The Role of Dendritic Cell Genotype in the
Induction of T-cell Responses against
Hematopoietic Malignant Cells after
Allogeneic Stem Cell Transplantation
Reinier Raymakers and Harry Dolstra
Radboud University Medical Center Nijmegen
The Netherlands
10th myeloma workshop, Sydney april 14 2005

Allogeneic transplantation in Multiple Myeloma
(upfront transplantation, cyclo-TBI, T-cell depletion, pre-emptive DLI)
Survival
1
0,8
0,6
BMT
0,4
0,2
0
0
12
2436
4860
7284
96
months
10th myeloma workshop, Sydney april 14 2005

Allogeneic transplantation in Multiple Myeloma
· Myelo-ablative, T-cell depleted: 23 patients treated
(1997-2002), 11 pre-emptive DLI
· 2/23: Molecular CR after therapeutic DLI
· 5/11: Molecular CR after pre-emptive DLI
· 1/11: CR, PCR at detection level (1:10*5)
· 4/11: late relapses, died; 1/11: late relapse, alive
10th myeloma workshop, Sydney april 14 2005

Allogeneic transplantation in Multiple Myeloma
· DLI can be effective, if instituted early after T-cell
depleted transplantation
· In non-T-cell depleted and reduced intensity
transplantation the data suggest less effect of DLI,
given later on, at relapse
10th myeloma workshop, Sydney april 14 2005

Response to Donor Lymphocyte Infusion at
relapse
· CMLcp
80-100%
· MM / AML / MDS
15-20%
· ALL
5-10%
10th myeloma workshop, Sydney april 14 2005

Why does CML show a high DLI response at
relapse
· sensitivity to CTL-mediated killing
· expression co-stimulatory molecules
· secretion immune-stimulatory/suppressive cytokines
· DC as professional APC are of tumor /recipient
genotype
10th myeloma workshop, Sydney april 14 2005

Target antigens for the graft versus tumor
response
Minor Histocompatibility Antigens:
· Male-specific HY antigens
· Autosomal gene-encoded antigens: HA-1,
HA-2, HB-1, ......
Differentially over-expressed proteins:
· Proteinase 3, WT1, ......
Tumor-specific fusion proteins:
· bcr-abl, pml-rara, aml1-eto, ......
10th myeloma workshop, Sydney april 14 2005

Minor H antigen specific T cells in blood in relation to clinical response to DLI
WBC
CD8+HA-2
CD8+HA-1
CD8+HY
GVHD I skin/mouth
tDLI (0.7x108/kg)
Marijt et al. PNAS 2003
10th myeloma workshop, Sydney april 14 2005

Allogeneic T-cell responses after HLA-matched SCT
Phase 3: Lysis and apoptosis
of recipient target cells
Phase 1:
Phase 2:
Presentation of
Clonal expansion
Leukemia
GVT
minor antigens
and migration to
to donor T cells
target tissue
T
T
T
Hematopoietic
GVM
T
T
DC
T
GVHD
Epithelial
10th myeloma workshop, Sydney april 14 2005

Role DC genotype in alloimmune response
Cross-
Direct
presentation
presentation
Recipient
Donor
antigens
CD8+ T cells
T
Recipient DC
Donor DC
10th myeloma workshop, Sydney april 14 2005

Inactivation of Recipient APC and Replacement with Donor APC is
associated with reduction of CD8+ T cell-mediated GVHD!
C3H.SW BM (T-depleted)
9.5 Gy
+ CD8+ T cells
GVHD
Recipient:
1.
B6
+++
2.
B6 b2m-/- B6 (recipient APC no class I)
-
3.
B6 B6
+++
4.
C3H.SW BM B6 (no T-cells)
-
Shlomchik et al. Science 1999; Zhang et al. J. Clin. Invest. 2002;
Shlomchik et al. Exp. Hematol. 2003
10th myeloma workshop, Sydney april 14 2005

Role APC genotype in mouse BMT models
Inactivation of Host APC and replacement with Donor APC is associated with
reduction of CD8+ T cell-mediated GVHD
Shlomchik et al. Science 1999; Zhang et al. J. Clin. Invest. 2002
Once initiated, CD8-mediated GVHD is intensified by donor APCs
But, donor APCs are not required for CD8-mediated GVL
Matte et al. Nat Med. 2004
DLI mediate superior GVT response in mixed compared to full chimeras:
a criticale role for host APCs
Mapara et al. Blood 2002
10th myeloma workshop, Sydney april 14 2005

Patient HB
Chimerism
Serum Free Light chains
120
Free Kappa mg/l
100
100
Free Lambda mg/l
T cells
80
80
NK cells
60
Mono
60
cells
40
MDC
40
donor
PDC
%
20
RIC-SCT
20
Granulo
0
0
0,5
1
269
3
4
4
4
4
4
4
4
4
5
-0
-0
-0
-0
-0
-0
-0
-0
-0
-0
1
1
3
4
6
8
9
1
2
2
-1
-0
-0
-0
-0
-0
-0
-1
-1
-0
months after RIC-SCT
5
4
4
3
2
1
0
9
9
7
2
1
0
2
1
0
2
0
2
1
Datum
GVHD status: ext. GVHD
10th myeloma workshop, Sydney april 14 2005

