Blood, 15 November 2001, Vol. 98, No. 10, pp. 3162-3164
BRIEF REPORT
Correlation of peripheral blood
OX40+(CD134+) T
cells with chronic graft-versus-host disease in patients who underwent
allogeneic hematopoietic stem cell transplantation
Ai Kotani,
Takayuki Ishikawa,
Yumi Matsumura,
Tatsuo Ichinohe,
Hitoshi Ohno,
Toshiyuki Hori, and
Takashi Uchiyama
From the Department of Hematology/Oncology, Department
of Dermatology, Graduate School of Medicine, Kyoto University, Kyoto,
Japan.
 |
Abstract |
There is no reliable laboratory indicator of the onset of chronic
graft-versus-host disease (cGVHD). This study looks at whether the
expression of OX40, a member of the tumor necrosis factor receptor
family, is related to the development of cGVHD in patients who
underwent allogeneic hematopoietic stem cell transplantation. Peripheral blood mononuclear cells from 22 patients after day 100 were
subjected to multicolor flow cytometry. The percentages of both
OX40+CD4+ and OX40+CD8+
T cells were significantly higher in patients with cGVHD than those
without (P < .0001 and P = .001,
respectively). Serial analyses showed that
OX40+CD4+ T cells elevated before the onset of
cGVHD and closely correlated with the therapeutic response. The
expression of CD25, CD69, and HLA-DR was partially detectable on
OX40+ T cells. These results indicate that serial
measurement of OX40+ T cells is useful for predicting the
onset as well as the therapeutic response of cGVHD and raise a
possibility that the OX40/gp34 system is involved in the pathogenesis
of cGVHD.
(Blood. 2001;98:3162-3164)
© 2001 by The American Society of Hematology.
| |
Introduction |
The increase in allogeneic hematopoietic stem cell
transplantations (allo-HSCTs), particularly those using peripheral
blood stem cells and involving donors other than HLA-identical
siblings, has made the management of graft-versus-host disease (GVHD) a continuing problem. Although there have been advances in the prevention of acute (a) GVHD with the introduction of tacrolimus (FK506) and
T-cell depletion, the incidence of chronic (c) GVHD has not decreased1 and the absence of reliable markers for
predicting and monitoring cGVHD makes its control difficult.
The clinical manifestations of aGVHD and cGVHD are somewhat different.
aGVHD is characterized by a triad of dermatitis, gastroenteritis, and
hepatitis, whereas cGVHD is a more diverse syndrome, usually presenting
with multiorgan involvement and closely related to autoimmune disease.
Unlike aGVHD, T cells, which newly differentiate from donor
hematopoietic stem cells, are thought to play more important roles
in the development of cGVHD.2
OX40, a member of the tumor necrosis factor receptor family, is
expressed on activated T cells. The interaction of OX40 with its
ligand, gp34, is shown to enhance T-cell proliferation.3 In murine models, several reports showed that the OX40/gp34 system is
involved in the process of GVHD4-7 as well as several
autoimmune disease.8-10 In addition, studies with OX40- or
gp34-deficient mice revealed that this system has a critical
costimulatory function in dendritic cell/CD4+ T-cell
interactions.11
In this study, we measured the expression of OX40 on T cells after
allo-HSCT to determine its correlation with the manifestations of cGVHD.
| |
Study design |
Twenty-two patients with hematologic malignancies and severe
aplastic anemia undergoing bone marrow transplantation between 1998 and
2000 were included in this study. All patients were conditioned with
myeloablative regimens. No patients received T-cell-depleted bone
marrow or prophylactic anti-thymocyte globulin. Ten donors were related
and 12 donors were unrelated, and HLA types were serologically 5- or
6-antigen matched between all pairs of patients and donors. Other
characteristics of patients with cGVHD are shown in Table
1. Peripheral blood mononuclear
cells obtained more than 100 days after transplantation were
subjected to 3-color flow cytometry by using FACScan (Becton Dickinson,
San Jose, CA).
Cells were stained with fluorescein isothiocyanate-conjugated
monoclonal antibodies (mAbs) against CD4, CD8, CD25, CD69, or HLA-DR
(Becton Dickinson), peridinin chlorophyll protein-conjugated mAb
recognizing CD3 (Becton Dickinson), and biotinylated 31512 (anti-human OX40) in combination with phycoerythrin-conjugated streptavidin (Becton Dickinson). After gating T cells according to CD3
positivity, the ratio of OX40+ cells in CD4+ or
CD8+ cells were calculated, and the relations of
OX40+ cells with CD25+ cells, CD69+
cells, and HLA-DR+ cells were determined. All data
were analyzed with STATCEL program (Hisae Yanai). Two-group comparison
was done on the analysis of variance and Student t test.
Differences with P < .05 were considered to be
statistically significant.
| |
Results and discussion |
Of 22 patients enrolled, 11 patients (UPN 1-11) developed cGVHD
and 6 of them were successfully treated with conventional immunosuppressive therapy such as prednisone. Among the remaining 5 patients, one patient (UPN 1) died of progressive cGVHD. Although the
absolute numbers of CD4+ and CD8+ T cells
varied considerably in each assay (data not shown), the percentages of
both OX40+CD4+ T cells and OX40+
CD8+ T cells in patients with cGVHD just before the
introduction of immunosuppressive therapy were significantly higher
than the peak value of OX40+ cells in patients without
cGVHD (47.5% ± 17.9% versus 14.8% ± 11.6%,
P < .0001; 35.3% ± 25.0% versus 5.8% ± 4.2%,
P = .001; Figure 1A). The
mean interval between the analysis and stem cell transplantation (SCT)
is 233 days for patients with cGVHD and 274 days for those without
cGVHD. Neither the severity of cGVHD (extensive type or limited type)
nor the organ involved was correlated with the percentages of
OX40+ cells (data not shown). CD25, CD69, and HLA-DR, which
are expressed on activated T cells, were hardly or partially expressed
on OX40+CD3+ T cells (Figure 1B). Neither the
interval after transplantation, presence of aGVHD, nor
immunosuppressive drugs for GVHD prophylaxis affected the expression of
OX40 statistically (data not shown).
