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Alloreattività e Tolleranza nei
Trapianti di Cellule Staminali
Emopoietiche Allogeniche
Massimo Fabrizio Martelli
41° Congresso Nazionale SIE14-17 Ottobre 2007
BOLOGNA
Ematologia ed Immunologia ClinicaEmatologia ed Immunologia Clinica
Università degli Studi di Università degli Studi di PerugiaPerugia
Alloreattività e Tolleranza nei
Trapianti di Cellule Staminali
Emopoietiche Allogeniche
Massimo Fabrizio Martelli
41° Congresso Nazionale SIE14-17 Ottobre 2007
BOLOGNA
Ematologia ed Immunologia ClinicaEmatologia ed Immunologia Clinica
Università degli Studi di Università degli Studi di PerugiaPerugia
E. DONNALL THOMAS
1990 Nobel Laureate in Medicine
for their discoveries concerning organ and cell
transplantation in the treatment of human disease
Preparative Regimens
(chemotherapy + TBI)
Immunosuppression
Malignant Cell Eradication
I.V.INFUSION
of HSCs
Hematopoietic and
Immunological
Recovery
Graft versus Host
Disease Prophylaxis
Supportive
Care
Allogeneic Hematopoietic Stem Cell Transplant
Bone marrow
harvest from
HLA identical
sibling
In the contest of conventional allogeneic HSCT
donor T-cell alloresponses are responsible for
three major transplant events:
• Engraftment
• Graft versus Host Disease
• Graft versus Leukemia Effect
Presentation of recipient antigens to donor T-cells
by host antigen presenting cell
+
Donor Donor
Recipient
APC
Recipient
APC
Current immunosuppressive armamentarium
Inhibitors of DNA synthesis
Methotrexate
Azathioprine
Mycophenolate mofetil
Inhibitors of cytokine production
Cyclosporine
Tacrolimus
Inhibitors of cytokine binding
IL-2 receptor-specific monoclonal antibody
Inhibitiors of cytokine receptor signal transduction
Rapamaycin
They target the cascade of events leading from antigen
recognition, processing and presentation to clonal proliferation
of immune effectors cells
GvHD after standard prophylaxis
• Acute, grade II-IV • Chronic
79%51-62 y
30%45-50 y
20%< 20 y
20-50%MUD
10-35%sibling
30-70%MUD
10-33%sibling
Duration of immunosuppressive treatment for chronic GvHD
Stewart BL Blood, 104, 3501, 2004
Time to Discontinuation of Immunosuppression
Cumulative incidence of discontinued immunosuppressive treatment without
relapse among all patients (panel A), among patients transplanted with mobilized
peripheral blood (B), among male recipients with female donors (C) and among
patients with HLA disparity (D)
Development of Tolerance
Donor T cells that develop in the recipientthymus after HSCT do not cause acute GvHD
Those that express receptors for recipients alloantigensare eliminated through interactions with thymicepithelial cells and also with dendritic cells of therecipient that may survive for several months aftertransplantation.
The presence of donor marrow-derived dendritic cells inthe thymus prevents the development of T-cells thatcould recognize and destroy the graft.
