IL-4-producing CD8(+) T cells may be an immunological hallmark of chronic GVHD.

Abstract

Chronic graft-versus-host disease (cGVHD) occurs in approximately 60-80% of those who survive over 100 days after allogeneic hematopoietic stem cell transplantation (allo-HSCT). However, the pathophysiology of cGVHD is poorly understood. To gain more insight into the immunological mechanism of cGVHD, we examine cytokine production of peripheral blood T cells from 19 patients in the chronic phase of allo-HSCT. The percentage of IFN-gamma-producing CD8(+) T cells among CD8(+) T cells was significantly higher in patients with or without cGVHD than in normal control subjects (P<0.001). On the other hand, the percentage of IL-4-producing CD8(+) T cells among CD8(+) T cells was significantly higher in patients with cGVHD (mean 3.3%; range 1.3-8.2%) than in patients without cGVHD (mean 1.2%; range 0.8-1.7%) and normal control subjects (mean 1.1%; range 0.1-1.6%) (both P<0.001). By contrast, the percentage of IL-4-producing CD4(+) T cells was not different among patients with and without cGVHD and normal controls. These findings suggest that IL-4-producing CD8(+) T cells may be an immunological marker of cGVHD.

Full text

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IL-4-producing CD8þT cells may be an immunological hallmark
of chronic GVHD
K Nakamura, R Amakawa, M Takebayashi, Y Son, M Miyaji, K Tajima, K Nakai, T Ito,
N Matsumoto, K Zen, Y Kishimoto and S Fukuhara
1st Department of Internal Medicine, Kansai Medical University, Moriguchi, Osaka, Japan
Summary:
Chronic graft-versus-host disease (cGVHD) occurs in
approximately 60–80% of those who survive over 100
days after allogeneic hematopoietic stem cell transplant-
ation (allo-HSCT). However, the pathophysiology of
cGVHD is poorly understood. To gain more insight into
the immunological mechanism of cGVHD, we examine
cytokine production of peripheral blood T cells from
19 patients in the chronic phase of allo-HSCT. The
percentage of IFN-c-producing CD8þT cells among
CD8þT cells was significantly higher in patients with
or without cGVHD than in normal control subjects
(Po0.001). On the other hand, the percentage of IL-4-
producing CD8þT cells among CD8þT cells was
significantly higher in patients with cGVHD (mean 3.3%;
range 1.3–8.2%) than in patients without cGVHD (mean
1.2%; range 0.8–1.7%) and normal control subjects
(mean 1.1%; range 0.1–1.6%) (both Po0.001). By
contrast, the percentage of IL-4-producing CD4þT cells
was not different among patients with and without
cGVHD and normal controls. These findings suggest that
IL-4-producing CD8þT cells may be an immunological
marker of cGVHD.
Bone Marrow Transplantation advance online publication,
25 July 2005; doi:10.1038/sj.bmt.1705107
Keywords:
IL-4-producing
GVHD; HSCT; cytokines
CD8þ
Tcells; chronic
Graft-versus-host disease (GVHD) is a major cause of
morbidity and mortality after allogeneic hematopoietic
stem cell transplantation (allo-HSCT),1which is commonly
classified into acute or chronic GVHD Acute GVHD
(aGVHD) mainly develops in the skin, gut, and liver and
directly contributes to the higher mortality rate in the early
phase after allo-HSCT.2On the other hand, chronic
GVHD (cGVHD) usually develops in systemic organs
and decreases the quality of life in the late phase after allo-
HSCT.3–6Furthermore, elevated mortality is associated
with the complications following cGVHD such as inter-
stitial pneumonia and infections.7
GVHD is a complicated post transplant condition, and a
myriad of factors are suggested to be involved in this
process. The essential basis of GVHD is recognized to be
primarily the immune responses that are evoked by the
existence of allogeneic disparities between donor and
recipient and that subsequently attack the recipient
organs.1The first process might be allo-antigen present-
ation to the donor T cells by antigen-presenting cells
(APCs), especially dendritic cells. This interaction between
APCs and T cells in microenvironments such as cytokines
dictate the development of type 1 helper T-cells (Th1) or
type 2 helper T-cells (Th2).8,9It is well established that Th1
cells produce cytokines such as IL-1, IL-2 and IFN-g, while
Th2 cells produce cytokines such as IL-4 and IL-10.
