Baird, K.

Beck, S.L.

Cole, M.L.

Dickinson, J.

Dudley, W.N.

Gress, R.E.

Hakim, F.T.

Kline Leidy, N.

Mitchell, S.A.

Mooney, K.H.

Palit, P.

Pavletic, S.Z.

Rehman, N.K.

Wayne, A.S.

Elsevier - Publisher Connector 

118 Poster Session-IIIL-2. IL-2-expanded CB NK cells formed increased immune syn-
apses with K562 tumor cells (mean 65%; 60–71%) and primary hu-
man AML blasts (mean 48%, range 39–55%), comparable to the
levels generated with peripheral blood NK cells. Additionally, we
demonstrated that ex vivo expanded CB NK cells could efficiently
kill human AML engrafted in a NOD/scid/IL-2Rg-null mouse
model. A mean 50% (37–66%) reduction in AML blasts was
observed in comparison to control groups (mean 3%;range 2.4–
4.7%) by 6 weeks post NK infusion (p\0.05). Our results suggest
that ex vivo expansion of CB NK cells is a feasible and effective
strategy for the treatment of AML.328
CHARACTERIZATION OF CTL CLONES ISOLATED FROM BONE MARROW
TRANSPLANT RECIPIENT WITH HLA-CW-MISMATCHED DONOR
Murata, M., Sugimoto, K., Terakura, S., Naoe, T. Nagoya University
Graduate School of Medicine, Nagoya, Aichi, Japan
HLA-Cw disparity in a donor increases the risk of severe acute
GVHD after BMT. However, HLA-Cw-specific cytotoxic T lym-
phocytes (CTLs) generated in post-transplant recipients who de-
velop acute GVHD have not been characterized in detail. Here,
we characterized CTL clones isolated from a recipient at the on-
set of grade II acute GVHD who was transplanted from an HLA-
A, B and DRB1-matched, Cw-mismatched (recipient, Cw*0303/
Cw*0702; donor, Cw*0801/Cw*0702) unrelated donor. Seven
CTLs were isolated. Four CTLs were CD8-positive, one was
CD4-positive, and two were CD4/CD8-double positive. Three
of those including a CD8-positive CTL, a CD4-positive CTL
and a CD4/CD8-double positive CTL had exactly the same nu-
cleotide sequences in the CDR3 region of their T cell receptors,
suggesting these three CTLs with variable phenotypes originated
from a single clone. All CTLs lysed Epstein Barr virus-trans-
formed lymphoblastoid cell (B-LCL) from the recipient and donor
B-LCL transfected with the HLA-Cw*0303 cDNA construct, but
failed to lyse B-LCL from the donor. COS cells transfected with
HLA-Cw*0303 cDNA construct stimulated IFNg production by
all CTLs. Thus, all isolated CTLs recognized HLA-Cw*0303
molecule as an alloantigen. COS cells transfected with Cw*0303
mutants in which amino acids constituting peptide-binding
pockets (aa position: 114, 116, 152, or 163) were substituted
with Cw*0801-amino acids could not stimulate IFNg production
by any CTLs, whereas COS cells transfected with Cw*0303 mu-
tants bearing Cw*0801-amino acids outside the positions consti-
tuting peptide-binding pockets stimulated all CTLs as well as
the wild-type Cw*0303 construct. These data indicated that pep-
tides bound to Cw*0303 molecules influence the allorecognition
of Cw*0303-specific CTLs. Taken together, our findings suggest,
although the immunogenicity of HLA-Cw antigens is considered
to be low as a consequence of their low level of cell surface ex-
pression, HLA-Cw-specific CTL clones with a variable phenotype
can naturally arise in post-transplant recipients and cause acute
GVHD through recognition of the peptides bound to Cw mole-
cules. This is consistent with a recent statistical study showing
a significant association between some specific amino acid substi-
