Transcript Berlino

Treg adoptive immunotherapy
in full-haplotype mismatched HSCT
Massimo Fabrizio Martelli
Hematology and Clinical Immunology Section
University of Perugia, Italy
Factors involved in engraftment of
T-cell depleted Haploidentical HSCs
Conditioning
Conditioning
sTBI
Thiotepa
Fludara
ATG
8 Gy TBI in a single fraction
at 16 cGy/m
Thiotepa
ATG
Fludarabine
Graft
Median
CD34+Cells
MedianDose
dose of CD3+
cells
6
4 / kg
12,8x10
b.w.
1.5 x 10/kg
Stem
Stem
Stem Median Dose of CD3+ Cells
Stem
Stem
1x104/kg b.w.
Stem
Stem
Stem
Stem
Stem
T cell
Median Dose of CD20+ Cells
4.1x10
b.w. cells
Median
dose4/kg
of CD34+
12 x 106/ kg
Reisner Y and Martelli MF: Immunol.Today 1995, 16, 437
Engraftment
GvHD
100
No of patients=196
90
80
70
60
50
40
92%
98%
No post-transplant
immunosuppression
30
20
10
0
2%
Primary
Overall
Acute
Aversa F. et al., N Engl J Med 1998;339:1186-1193
Aversa F. et al., J Cln Oncol 2005;23:3447-3454
3%
Chronic
Post-transplant generation of alloreactive NK
repertoire (Ruggeri L., Velardi A. Blood 1999)
NK repertoire Host targets
cells/cmm
High-intensity
conditioning
600
CD16
400
KIR2DL1
HLA-C
group 1
Cw2/Cw4
KIR2DL
2/3
Missing
self
KIR3DL1
HLA-Bw4
CD8
200
CD4
0
0 50 100150200
Stem
Stem
T cell Stem
Stem Stem
Stem Stem
Stem Stem
Stem
Stem
The reconstituting NK cells have the
same repertoire as the donor,
including high frequency of donor
versus recipient alloreactive NK clones
A potentially NK alloreactive donor
Mega-dose stem cells from “allo NK” donor
occurs in nearly 50% of transplant pairs
No post-transplant immune suppression
Post-transplant leukemia relapse in T-cell depleted haplo HSCT
64 AML Patients (CR I #24, CR II #28, CR≥III #12 )
Cumul. incidence of relapse
1,0
0,8
0,6
Non-NK alloreactive (n=32)
0,4
0,2
P = 0.03
NK alloreactive (n=32)
0,0
0
5
10
15
20
Years
Ruggeri et al., Science 2002; Blood 2007; Stern et al., Blood 2008; updated 2011
Disease free survival in high-risk AML
T-cell depleted haplo HSCT
0.50± 0.09
0.35± 0.07
CR 1 (n=34)
CR ≥ 2 (n=49)
Prob of DFS
Prob of DFS
T-cell depleted haplo HSCT
0.48 CR 1
CR ≥ 2
EBMT-ALWP survey
Aversa F. et al., N Engl J Med 1998
Aversa F. et al., J Cln Oncol. 2005
Ciceri et al. Blood 2008
High-risk AML
Diagnosis and HLA-typing
No HLA-identical
sibling
Search for
alternative donors
the perplexing question:
which is the best option?
URD, partially matched
No well-matched
URD
UCB unit
Haplo family member
Post-transplant immune recovery in T-cell depleted
full haplotype mismatched HSCT
Very narrow T- cell repertoire
• Few T- cells in the graft so as to prevent GvHD
• Extra in vivo T- cell depletion by ATG which also
antagonizes T-cell homeostatic expansion
Cumulative incidence of TRM
0.36 for 145 AL patients in any CR
CNS Others
toxicity
MOF
GvHD
Rejection
TOXO
1
E.COLI
1
Fatal Infections
2
STREPTO
4
PSEUDOM
I.P.
