Download Aplastic anemia (AA) is a bone marrow failure disease, which mainly

The preliminary research of immune function monitoring before and
after allogeneic hematopoietic stem cell transplantation in children
with aplastic anemia
TONG Chun1
，
2
,GUO Zhi2,LOU Jing-Xing2,LIU Xiao-Dong2,YANG Kai2,HE Xue-Peng2,
ZHANG Yuan2,CHEN Peng2,CHEN Hui-Rren2*
1
Clinical Medical College of Anhui Medical University,General Hospital of Beijing Military
Command，Beijing 100700,China. 2Department of Hematology,General Hospital,Beijing Military
Area,Beijing 100700,China
*Corresponding
author: CHEN Hui-Ren, Senior Physician, Professor, Tutor of Doctorial
Postgraduated. Email: [email protected]
Abstract Objective: To explore the clinical significance of the relationship between the immune
function and the pathogenesis of aplastic anemia in children with aplastic anemia, along with
the incidence of graft-versus-host disease (GVHD) by monitoring the changes of T lymphocyte
subsets
dynamicly
cell transplantation.
on
+1, +3, +6, +12 month
Methods:
12
cases
after
allogeneic
hematopoietic
stem
received
allogeneic
hematopoietic
stem
cell transplantation in the department of Hematology of General Hospital of Beijing Military
Region from January 2013 to January 2014, including 4 males and 8 females, average age of 7.92
(3-14 ) years old with 5 cases of HLA matched and 7 cases of HLA mismatched. Monitoring the
level of T lymphocyte subsets including CD3+, CD4+, CD8+, CD4+/CD8+, CD56+,
CD4+CD25high+FOXP3+ with flow cytometry (FCM) on +1, +3, +6, +12 month after and before
transplantation dynamicly in the peripheral blood. While monitoring T lymphocyte subsets level in
12 cases healthy children at the same period as control. Results: Follow up to March 2015, 10
cases have abnormal cellular immunity (CD4+/CD8+ inversion) in the 12 cases. Compared with the
control group, CD3+ slightly higher, CD4+ decreased, CD8+ increased, CD4+/CD8+ decreased and
CD56+ decreased, CD4+CD25high+FOXP3+ decreased in AA patients before transplantation, with
the difference were statistically significant (P< 0.05). The lever of CD3+, CD4+, CD8+,
CD4+/CD8+, CD56+, CD4+CD25high+FOXP3+ had a different degree of recovery after
transplantation for all cases and returned to normal on +12 month basically. The level of
CD4+CD25high+FOXP3+ for patients with acute GVHD was lower than that of non acute GVHD
cases. Conclusion: 1.Compared with healthy control group, abnormal cell immune function in
some cases with AA; 2.Compared with pre-transplantation, the level of CD3+, CD4+, CD8+,
CD4+/CD8+, CD56+ and CD4+CD25high+FOXP3+ reduced after allogeneic hematopoietic stem
cell transplantation; CD8+ T cells recovered earlier than CD4+ T cells, and returned to normal on
+12 month gradually, then achieved stable on +18 month finally; While the decrease of CD4+ T
cells lasted more than 1 year; The proportion of CD4+/CD8+ inversion also lasted for more than 1
year; 3.After transplantation, the level of CD4+CD25high+FOXP3+ in GVHD positive group was
significantly lower than the negative group, which can be used to predict the occurrence of
GVHD.
Key words: Aplastic anemia; Allogeneic hematopoietic stem cell transplantation; cellular immune
function; flow cytometry; T lymphocyte subsets; graft versus host disease
Aplastic anemia (AA) is a bone marrow failure disease, which mainly involving inner defects
of hematopoietic stem cells, abnormal immune response in hematopoietic stem cells,
hematopoietic microenvironment defect[1], especially for AA (severe aplastic anemia, SAA) with
poor clinical prognosis. It should be performed blood component transfusion and special
treatment immediately when diagnosed, otherwise most children died of infection or
complications such as bleeding about one year [2]. At present, the immune mediated hematopoietic
function inhibition plays an important role in the pathogenesis of AA, and T lymphocytes are the
major effector cells in the immune response[3]. The first-line treatment of SAA includes
immunosuppressive therapy (IST) and hematopoietic stem cell transplantation (HSCT). IST could
make blood completely back to normal in about 20%~40% patients, but which effects slowly, and
still need blood transfusion, prevention and treatment of infection and so on, with greater risk.