Accumulating evidence has highlighted the importance
of Th1 balance in aGVHD.10–19However, the pathophy-
siology of cGVHD is much less well understood than that
of aGVHD, and the current understanding of cGVHD is
largely the result of murine studies. In the murine system,
it has been shown that post-thymic CD4þT cells are
important in inducing cGVHD.20In addition, several
reports have demonstrated that type 2 cytokines are
associated with cGVHD,21,22whereas other studies favor
the significance of Th1 balance in cGVHD.23–26
In this study, we analyzed the cytokine production of
T cells in the chronic phase of patients who underwent
allo-HSCT and found that IL-4-producing CD8þT cells
are closely associated with cGVHD.
Patients and methods
Patients
A total of 19 patients who underwent allo-HSCT between
June 1994 and March 2001 and were alive over 100 days
after allo-HSCT without relapse were enrolled in this study,
for which informed consent was obtained from all patients.
The patient characteristics are shown in Table 1. The group
of patients with cGVHD showed some sign of cGVHD,
while the group of patients without cGVHD did not show
any sign of cGVHD at the time of examination. Of them 10
Received 19 November 2004; accepted 16 June 2005
Correspondence: Dr R Amakawa, First Department of Internal
Medicine, Kansai Medical University, 10-15 Fumizono-cho, Moriguchi,
Osaka 570-8506, Japan; E-mail: amakawa@takii.kmu.ac.jp
Bone Marrow Transplantation (2005), 1–9
& 2005 Nature Publishing Group All rights reserved 0268-3369/05 $30.00
www.nature.com/bmt

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patients (six men and four women, mean age 33 years,
range 20–47 years) developed cGVHD, while the remaining
nine patients (six men and three women, mean age 29 years,
range 13–39 years) did not show any clinical signs of
cGVHD. The patients with or without cGVHD were
examined at the median time of 1204 days (range 522–2840
days) and 1153 days (range 571–2798 days) after allo-
HSCT, respectively. Five patients with cGVHD had acute
myelogenous leukemia (AML), two had chronic myelogen-
ous leukemia (CML), two had acute lymphoblastic
leukemia (ALL) and one had severe aplastic anemia. Six
patients without cGVHD had AML, one had CML and
two had ALL. Eight patients with cGVHD received bone
marrow transplantation (BMT) from their siblings, one
patient received unrelated BMT and one patient received
peripheral blood stem cell transplantation (PBSCT) from
their siblings. On the other hand, seven patients without
cGVHD received related BMT, one patient received
unrelated BMT and one patient received related PBSCT.
One patient with cGVHD and one patient without cGVHD
received allo-HSCT from human leukocyte antigen (HLA)
mismatched donors (both one locus; HLA-DR), while the
remaining patients received HLA-full-matched grafts. As a
conditioning regimen, seven patients with cGVHD received
total body irradiation (TBI total 12Gy), cyclophosphamide
(CY) and cyclosporin A (CyA), two patients received TBI
and CY and the remaining one patient was administered
combinations of high-dose chemotherapy; busulfan (BU)
and CY. On the other hand, six patients without cGVHD
received TBI, CY and CyA, two patients received TBI and
CY and one patient BU, CY and TBI. Three patients with
cGVHD and two patients without cGVHD received a
combination of tacrolimus (FK) and methotrexate (MTX)
for prophylaxis of aGVHD, while others received combi-
nation of CyA and MTX. Five patients with cGVHD and
four patients without cGVHD had previously developed
grade II–IV aGVHD.27Of 10 patients who had cGVHD at
the time of examination, four with extensive cGVHD were
being administered immunosuppressive agents or corticos-
teroids at the time of examination (one had received CyA,
one had prednisolone (PSL) and two had a combination of
PSL and FK). The cGVHD in these four patients was thus
ameliorated and manifested as of the limited type (although
their cGVHD is shown as ‘extensive’ in Table 1)28at the
time of examination. By contrast, the remaining six showed
limited-type cGVHD and had not been administered any
immunosuppressive agents or corticosteroids for at least 2
months before the examination. On the other hand, of nine
patients who did not have cGVHD at the time of
examination, three had limited type cGVHD prior to this
study and had been successfully treated with immuno-
suppressive agents or corticosteroids (one had received FK
and two had PSL), whereas the remaining six had not been
received any immunosuppressive agents or corticosteroids
for at least 2 months before the examination. All 19
patients were alive at the time of this analysis.
