Tolerogenic Vaccination Reduced Effector Memory CD4 T Cells and Induced Effector Memory Treg Cells for Type I Diabetes Treatment
et al. (2013) Tolerogenic Vaccination Reduced Effector Memory CD4 T Cells and Induced Effector Memory Treg
Cells for Type I Diabetes Treatment. PLoS ONE 8(7): e70056. doi:10.1371/journal.pone.0070056
Tolerogenic Vaccination Reduced Effector Memory CD4 T Cells and Induced Effector Memory Treg Cells for Type I Diabetes Treatment
Jingyao Zhang 0
Wenjuan Gao 0
Xu Yang 0
Jingjing Kang 0
Yongliang Zhang 0
Qirui Guo 0
Yanxin Hu 0
Guoliang Xia 0
Youmin Kang 0
Ciriaco A. Piccirillo, McGill University Health Center, Canada
0 1 State Key Laboratory for Agro-Biotechnology, College of Biological Science, China Agricultural University , Beijing , China , 2 Department of Modern Sciences & Technology, Agricultural University of Hebei , Baoding , China , 3 College of Veterinary Medicine, China Agricultural University , Beijing , China
Background: Vaccination could induce immune tolerance and protected NOD mice from the development of type I diabetes (T1D). We previously demonstrated that insulin peptide (B9-23) combined with dexamethasone (DEX) stimulated the expansion of antigen specific regulatory T (Treg) cells which in turn effectively prevented T1D in NOD mice. Here, we aimed to investigate the therapeutic effect of tolerogenic vaccination for T1D treatment. Methodology/Principal Findings: The diabetic NOD mice (Blood glucose level 250 mg/dl) were treated with B9-23 and DEX twice. The tolerance was restored by blocking maturation of dendritic cells (DCs) and inducing Treg cells in treated NOD mice. Remarkably, the reduction of autoreactive effector memory CD4 T (Tm) cells and the induction of functional effector memory Treg (mTreg) cells contributed to the improvement of T1D in treated NOD mice. Conclusions/Significance: Tolerogenic vaccination restored tolerance and ameliorated T1D by suppressing effector CD4 Tm cells and inducing effector mTreg cells. Our findings implicate the potential of tolerogenic vaccination for T1D treatment.
Funding: This work was supported by National Natural Science Foundation of China (Project: 31270956), the National Natural Science Foundation (Project:
J1103520) for Undergraduates and State Experiment Innovation for Undergraduates. The funders had no role in study design, data collection and analysis,
decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
T1D results from a chronic destruction of insulin-producing b
cells, presumably mediated by autoreactive CD4 T cells .
Interventions are less effective on activated T cells, including Tm
cells in pancreatic islets, as the pathogenic response becomes
established . Autoreactive T cells are important mediators of
T1D and have been shown to be antigen-specific Tm cells
targeting islet antigen in T1D patients . Self-antigen specific
Tm cells were observed in diabetic patients, but not in healthy
individuals . When naive T lymphocytes are antigen activated,
the expressions of several adhesion and homing molecules increase
or decrease, leading to an activated effector memory cell
phenotype of CD44HighCD62LLow . In T1D mice,
isletinfiltrating cells were characterized as CD44HighCD62LLow which
appeared to be memory cells and able to transfer insulitis and
diabetes . Using MHC class II tetramers, autoantigen-specific
CD4 Tm cells are prevalent in the early progression to T1D . In
this study, CD44HighCD62LLow cells were used as markers of
effector Tm cells in T1D mice.
More than 400 agents or agent combinations have been
investigated in preclinical T1D, such as cyclosporine, anti-CD3
antibody for T cells or anti-CD20 antibody for B cells, and TNF-a
or IL-1 blocking agents. These agents broadly inhibit the immune
response. However, responses to infections could be
inappropriately suppressed . The self-antigen induced Treg cells have
been shown potential in maintaining immunological self-tolerance
as prevention or therapy for autoimmune diseases [2,9]. The
expression of transcription factor Foxp3 and cytokine IL-10 play
critical roles in suppressive function of Treg cells [10,11]. The
deliberate induction of Tregs has generally been difficult to
achieve in vivo, and there is a pressing need to develop effective
methods for generating Tregs in a predictable way.
Vaccination with autoreactive antigen or peptides could
suppress the immune response by inducing Treg cells for
prevention or therapy of autoimmune disease. Several
vaccination strategies using islet antigens had been shown to
modify the time of onset and severity of T1D in mice . When
incomplete Freunds adjuvant (IFA) was co-administered with
insulin peptides subcutaneously, T1D development was inhibited.