HSCT has rapid hematopoietic recovery, complete remission generally which needs no
maintenance treatment, can reduce the mortality rate of graft-versus-host disease (GVHD) with
survival rate can be improved distinctly[4]. In 2000, Horowitz MM[5] reported that allogeneic stem
cell transplantation could make the 5 years survival rate of AA to 77%, and 90% for children. It is
important to monitor the reconstruction of immune function in SAA children dynamically after
transplantation. It was confirmed[6]that the T lymphocyte subsets are the main regulatory cells in
the reconstruction of immune function. This study by monitoring T lymphocyte subsets level of
patients with SAA pre-transplantation and on + 1, + 3, + 6 and 12 months post-transplantation
with flow cytometry and explore the change rule. By comparing T lymphocyte subsets of AA
children and healthy control group to investigate immune factors in the pathogenesis of AA, also
compared between GVHD positive and negative to explore the relationship between GVHD and T
lymphocyte subsets.
1 materials and methods
1.1 Basic data
12 cases with SAA underwent allogeneic hematopoietic stem cell transplantation in the
department of hematology of General Hospital of Beijing Military Region from January 2013 to
January 2014, including 4 males and 8 females, average age of 7.92 (3-14)years old, randomly
selected 12 cases healthy children in our hospital physical examination center as control over the
same period, 6 males and 6 females, with the median age of 6.83 (2-12) years old. SAA diagnostic
criteria is in accordance with the third edition "blood disease diagnosis and efficacy standards"
which edited by Zhang Zhinan[7]. The transplantation methods included 5 cases of HLA matched,
other 7 cases of HLA mismatched. According to the graft-versus-host disease (GVHD) occurrence
after transplantation, cases were divided into GVHD positive group and negative group, and the
gender, age, diagnosis, HLA matching, pre-treatment regimen and GVHD prophylaxis had
comparability in the two groups. All cases were examed the functions of important organs
comprehensively before transplantation and were not associated with Basic disease or important
organ dysfunction. Children's families were fully informed and signed informed consent, and the
transplantation program was approved by the hospital ethics committee. The basic information of
the children is in table 1.
Table 1
Group
Gender
Age (years)
The basic information of cases
HLA matching
Infusion of
Follow-up
CD34+ cells
time
（×106/kg）
(month)
3.25
6
Complication
outcome
Pneumonia、
Dead
Infusion of nuclear
cells（×108/kg）
1
M
3
5/10
8.47
Intestinal GVHD
2
F
2
6/10
5.38
2.52
12
3
F
12
10/10
10.84
2.97
12
Pneumonia
Alive
Pneumonia、
Dead
Intestinal GVHD
4
M
8
10/10
5.56
6.81
14
Pneumonia
Alive
5
M
1
5/10
7.99
3.37
15
Hemorrhagic
Alive
cystitis、
Septicemia
6
F
8
5/10
15.54
14.73
16
Septicemia、Skin
Alive
GVHD
7
M
9
10/10
14.22
10.16
17
Hemorrhagic
Alive
cystitis、Skin
GVHD
8
F
11
10/10
16.39
9.09
18
cytomegalovirus
Alive
viremia、
Intestinal GVHD
9
F
14
6/10
8.96
4.12
22
Hepatic GVHD
Alive
10
F
6
10/10
11.16
2.90
24
Pneumonia、Oral
Alive
infection
11
F
10
5/10
12.45
8.63
25
Skin GVHD
Alive
12
F
11
7/10
11.17
9.37
26
Pneumonia、
Alive
Intestinal GVHD
1.2 Preprocessing scheme
All patients accepted the reduced intensity conditioning regimen, which using fludarabine
(Flu), cyclophosphamide (CTX) and anti lymphocyte globulin (ATG) for 5 cases had HLA
haploidentical donor, specifically: Flu 30 mg/(m2·d), -5d, -4d, -3d, -2d; CTX 40 mg/(m2·d), -4d,
-3d; ATG 5 mg/(m2·d), -4d, -2d, -3d, -1d; Bai Shufei (Bu), CTX, ATG and Flu was used in 7 cases
with HLA mismatched donors, specifically: Bu 3.2mg/(kg·d), -6 day; Flu 30 mg/(m2·d), - 5d, - 4d,
- 3d, - 2d; CTX 40 mg/(kg·d), - 5d, - 4 d; ATG 5 mg/(kg·d), - 4d, - 3d, - 2d, - 1 d.