Controls
For normal controls, we collected blood from 10 healthy
adult volunteers (six men and four women, mean age 29
Table 1
Patient characteristics
Case
Diagnosis
Disease
status
Sex
Age
Graft
Donor
type
HLA
corresponding
Pre-transplant
conditioning
GVHD
profiraxisis
aGVHD
grade
aGVHD
onset
Days post
HSCT
cGVHD at the
time of examination
cGVHD
type
Treatment against
cGVHD
Outcome
1
AA
M
37
BM
R
6/6
CA/CY/TBI
CyA/MTX
0
2840
+ (liver)
Limited
None
Alive
2
CML
CP
F
47
BM
R
6/6
BU/CY
CyA/MTX
II
30
1818
+ (liver)
Limited
None
Alive
3
AML (M2)
1st CR
M
34
BM
R
6/6
CA/CY/TBI
CyA/MTX
II
14
1566
+ (skin)
Limited
None
Alive
4
ALL (L2)
1st CR
M
32
BM
R
6/6
CA/CY/TBI
CyA/MTX
II
18
1453
+ (skin, liver)
Limited
None
Alive
5
AML (M3)
2nd CR
F
33
BM
UR
5/6
CA/CY/TBI
FK/MTX
II
11
1057
+ (liver)
Limited
None
Alive
6
AML (M3)
1st CR
M
25
BM
R
6/6
CY/TBI
FK/MTX
0
522
+ (liver)
Limited
None
Alive
7
ALL (L2)
1st CR
F
49
BM
R
6/6
CA/CY/TBI
CyA/MTX
I
32
1285
+ (liver)
Extensive
CyA (50mg)
Alive
8
AML (M5a)
1st CR
M
27
BM
R
6/6
CA/CY/TBI
CyA/MTX
I
5
1124
+ (skin)
Extensive
PSL (10mg)
Alive
9
AML (M4)
Non-CR
M
20
BM
R
6/6
CA/CY/TBI
CyA/MTX
0
669
+ (liver)
Extensive
PSL (2.5mg)/FK (1mg)
Alive
10
CML
AP
F
44
PB
R
6/6
CY/TBI
FK/MTX
III
34
579
+ (skin)
Extensive
PSL (12.5mg)/FK (1mg)
Alive
11
AML (M4)
3rd CR
F
39
BM
R
6/6
CA/CY/TBI
CyA/MTX
I
19
2798
None
Alive
12
AML (M1)
1st CR
F
27
BM
R
6/6
CA/CY/TBI
CyA/MTX
0
1832
None
Alive
13
CML
CP
M
29
BM
R
6/6
CA/CY/TBI
CyA/MTX
III
8
1545
None
Alive
14
AML (M2)
2nd CR
M
32
BM
R
6/6
CA/CY/TBI
CyA/MTX
I
18
986
None
Alive
15
ALL (L2)
1st CR
M
30
BM
R
6/6
CY/TBI
CyA/MTX
II
18
739
None
Alive
16
AML (M5a)
1st CR
M
13
BM
R
6/6
CA/CY/TBI
CyA/MTX
I
12
571
FK(1mg)
Alive
17
ALL (L2)
Non-CR
M
18
BM
R
6/6
CA/CY/TBI
CyA/MTX
II
25
1154
PSL (2.5mg)
Alive
18
AML (M3)
2nd CR
M
29
BM
UR
6/6
CY/TBI
FK/MTX
0
783
PSL (10mg)
Alive
19
AML (M1)
2nd CR
F
34
PB
R
5/6
BU/CY/TBI
FK/MTX
II
12
1153
None
Alive
AA¼aplastic anemia; CML¼chronic myelogenous leukemia; AML¼acute myelogenous leukemia; ALL¼acute lymphoblastic leukemia; CP¼chronic phase. CR¼complete remission; M¼male; F¼female;
BM¼bone marrow; PB¼peripheral blood stem cell; R¼related donor; UR¼unrelated donor; HLA¼human leukocyte antigen; CA¼cytosine arabinoside; CY¼cyclophosphamide; TBI¼total body
irradiation; BU¼busulphan; CyA¼cyclosporin A; FK¼tacrolimus; MTX¼methotrexate; cGVHD¼chronic graft-versus-host disease; aGVHD¼acute graft-versus-host disease; PSL¼prednisolone.