T-cell alloresponses against the
recipient’s lympho-hematopoietic
system favor
• engraftment
• eradication of minimal residual disease
Chimerism analysis of a patient using primers M551 and 33.6.Posttransplant samples were sorted into myeloid cells (M) and T cells (T)
on days 56,70, and 100 and showed donor T-cell engraftment preceding
donor myeloid engraftment
Childs R et al Blood 94, 3234, 1999
RIC HLA-identical transplant
Allogeneic HSCT cures leukemia through
the concerted action of two mechanisms:
1) the myeloablative effect of a conditioning
regimen
2) immune cells in the graft eliminating
residual leukemic cells (GvL effect)
As a rule the GvL effect is mediated by T-cell
alloreactions directed against minor recipient
antigens
Horowitz et al, Blood 1990
Pro
bab
ilit
yo
fR
ela
pse
GVL effects in clinical HSC transplants
Less relapse in patients
with aGvHD and/or
cGvHD than in those
without GvHD
More relapses after
identical twin than
allo HSCT
High relapse rates after
T-cell depleted HSCT
GVL effects in HSCT
Donor lymphocyte infusions induce cytogenetic
and molecular remissions after post-HSCT
relapse
Non-myeloablative conditioning which has
little anti-tumor activity gradually achieves
durable complete remissions months after
HSCT
Graft-versus-leukemia effect of DLT: EBMT-95 survey
Kolb HJ et al Blood 103, 767, 2004
Multivariate analyses of alloSCT in
patients(>50yrs) with AML
Reduced Intensity (n=315) versus
Myeloablative Conditioning (n=407)
TRM 0.48 <0.001
Relapse 1.78 <0.001
LFS 1.15 0.24
RIC versus MA Relative
risk
P-value
Aoudjhane et al Leukemia 2005
GvL effect and GvHD
mHAs dominate allogeneic T-cell recognition
of leukemic cells which is not entirely
specific for leukemia-associated antigens.
Therefore, any specific GvL response is
largely masked by GVHD-related GVL effect.
Graft T-Cell Depletion for GvHD
Prevention in HLA-Matched HSCT
Overcoming graft rejection and
post-HSCT leukemia relapse
Graft composition Recipient immunity
after T-cell depletion after standard
of the HSCs conditioning regimen
TT
GvHRGvHR
HvGRHvGRTT
T-CellT-Cell DepletedDepleted HSCTHSCT fromfrom HLA-IdenticalHLA-Identical SiblingSibling
Donor Recipient
CD34CD34
CD34CD34
CD34CD34
TT
TT
TTTT
TT
CD34CD34
Adverse Effects
Graft
Rejection
ImmunologicalImmunological BalanceBalance
Adverse Effects
Rejection
LeukemiaLack of the Graft versus Host
Disease Related
Graft versus Leukemia Effect
T-CellT-Cell DepletedDepleted HSCTHSCT fromfrom HLA-IdenticalHLA-Identical SiblingSibling
Relapse
Adverse Effects
Graft
Leukemia
Myeloablation
Immunosuppression
Myeloablation
Conditioning
Conditioning
T-CellT-Cell DepletedDepleted HSCTHSCT fromfrom HLA-IdenticalHLA-Identical SiblingSibling
Counter Measures
Rejection
Relapse
CONDITIONING REGIMEN
HFTBI TT CY CY
14.4 Gy HFTBI
TT : thiotepa 10 mg/kg
CY: cyclophosphamide 50 mg/kg/day
TheThe effectseffects ofof addingadding myeloablativemyeloablative agentsagents toto
TBI in a mouse model ofTBI in a mouse model of stemstem cellcell competitioncompetition
Treatment Survival 30 days Chimerism status 2 mo
post-transplant post-transplant (donor %)
TBI 44/47 (93%) 3/44 (6.8%)
TBI + CY 46/47 (97%) 5/46 (10.8%)
TBI + TT 26/33 (78%) 21/26 (80%)
TBI + BU 28/34 (82%) 27/28 (96%)
TBI + DMM 14/18 (77%) 13/14 (93%)
Terenzi A. et al. Transplantation 1990,58,717
CONDITIONING REGIMEN
ATG
HFTBI TT CY CY
14.4 Gy HFTBI
TT : thiotepa 10 mg/kg
CY: cyclophosphamide 50 mg/kg/day
ATG Merieux
SBA-E- BM
No post-transplant
immunosuppression
Aversa F. et al. JCO 1999;17:1545
T-Cell-Depleted HLA-Matched Bone Marrow
Transplantation in Acute Leukemia Adult Patients
74%
59
36
1412 %
28
33
79
Disease-free Survival Leukemia Relapse
GraftGraft rejectionrejection 0%;0%; GvHDGvHD 0%0%
Conditioning: 14.4 HfTBI, cyclo, ATG, TT
Inoculum: SBA-E- BM
No post-transplant immunosuppression
Papadopoulos et al. Blood 1998;91:1083
T-cell-depleted HLA-matched Bone Marrow
Transplantation in acute myeloid leukemia adult patients
Disease-free Survival Relapse
These studies showed :
• the drawbacks of T-cell depletion
need to be counterbalanced
• appropriate modifications to the
conditioning regimen must not
significantly increase extra-
hematological toxicity
T-Cell-Depleted HSCT from
HLA-Identical Sibling
Is it still alive?