1.3 Stem cell collection and GVHD prevention
Transplantation method was bone marrow combined with peripheral blood stem cell
transplantation. Donors received mobilization by granulocyte colony stimulating factor, 5-10 μ
g/(kg·d), for 5-6 days and collected bone marrow suspension on the 5 day, 8-12 ml/kg, then
separated peripheral blood stem cells by blood cell separator on the 6 day. Using flow cytometry
counted CD34 + cells to ensure the mononuclear cell number ≥5×108/kg, and the median CD34
+ cells ≥2.0×106 / kg. Cyclosporin A (CSA), methotrexate (MTX), mycophenolate mofetil
(MMF) and tacrolimus (FK506) for GVHD prophylaxis. If there was any GVHD, according to the
severity, added ATG, sugar cortical hormone, and CD25 monoclonal antibody. If with no GVHD
the immunosuppressant reduced gradually in the half year after transplantation.
1.4 The method of lymphocyte subsets detection
T lymphocyte subsets were detected by 3-4ml venous blood from children.The main reagents
and instruments were monoclonal antibodies, red cell lysis and FACSCantoII type flow cytometry.
Take two FACS tubes with anticoagulant blood 100μl, joined CD3-APC, CD8-APC, CD4-FITC,
CD45-VioGREENA, CD56-PE monoclonal antibody each 10μl into one tube, mixed and
incubated protect form light at room temperature for 20 min, then add the FACS Lysing solution
lysis to be measured. CD4-FITC, CD45-VioGREENA, CD25-APC, 10μl respectively, were added
into the other tube. All of the above antibodies were cell membrane antibodies.
1.5 Intracellular markers FOXP3
Using intracellular staining method for the detection of Foxp3 antibody, specific steps as
follow, in accordance with the above methods with membrane labeled antibody, incubation,
hemolysis, centrifugal, discard supernatant, then add 2ml PBS to mix, 1050rpm centrifugation for
5 minutes, add 750 mu IF/P/D and 250 mu IF/P to mix after discard supernatant, 4℃ for 30
minutes, and add 2 ml buffer to mix, then centrifuge 5 minutes to discard the supernatant, add 3ul
cytoplasmic antibody FOXP3-PE, in dark at room temperature light incubation then add 2 ml
buffer to mix after incubation for 30 minutes at room temperature. Finally, add 0.5ml PBS to mix
then detected on the machine.
1.6 Follow up and statistics
Follow up to March 2015, the average follow-up time of 17.3 months (6-26 months), the
follow-up period of survival children were more than 1 year, and the longest follow-up period was
up 26 months. Analyse implantation, transplantation related complications and disease-free
survival situation of all children, then use flow cytometry (FCM) dynamic to monitor the lever of
CD3+, CD4+, CD8+, CD4+/CD8+ and CD56+, CD4+CD25high+FOXP3+ in aplastic anemia patients
before transplantation and on + 1, + 3, + 6 and + 12 month after transplantation, and statisticsthe
overall survival rate of the whole group. Monitor T lymphocyte subsets in healthy children as
control group at the corresponding period. Using SPSS13.0 statistical software for statistical
processing. The measurement data were expressed by mean± standard deviation( x ±SD), P<0.05
was statistically significant, and survival analysis was used Kaplan-Meier method.