tutions at positions constituting peptide-binding pockets of the
HLA-Cw molecule and the occurrence of severe acute GVHD
after unrelated BMT.329
A ROLE FOR NF-KB INDUCING KINASE (NIK) IN GRAFT VERSUS HOST RE-
ACTIONS
Sanchez-Valdepenas, C.2, Casanova, L.1, Colmenero, I.3, Gonzalez-
Vicent, M.1, Madero, L.1, Diaz, M.A.1, Fresno, M.2, Ramirez, M.1 1 Hos-
pital Universitario Nin˜o Jesus, Madrid, Spain; 2 Centro de Biologıa Molec-
ular, Madrid, Spain; 3 Hospital Universitario Nin˜o Jesus, Madrid, Spain
NF-KB is a family of transcription factors with an important role
in immune and inflammatory responses, and represents a target for
the treatment of immune diseases. NF-KB inducing kinase (NIK) is
required for the activation of the NF-KB noncanonical (or alterna-
tive) pathway. We have studied the role of NIK in graft versus hostdisease (GVHD) in humans and in a murine model of GVHD.
Human T lymphocytes activated in vitro (CD3 1 CD28 anti-
bodies, and/or TNF-a) showed an initial downregulation of
NIK, followed by an increase above basal levels, during the first
48 hours. On the other hand, circulating T lymphocytes from pa-
tients with acute GVHD (aGVHD) expressed NIK while T cells
from healthy donors did not, and T lymphocytes infiltrating the
dermis of pathological lesions of those patients expressed high level
of NIK. We next explored the effects of suppressing NIK in a mu-
rine model of GVHD in mice: H2Db T lymphocytes transplanted
into fully mismatched H2Dd recipients. Donor mice were either
aly/aly (NIK deficient) or C57BL/6 (control), and recipient mice
were Balb/c. Mice recipient of BL6 cells developed a severe form
of aGVHD and died in the third week after transplant. On the con-
trary, mice transplanted with aly T lymphocytes survived for 3
months and did not develop GVHD Histopathological analysis of
skin, gut and liver of these surviving mice showed no sign of
GVHD. We next studied the fate of the infused T lymphocytes
in the early days postransplant, in order to ascertain the cause of
the difference in GVHD survival. We found that aly and BL6 T
lymphocytes had the same capacity for homing into the spleens,
but the clonal expansion of aly T lymphocytes was significantly im-
paired at day 5. Analysis of in vivo T cell divisions showed the same
kinetics for aly and BL6 T lymphocytes at 1, 3 and 5 days after
transplant. Therefore, aly T cells divided in vivo but the process
was not productive. Finally, the proportions of apoptotic aly T lym-
phocytes were higher than those of BL6, at days 1 and 3, but not at
day 5. We also detected lower amounts of Th1 cytokines levels in
the serum of mice receiving aly vs. BL6 T cells, 5 days after trans-
plant. In summary, NIK-deficient T lymphocytes had a normal
proliferative capacity but an increased apoptotic rate early after
transplant compared to controls, and were unable of mounting
a successful GVH reaction in the fully mismatched allogeneic
setting.330
PLASMA CYTOKINE LEVELS DISTINGUISH CHRONIC GRAFT VERSUS
HOST DISEASE (CGVHD) SYMPTOM PROFILE GROUPS
Mitchell, S.A.1,2, Hakim, F.T.3, Baird, K.4, Wayne, A.S.4,
Mooney, K.H.2, Rehman, N.K.3, Dickinson, J.5, Palit, P.1, Beck, S.L.2,
Kline Leidy, N.6, Cole, M.L.7, Dudley, W.N.2, Gress, R.E.3,
Pavletic, S.Z.3 1 National Institutes of Health, Bethesda, MD; 2 University
of Utah, College of Nursing, Salt Lake City, UT; 3 National Cancer Insti-