1
CANDIDA
10
ASPERG
Infection
66%
1
HHV6
EBV
2
ADENO
2
16
CMV
0
2
4
6
8
10
12
14
16
18
Improving post-transplant immunity after HLA-haploidentical HSCT
Adding back mature donor T-cells
that are pathogen specific
a) anti CMV or anti Aspergillus CD4+ clones after
screening for cross-reactivity to host alloantigens
b) anti EBV
Adding back mature donor T-cells
with a broad repertoire
Clinical techniques to prevent graft versus host disease
a) suicide gene insertion (i.e. HSV-tk) into T-cells
allows switch-off of GvHD
b) donor T cells ex vivo depleted of anti-host alloreactivity
( i.e. photodynamic purging of alloreactive T cells; depletion
of activated T cells with anti-CD25 bound to beads)
Rebuilding post-transplant immunity
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
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
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
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
In vivo dynamics of regulatory T-cell trafficking and survival predict effective
strategies to control graft-versus-host disease following allogeneic transplantation
Nguyen et al. Blood 2007, 109:2646-2656
The impact of regulatory T cells on T-cell immunity following hematopoietic cell
transplantation
Nguyen et al., Blood 2008, 111:945-953
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
Selection and Characterization of CD4+CD25+ Regulatory T Cells
1st step:
Depletion of
CD8+/CD19+cells
Leukapheresis product
Fully Automated
Immunomagnetic
Selection of
CD4+CD25+Cells
2nd step:
Enrichment of
CD25+ cells
Gate on CD4CD25+high
Starting fraction
CD25
Gate on CD4CD25+
CD4
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)
FoxP3
Final fraction
%CD4CD25high
256 (185.6-365.4)
0.3 (0.12- 0.89)
N° cells (x 106) 36.12 (19.98 - 84)
CD127
Fox P3+ cells
71.9 ± 15 %
33.6 (14.4-39.6)
68.6 (20.9-143)
Di Ianni M et al. Blood 2011; 117:3921-3928
Selection and Characterization of CD4+CD25+ Regulatory T Cells
1st step:
Depletion of
CD8+/CD19+cells
Leukapheresis product
Fully Automated
Immunomagnetic
Selection of
CD4+CD25+Cells
2nd step:
Enrichment of
CD25+ cells
Starting fraction
Final Cell Fraction
Final fraction
Cells (x109)
1060 (540-1370)
280 (202- 390)
%CD4CD25
3.0 (1.5-7.45)
92.4 (90-97.1)
Enhanced expression of CD62L, CD39,
GITR, CTLA4
N° cells (x 106) 330 (221-1020)
256 (185.6-365.4)
CD45RO was the predominant isoform
while the CD45RA was around 10%.
%CD4CD25high
0.3 (0.12- 0.89)
N° cells (x 106) 36.12 (19.98 - 84)
33.6 (14.4-39.6)
68.6 (20.9-143)
Harvesting Tregs from peripheral blood before stem cell
collection increases cell number in starting fraction and
significantly enhances the Treg count in the final fraction
Post HSC collection
Total n° Cells (x 106)* 163.8 (58.2 - 387)
%CD4CD25
N° cells (x 106)*
%CD4CD25high
N° cells (x 106)*
N=8
88.7 (76.2 – 91.7)
149.9 (57.2 – 321)
11.8 (0.9 – 25.5 )**
17.0 (0.7 - 41.8)**
Pre HSC collection
Total n° Cells (x 106)*
280 (202 - 390)
%CD4CD25
N° cells (x 106)*
92.4 (90 – 97.1)
256 (185.6 – 365.4)
%CD4CD25high
N° cells (x 106)*
33.6 (14.4 – 39.6)**
68.6 (20.9 - 143)**
N=15
* median+range
** p<0.05
Tregs inhibit MLR in a dose dependent way
100
% of Inhibition
80
60
40
20
0
T resp + Tregs 1:2
T resp + Tregs 1:1
T resp + Tregs 1:0,1
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.
Treg and Tcon adoptive immunotherapy in haplo HSCT
for patients with high risk AL
10x106/k
g
2x106/kg
CD34+
days
1st clinical trial
Sept 2008-Oct 2009
Tcons
8 Gy TBI
Thiotepa
Fludarabine
Cyclophosphamide
Blood 2011; 117:3921
Di Ianni M et al.
