Blazar, B.R.

Lee, A.

McCullar, V.

Miller, J.S.

Verneris, M.R.

Wang, H.

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transplant protocols such as the type of GVHD prophylaxis used.
Nevertheless, the skin explant model remains a unique in vitro
system which provides an in situ histopathological read-out for
studying alloreactivity and human GVHD. The model has also the
potential to aid the development of novel prophylaxis and treat-
ment for GVHD.
170
INTERFERON-	 THERAPY FOR PATIENTS WITH MOLECULAR RELAPSE
IN LEUKEMIA AFTER ALLOGENEIC STEM CELL TRANSPLANTATION
Murai, K., Sugawara, T., Oyake, T., Kowata, S., Numaoka, H.,
Suzuki, K., Ishida, Y. Hematology and Oncology, Iwate Medical Uni-
versity, Morioka, Iwate, Japan.
One of the problems in allogeneic stem cell transplantation
(alloSCT) for acute leukemia (AL) is the recurrence of the disease.
Wilms’ tumor gene (WT1) expression has been reported to be
expressed in leukemic cells and used as a prediction marker of
leukemia relapse after chemotherapy (CT) in patients with AL.
Recently, Ogawa et al. described that the quantitative measure-
ment of WT1 expression in bone marrow cells (BM cells) assisted
the prediction of leukemia recurrence after allo-SCT (Blood 2003).
Several reports showed that interferon 
 (IFN
) administration
was effective in the prevention of leukemia recurrence in patients
after alloSCT. Based on this evidence, we tried to prevent the
recurrence of AL by IFN
 administration in patients with
alloSCT. We reported here three patients with molecular relapsed
AL after alloSCT who received the administration of IFN
 to get
and maintain complete remission (CR). The molecular relapse was
deﬁned as the criteria, described by Ogawa et al. They could not
receive donor lymphocyte infusion (DLI) because of no acceptance
of donor. Case 1 (SI): The patient with refractory AML received
BMT from unrelated donor (uBMT). He began to receive IFN
 (2
MU/m2 , four times per week) on day 84, when the molecular
relapse was diagnosed, and maintained the administration for 1
month. WT1 expression in BM cells decreased rapidly (32003 82
copies/mg RNA). He has achieved CR more than 30 months. Case
2 (TM): The patient with high risk AML received PBSCT from
sibling donor. She began to receive IFN
 (2 MU/m2 , two times
per week) on day 384, when the molecular relapse was diagnosed,
and had kept the administrations for 4 months. WT1 expression in
BM cells decreased rapidly (4100 3 1100 copies/mg RNA). She
has maintained CR for 21 months. Case 3 (HK): The patient with
high risk AML received uBMT. He began to receive IFN
 (2
MU/m2 , two times per week) on day 209, when the molecular
relapse was diagnosed, and had kept the administration for 2
months. WT1 expression in BM cells decreased rapidly (110003
1100 copies/mg RNA). He had maintained CR for 12 months. His
WT1 expression level re-increased on day 366, and a BM aspirate
showed 18.5% leukemic cells on day 387. He again received the
CT and alloSCT. In each case, WT1 expression in BM cells
decreased rapidly. They could keep CR more than 5 months (30,
21 and 5 months). These results strongly suggest that IFN
 might
be useful in treating molecular relapsed leukemia after alloSCT.
171
IMPORTANCE OF DETERMINING TRANSFORMING GROWTH FACTOR
1 PROMOTER ALLELES TO PREDICT EXPRESSION PHENOTYPE AND
ITS POTENTIAL IMPACT ON BONE MARROW TRANSPLANTATION
Shah, R., Selby, S.T., Rahaman, B., Posch, P.E., Hurley, C.K. George-
town University Medical Center, Washington, DC.
Transforming growth factor beta 1 (TGF-1) is a multifunc-
tional cytokine that plays a crucial role in immune regulation.
Three of eight known polymorphic sites in the human TGF-1 5
regulatory and signal peptide regions have been associated with
higher secreted levels of TGF1. These single nucleotide poly-
morphisms (SNPs) have been linked to bone marrow transplant
(BMT) outcome but the results are inconsistent. As each of these
studies examined single SNPs, the conﬂict could be due to differ-
ent linkages between these SNPs and other functional SNPs and
the corresponding phenotypes. A more comprehensive study of
diversity and SNP linkages was undertaken here. Ten novel poly-
morphisms and 14 novel alleles were identiﬁed by sequence char-
acterization of 38 unrelated individuals. The TGF-1 alleles clus-