IL-4-producing CD8þT cells in chronic GVHD
K Nakamura et al
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years, range 22–46 years). No patients or volunteers
showed evidence of infection at the time of study.
Media
RPMI 1640 medium supplemented with 2mM L-glutamine,
100 units/ml penicillin, 100ng/ml streptomycin, and heat-
inactivated 10% fetal bovine serum (Irvine Scientific, Santa
Ana, CA, USA) was used throughout the experiments.
Count of absolute number of CD4þor CD8þT cells
The peripheral blood mononuclear cells (PBMCs) were
freshly isolated by Lymphoprep (Nycomed Pharma, Oslo,
Norway) gradient centrifugation of heparinized blood. The
isolated PBMCs were stained with fluorescein isothio-
cyanate (FITC)-labeled CD4 (RPA-T4; Becton Dickinson,
San Jose, CA, USA), phycoerythrin (PE)-labeled CD8
(HIT8a; Becton Dickinson) and phycoerythrin-cyanin 5.1
(PC5)-labeled CD3 (UCHT1; Beckman Coulter, Mar-
seilles, France). These cells were washed with phosphate
buffered saline (PBS) containing 0.2% bovine serum
albumin, and the percentages of CD4þor CD8þT cells
were then measured using a FACScan flow cytometer
(Becton Dickinson). The absolute numbers of CD4þor
CD8þT cells (/ml) were calculated by multiplication of the
percentage of each cell-type with total PBMCs (/ml).
Detection of cytokines from T cells by intracellular staining
PBMCs were suspended in RPMI 1640 at a concentration
of 3?106/ml. Cells were stimulated with 50ng/ml phorbol-
12-myristate-13-acetate (PMA) (Sigma, St Louise, MO,
USA) and 1mg/ml ionomycine (Sigma) in the presence of
2mg/ml brefeldin A (ICN Biomedicals, Aurora, OH, USA)
for 4h at 371C. The cells were then washed with PBS
containing 0.2% bovine serum albumin and stained with
PC5-labeled anti-CD4 (13B8.2; Beckman Coulter), or anti-
CD8 (B9.11; Beckman Coulter) for 30min in the dark. To
evaluate the cytokine production, these cells were further
manipulated using Fix & Perm cell Permeabilization Kit
(CALTAG, Burlingame, CA, USA) and were stained with
FITC-labeled anti-IFN-g (4S.B3; Becton Dickinson) com-
bined with PE-labeled anti-IL-4 (8D4-8; Becton Dickin-
son), or IL-10 (JES3-19F1; Becton Dickinson). These cells
were washed with PBS and resuspended in 200ml of 1%
formaldehyde (in PBS, pH 7.2), and were then measured
using a FACScan flow cytometer.