HighlyHighly MyeloablativeMyeloablative ConditioningConditioning RegimenRegimen
Present strategy in T-cell depleted HSCT
from HLA-identical sibling
GraftGraft Processing Processing
Peripheral blood CD 34+ cells positively
immunoselected using the Milteny device
GraftGraft ContentContent
NoNo post-HSCTpost-HSCT ImmunosuppressionImmunosuppression
CD34+ 10x106 /kg b.w.; CD3+ 1x104 /kg b.w.
Conditioning Regimen and Graft Composition
ATG
TBI TT FLUDARABINE
TBI: 8 Gy in a single fraction at 16 cGy/m
TT : thiotepa 10 mg/kg
Fludarabine: 40 mg/sqm/day
ATG Merieux
CD34+CD34+
No post-transplant
immunosuppression
GraftGraft contentcontent
CD34+ 10x106/kg b.w.
CD3+ 1x104/kg b.w.
T-cell depleted HLA-identical HSCT
T-cell-depletedT-cell-depleted HSCTHSCT fromfrom aa HLA-matchedHLA-matched
donor afterdonor after TBI-Thiotepa-FludarabineTBI-Thiotepa-Fludarabine
10 (3.8-18.7)
1 (0.3-2.3)
Graft composition
CD34+ (x 106/kg)
CD3+ (x 104/kg)
11
4
Disease status at Tx:
CR
Relapse
10/2
2/1
Disease:
AML/ALL
NHL/HD
15
45 (26-63)
Patients
Age, median in yrs (range)
Aversa F. et al
RICOSTITUZIONE POST-TRAPIANTOResults
Globuli Bianchi
0
0,5
1
1,5
2
2,5
3
3,5
4
4,5
5
0 3 7 11 15 17 20 24 27 31 34 37 40 47 54 60 68 75
Piastrine
0
50
100
150
200
250
0 3 7 9 12 15 22 26 33 36 41 48 55 58 65 83 96
Neutrophils Platelets
MedianMedian 12 (12 (rangerange 10-17)10-17) MedianMedian 14 (14 (rangerange 9-26)9-26)
10 / 9 (1-25 months after Tx)
4 (3,6,12,13 months after Tx)
2 (1 GvHD, 1 IP)
Overall / Event-free Survivors
Relapse
Non-relapse mortality
1/15 (DRB1 #)
0/11
Acute GvHD � II
Chronic GvHD
15 (100%)Engraftment
Aversa F. et al
0
100
200
300
400
500
1 2 3 4 5 6 7 8 9 10 11 12
0
100
200
300
400
500
1 2 3 4 5 6 7 8 9 10 11 12
CD4+ cell recovery
Without ATG in the conditioning
With ATG in the conditioning
months
HLA-identical CD34+ cell transplantation
Aversa F. et al
0
1
2
3
4
5
6
7
8
9
10
0
2
4
6
8
10
12
0-30
0-30
31-60
31-60
61-90
61-90
91-120
91-120
121-150
121-150 151-180
151-180
181-210
181-210
241-270211-240 gg TMO
gg TMO
No ATGNo ATG
ATGATG
Evaluable
CMV-positivity
1010
3
8
9
4
8
2
6
3
5
1 1 1
4
3
2
11
8
2
6
5 5 5
2
CMVCMV
AntigenemiaAntigenemia
Aversa F. et al
T-Cell Depleted HSCT
across MHC Barriers
PROBABILITY OF HAVING A BONE MARROW DONOR
Matched
sibling
No Donor
30%
HSCT from an
alternative donor
is the only option
• matched unrelated donor
• unrelated cord blood unit
• mismatched family donor
Matched unrelated donor HSCT
Disadvantages
•• ManyMany high-riskhigh-risk acuteacute leukemialeukemia patientspatients whowho mightmight findfind
anan appropriate donorappropriate donor oftenoften relapserelapse whilewhile the HLAthe HLA
typingtyping isis in progress orin progress or whilewhile waitingwaiting toto start thestart the
transplanttransplant proceduresprocedures
•• More accurateMore accurate matchingmatching inevitablyinevitably reducesreduces thethe
probabilityprobability ofof identifyingidentifying aa suitablesuitable donordonor