tered into three phylogenetic groups based on the common
functional SNPs 509C-T and 869T-C suggesting three phe-
notypic groups. However, the 509 and 869 SNP positions
might not be as informative for predicting TGF-1 phenotypes as
suggested by the allelic groups. For example, individuals who carry
allele p014 (intermediate phenotype) are more likely to have a low
production phenotype due to the presence of 915C (decreased
TGF-1 expression) in this allele. This observation highlights why
limited genotyping to predict phenotypes may not be deﬁnitive as
linked SNPs likely affect the expected phenotypes that would be
attributed to single SNPs. To assess impact of TGF-1 promoter
genotype on likelihood of developing and/or severity of graft-
versus-host-disease (GVHD) in bone marrow transplant patients,
we are characterizing 40 unrelated donor/recipient pairs in a pilot
study. The genotype, p001/p003, was frequent (12/17 70.5%) in
recipients with grade 3 and 4 GVHD in comparison to recipients
with GVHD grades 0-2 (7/16 43.7%). These data target certain
TGF-1 promoter alleles for further study.
172
THE UNEXPECTED AND DIFFERENTIAL EFFECT OF CYCLOSPORIN A ON
CD56bright AND CD56dim NK CELL EXPANSION, PHENOTYPE, FUNC-
TION AND DEVELOPMENT FROM PROGENITOR CELLS
Wang, H., Lee, A., McCullar, V., Blazar, B.R., Miller, J.S.,
Verneris, M.R. University of Minnesota, Minneapolis, MN.
Following allo-HCT, NK cells play important roles in engraft-
ment, anti-viral responses, GVHD and GVL. Cyclosporine A
(CsA), is frequently administered to prevent or treat GVHD. It is
generally considered that CsA inhibits GVL. To date, little is
known about the impact of CsA on NK cell function. To investi-
gate this, NK cells (from healthy donors) were cultured with IL-2,
IL-15 and either physiological levels of CsA (1 g/ml) or vehicle
control. CsA treated cultures showed reduced NK cell expansion at
one week (4.88 vs. 1.87 fold expansion, n  10). The phenotype of
CsA treated NK cells was markedly different than controls. After
7-10 days of culture with CsA there were signiﬁcantly more
CD56brightCD16 cells and signiﬁcantly less CD56dimCD16 cells
(P  .001 for both). The percentage of KIR receptor (CD158a/h,
CD158b/j and CD158e) expressing cells was signiﬁcantly less in
CsA treated cultures. No changes were detected in NKG2D,
NKp30, NKp44, or NKp46. To further investigate the inﬂuence
of CsA on NK cell subset expansion, freshly isolated NK cells were
stained with CFSE then cultured with CsA or vehicle control for 1
week. Using FACS we monitored CD56dimCD16 and
CD56brightCD16 cell division. There was no difference in the
proliferation of CD56brightCD16 cells between the CsA and con-
trol treated cultures. In contrast, the CsA treated CD56dimCD16
cells had fewer cell divisions, demonstrating that CsA selectively
inhibits CD56dimCD16 cell proliferation. To investigate the cy-
totoxicity of CsA treated NK cells, we performed killing assays.
Surprisingly, we found higher cytotoxicity in CsA treated NK cells
compared to controls (K562, P .05 and Raji, P .05). To further
evaluate CsA treated NK cells, we investigated the intracellular
IFN- secretion following IL-12/IL-18 stimulation. In 5 consec-
utive experiments the percentage of IFN- secreting cells was
higher in CsA treated NK cells (44% vs. 24%, P  .05). Lastly, we
determined the effect of CsA on NK cell differentiation from
progenitor cells (CD34LinCD38) using an in vitro differenti-
ation system. Brieﬂy, progenitor cells were cultured on a murine
feeder cell line (AFT-024) for 42 days in the presence of IL-3,
IL-7, IL-15, SCF and FLT3L  CsA. CsA treated cultures had
less KIR expressing cells compared to controls. Collectively these
results show that physiological levels of CsA results in NK cells
that have less KIR receptors, higher cytotoxicity and more cytokine
secretion.
Poster Session I
61BB&MT


The unexpected and differential effect of cyclosporin A on CD56Bright and CD56Dim NK cell expansion, phenotype, function and development from progenitor cells 

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The unexpected and differential effect of cyclosporin A on CD56Bright and CD56Dim NK cell expansion, phenotype, function and development from progenitor cells

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