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| Figure 1.
OX40 expression with cGVHD.
(A) Comparison of OX40 expression between patients with cGVHD
(n = 11; closed circles and triangles) and without GVHD (n = 11;
open circles and triangles). The mean interval between the analysis and
SCT is 233 days for patients with cGVHD and 274 days for those without
cGVHD. Each bar indicates the mean ± SD in each patient group.
(B) Surface phenotype of T cells of a representative case with cGVHD.
CD3+ T cells were gated and analyzed for expression of
OX40, CD25, CD69, and HLA-DR. (C) Serial changes in the percentage of
OX40+CD4+ T cells at 4 different time points
around the onset of cGVHD. The first point is the day when patients
visited the outpatient clinic 2 months before the onset of GVHD. The
second point is the latest visit about 1 month before the onset. The
third point is the time point at the onset of cGVHD just before
immunosuppressive therapy. The fourth point is the next visit 1 to 2 weeks after the immunosuppressive therapy. The changes between the
first 2 points and the comparison between the nonresponders (n = 5;
closed point) and the responders (n = 5; open point) at the onset are
depicted separately.
|
|
In the 11 patients who developed cGVHD, we could serially follow
the expression kinetics of OX40+ T cells before and after
the onset of cGVHD and introduction of immunosuppressive therapy
(Figure 1C). OX40+CD4+ T cells gradually
increased before the appearance of clinical manifestations of cGVHD. In
addition, the percentages of OX40+ cells in
CD4+ T cells were significantly higher 1 month before the
onset than 2 months before (23.7% ± 6.9% versus 12.3% ± 3.9%,
P = .0007; Figure 1C). The percentage of OX40+
cells just before the immunosuppressive therapy was closely correlated with the therapeutic response. These patients who did not respond to
first line therapy for cGVHD tended to show a more highly elevated value than responders (nonresponders versus responders,
59.8% ± 17.9% versus 36.9% ± 10.2%, P = 0.037;
Figure 1C). When the percentage of OX40+CD4+ T
cells was elevated above 50%, the conventional immunosuppressive therapy was not effective, and more intensive salvage regimens for
cGVHD, such as mycophenolate mofetil, were required. In contrast to
OX40+CD4+ T cells, the percentage of
OX40+CD8+ T cells did not correlate with the
clinical response, although it elevated coincidentally with the
onset of cGVHD (data not shown).
Our findings that the expression of CD25, CD69, and HLA-DR were hardly
or partially expressed on OX40+CD3+ T cells
implies that OX40+CD4+ T cells in patients with
cGVHD might be distinct from recently activated T cells. Furthermore,
we detected OX40+CD8+ T cells in almost all
cGVHD patients. In mice ex vivo OX40 staining on tumor infiltrating
CD8+ T cells was described.16 As far as we
know, the present study first established that the expression of OX40
is inducible on human CD8+ T cells. The relationship
between this subset and the effector cells of cGVHD is to be determined.
Our study for the first time showed a strong correlation between the
expression of OX40 on T cells and cGVHD. In contrast, previous studies
showed that monitoring OX40+ T cells was not useful for the
prediction of aGVHD.17 We assume that the pathogenesis of
aGVHD and cGVHD may not be identical2 and infections that
are more common within 100 days after allo-HSCT18 might
modify the expression of OX40.
Because accumulating evidence has indicated that OX40+ T
cells play crucial roles in the development of several autoimmune diseases,9,10,13-15 the immune cell activation by the
OX40/gp34 system might underlie more closely the pathogenesis of cGVHD. Considering that administration of anti-CD134L mAb significantly ameliorated aGVHD in mice,6 the selective blockade of
OX40/gp34 system might be effective for GVHD in humans.
In conclusion, expression of OX40 in peripheral blood T cells can be
used as a sensitive indicator of cGVHD after allo-HSCT and might be
important for which chemotherapeutic drugs could be used. Modification
of the OX40/gp34 system may enable us to better control cGVHD.
| |
Acknowledgments |
We thank Dr M. Sasada, Dr T. Okazaki, Dr A. Takahashi, Dr K. Imada,
Dr N. Kadowaki, and Dr C. Ueda for their cooperation in collecting the
blood samples, and we thank Ms. K. Fukunaga for expert technical assistance.
| |
Footnotes |
Submitted March 20, 2001; accepted July 9, 2001.
Supported by grants-in-aid from the Ministry of Education, Science,
Sports and Culture of Japan.
The publication costs of this
article were defrayed in part by
page charge payment. Therefore,
and solely to indicate this fact,
this article is hereby marked
"advertisement"
in accordance with 18 U.S.C.
section 1734.
Presented in part at the 42nd Annual Meeting and Exposition of the
American Society of Hematology.
Reprints: Toshiyuki Hori, Department of Hematology/Oncology,
Graduate School of Medicine, Kyoto University, Kyoto 606-8507, Japan;
e-mail thori{at}kuhp.kyoto-u.ac.jp.
| |
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Abstract
Introduction