Detection of cytokine production from T cells
incubated for 24h
PBMCs were divided into CD4þT cells and CD8þT cells
using anti-CD4-coated magnetic beads (Miltenyi Biotec,
Bergisch Gladbach, Germany) and anti-CD8-coated mag-
netic beads (Miltenyi Biotec) among patients with cGVHD,
patients without cGVHD and the normal control subjects.
The 498% purity of these cells was proved by FACS
analysis using anti-CD4 or anti-CD8mAb. The isolated
CD4þand CD8þT cells (1?106cells) were cultured
separately with 50ng/ml PMA and 2mg/ml ionomycine in
24-well flat-bottomed culture plates in 1000ml of medium
per well. After 24h, the cytokines (IL-4, IL-10 and IFN-g)
in each supernatant were measured by ELISA using
commercially available kits (Endogen, Woburn, MA,
USA).
Statistical analysis
Continuous data were compared using Kruskal–Wallis test.
When the difference was significant, we evaluated each
two variables using Mann–Whitney U-test with the Stat
View statistical program (Abacus Concepts, Berkeley, CA,
USA). Differences were considered significant when tied
P-values were less than 0.05.
Results
Absolute number of CD4þT cells and CD8þT cells
The absolute number of CD4þT cells was not significantly
different among normal
cGVHD, and patients with cGVHD (control; mean
7.4?105/ml, range 5.3–11.9?105/ml, patients without
cGVHD; mean6.5?105/ml,
patients with cGVHD; mean5.9?105/ml, range 3.3–8.6?
105/ml) (Figure 1a). Furthermore, the absolute number
of CD8þT cells was not significantly different among
the three groups (normal controls; mean 4.4?105/ml,
range 2.2–5.9?105/ml, patients without cGVHD; mean
6.5?105/ml,
cGVHD; mean 6.3?105/ml, range 2.3–11.3?105/ml)
(Figure 1b). In addition, the absolute number of CD4þ
or CD8þT cells in patients with cGVHD or without
cGVHD was not significantly affected by the use of
immunosuppressive agents or corticosteroids (Figure 2a, b).
controls, patients without
range 3.4–9.7?105/ml,
range3.3–9.8?105/ml, patients with
Increased percentage of IFN-g-producing peripheral blood
CD4þT cells in two patient groups
We analyzed the cytokine production of the peripheral
blood CD4þ
T cells and CD8þ
intracellular cytokine staining method. The percentage
of IL-4-producing CD4þT cells among total CD4þT
cells was not significantly different among patients with
cGVHD (mean 3.9%; range 2.4–7.7%), without cGVHD
(mean 3.6%; range 0.7–7.6%) and normal control subjects
(mean 2.8%; range 0.2–4.9%) (Figure 3a). On the other
hand, the percentage of IFN-g-producing CD4þT cells
was significantly higher in patients with cGVHD (mean
51.1%; range 18.6–80.7%) and without cGVHD (mean
37.7%; range 9.5–63.4%) than in the normal control
subjects (mean 16.7%; range 6.7–38.9%) (P¼0.0001,
P¼0.0073) (Figure 3b). However, the percentage of
IFN-g-producing CD4þ
T cells was not statistically
different between the two patient groups. To confirm the
results from intracellular cytokine staining, we examined
the titers of cytokines in the supernatants of 24-h-cultured
CD4þT cells by ELISA. Although the titer of IL-4
and IFN-g from CD4þ
T cells was not statistically
different among patients with and without cGVHD and
T cells using the
IL-4-producing CD8þT cells in chronic GVHD
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normal control subjects, the ELISA data were basically
similar to that of intracellular cytokine staining (data not
shown).
Increased percentage of IL-4-producing peripheral blood
CD8þT cells in patients with cGVHD
The percentage of IL-4-producing CD8þT cells was
significantly higher in patients with cGVHD (mean 3.3%;
range 1.3–8.2%) than in patients without cGVHD (mean
1.2%; range 0.8–1.7%) (P¼0.0002) and normal control
subjects(mean1.1%;range
(Figure 4a). There was no significant difference in the
percentage of IL-4-producing CD8þ
0.1–1.6%)(P¼0.0006)
T cells between
patients without cGVHD and normal control subjects.
