology Volume 153, Issue 1, July 2014, Pages 23–30
• ONE STUDY ongoing…
• Thril (ClinicalTrials.gov Identifier: NCT02166177) ongoing...
HD
Lymphocyte
%
CD4+CD25+
%
CD4+CD25high
%
CD4+CD25high FoxP3+
%
1 38,6 3,1 0,7 0,6
2 29 4,7 0,9 0,5
3 22,5 4,7 0,6 0,3
4 29 4,8 0,9 0,3
5 30 3 0,8 0,5
6 32 5,7 0,9 0,3
7 22,4 3 0,7 0,6
8 27,6 3,3 0,6 0,4
mean 28,89 4,04 0,76 0,44
St. Dev 5,21 1,06 0,13 0,13
Peripheral Blood analysis: Healthy Donors
Percentages are expressed as proportion of white blood cells
Patients
Lymphocyte
%
CD4+CD25+
%
CD4+CD25high
%
CD4+CD25high FoxP3+
%
1 39 9,7 1,5 0,9
2 14 2 0,4 0,2
3 24,5 3,6 0,7 0,4
4 28,6 7 1 0,7
5 23 7,5 0,9 0,6
6 15 2,6 0,3 0,3
7 20 3,7 0,6 0,5
8 18 5,6 0,7 0,4
9 30 6,8 0,8 0,4
10 20 5,9 1 0,9
11 15,5 7 0,6 0,5
12 4,9 4 0,8 0,8
mean 21,04 5,45 0,78 0,55
St. Dev 8,86 2,29 0,31 0,23
Peripheral Blood analysis: patients in waiting list for liver transplantation
Percentages are expressed as proportion of white blood cells
Circulating Tregs: HD Vs Patients
HD
Liver
Pts
0
2000
4000
6000
8000
10000
WB
C/u
L
HD
Liver
Pts
0
10
20
30
40
50
Tre
gs/u
L
Phenotypic evaluation
Cryopreservation
Treg function
Tregs purification
Phenotyphic characterization
Gating strategy used to characterize Treg cells pre and post thawing
Cryopreservation’ strategy
Medium: 50% Plasma AB;
40% CliniMacs Buffer
10% DMSO
Cells were cryopreserved using Planer Kryo 560-16
Yeld
Post-thawing0
20
40
60
80
100Patients
Healthy Donors
%
Yield
A
C
B
A: only stimulus
B: Treg/Tresp 1/4
C: Treg/Tresp 1/10
Suppression assay Method
ModFit LTTM 3.1Verify software house
Co-colture (4 days) of
autologous Tregs with CD4+
CD25- (Tresp) stimulated with
anti-CD3 and anti CD28
(5ug/ml) and labelled with
CFSE.
stim
ulus
Treg/
Tresp
1/2
5
Treg/
Tresp
1/1
0
Treg/
Tresp
1/4
0.0
0.2
0.4
0.6
0.8
1.0
*
***
Pro
life
rati
on
(Fo
ld-c
ha
ng
e)
FOLD-CHANGE was calculated as ratio between the” upper generation proliferation index” of Tresp
cultured in the presence of increasing ratios of Tregs and the” upper generation proliferation index” of
CTR colture.
Function of freshly isolated and thawed
Tregs from healthly donors
Stim
ulus
Treg/
Tresp
1/2
5
Treg/
Tresp
1/1
0
Treg/
Tresp
1/4
0.0
0.2
0.4
0.6
0.8
1.0***
Pro
life
rati
on
(Fo
ld c
ha
ng
e)
Fresh Thawed
CD4+CD25+FOXP3+
86,14 %
CD4+CD25+ FOXP3+
89,60
CD4+CD25+127 low
92,43 %
CD4+CD25+127 low
94,43 %
Thawed
STIM
ULU
S
1/10
Tre
g/Tre
sp
1/4
Treg/
Tresp
1/2
Treg/
Tresp
0.0
0.2
0.4
0.6
0.8
1.0
1.2
1.4
1.6
Pro
life
rati
on
(Fo
ld-c
ha
ng
e)
STIMULU
S
Treg/
Tresp
1/2
5
Treg/
Tresp
1/1
0
Treg/
Tresp
1/4
0.0
0.2
0.4
0.6
0.8
1.0
**
**
Fresh
Pro
life
rati
on
(Fo
ld-c
ha
ng
e)
Function of freshly isolated and thawed Tregs
from patients in waiting list for liver
transplantation
CD4+CD25+ FOXP3+
91,93 %
CD4+CD25+FOXP3+
85,93 %
CD4+CD25+127 low
95,43 %
CD4+CD25+127 low
92 %
FOLD-CHANGE was calculated as ratio between the” upper generation proliferation index” of Tresp cultured in
the presence of increasing ratios of Tregs and the” upper generation proliferation index” of CTR colture.
