utilizzo dei linfociti t regolatori nel trapianto da
TRANSCRIPT
UTILIZZO DEI LINFOCITI T REGOLATORI NEL TRAPIANTO DA DONATORE APLOIDENTICO
Mauro Di Ianni
SIES, Discutiamone Insieme, Firenze 20 Novembre 2014
CD4+CD25+ regulatory T cells preserve graft versus-tumor-activity while inhibiting graft-versus host disease
after bone marrow transplantation
Edinger et al., Nat Med 2003, 9:1144-1150
Naturally arising CD4+ regulatory T cells for immunologic self-tolerance and negative
control of immune responses
Sakaguchi et al., Ann Rev Immunol, 2004, 22:531-562
The impact of regulatory T cells on T-cell immunity following hematopoietic cell
transplantation
Nguyen et al., Blood 2008, 111:945-953
Donor type CD4+CD25+ regulatory T cells suppress lethal acute graft-versu-host disease
after allogeneic bone marrow transplantation
Hoffmann et al., J Exp Med, 2002, 196:389-399
Lessons from animal models
Adoptive transfer of naturally arising CD4+CD25+
regulatory T cells (Tregs), when coinfused with
conventional T lymphocytes (Tcons), prevents GvHD,
while favoring immune reconstitution
Nguyen et al. Blood 2007, 109:2646-2656
In vivo dynamics of regulatory T-cell trafficking and survival predict effective
strategies to control graft-versus-host disease following allogeneic transplantation
Rebuilding post-transplant immunity
Immune reconstitution is preserved in hematopoietic stem cell transplantation coadministered
with regulatory T cells for GvHD prevention
Gaidot et al. Blood 20010, 117:2975-2983
FOXP3
GITR
CTLA-4
CD 62L
CD 39
DONOR
mismatched HSC transplant
RECIPIENT
expansion of
alloantigen
specific Tregs
Treg lymph node APCs
IL 2
activation of
specific donor
Tregs for recipient
alloantigens
migration of
alloantigen
specific Tregs
Tregs
Skin
Gut
Lung
Bone
marrow
CD45RO+ 100%
Nguyen et al. Blood 109, 2646, 2007
Alloantigen
specific Treg
Alloantigen
specific Tcon
Recipient
dendritic cell
Alloantigen-specific Tregs
act in an antigen-specific
manner in vivo during their
effector phase, thus …..
, …
… they control activation and
early proliferation of alloreactive
donor T cells via intereaction
with APCs in priming sites.
Conversely they do not cross
inhibit pathogen-specific Tcon
expansion and response.
First “check point” in lymph nodes
Second “check point”
in peripheral tissues
(skin, gut, liver,lung)
Alloantigen specific Tregs control Tcon alloreactivity
T reg cell Isolation • Magnetic bead separation: Significant enrichment of the CD4+CD25+
FoxP3+Treg cells
• Cell Sorting techniques: Sort CD25 high positive population; significant loss
of FoxP3+ cells.
Sort CD127-/CD4+CD25+ cells; isolating the majority of FoxP3+ cells within an apheresis sample
• Major limitation:
Paucity of T reg cells in the peripheral blood
Expansion of T reg cells may be necessary
1st step:
Depletion of
CD8+/CD19+cells
2ndstep:
Enrichment of
CD25+ cells
Selection of
CD4+CD25+Cells
Selection and Characterization of CD4+CD25+ Regulatory T Cells
Cells (x109) 1060 (540-1370) 280 (202- 390)
%CD4CD25 3.0 (1.5-7.45) 92.4 (90-97.1)
N° cells (x 106) 330 (221-1020) 256 (185.6-365.4)
%CD4CD25high 0.3 (0.12- 0.89) 33.6 (14.4-39.6)
N° cells (x 106) 36.12 (19.98 - 84) 68.6 (20.9-143)
Starting fraction Final fraction
CD
25
CD127
CD4
FoxP3
Gate on CD4CD25+high
Gate on CD4CD25+
Leukapheresis product
Fox P3+ cells
92.05 ± 15 %
Di Ianni et al., Exp. Hematology 2008
