Dickinson, A.M.

Hilkens, C.M.U.

Holtick, U.

Marshall, S.R.

Wang, X.N.

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Investigating the role of dendritic cells in extracorporeal photopheresis using an in vitro model 

low-up of 21 adult patients with acute leukemia treated with pe-
ripheral blood HPCT following RIC after being deemed
inadequate for fully myeloablative conditioning. A matched com-
parison arm of 42 consecutive patients transplanted after full my-
eloablative (FMA) conditioning is also reported. Methods: Be-
tween 1999–2004, 21 patients were treated with Fludarabine-based
conditioning prior to infusion of hematopoietic stem cells. Patients
were followed for toxicities and responses. IRB approval was ob-
tained prior to data collection. Kaplan-Meier estimates of survival
were performed. Patient disease characteristics were analyzed us-
ing T-tests to determine any factors that may be associated with
outcome and relapse. Signiﬁcant factors on univariate analysis were
placed into a Cox regression model for multivariable analysis.
Results: Patient characteristics: 76% of patients had AML and
24% had ALL. 14% of patients had relapsed or refractory disease
at transplant. A greater proportion of patients undergoing RIC,
had poor-risk cytogenetics. Median age was 56 yrs. 81% of patients
experienced clinically signiﬁcant acute or chronic GVHD. Overall
post-transplant survival was 29%. Twenty-nine percent (6/21) pa-
tients suffered relapse at a median of 4 months post-transplant.
Cox proportional hazards models for overall post-transplant sur-
vival and logistic regression models for relapse and overall survival
(N  63), showed that the only signiﬁcant predictor for OS and
relapse was cytogenetic risk group (P  .000). Of note, none of the
5 ALL patients transplanted after RIC have relapsed with a median
follow-up of 2.2 years (0.1–3.9 years); among 16 AML pts., 5 have
relapsed with median follow-up of 0.4 years (.1–2.2 years). These
results compare favorably to our cohort of pts. transplanted after
full myeloablative conditioning. Conclusion: The role of RIC
transplants for the treatment of high risk acute leukemia in older
adults remains a promising therapy and warrants further study
(Table).
Patient Characteristics
Characteristics
RIC pts.
N  21 (%)
Comparison
arm (FMA)
N  42 (%)
ALL 5 (24%) 10 (24%)
AML 16 (76%) 32 (76%)
Median age 56 (45–68) 52 (41–62)
Male 14 (67%) 20 (48%)
Disease status at
transplant:
CR1 13 (62%) 30 (71%)
CR2–3 4 (19%) 7 (17%)
Relapsed-
Refractory
3 (14%) 5 (12%)
Donor: Related 12 (57%) 34 (81%)
Unrelated 9 (43%) 8 (19%)
Cytogenetic risk: *
Favorable 1 (5%) 4 (10%)
Intermediate 6 (28%) 19 (45%)
Poor 14 (76%) 18 (43%)
*1 Pt, cytogenetics were unknown
199
INVESTIGATING THE ROLE OF DENDRITIC CELLS IN EXTRACORPO-
REAL PHOTOPHERESIS USING AN IN VITRO MODEL
Holtick, U.1, Marshall, S.R.1, Wang, X.N.1, Hilkens, C.M.U.2,
Dickinson, A.M.1 1. Haematological Sciences; 2. Rheumatology, The
Medical School, University of Newcastle upon Tyne, United Kingdom.
Graft-versus-host disease (GvHD) is the major complication
after allogeneic transplantation and contributes signiﬁcantly to
transplant related mortality and morbidity. Especially steroid
refractory or steroid depending GvHD is linked to poor survival
or life quality. Conventional immunosuppression has very lim-
ited success in these conditions and increases susceptibility for
infection and relapse. Extracorporeal Photopheresis (ECP) is a
promising therapy for acute and chronic GvHD not responding
to conventional immunosuppressive therapy. ECP treatment
seems not to result in a pan-immunosuppression but has quite
selective effects on the pathogenic process in GvHD. The
mechanisms of action of ECP in GvHD known so far include
lymphocyte senescence or apoptosis and cytokine modulation.