Tregs expansion
Treg Expansion is based on Beads pre-loaded with CD3 and CD28 antibodies
(Miltenyi, Germany) at a bead-to-cell ratio of 4:1. Best expansion is achieved using
medium supplemented with recombinant Interleukin-2 and Rapamicyn
(Miltenyi, Germany)
CD3
CD28
Treg cell MACSiBead
+ rhIL-2 (1000 U/mL)
Rapamicyn (100nM)
DAY 0 7 14 21
IL-2
2
1 x 106 Tregs
Second stimulation Third stimulation
Infusion/
criopreservation
CD8+ depleted
CD25+ enriched
Manufacture
Protocol
One Study
CD8+ depleted
CD25+ enriched
5 Feedings
Medium 50%
increase
Rapa
IL-2 feed 1-4
Cryopreservation
Stimul 1 Stimul 2 Stimul 3 Final Harvest
Phenotype >60%
Bead removal <100b/3x106
cells
Viability >70%
Potency >60%
Sterility
Endotoxin
Mycoplasma
TexMACS +5%hAB
100nM Rapamycin
1000U/ml IL-2
ExpAct Treg beads CD3/28
5 Feedings
Medium 50%
increase
Rapa
IL-2 feed 3-4
5 Feedings
Medium 50%
increase
Rapa
IL-2 feed 1-4
Day -49 Day -13 Day 0 Day +5
Blood procurement
Manufacture Day 0
Final harvest
Tx
DP infusion
Storage of frozen DPDP manufactureTransport Max 24h
Manufacture Day -1
Day 0 Day +37 Day +92Month 3
Starting material
procurement
Manufacture Day 0
Final harvest
Tx
DP infusion
Storage of frozen DPDP manufactureTransport Max 24h
Kidney
Liver
Manufacture Timelines
Why RAPAMYCIN
Immunoregulatory functions of mTOR inhibition . Nat Rev Immunol 2009; Angus W. et al.
DAY 0
DAY 7
DAY 1
4
DAY 2
1
1.0×10 05
1.0×10 06
1.0×10 07
1.0×10 08
1.0×10 09
1.0×10 10
Cell
nu
mb
er
DAY 0
DAY 7
DAY 1
4
DAY 2
1
0.1
1
10
100
1000Donor A
Donor B
Donor C
FO
LD
IN
CR
EA
SE
Tregs Expansion from
Healthy Donors
Phenotypic evaluation of rapamycin
expanded Tregs from HD
DAY 0
DAY 7
DAY 1
4
DAY 2
1
50
60
70
80
90
100
110
CD4+CD25+
CD4+ CD25+ FOXP3+
CD4+CD25+CD127-
%
DAY 0
DAY 7
DAY 1
4
DAY 2
1
0.0
0.5
1.0
1.5
2.0
2.5
5
10
15CD8+ cells
Th17 cells
CD19+ cells
CD14+ cells
CD3+ CD56+ cells%
Function of rapamycin expanded Tregs from
healthy donors
FOLD-CHANGE was calculated as ratio between the” upper generation proliferation index” of Tresp
cultured in the presence of increasing ratios of Tregs and the” upper generation proliferation index” of
CTR colture.
Stim
ulus
1/10
Treg/T
resp
1/4
Treg/T
resp
1/2
Treg/T
resp
0.0
0.2
0.4
0.6
0.8
1.0
**
*
Pro
life
rati
on
(Fo
ld-c
ha
ng
e)
Next step…
Treg stability in the presence of 2 pro-inflammatory milieu:
A: IL-2, IL-1β, IL-6, TGF-β. B: IL-2, IL-21, IL-23, TGF-β.
IL-17 and IFN-γ
production
CTLA-4, GITR and
CD39
TSDR analysis
Differential effects of rapamycin and retinoic acid on expansion, stability and suppressive qualities of
human CD4+CD25+FOXP3+ T regulatory cell subpopulations. Scottà et al. Haematologica. 2013
Aug;98(8):1291-9.
Evaluation of
Impact of immunosuppressive regimen on ex
vivo expanded human regulatory T cells
Cristiano Scottà, Giorgia Fanelli, Julie Hoong, Marco Romano, Mitalee Sukthankar, Giuliana
Guggino, Henrieta Fazekasova, Ratnasothy Kulachelvy, Pablo Becker, Ben Afzali, Robert Lechler
and Giovanna Lombardi
Question:
Can immunosuppressive drugs affect Treg viability,function and trafficking?