2 Result
2.1 The effect of transplantation
Follow up to March 2015, all patients obtained hematopoietic reconstitution after
transplantation and the mean time of neutrophil ≥ 0.5 *109 / L and platelet ≥ 20*109/L were 16.8d
and 18.5d respectively. After transplantation, 8 cases of acute GVHD, no chronic GVHD occurred,
there were 6 cases of pulmonary infection, 1 case of septicemia, 2 cases of cytomegalovirus
viremia, 2 cases of bleeding cystitis, 1 case of oral infection. 1 case died of acute GVHD, 1 case
died of pulmonary infection, and the remaining 10 cases were alive. (Figure 1)
Figure 1 Survival curves of all cases
2.2 The comparison of T lymphocyte subsets between before and post transplantation
Compared to the control group, CD3+ cells of AA children was slightly higher after
transplantation, CD4+ cells decreased, CD8+ cells increased, CD4+/CD8+ decreased, CD56+ cells
decreased, CD4+CD25high+FOXP3+ cells reduced, and the differences above were statistically
significant (P< 0.05). 10 patients existed abnormal immune cells (CD4+/CD8+ inverted ) in 12
cases with AA. (Table 2)
Table 2 The comparison of peripheral blood T lymphocyte subsets betweeen AA patients
and control group ( x ±SD)
Group
AA group
Cases
CD3+
CD4+
12
（66.79±7.35)%
（33.73±7.26)%
（35.69±6.78)% （1.23±0.56)%
(7.46±2.8)%
(3.3±1.5)%
(62.74±5.58)%
(39.54±3.46)%
(25.34±4.36)%
(16.73±3.7)%
(8.1±1.3)%
0.043
0.037
Control group 12
P
CD8+
CD4+/CD8+
CD56+
(1.78±0.34)%
0.000
0.001
CD4+CD25high+FOXP3+
0.000
0.003
2.3 Changes of T lymphocyte subsets after transplantation
This study showed that 1 month after transplantation, the proportion of CD3+ cells decreased,
the difference was statistically significant(P< 0.05), CD3+ cells gradually returned to the level of
before transplantation on the 3 month after transplantation and CD8+ cells had been restored and
even more than the level of before transplantation, CD4+ cells recovery delayed, the ratio of CD4+/
CD8+ sustained inversion more than 1 year; CD56+ cells were significantly lower on 1 month after
transplantation, and gradually restored on 3 month. CD4+CD25high+FOXP3+ cells were lower in
early stage after transplantation. (Table 3)
Table 3 The level of T lymphocyte subsets in AA children before and after transplantation
( x ±SD)
Group
Pre
CD3+
Cases
CD4+
CD8+
CD4+/CD8+
CD56+
CD4+CD25high+FOXP3+
12
(66.79±7.35)%
(33.73±7.26)% (35.69±6.78)%
(1.23±0.56)%
(7.46±2.8)%
(3.3±1.5)%
Post/+1M 12
(48.0±28.2)%*
(20.8±23.7)%* (20.2±23.8)%*
(1.5±2.7)%*
(5.35±3.2)%*
(1.2±1.1)%*
(1.1±0.9)%
+3M
12
(58.1±27.1)%
(10.8±7.7)%
(51.3±18.8)%
(0.2±0.2)%
(12.65±3.6)%
+6M
12
(63.9±28.4)%
(23.7±7.6)%
(37.5±28.3)%
(1.0±0.9)%
(13.78±2.1)%
(1.6±1.7)%
(79.1±6.5)%
(28.0±2.9)%
(47.4±6.1)%
(0.6±0.1)%
(15.73±4.7)%
(1.3±0.6)%
+12M 12
Note:* Compared with befor e transplantation，P<0.05
2.4 The correlation between GVHD and CD4+CD25high+FOXP3+
8 cases developed acute GVHD after transplantation in this group, defined as GVHD positive
group, 4 cases with no acute GVHD, defined as GVHD negative group. The difference of
CD4+CD25high+FOXP3+ in GVHD positive group and negative GVHD group on+ 1, + 3, + 6 and
+12 month after transplantation statistically significant (P < 0.01).(Table 4)
Table 4 The comparison of CD4+CD25high+FOXP3+ between GVHD positive group and
negative group ( x ±SD)
Group
CD4+CD25high+FOXP3+
Cases
+1M
GVHD positive
group
8
(0.4±0.6)%
+3M
+6M
(0.7±0.3)%
(1.1±0.5)%
+12M
(1.4±0.3)%
GVHD negative
P
group
4
(1.6±0.7)%
(2.7±0.4)%
0.007
0.000
(2.9±0.7)%
0.005
(3.6±0.2)%
0.006
3 Discussion
With the continuous improvement of allogeneic hematopoietic stem cell transplantation
technology, HSCT has become the most effective for children with SAA[7-8]. The reconstruction of
immune function in children requires a long time after transplantation, which means the
recovery of various immune effector cells. Hematopoietic stem cells produce lymphatic
hematopoietic stem/progenitor cells in the bone marrow after HSCT,then migrate to the thymus,
and proliferate, differentiate and maturate[9]. Generally, CD3+ cells gradually returned to normal
on +3 month after transplantation, and the reduction of CD4+ cells was more than 1 year, CD8+
cells recovered quickly, and the inversion of CD4+/CD8+ was more than 1 year. The reason may
be that the generation of CD4+ cells depend mainly on the thymus, while the formation and
maturation of CD8+ cells are not completely dependent on the thymus[10]. Related research[11]
showed that, lymphocyte subsets especially the dynamic balance of T lymphocyte subsets was
related to immune function closely. Allogeneic hematopoietic stem cell transplantation technique
significantly improves the disease free survival rate of the SAA patients. Infection, GVHD,
transplantation related complications concern the efficacy after transplantation, and the immune
function recovery can reduce transplantation related complications[12]. Therefore, it is very
important to monitor the immune reconstitution after transplantation, which has important
significance to improve the reconstitution of immune function after transplantation.