•• OtherOther drawbacksdrawbacks::
Age > 50 yrs ,Age > 50 yrs , advancedadvanced stagestage diseasedisease
•• 30% of patients do not find a matched donor
Advantages Disadvantages
Risk of congenitaldisease
Naif status of cordblood lymphocytes
Median search time <1month
Rare haplotypes 20%
Low incidence of GvHD
HLA mismatches
No risk to donor
Low stem cell dose
Slow hematological
recovery
High risk of graft
rejection in adults
UNRELATED CORD BLOOD TRANSPLANT
A2 B12 Dw4A1
A3
B8
B7
Dw3
Dw2
Recipient Donor
B7A3 Dw2
Overall incompatibility : “3 loci” (A+B+D)
HLA-haploidentical three loci mismatched family members
ONE HAPLOTYPE MISMATCHED HSCT
Obvious Advantages
a family donor for almost every patient
no undue delay
HLA-Haploidentical Transplants
A2 B12 Dw4A1
A3
B8
B7
Dw3
Dw2
Re
cip
ien
t
Do
no
rB7A3 Dw2
Overall incompatibility : “3 loci” (A+B+D)
HLA-haploidentical three loci mismatched family membersR
ecip
ien
t
An
tig
en
Pre
sen
tin
g
Cell
Do
no
rT
-lym
ph
ocyte 2% or more of the total T-cells
may be reactive with an
allogeneic MHC determinant.
Only one in 10.000 of the same
T-cell pool is reactive with an
exogenous protein.
In the setting of MHC disparity,
GvH and HvG alloresponses are the strongest
ONE HAPLOTYPE MISMATCHED HSCT
ObstaclesObstacles
T-replete BMT T-depleted BMT
High incidence High incidence
of severe GvHD* of rejection*
*mediated by the high
frequency of anti-host
alloreactive T cells
in unmanipulated grafts
*mediated by residual
anti-donor CTL-p’s
which survive the
conditioning
T-cell Depleted Mismatched HSCT
Experimental Data*
Escalating doses of T-cell-depleted mismatched marrow
cells ensure full donor type engraftment in mice:
• presensitized with donor lymphocytes
• partially reconstituted with graduated number of
host T cells before the transplant
• pretreated with sublethal doses of TBI
*reviewed in Reisner Y and Martelli MF
Immunol Today 1999;20:343-347
‡Lapidot et al Blood 73, 1989
*Bachar-Lustig et al Nat. Med. 1,1995
‡
‡
*
G-CSF Mobilized Peripheral Blood
Hematopoietic Progenitor CellsT-Cell Depletion Procedures
Soybean agglutination and E-rosetting
(1993-95)
E-rosetting and CD34+ selection
(1996-98)
One Step Automated CD34+selection
(1999-2007)
CliniMacs
Megadose of T-Cell Depleted CD34+ Cells
CD3+cells logCD3+cells log depletiondepletion 4.54.5
After Before
CD 34+ Cells
Purity 95%
Recovery 78%
Aversa F. et al., Blood 1994; 84:3948-3955
Aversa F. et al., N Engl J Med 1998;339:1186-1193
Aversa F. et al., J Cln Oncol. 2005;23:3447-3454
Factors involved in engraftment of
T-cell depleted Haploidentical HSCs
Conditioning GraftConditioning Graft
T cellT cell StemStemStemStem
StemStemStemStem
StemStemStemStem
StemStemStemStem
StemStemStemStem
sTBI
Thiotepa
Fludara
ATG
Median dose of CD34+ cells
12 x 106/ kg
Median dose of CD3+ cells
1.5 x 104 / kg
NoNo post-transplantpost-transplant
immunosuppressionimmunosuppression
Median Dose of CD3+ Cells
1x104/kg b.w.