Patient SPA
Chimerism
Serum Free Light chains
300
Free Kappa mg/l
100
250
Free Lambda mg/l
80
T cells
200
12 mo after
NK cells
60
cells
RIC-SCT
150
Mono
40
MDC
100
donor
6 mo after
%
RIC-SCT
20
PDC
50
0
0
0,5
1
23
69
4
4
4
4
4
4
4
5
-0
-0
-4
-0
-0
-0
-0
-0
1
3
4
-0
6
8
9
1
2
2
-0
-0
-0
-0
-0
-0
-1
-1
-0
4
4
3
2
1
9
9
7
months after RIC-SCT
1
0
2
0
1
0
2
0
2
1
Time
GVHD status: NO
10th myeloma workshop, Sydney april 14 2005

Efficacy of Recipient and Donor MODC in T cell activation
1.0
Target cells:
medium
nm)
DC recipient
DC stem cell donor
release 450 0.5
at
DC HLA-mismatched donor
IFN- D(O
0.0
T cells
T cells
T cells
primed with
primed with
primed with
donor DC
recipient DC
HLA-mismatched DC
Expansion
0
3.5
4.5
10th myeloma workshop, Sydney april 14 2005

Efficacy of Recipient and Donor MODC in T cell activation
Donor CD8+ T cells
stimulated 2x with Recipient MODC
medium
DC donor
EBV-LCL donor
DC recipient
EBV-LCL recipient
Limiting dilution
A2
restricted
clones
35 clones:
19 recipient specific
B/C
1 EBV specific
restricted
15 non-specific
clones
0.0
0.5
1.0
1.5
2.0
2.5
IFN- release (OD at 450 nm)
10th myeloma workshop, Sydney april 14 2005

HOVON 54 MM patient;
Treated with RIC allo-SCT
Clinical feasibility study:
Host DC vaccination
In MM patients
Evaluation for
after mini allo-SCT
GVHD, chimerism, disease status
M-component /ASO-PCR +
M-component
No or Lim cGVHD
ASO-PCR neg
Recipient DC
Follow up
at >6 months
Recipient DC + DLI
Increase M-component
3 months later
ASO-PCR
Evaluation for
GVHD, chimerism, disease status, GVM
10th myeloma workshop, Sydney april 14 2005

Production DC vaccine
Apheresis procedure prior to RIC-alloSCT
Removal of platelets
Storage of PBMC in liquid nitrogen tank: 4 small bags of 3x109 PBMC/bag
Inclusion patient into the study
Generation DC vaccine from thawed PBMC
GM-CSF, IL-4
IL-1, IL-6, TNF-, PGE2
6 days
2 days
CD14+ monocytes
Immature DC
Mature DC
10th myeloma workshop, Sydney april 14 2005

MoDC generated from apheresis material:
Fresh vs cryopreserved
100
75
cells
50
positive
%
25
0
CD3
CD19
CD14
CD80
CD83
CD86
CD11c
CD40
CCR7
fresh 6-well plate
fresh teflon bag
cryopreserved 6-well plate
cryopreserved teflon bag
Maturation: 3 days with IL-1, IL-6, TNF- and PGE2
10th myeloma workshop, Sydney april 14 2005

DC genotype and activation after SCT
SCT
pDLI
tDLI
Relapse
IS
0
3
6
months after SCT
Recipient
Donor
?
DC chimerism
DC chimerism
Cytokine release
+++
++
+
-
-
Tissue damage
Recipient Ag
presentation
10th myeloma workshop, Sydney april 14 2005

Conclusions:
· The goal of alloSCT should be a molecular / M-
component neg CR
· If no GVHD /GVM effect and DC's are of donor origin
DLI alone will be ineffective to induce GVHD / GVM
· In vitro recipient DC's, not donor DC's are capable to
induce GVHD /GVM
· The possibility of recipient DC to induce GVHD / GVM
effect has to be proven in vivo in a clinical study
· We showed DC's can be generated from frozen
PBMC's collected before allo transplantation
10th myeloma workshop, Sydney april 14 2005

Acknowledgements
Department of Hematology
Central Hematology Laboratory
Michel Schaap
Frans Maas
Henriette Levenga
Hanny Fredrix
Theo de Witte
Ingrid Overes
Rob Woestenenk
Agnes van Horsen
Tumor Immunology Laboratory
Bennie Esendam
Jolanda de Vries
Marianne Bakker
Gosse Adema
Laura Groothuis
Carl Figdor
Paul Ruijs
Frank Preijers
ABTI
Harry Dolstra
Joop Goertz
Ina Klasen
Dept. Hematologie, UMC Utrecht
Henk Lokhorst
Tuna Mutis
10th myeloma workshop, Sydney april 14 2005

Document Outline