Di Ianni et al., Clinical and Exp Immunology 2009
Di Ianni et al., Blood 2011
Di Ianni et al., Best Prac Res Clin Haematol 2011
Di Ianni et al., Transfusion Apheresis Science 2012
CD45RO
CD45RA
88.0% 85.9%
9.6% 8.2% 61.3%
31.8%
0.0% 0.3%
Final Cell Fraction
Murine T lymphocytes lacking CCR7 can generate attenuated GvHD besides robust
GvL responses
CCR7 negative Tregs strongly protect against lethal GvHD
Final Cell Fraction
CD4/CD25 CD4/CD25 CD4/CD25bright CD4/CD25bright
84.2% 93.2%
CD62L
SS
Lin
CD39
SS
Lin
64.9% 95.7%
SS
Lin
CD49d
37.7% 27%
GITR
SS
Lin
33.4% 58.1%
cyCD152
SS
Lin
15.1% 33.8%
Gate on CD4+CD25+ Gate on CD4+CD25+
CD127
Fo
xP
3
96% 3%
Fo
xP
3CD45RA
95% 4%
T regulatory cells characteristics
0
20
40
60
80
100
T resp + Tregs 1:2 T resp + Tregs 1:1 T resp + Tregs 1:0,1
% o
f In
hib
itio
n
Mean SD
T resp + Tregs 1:2 72,4 8,9
T resp + Tregs 1:1 60,8 20,5
T resp + Tregs 1:0,1 25,6 19,1
Tregs inhibit MLR in a dose dependent way
0
10
20
30
40
50
60
70
80
90
T resp allo + T regs T resp auto + T regs
% In
hib
itio
n
HLA identity between Tregs and Tcons is required
for maximun suppression of alloresponses
Donor Tregs do not prevent graft rejection in mismatched transplant
Tregs from a third party donor might not prevent GvHD after HSCT
Alloantigen APCs pre-incubation enhances Treg-mediated immunosuppression
Freshly selected polyclonal
T-reg cells were
co-cultured in the presence
of allogeneic APCs for 48 hours.
Cells were plated under
limiting dilution conditions and
proliferating T cell clones counted.
Inhibition of xenogenic acute GvHD in vivo (NOD/scid IL-2 Rγnull mice)
% f
ree o
f G
vH
D
▀ CD25- (Group I)
▲Tregs (Group II)
▀ CD25- & Tregs (Group III) 0
20
40
60
80
100
1 3 5 7 9 11 13 15 17 19 21
%CD45
Bone marrow Spleen Liver Skin
Days +4 +12 +4 +12 +4 +12 +4 +12
Group I
(Tcon)
5.5 77.4 70.3 91.6 8.1 46 0 7.5
Group II
(Tregs)
3 13.8 80 20.8 1.7 2.8 0 0
Group III
(Tcon+Tregs)
3.1 46.5 47 71.2 3.7 3.75 0 0
Stable composition according to:
CD25 expression -35-40% high
-55-60% int
-5-10% low
FoxP3 expression
-90%
CD127 expression
<20%
Function:
-in vitro inhibition of MLR
-inhibition is enhanced by 48 hrs APC exposure
-in vivo prevention of GvHD
-MHC identity is required for suppression
Next step: clinical application
Characteristics of immunoselected Tregs
Sept 2008-Oct 2009
CD34+ No
po
st-tra
nsp
lan
t
imm
un
osu
pp
ressio
n
T regs
Tcons
days
Treg and Tcon adoptive immunotherapy in haplo HSCT
for patients with high risk AL
2x106/kg
10x106/kg
1x106/kg
Thymoglobuline
3-7.5 mg/kg;
day-21 before Tregs
8 Gy TBI
Thiotepa
Fludarabine
Jan 2012
May 2010-Jan 2011
Alemtuzumab 20mg/sqm;
day-21 before Tregs
8 Gy TBI
Thiotepa
Fludarabine
8 Gy TBI
Thiotepa 4 mg/kg/day
Cyclophosphamide 35 mg/kg/day
Fludarabine 40 mg/sqm/day
1st clinical trial*
2nd clinical trial**
In vivo activation
and expansion
of alloantigen
specific Tregs
* Di Ianni et al., Blood 2011; **Martelli, Di Ianni et al., Blood 2014
Demographics
No of patients 45
Median age (years, range) 40 (20-59)
Gender (male/female) 21/24
Disease and status at transplant
Acute myeloid leukemia 33
CR1 18
≥CR2 15
Acute lymphoid leukemia
12
CR1 8
≥CR2
4
# 25 in the 1st trial (Sept 2008-Oct 2009)
# 20 in the 2nd trial (May 2010
>500 >10000
10
20
30
ANC/l
Days p
ost
BM
T
>25000 >500000
20
40
60
PLT/mm3
Day
s p
ost
BM
T
PRIMARY ENGRAFTMENT