Some groups report that antigen presenting cells like dendritic
cells (Dc) might be important for ECP mechanisms. We have
developed an in vitro model of ECP (in vitro PUVA) to inves-
tigate ECP effects on dendritic cells. Initial experiments have
shown the maturation of monocyte derived Dcs treated with in
vitro PUVA (upregulation of CD83, CD86, HLA-DR as well as
reduced endocytosis capacity), but also the induction of apopto-
sis. The stimulatory capacity of in vitro PUVA treated Dcs was
strongly inhibited in autologous and allogeneic MLR. However,
treatment of antigen-primed Dcs resulted in less inhibition,
suggesting factors that might preserve Dc stimulatory capaci-
ties. Immature Dcs, retrieved after coculture with in vitro
PUVA treated lymphocytes, show inhibited stimulatory capacity
on autologous and allogeneic T cells. Currently, we are inves-
tigating the changes in phenotype and cytokine pattern which
could transfer anergy or promote tolerance induction. In par-
allel, we are analyzing effects on monocyte-derived dendritic
cells from patients undergoing ECP treatment for chronic
GvHD. Dcs rendered tolerogenic could play a major role in
ECP mechanisms.
200
HUMAN T LYMPHOCYTE ACTIVATION KINETICS FOR IDENTIFYING AND
TARGETING ALLOREACTIVE T CELLS
Bajwa, R.P.S., McCarthy, P.L., Wallace, P.K., Wallace, S., Wu, Y.,
Battiwalla, M. Roswell Park Cancer Institute, Buffalo, NY.
Selective depletion of alloreactive T cells from a stem cell graft
has the potential of reducing graft-versus-host disease (GvHD) while
preserving graft-versus-leukemia (GvL) and third party responses. For
this purpose several techniques generate and deplete alloreactive cells,
which are donor-derived T cells activated by recipient tissue. The
kinetics of T cell activation in donor-recipient co-culture systems is
critical in optimizing the timing of depletion of alloreactive T cells.
We present the T cell activation kinetics in our preclinical system.
Peripheral blood mononuclear cells (PBMCs) were derived form
several pairs of unrelated healthy human volunteers. 2500 cGy irra-
diated cells (stimulators) were co-cultured with PBMCs (responders)
in a 1:1 ratio and a concentration of 5  106/ml in serum free
medium. Stimulator cells were labeled with PKH67 and the co-
cultures were, analyzed for CD3, CD4, and CD25, expression by ﬂow
cytometry on days 0 through 7, using Topro-3 to exclude dead cells.
Our results show that CD3, CD4, CD25 cells (alloreactive CD8
cells) increased from 1 % on day 0, to 6.5 percent by day 5; the
addition of IL2 ampliﬁed the increment to nearly 10% by day 5.
CD3, CD4, CD25dim or total cells (CD4 activated T cells)
did not appreciably increase over time and ranged between 2 to
5%; the addition of IL2 did not have any effect. The CD3,
CD4, CD25bright cells (T regulatory cells) increased from
1% at baseline to 5% by day 5 and these were unaffected by
the addition of IL2. The proportion of non-activated T lym-
phocytes, decreased with time of co-culture progression. Our
results show that T lymphocyte activation, deﬁned by CD25
expression, progressively increases through the ﬁrst week of
co-culture. This important observation will help in establishing
the timing of allograft manipulation, for the selective depletion
of alloreactive T cells in clinical hematopoietic stem cell trans-
plantation.
201
SIROLIMUS/MYCOPHENOLATE MOFETIL (MMF) AS TREATMENT FOR
GRAFT-VERSUS-HOST-DISEASE IN TWO CHILDREN WITH SEVERE RE-
NAL AND CALCINEURIN-INHIBITOR-ASSOCIATED CENTRAL NERVOUS
SYSTEM (CNS) TOXICITY
Ehlert, K., Groll, A.H., Vormoor, J. University Hospital, Department of
Pediatric Hematology and Oncology, Muenster, Germany.
Poster Session I
70


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Investigating the role of dendritic cells in extracorporeal photopheresis using an in vitro model

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