Median Dose of CD20+ Cells
4.1x104/kg b.w.
Median Dose of CD34+Cells
12,8x106/kg b.w.
CONDITIONING REGIMENS
-5 -4 -3 -2 -1 0 -8 -7 -6 -5 -4 -3 -2 -1 0
ATG
TBI TT CY CY TBI TT
FLUDARABINE
ATG
March 1993 - August 1995 October 1995
TBI: 8 Gy in a single fraction at 16 cGy/m
TT : thiotepa 10 mg/kg
ATG (Fresenius): 5 mg/kg/day
Cyclophosphamide 50 mg/kg/day
Fludarabine 40 mg/sqm/day
days
HSCHSC
Martelli MF et al ASH 1995
Fludarabine as an Immunosuppressor
for HSCT Conditioning
Cyclophosphamide, at high dosages combined
with chemo and/or radiotherapy induces:
• haemorrhagic cystytis
• interstitial pneumonia
• cardiotoxicity
• VOD
Fludarabine is highly immunosuppressive and
does not induce notable extra-hematological
complications
1500
1250
1000
750
500
250
0
Clo
nab
lec
ell
s
TBI FLU + TBI TBI + CY TBI + FLU
FludarabineFludarabineAnAn alternativealternative toto CyclophosphamideCyclophosphamide in thein the
conditioningconditioning regimenregimen forfor allo HSCTallo HSCT
Terenzi A. et al. Transplant Proc 1996;28:3101
EngraftmentEngraftment && GvHDGvHD
255255 PatientsPatients withwith AcuteAcute LeukemiaLeukemia
Days after transplantation
17%
6%
0P= 0.01
Acute GvHD grade � II
1993-1995
1995-1998
1999-2005
Days to engraft (>500 N)
SBA n=36
Cellpro n=44
Clinimacs n=175
84%
95%
96%P= 0.0004
Primary Engraftment
G-CSF-
G-CSF+
Overall Engraftment =98% Aversa F. et al., Blood 1994
Aversa F. et al., N Engl J Med 1998
Aversa F. et al., J Clin Oncol. 2005
EVENT FREE SURVIVALEVENT FREE SURVIVAL
ALL (n=108) AML (n=147)
CR 1 (n=23)
CR 1 (n=34)
CR � 2 (n=39)
CR � 2 (n=49)
Relapse (n=46)
Relapse (n=64)
0.27± 0.10
0.25± 0.08
0.05± 0.04
0.50± 0.09
0.35± 0.07
0.14± 0.04
P=0.0003 P=0.0006
Aversa F. et al., Blood 1994; 84:3948-3955Aversa F. et al., N Engl J Med 1998;339:1186-1193Aversa F. et al., J Cln Oncol. 2005;23:3447-3454
Haplo-TransplantHaplo-Transplant in 255 in 255 PatientsPatients WithWith AcuteAcute LeukemiaLeukemia
Comparison of outcomes afterComparison of outcomes after
Unrelated Cord Blood orUnrelated Cord Blood or
HaploidenticalHaploidentical T-cell depleted PeripheralT-cell depleted Peripheral
Blood Stem Cells in Adults with High RiskBlood Stem Cells in Adults with High Risk
Acute LeukemiaAcute Leukemia
V Rocha, F Aversa, M Labopin, G Sanz, F Ciceri, W Arcese, DBunjes, J Rowe, P Di Bartolomeo, F Frassoni, M Martelli and EGluckman on behalf of the Eurocord Group and AcuteLeukemia Working Party EBMT
5° Workshop on Haploidentical Stem Cell Transplantation
Catania October 4-6,2007