43/45 (95%)
Acute Graft vs Host Disease
Grade ≥II 6/43 (13%)
0 60 120 180 240
0
450
900
Recovery of CD4+ T cells
days post transplant
cell
s/μ
l
Treg Haplo
Standard Haplo
Pattern of immunoreconstitution
0
50
100
150
200
250
1 2 3 4 5 6 7 8 9 10 11 12
Sp
ectr
aty
pe c
om
pexit
y S
co
reC
om
ple
xit
y s
co
re
months post transplant
Spectratyping
Months after transplant
0 2 4 6 8 10 12
1
10
100
1000
10000
0 2 4 6 8 10 12
1
10
100
T-reg Haplo (Campath1+ ATG)
(n=20)
0 2 4 6 8 10 12
1
10
100
1000
10000
Standard Haplo (n=150)
Pro
life
rati
ng
CD
4+
path
og
en
-sp
ecif
icT
cells i
n 1
06 c
ell
s
Pro
life
rati
ng
CD
8+
path
og
en
-sp
ecif
icT
cells i
n 1
06 c
ell
s
0 2 4 6 8 10 12
1
10
100
Reconstitution of pathogen-specific T cells after
HLA-haploidentical transplantation
ASP
Cand
CMV
ADV
HSV
VZV
Toxo
1
10
100
1000
10000
1
10
100
Donors
Months after transplant
0
50
100
0
10
20
1 2 3 4 5 6 7-12 >12 months
CMV reactivation episodes Evaluable Patients
100
28
96
50
82
34
75
22
67
9
56
9
48
1 1
29
20
3
19
6
18
1
18
2
17
1
17
0 0
2
Sta
nd
ard
Ha
plo
T
reg
Ha
plo
CMV reactivations
17 17
Naive and Memory CD4+ and CD8+ reconstitution
1 3 6 9 120
50
100
150
200
250
Months after transplant
CD
4+C
D45R
A+cell
s/
l
1 3 6 9 120
500
1000
1500
Months after transplant
CD
4+C
D45R
O+cell
s/
l
1 3 6 9 120
100
200
300
400
500
Months after transplant
CD
8+C
D45R
A+cell
s/
l
1 3 6 9 120
500
1000
1500
2000
2500
Months after transplant
CD
8+C
D45R
O+cell
s/
l
Naive and Memory CD4+ and CD8+ Reconstitution
Fr1-JH Fr1-JH
Fr2-JH
Fr3-JH
Fr2-JH
Fr3-JH
Day + 30 post BMT Day + 90 post BMT
Reconstitution of peripheral blood B cell number, B cell repertoire
and seric levels of immunoglobulins
30 60 90 1200
250
500
750
1000
1250
Days post BMT
CD
20/
l
IgG IgA IgM
3 months
6 months
733 (80-1530) 31 (1-72) 71 (17-229)
692 (411-876) 44(8-111) 63 (45-140)
0
5
10
15
20
25
0
15
30
45
60
Donor 1 2 3 4 5 6 7 8 9 10 11 12
months post transplant
0
5
10
15
20
25
0
15
30
45
60
Donor 1 2 3 4 5 6 7 8 9 10 11 12
Donor 1 3 6 12
cell
s/c
mm
cell
s/c
mm
% a
llo
rea
cti
ve
NK
clo
ne
s
% a
llo
rea
cti
ve
NK
clo
ne
s
“Standard” haplo Lysis of KIR ligand-mismatched targets
“T-reg” haplo
Treg immunotherapy did not impair regeneration of
donor vs recipient alloreactive NK cell repertoires
(single) KIR2DL1+
(single) KIR2DL2/3+
(single) KIR3DL1+
C2 mismatch
C1 mismatch
Bw4 mismatch
Donor 1 3 6 12
months post transplant
0 1 2 3 4 5
0.0
0
.2
0.4
0
.6
0.8
1
.0
Cumulative incidences of transplant-related mortality P
rob
ab
ilit
y
Years post transplant
All the patients
CTX in the conditioning
Anti-T antibodies
in the conditioning
0 1 2 3 4 5
Years post transplant
0,0
0,2
0,4
0,6
0,8
1,0 P
rob
ab
ilit
y
Disease-free survival
All the patients
Anti T antibodies
In the conditioning
Cyclophosphamide
In the conditioning
Is the graft vs leukemia effect maintained
in patients who received adoptive
immunotherapy with Tregs and Tcons?
FOXP3+ Tregs are indispensable for the maintenance of
immune homeostasis. However, as a double-edged sword,
they can also suppress antitumor immune responses
Treg and Tcon adoptive immunotherapy
in full haplotype mismatched HSCT
A crucial point
Trenado A, Charlotte F, Fisson S, et al. Recipient-type specific CD4+CD25+ regulatory T cells favor
immune reconstitution and control graft-versus-host disease
while maintaining graft-versus-leukemia.