tute, NIH, Bethesda, MD; 4 National Cancer Institute, NIH, Bethesda,
MD; 5 Washington University School of Medicine, St. Louis, MO; 6 United
BioSource, Bethesda, MD; 7 Lawrence Technological University, South-
field, MI
Background:Understanding the biologic milieu associated with
differing symptom profiles in allogeneic HSCT survivors with
cGVHD has the potential to clarify the pathogenesis of cGVHD
activity and tissue damage, and may direct the development of
new strategies to ameliorate symptoms and improve quality of
life.
Objective: To determine if differing cGVHD symptom profiles
are associated with unique patterns of pro-inflammatory cytokine ex-
pression and markers of systemic immune activation.
Methods: Plasma levels of sBAFF, IL-1b, IL-1 receptor antago-
nist, IL-6, soluble IL-6 receptor, soluble TNF-receptor II, MCP-
1, and MIG were assayed by sandwich ELISA. Lymphocyte subset
quantities were measured. Concurrently, the Lee Chronic GVHD
Symptom Scale, a self-report measure of cGVHD symptoms, and
clinical data were obtained. Data from 79 patients were analyzed us-
ing descriptive statistics, latent profile analysis, and multinomial lo-
gistic regression.
Results: Three distinct symptom subgroups were identified
through latent profile analysis: Group 1 (low on all symptoms),
Group 2 (prominent oral, upper GI, and weight loss symptoms);
and Group 3 (eye, muscle/joint, fatigue and mood symptoms). Mul-
tinomial logistic regression, controlling for age, length of time since
transplant, cGVHD severity, intensity of immunosuppression and
post-transplant lymphocyte reconstitution, revealed that IL-6,
TNF-RII, MCP-1 and sBAFF levels significantly distinguished the
three symptom subgroups.
Poster Session-II 119Odds Ratios and 95% Confidence Intervals for the Multinomial
Logistic Regression of Symptom Latent Class Membership on
Cytokines † Controlling for Age, Time Since Transplant,
cGVHD Severity, Intensity of Current Immunosuppression
and Absolute Lymphocyte Counts †
Symptom Profile Symptom ProfileSymptom Profile 1
(low on all symptoms)
vs. Symptom Profile
2 (prominent oral/
GI symptoms)
OR (95% CI)1 (low on all
symptoms) vs.
Symptom Profile
3 (prominent
eye, muscle/joint,
fatigue and
mood symptoms)
OR (95% CI)2 (prominent oral/
GI symptoms) vs.
Symptom Profile 3
(prominent eye,
muscle/joint,
fatigue and mood
symptoms)
OR (95% CI)IL-1 b 1.59 (0.30–8.45) 0.92 (0.32–2.60) 0.58 (0.10–3.32)
IL-6 0.27 (0.09–0.79)* 1.15 (0.52–2.52) 4.26 (1.32–13.82)*
IL-1 RA 0.69 (0.22–2.14) 0.55 (0.29–1.07) 0.81 (0.26–2.48)
sIL-6R 0.50 (0.12–2.13) 1.78 (0.54–5.90) 3.53 (0.83–15.13)
TNF-RII 0.29 (0.04–2.42) 0.28 (0.08–0.99)* 0.95 (0.12–7.59)
MCP-1 8.70 (1.46–51.66)* 0.40 (0.11–1.40) 0.05 (0.01–0.36)**
MIG 1.33 (0.73–2.44) 1.02 (0.63–1.65) 0.77 (0.42–1.39)
sBAFF 0.40 (0.13–1.30) 1.86 (0.88–3.93) 4.61 (1.39–15.31)**† values were log normal transformed prior to analysis
**or *p\ .01 or p\ .05, respectively, multinomial logistic regression.
Higher IL-6 levels significantly differentiated participants in
Group 2 from those in Groups 1 and 3. Group 3 had higher TNF-
RII compared to Group 1. Those with higher sBAFF levels were sig-
nificantly more likely to be in Group 2, while those with higher
MCP-1 levels were significantly more likely to be in Group 3.
Conclusion: Allogeneic HSCT survivors with differing cGVHD
symptom profiles were distinguished by significantly different levels