Tacrolimus Tacrolimus is used after allogeneic organ transplant to reduce the activity of the patient's immune system and lower the risk of organ rejection. Tacrolimus inhibits calcineurin and prevents the dephosphorylation of NF-AT. Thus it negatively affects both T-lymphocyte signal transduction and IL-2 transcription.
Prednisolone Prednisolone irreversibly binds with glucocorticoid receptors. Its interaction with DNA modifies gene transcription. It induces synthesis of some proteins, and inhibit synthesis of others. Regulation of gene suppression leads to systemic suppression of inflammation and immune response.
Mycophenolate Mofetil (MMF) - MPA
MMF reversibly inhibits inosine monophosphate dehydrogenase (IMPDH) in purine biosynthesis which is necessary for the growth of T cells and B cells. It is extensively used in transplant medicine to suppress T and B cells from attacking donor’s graft.
Immunesuppressive Drugs
Treg Viability in the Presence of Immunosuppressants
Reagent Amount
IL-2 20 IU/ml
anti-CD3/CD28 beads 1:2 (Stimulation)
Rapamycin 100 nM
Methyl-Prednisolone
(mPred)
0-4 ug/ml
Mycophenolate (MPA) 0-2.5 ug/ml
Tacrolimus (Tacro) 0-20 ng/ml
0
1:25
6
1:12
81:
641:
321:
16 1:8
1:4
1:2
Max
0
20
40
60
80
100
Dilution from highest conc
Via
ble
cell
(%
)
In vivo drug distribution
Tacrolimus * : Erythrocyte fraction 70-80%
Plasma fraction 20%
Leukocytes 1%
Mycophenolate * * : Albumin bound ~ 97%
*Zahir et al. The Drug Monit 2004, * * Bullingham et al. Clinical Pharm 1998
Tacrolimus
00.
080.
160.
310.
621.
25 2.5 5 10 20
0
20
40
60
80
100EC50: 2ng/ml
ng/ml
Via
ble
cell
(%
)
MPA
00.
080.
160.
310.
621.
25 2.5
0
20
40
60
80
100EC50: 1ug/ml
ug/ml
Via
ble
cell
(%
)
methyl-Prednisolone
0
0.01
50.
030.
060.
120.
25 0.5 1 2 4
0
20
40
60
80
100EC50: 0.5ug/ml
ug/ml
Via
ble
cell
(%
)
Dose Response Curves to Derive the Concentration of
Immunosuppressant
Immunosuppressants Can Reduce the Proliferative
Ability of Treg Preparations after 5 Days of Culture
CTR
TAC
MPA
mPr
IS M
ix
1
10
100
1000
10000
TAC
CTR
MPA
mPr
IS Mix
**
**
*N
um
ber
of
cell
s (
x10
3)
0
20
40
60
80
100
Pro
life
rati
on
(%
)CTR
TAC
MPA
mPr
IS M
ix
0
20
40
60
80
1001:10
1:3
1:1
Pro
life
rati
on
(%
)Tregs Maintain Full Suppressive Capacity
after 5 days of Culture with Drugs
FOXP3
CD
25
1
0
1000
2000
3000
4000
CTR
MPA
mPr
TAC
IS Mix
CD25 ExpressionM
FI
1
0
500
1000
1500
FOXP3 Expression
MFIIS Mix
(5d)
IS Mix
(5d)
97.3%
98.8%
88.4%
87.4%
HD1
HD2
Immunosuppressants Do Not Affect FOXP3
Expression after 5 Days of Culture
CD39
GIT
R
HLA
-DR
CD95
PD-L
1
CTLA
4
ICOS
TGFbet
a-LA
P
0
20
40
60
80
100
CTR
TAC
MPA
mPr
IS Mix
Nu
mb
er
of
cell
s (
%)
Functional Treg Molecules are not Affected
by Immunosuppressants (5 days)
Acknowledgement
Laboratorio Terapia cellulare, istituto
Seragnoli:
•Francesca Ulbar
•L. Catani
•S. Rizzi,
•M. Motta,
•E. Dan
•D. Sollazzo,
•M. Arpinati
•Prof. RM Lemoli
Immuneregulation Lab
Cristiano Scottà
Prof. Giovanna Lombardi
Prof. Robert Lechler
Giorgia Fanelli
Julie Hoong
Estefania Nova Lamperti
Ratnasothy Kulachelvy
Giuliana Guggino
Ben Afzali
Reuben McGregor
Pablo Becker