J Clin Invest. 2003;112(11):1688-1696.
Edinger M, Hoffmann P, Ermann J, et al. CD4+CD25+ regulatory T cells preserve graft-versus-tumor
activity while inhibiting graft-versus-host disease
after bone marrow transplantation.
Nat Med. 2003;9(9):1144-1150
Studies in mouse models of mismatched HSCT
0 50 100 1500
20
40
60
80
100
Days
% s
urv
ival
Leuk. + T cons = no leuk, GvHD
Leuk. + T regs/Tcons = survival
w/o leukemia and GvHD
Leuk. + T regs = leukemia
Leukemia
AML primary
cells (7x106)
T cells
(3x106)
Clearance of human leukemia by human
T regs + T cons in immunodeficient (NSG) mice
Martelli, Di Ianni et al., Blood 2014
0
50
100
% l
ys
is
0 50 100 1500
20
40
60
80
100
days
0 50 100 1500
20
40
60
80
100
% s
urv
iva
l Clearance of human leukemia by human
T regs + T cons in immunodeficient (NSG) mice
ALL
Burkitt-like cell line
days
Years post transplant
Pro
bab
ility
Cumulative Incidence of Relapse in High Risk AL Patients
Treg and Tcon adoptive immunotherapy in full haplotype mismatched HSCT
0.05 at median follow-up of 46 months
0 1 2 3 4 5 6
0.0
0
.2
0.4
0
.6
0.8
1
.0
P=0.03
0 5 10 15 20
0.0
0
.2
0.4
0
.6
0.8
1.0
0.0
0
.2
0.4
0
.6
0.8
1
.0
0 1 2 3 4 5
0.0
0
.2
0.4
0
.6
0.8
1
.0
0 1 2 3 4 5
Pro
bab
ilit
y
0.0
0
.2
0.4
0
.6
0.8
1
.0
Non-NK allo
NK allo
Years post transplant
0 5 10 15 20
Years post transplant
Pro
bab
ilit
y
Non-NK allo
NK allo
Non-NK allo
NK allo
Standard Haplo T cell depleted
ALL ALL
Treg Haplo
AML AML
Non-NK allo
NK allo
Cumulative Incidence of Post-transplant Relapse
The GvL Effect
What are the underlying mechanism/s?
2) No post-transplant pharmacological immunosuppression
1)High number of infused Tcons (1x106/kg)
which is non-specific immunologically,
only partially successful in preventing GvHD
and may compromise the T-cell induced GvL effect.
3) Tregs suppress GvHD at the periphery and unleash
the GVL effect in the bone marrow Ruggeri et al., ASH meeting 2014
Adoptive immunotherapy with naturally occurring
polyclonal Tregs is a feasible option in
HLA-haploidentical HSCT since this strategy
controls alloreactivity of a significant number of
coinfused Tcons with low incidence of GvHD
favors immune reconstitution and reduces the risk
of CMV reactivation
is associated with a powerful GvL effect
nTregs for future protocols: which separation and/or expansion strategy?
CD4/CD25/CD45RA is the strongest
immunosuppressive nTreg
and the more stable in culture (Edinger et al, Blood 2006)
Separation and infusion Negative selection with CD8,CD19 and
CD45RO followed by positive selection
with CD25 (CD45RO beads need to be validated)
Separation and in vitro expansion -Polyclonal expansion
(anti-CD28+anti CD3 +IL-2 ± rapamycine)
-Antigen-specific expansion
(more effective than polyclonal)
-Expansion of genetically modified Tregs (TCR with known specificity)
Advantage: no in vitro manipulation
Disadvantage: low Treg number?
Advantage: high Treg number
Disadvantage: in vitro manipulation
Clinical protocol
Phenotype
Immunosuppressive assay
TSDR
Mouse model
Translational Research in Full-Haplotype Mismatched Transplant
UNIVERSITY OF PERUGIA
HEMATOLOGY AND IMMUNOLOGY SECTION
Head: Brunangelo Falini
Alessandra Carotti
Loredana Ruggeri
Adelmo Terenzi
Antonio Pierini Maria Speranza Massei
Elena Urbani
Fabiana Topini
Antonella Tosti
Franca Falzetti
Mauro Di Ianni Lucia Amico
Tiziana Zei
Roberta Iacucci
Beatrice Del Papa
Debora Cecchini
Alain Bell
HSCT Unit and Transplantation
Immunology
Head: Andrea Velardi Department of Immunology
Weizmann Institute of Science
Head: Yair Reisner
Massimo Fabrizio Martelli
Professor Emeritus