1x106/kg
In vivo expansion
of donor Tregs
in the setting
of HLA disparity
No post-transplant
immunosuppression
TBI based conditioning
Tregs
PRIMARY ENGRAFTMENT
26/28 (93%)
80
60
Days post BMT
Days post BMT
60
40
20
0
>500
>1000
40
20
0
>25000
>50000
3
ANC/l
PLT/mm
GRAFT vs HOST DISEASE
Grade ≥II
2/26
The two cases of GvHD were among the 5 patients who
had received 4x106 Kg/bw Tregs and 2x106 Kg/bw Tcons
Pattern of immunoreconstitution
Recovery of CD4+ and CD8+ T cell subpopulations
100
Days post BMT
150
100
50
0
>50
>100
80
60
40
20
0
>200
>50
>100
CD4/l
CD8/l
Spectratyping
250
Spectratype
compexity
Score
score
Complexity
Days post BMT
200
Donors
200
150
100
50
0
1
2
3
4
5
6
7
8
Months after transplant
9
10
11
12
>200
Proliferating CD4+ pathogen
-specific T cells per 106 cells
Reconstitution of pathogen-specific T-cell repertoire
Limiting dilution analyses of pathogen-specific CD4+ and CD8+ cells
1000
Standard Haplo (n=150)
Haplo with T-reg (n=26)
ASP
Cand
CMV
ADV
HSV
VZV
Toxo
750
500
250
INF-g producing CD8+ pathogen
-specific T cells per 106 cells
0
15
10
5
0
0
2
4
6
8
10
12
0
2
4
Months after transplant
6
8
10
12
T cell response to CMV: cytokine profiles
TNF-a
IL-2
IFN-y
IL-13
Sbj
TNF-a
IL-2
IFN-y
VE-SE 1
GI-MO 17
RO-DO 20
LE-AN 23
BA-SI 24
CD4+
CD8+
IL-13
Naive and Memory CD4+ and CD8+ reconstitution
1500
CD4+ CD45RO+ cells/ l
CD4+ CD45RA+ cells/ l
250
200
150
100
50
0
1000
500
0
1
3
6
9
12
1
Months after transplant
500
6
9
12
2500
CD8+ CD45RO+ cells/ l
CD8+ CD45RA+ cells/ l
3
Months after transplant
400
300
200
100
0
2000
1500
1000
500
0
1
3
6
9
Months after transplant
12
1
3
6
9
Months after transplant
12
Reconstitution of peripheral blood B cell number, B cell repertoire
and seric levels of immunoglobulins
1250
Fr1-JH
Fr1-JH
CD20/l
1000
750
Fr2-JH
Fr2-JH
500
250
Fr3-JH
0
30
60
90
Fr3-JH
120
Days post BMT
Day + 30 post BMT
IgG
IgA
IgM
3 months
733 (80-1530)
31 (1-72)
71 (17-229)
6 months
692 (411-876)
44(8-111)
63 (45-140)
Day + 90 post BMT
Are these patients immunologically competent ?
•
•
In accordance with ISS guidelines
7 subjects ( ≥ 3 months after stem cell transplantation) were vaccinated against
pandemic influenza with 2 doses of MF59-H1N1california. No vaccination for
seasonal flu.
6 months
Day
0
30
60
visit
1
2
3
240
4
Hemoagglutinin Inhibition Assay (HI)
STRAIN
ID samples
A/H1N1/CALIFORNIA/2009
VISIT 1
VISIT 2
A/H1N1/BRISBANE/2009
VISIT 3
1
5
5
5
5
5
6
20
20
40
40 NA
11
20
20
40
40
640
17
20
20
20
20
20
20
20
40
23
20
20
24
20
20
VISIT 1
5
VISIT 2
A/H3N2/BRISBANE/2009
VISIT 3
5
5
5
5
5
640
640 NA
640
10
10
320
320
320
40
40
40
40
320
320
40
40
40
40
40
80
40
40
80
80
20
20
40
40
160
160
40
40
NA
5
5
VISIT 1
5
VISIT 2
VISIT 3
5
5
5
5
5
5
5
5 NA
320
5
5
5
5
5
5
40
40
5
5
5
5
5
5
80
160
160
5
5
5
5
5
5
80
80
80
80
5
5
5
5
5
5
40
40
40
40
5
5
5
5
5
5
NA
HI titer ≥1:40 = protection against H1N1
HI ≥ 4 fold = vaccination efficacy
5
5
NA
Profile over time of Flu specific CD4+ after MF59H1N1
vaccination
california
% Ctk+ CD4+ / CD4+ T cells
0.1
0.08
0.06
0.04
0.02
0
Patient No.
6
11
17
pre immune
30 days post 1 dose
30 days post 2 dose
20
23
24
Rebuilding post-transplant immunity
Adoptive immunotherapy with naturally occurring
polyclonal Tregs
controls alloreactivity of a significant number of
coinfused Tcons
favors immune reconstitution since alloantigen-specific
Tregs do not cross-inhibit pathogen- specific Tcon
responses
Tregs and Tcons in haplo HSCT
for patients with high risk AL
0.8
Veno-occlusive disease (3)
Multi-organ failure(1)
Adenoviral infection (1)
Adenoviral infection and GvHD (1)
GvHD (1)
Bacterial sepsis (1)
Systemic toxoplasmosis (1)
Fungal pneumonia (4)
CNS aspergillosis (1)
0.8
0.6
0.2
0.4
0.4
0.6
1.0
TRM
0.0
0.0
0.2
Probability
1.0
Disease Free Survival
0
1
2
3
4
Years
Median follow up: 35 months (range 32 – 43)
0
1
2
3
Years
Updated 16-03-2012
4
5
Treg and Tcon adoptive immunotherapy in haplo HSCT
for patients with high risk AL
10x106/k
g
T regs
CD34+
days
1st clinical trial
2nd clinical trial
Sept 2008-Oct 2009
May 2010-Oct 2011
8 Gy TBI
Thiotepa
Fludarabine
Cyclophosphamide
Blood 2011; 117:3921
Tcons
Alemtuzumab
(20mg/sqm; day-21)
1x106/kg
8 Gy TBI
Thiotepa
Fludarabine
ASH 2011
Di Ianni M et al.