On the other hand, the percentage of IFN-g-producing
CD8þT cells was significantly higher in patients with
cGVHD (mean 74.5%; range 59.2–91.3%) and without
cGVHD (mean 68.9%; range 34.1–93.7%) than in normal
controlsubjects (mean
(Po0.0001, P¼0.0007) (Figure 4b). However, it was not
different between the two patient groups. Figure 5
shows representative data of intracellular cytokine staining
of IL-4 and IFN-g of CD8þT cells. IL-4-producing CD8þ
T cells were barely detected in the control subjects
(Figure 5a) and the patients without cGVHD (Figure 5b),
whereas a low but definite percentage of these cells were
detected in the patients with cGVHD (Figure 5c). The
35.3%;range 20–58.4%)
CD8+ T cells
CD4+ T cells
0
2.5
5
7.5
10
CD8+ T cells
0
2.5
5
7.5
10
a
b
(× 105/ml)
(× 105/ml)
Figure 1
(n¼9), and the black column is patients with cGVHD (n¼10). (a) The absolute number of CD4þT cells. (b) The absolute number of CD8þT cells.
The absolute counts of CD4þand CD8þT cells in PBMC. The open column is controls (n¼10), the dotted column is patients without cGVHD
0
2.5
5
7.5
10
With cGVHD
CD4 CD8
(× 10
5/ml)
a
0
2.5
5
7.5
10
12.5
Without cGVHD
CD4CD8
(× 10
5/ml)
b
Figure 2
immunosuppressive agents. The shaded column is patients who were receiving predonisolone and/or immunosuppressive agents. (a) The absolute counts of
CD4þand CD8þT cells of patients with cGVHD. Open column is n¼6, shaded column is n¼4. (b) The absolute counts of CD4þand CD8þT cells of
patients without cGVHD. Open column is n¼6, shaded column is n¼3.
The absolute counts of CD4þand CD8þT cells in PBMC. The open column is patients who were not receiving predonisolone or
IL-4-producing CD8þT cells in chronic GVHD
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percentages of IL-4-producing CD8þT cells among CD8þ
T cells in each patient were summarized in Table 2. The
percentage of IL-4-producing CD8þT cells in patients
with cGVHD was not significantly affected by the use
of immunosuppressive agents or corticosteroids, although
a patient with cGVHD receiving the agents showed a
relatively lower value.
Increased IL-4 production from CD8þT cells in patients
with cGVHD
The titer of IL-4 in the supernatants of 24-h-cultured
CD8þT cells was significantly higher in patients with
cGVHD (mean 593pg/ml; range 48–1446pg/ml) than
in patients without cGVHD (mean 109pg/ml; range
0
2
4
0
20
40
60
80
(%)
(%)
***
***
1
3
5
a
b
IL-4 IFN-?
Figure 3
column is patients without cGVHD (n¼9), and the black column is patients with cGVHD (n¼10). ***Po0.001. (a) The percentage of IL-4-producing
CD4þT cells among total CD4þT cells. (b) The percentage of IFN-g-producing CD4þT cells among total CD4þT cells.
The percentage of IL-4 and IFN-g -producing CD4þT cells by intracellular cytokine staining. The open column is controls (n¼10), the dotted
0
2
4
0
20
40
60
80
(%)
(%)
**
***
***
***
1
3
5
a
b
IL-4 IFN-?
Figure 4
column is patients without cGVHD (n¼9), and the black column is patients with cGVHD (n¼10). **Po0.01. ***Po0.001. (a) The percentage of
IL-4-producing CD8þT cells among total CD8þT cells. (b) The percentage of IFN-g-producing CD8þT cells among total CD8þT cells.
The percentage of IL-4 and IFN-g -producing CD8þT cells by intracellular cytokine staining. The open column is controls (n¼10), the dotted
IL-4-producing CD8þT cells in chronic GVHD
K Nakamura et al
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