of IL-6, TNF-RII, MCP-1 and sBAFF. These data validate this new
symptom grouping system based on the Lee Chronic GVHD Symp-
tom Scale as a measure of cGVHD disease burden. IL-6, TNF-RII,
MCP-1 and sBAFF appear to be important biomarkers that reflect
specific cGVHD manifestations and merit further study.331
DONOR DENDRITIC CELLS INITIATE COUNTER-REGULATORY IMMUNE
RESPONSES AND GVL EFFECTS IN ALLLOGENEIC BMT
Li, J.-M.1, Darlak, K.A.1, Lu, Y.1, Harris, W.1, Jaye, D.L.2,
Waller, E.K.1 1 Emory University, Atlanta, GA; 2 Emory University,
Atlanta, GA
Based on a clinical association of donor plasmacytoid dendritic cell
(DC) content with leukemia relapses after allo BMT (Waller, Blood
2001), we previously reported that donor CD11b- DC enhanced
IFN-g synthesis and GvL activity of donor T-cells, while CD11b1
DC resulted in increased IL-10 production and decreased GvL func-
tion by donor T-cells in allo BMT mouse models (Li, Blood 2007).
In this study, we tested mechanisms whereby donor DC in the graft
modulate donor T-cell activation in a MHC-mismatched (C57BL/
6/B10.BR) allo BMT. Recipients received 11 Gy irradiation fol-
lowed by tail vein injection of purified donor HSC, DC subsets
and T-cells two days later. Allografts consisted of 5 104 FACS-pu-
rified donor BM CD11b- DC or CD11b1 DC plus 3  103
c-kit1sca-11lineage- HSC in combination with either 3  105
T-cells or no additional T-cells. In vivo donor-derived T-cell prolif-
eration was assessed by CFSE staining. Serum and intracellular cyto-
kines (Th1: IL-12, IFN-g, IL-2, and TNF-a and Th2: IL-4, IL-5,
and IL-10) were tested by ELISA and flow cytometry. Costimulatory
molecule expression(CD40, CD80, ICOSL, PD-L1 and PD-L2)
was measured by flow cytometry following recovery of GFP1 donor
DC on day110 post-transplant. We found that donor CD8 T-cells
had higher levels of Ki-67 expression and proliferation than donor
CD4 T-cells 3 days post-transplant following transplantation with
CD11b- DC compared with CD11b1 DC, and without donor DC
(p\ 0.001). Both CD11b- DC and CD11b1 DC had similar level
of expression of CD40, CD80, and ICOSL in BM, in lymph nodes
and in spleen at 10 days post-transplant, but CD11b1 donor DC re-covered from BM had much higher levels of PD-L1 than CD11b-
DC, while CD11b- DC in all tissues had higher levels of PD-L2. Do-
nor CD11b- DC enhanced Th1 cytokine production of donor
T-cells, while donor CD11b1DC elevated Th2 cytokine production
compared with T-cell alone. In conclusion: Donor CD11b- DC en-
hanced donor T-cell proliferation, especially CD8 T-cells compared
with donor CD11b1 DC or recipients of T-cells alone. Homing of
donor DC to lymphoid organs and expression of costimulatory mol-
ecules on donor CD11b- and CD11b1 DC subsets in vivo were sim-
ilar, suggesting that the differences observed in donor T-cell
activation and proliferation in distinct donor DC subset is likely
due to local production of cytokines by donor DC. The limited
GvHD seen with T-cells injected with CD11b- DC may be due to
differential expression of PD-L2 compared with CD11b1 DC.332
C-FLIP EXPRESSION DETERMINES LOW SENSITIVITY OF TYPE 17 T
HELPER CELLS TO ACTIVATION-INDUCED CELL DEATH
Yu, Y.1, Iclozan, C.2, Anasetti, C.3, Dong, C.4, Yu, X.-Z.5 1 H. Lee Moffitt
Cancer Center and Research Institute, Tampa, FL; 2 M.D. Anderson Can-
cer Center, Houston, TX