In vivo expansion
of donor Tregs
in the setting
of HLA disparity
No post-transplant
immunosuppression
TBI based conditioning
2x106/kg
PRIMARY ENGRAFTMENT
23/23 (100%)
60
Days
Days
30
20
10
40
20
0
0
ANC>500
PLT>20000
GRAFT vs HOST DISEASE
Grade ≥II 4/23 (17%)
2/4 patients resolved GvHD,
2/4 patients died of GvHD
PLT>50000
Tregs and Tcons in haplo HSCT
for patients with high risk AL
CTX Prot
Alemtuzumab Prot
0.6
0,4
Fulminant Hepatitis (1)
GvHD (2)
HHV6 pneumonia (1)
HHV6 disease (1)
Fungal Pneumonia (3)
Fusariosis (1)
0.2
0,6
0.4
Probability
0,8
0.8
1,0
TRM
1.0
DFS
0,0
0.0
0,2
FU
0
10
20
30
40
Years
Median follow up: 19 months (range 11 – 24)
0
1
2
3
Years
Updated 16-03-2012
4
5
Tregs and Tcons in haplo HSCT
for patients with high risk AL
DFS
Alemtuzumab
Lot
#F8003H01(14 pt)
0.8
#84039D (9 pt)
Probability
0.4
0.0
0.0
0.2
0.2
Alemtuzumab Lot #84039D
Alemtuzumab Lot #F8003H0
0.6
Lot
0.4
Alemtuzumab
0.6
0.8
1.0
1.0
TRM
0
1
2
Years
3
4
5
0
1
2
Years
3
4
Pattern of immunoreconstitution
1st Alemtuzumab lot (# 84039D)
days after transplant
days after transplant
Recovery of CD4+ and CD8+ T cell subpopulations
>50
>100
CD4/mmc
>200
>50
>100
CD8/mmc
>200
Within 100 days, mean values of CD4+ and CD8+ T cells are more than 200/mmc
Treg and Tcon adoptive immunotherapy in haplo HSCT
for patients with high risk AL
10x106/k
g
T regs
CD34+
days
Jan 2012
Thymoglobuline
Tcons
(7.5mg/kg; day -21)
8 Gy TBI
1x106/kg
Thiotepa
Fludarabine
Gancyclovir
Foscarnet
Ambisome
ESA pentanoic acid
No post-transplant
immunosuppression
TBI based conditioning
2x106/kg
Treg and Tcon adoptive immunotherapy
in full haplotype mismatched HSCT
Post-Transplant Leukaemia Relapse
Demographics:
25 patients (NK allo 11/25):
22 AML (NK allo 10/22)
1 ALL
2 Biphenotypic AL
Relapse: 2/25
2/2 with FLT3-ITD+, NMPc+ AML
1/2 in molecular relapse at transplant
2/2 transplanted from a non-NK allo donor
Median follow up: 20 months (range 8 – 41)
Updated 01-03-2012
How is the GvL effect maintained?
Treg immunotherapy did not impact upon
post-transplant generation of donor vs recipient
alloreactive NK cell clones
High number of infused Tcons
No post-transplant immunosuppression
Translational Research in Full-Haplotype Mismatched Transplant
HEMATOLOGY AND IMMUNOLOGY SECTION
UNIVERSITY OF PERUGIA
Head:Prof Brunangelo Falini
BMT Unit
Franco Aversa
Alessandra Carotti
Adelmo Terenzi
Rita Felicini
Antonio Pierini
Valentina Zoi
Cinzia De Fazio
Maria Speranza Massei
Graft Processing
Franca Falzetti
Mauro Di Ianni
Paolo Sportoletti
Tiziana Zei
Roberta Iacucci
Elisabetta Bonifacio
Beatrice Del Papa
Debora Cecchini
Alain Bell
DEPARTMENT OF IMMUNOLOGY
WEIZMANN INSTITUTE OF SCIENCE
Head: Prof Yair Reisner
Immunology
Andrea Velardi
Loredana Ruggeri
Katia Perruccio
Elena Urbani
Antonella Tosti
Flora Castellino
Chimera of Arezzo
c.400 BC
A votive bronze dedicated to the supreme
Etruscan God of day, Tin orTina