For the role of T lymphocytes in the pathogenesis of SAA[13], now we consider that the CD4+
and CD8+ T cells regulate hematopoiesis. CD4+ T cells are helper inducer T lymphocyte subset(Th
cells) which can stimulate the hematopoiesis, and its increase means the immunologic function
enhance. CD8+ T cells are inhibited T lymphocyte subset(Ts cells) which can inhibit the
hematopoiesis, and its increase means immunologic function inhibit. The ratio of CD4+/CD8+
indicates the equilibrium state between Th and Ts, and is the most important index of the
environmental stability in human immune system,the lower ratio can cause immune function
reduced. The imbalance of CD4+ and CD8+ T cells is fundamental cause to inhibit the
hematopoietic function in AA children[14]. This study also showed that the T lymphocyte subsets
were unbalanced in the peripheral blood among a considerable part of cases. The main
performance were that CD8+ cells increased, the CD4+ cells decreased, and the ratio of CD4+
/CD8+ reversed. In addition, the number and activity of natural killer cells in most cases were
significantly decreased[15], and the same results were also obtained in this study. The most
important reason is that the existence of CD4+CD25+FOXP3+ regulatory T cells (Tregs) inhibits
the activation of T cells in the normal state which makes no autoimmune response under normal
condition[16]. Immune incompetence and immune suppression of CD4+CD25+FOXP3+ Tregs can
be activated to turn into inhibit effector T cells. Foxp3 was discovered in recent years which is the
new member of the forkhead/winged-helix transcription factor family, and plays an important role
in the Treg differentiation[17]. FOXP3 specifically expressed in the cytoplasm of CD4+CD25+Treg
cells, which mediates the development of Treg in the thymus and its expression in the periphery,
and maintains its immunologic suppression[18]. Magenau[19] confirmed
that
+
+
+
CD4 CD25high FOXP3 can be as a predictor of acute GVHD and a valuable biological marker
of prognosis. CD4+CD25+FOXP3+ Tregs reduced in most children with AA, this may weaken T
cell immune suppression function, to sustain the activation of the effector T cells, eventually led to
the imbalance of immune regulation.
This study aimed to explore the immune function recovery of the children with SAA after the
allogeneic hematopoietic stem cell transplantation through monitoring the T lymphocyte subsets
pretransplantation and on +1, +3, +6 and +12 month posttransplantation dynamicly. Our research
showed that CD4+CD25high+FOXP3+ increased gradually after transplantation in children withno
acute GVHD, and finally reached to the normal level, and CD4+CD25high+FOXP3+ relatively
reduced in children with acute GVHD. Compared to 4 cases withno acute GVHD,
CD4+CD25high+FOXP3+ lower in 8 cases with acute GVHD (P < 0.05), and no chronic GVHD
occurred which related the small sample.
In summary, the level of CD4+CD25high+FOXP3+ is closely related to the occurrence of
acute GVHD. We can detect and prevent the acute graft-versus-host disease (GVHD) before the
appearance of clinical symptoms through monitoring regularly the level of
CD4+CD25high+FOXP3+ after transplantation in peripheral blood, so as to improve the survival
rate of SAA patients with transplantation.
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