T cell apoptosis induced by repeated TCR stimulation, known as
activation-induced cell death (AICD), plays an important role in T
cell tolerance. Pro-inflammatory, IL-17-producing CD41 T cells
(Th17 cells) have been recently identified as a unique T helper sub-
set. Preliminary work from our lab indicated that Th17 cells partic-
ipate in the pathogenesis of acute graft-versus-host disease (GVHD).
In this study, we compared the susceptibility of polarized Th1 and
Th17 effectors to AICD in vitro and in vivo. We found that Th17 ef-
fectors were significantly less susceptible to AICD upon TCR re-
stimulation compared to Th1 effectors. It was further confirmed
by Th17 effectors generated from il17f/rfp knock-in mice, RFP1
(Th17) cells were indeed more resistant to AICD than RFP- (non-
Th17) cells. Resistance of Th17 cells to AICD was also observed
when Th17 cells were co-cultured with Th1 cells in vitro or co-in-
jected with Th1 cells in vivo in allogeneic recipients. To explore
the molecular mechanism of AICD resistance in Th17 cells, we
found that Th17 cells were defective in expression of FasL and in ac-
tivation of caspases, but highly expressed anti-apoptotic protein c-
FLIP as compared to Th1 cells. Given that Th17 cells were resistant
to AICD when co-cultured with Th1 cells that express high levels of
FasL, we hypothesized that Fas-signaling was impaired on Th17 ef-
fectors. After knocking down c-FLIP with its specific siRNA, Th17
cells upregulated FasL and underwent rapid apoptosis upon TCR re-
stimulation, indicating that Th17 cells are resistant to AICD likely
because the high level of c-FLIP prevents Fas-mediated apoptosis.
These results suggest the resistance of Th17 cells to AICD as addi-
tional mechanism that contributes to the high pathogenicity of Th17
cells in autoimmune diseases and GVHD. Our finding also
strengthens the rationale to use tumor-specific Th17 cells for adop-
tive cell therapy in cancer.333
MESENCHYMAL STEM CELL INFUSION AS PREVENTION FOR GRAFT RE-
JECTION AND GRAFT-VERSUS-HOST DISEASE AFTER ALLOGENEIC HEMA-
TOPOIETIC CELL TRANSPLANTATION WITH NONMYELOABLATIVE
CONDITIONING FROM HLA-MISMATCHED DONORS: A PILOT STUDY
Baron, F., Willems, E., Lechanteur, C., Baudoux, E., Frere, P.,
Hafraoui, K., Fillet, G., Beguin, Y. University of Lie`ge, Belgium
Background: Allogeneic hematopoietic cell transplantation
(HCT) following nonmyeloablative conditioning has been an effec-
tive treatment for many pts with hematological malignancies who
have a HLA-matched related or unrelated donor. However, results
of nonmyeloablative HCT in pts with HLA-mismatched donors
have been disappointing due to high incidence of graft rejection
and severe acute GVHD. Recent studies have suggested that infusion
of mesynchymal stem cells (MSC) the day of HCT might promote
engraftment and prevent acute GVHD after myeloablative alloge-
neic HCT. This prompted us to investigate whether MSC infusion
a few hours before HCT could allow non-myeloablative HCT
from HLA-mismatched donors to be performed safely (i.e. with
a 100-day incidence of nonrelapse mortality\35%).


Plasma Cytokine Levels Distinguish Chronic Graft Versus Host Disease (cGVHD) Symptom Profile Groups 

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Plasma Cytokine Levels Distinguish Chronic Graft Versus Host Disease (cGVHD) Symptom Profile Groups

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