However, when it was given intraperitoneally, the disease was not
modified . Co-immunization with insulin and DNA encoding
proinsulin induced CD4+CD25 islet-specific Treg cells and
prevented T1D onset . Our previous study demonstrated that
tolerogenic vaccination, insulin peptide B9-23 combined with
dexamethasone (DEX), could induce antigen specific Treg cells
and effectively prevented development of T1D .
Currently, there is no effective treatment strategy to preserve
residual b-cells and restore tolerance for T1D . Therefore, there
is an immediate need to restore both b cell function and immune
tolerance to control disease progression and ultimately cure T1D.
Based on these observations, we sought to investigate the
therapeutic effect of tolerogenic vaccination on T1D treatment.
Our results demonstrate that tolerogenic treatment restored
tolerance and ameliorated T1D by reducing CD4 Tm cells and
producing activated and memory T regulatory (mTreg) cells.
These results suggest a new method for T1D treatment.
Therapeutic Effect of Tolerogenic Treatment on T1D
Vaccination with autoantigen or peptides could induce
tolerance and effectively prevented autoimmune diseases [13,15,18].
We previously demonstrated that B9-23 combined with DEX
could effectively prevented T1D in NOD mice . To investigate
the therapeutic effect of this method, the diabetic NOD mice were
treated with B9-23/DEX twice. On day 7 after the second
treatment, the pancreases were prepared for histological section.
The lowest level of infiltration was noted in pancreas of mice
treated with B9-23/DEX compared with other groups. The
Figure 1. Tolerogenic treatment ameliorated T1D. A. On day 7 after the second treatment, pancreases of treated NOD mice were collected and
fixed in 4% formaldehyde for 24 h before being embedded in paraffin. Serial sections were cut and stained with H&E (2006). Data shown are
representative of 3 independent experiments. B. Pancreatic sections from each group were scored for islet inflammation. Shown is the average of
three independent experiments with similar results. For statistical analysis, mice treated with B9-23/DEX were compared with other control groups
and ANOVA were used; *p,0.05. C. The survival diabetic mice were counted weekly after treatment. Shown is the summary of three independent
experiments with similar results. For statistical analysis, mice treated with B9-23/DEX were compared with other control groups and ANOVA were
highest level of infiltration was observed In T1D control group, as
compared to mice treated with DEX, B9-23, or B9-23/DEX
(Figure 1A). Since DEX has immunosuppressive function, the
infiltrated lymphocytes in pancreas of DEX treated mice were less
than that in T1D control group (Figure 1A). For insulitis, the
lowest score was shown in B9-23/DEX treated mice compared
with that in other control groups (p,0.05). The score of insulitis in
T1D control mice was higher than that in other groups. The score
of insulitis in DEX treated mice was lower than that in T1D
control and B9-23 treated mice (Figure 1B). The treated diabetic
mice were monitored and counted weekly for survival rate
analysis. On 6 weeks after treatment, statistical analysis of survival
diabetic mice in B9-23/DEX group reached significant difference
compared with that in other groups (Figure 1C). The treated mice
were also monitored for glycemia analysis. There were no
significant differences of glycemia level among all groups since
T1D was developed (Data not shown). These results demonstrate
that B9-23/DEX treatment ameliorated T1D and improved the
survival time of diabetic mice.
Restoration of Immune Tolerance in Treated Diabetic
Many studies confirm the function of Treg cells in suppressing
pathologic immune responses of autoimmune diseases . Treg
cells had been induced under certain therapeutic interventions in
autoimmune disease [19,20]. Our previous study demonstrated
that tolerogenic vaccination could expand Treg cells in
nondiabetic NOD mice and prevented T1D of NOD mice . To test
whether tolerogenic vaccination can induce tolerance for T1D
treatment, the splenocytes of treated diabetic mice were prepared
and stained with anti-CD4 and anti-CD25 mAbs, then were
intracellularly stained with anti-Foxp3 mAb for Treg cells. For
IL10 expression in Treg cells, the samples were stained with
antiCD4 and anti-CD25 mAbs, then were intracellularly stained with
anti-Foxp3 mAb and anti-IL-10 mAbs. Gating on CD4+ T cells
(Figure 2A), the percentage of Treg cells (CD4+CD25+Foxp3+) was
counted relatively to total CD4 T cells. As shown in Figure 2A, the
number and percentage of Treg cells were increased significantly
in B9-23/DEX treated mice compared with that in other groups
(p,0.05). Furthermore, the percentage of IL-10+ Treg cells
(CD4+CD25+ Foxp3+IL-10+) to total Treg cells (CD4+ CD25+ T
cells, R1 in Figure 2B) was increased significantly in B9-23/DEX
treated mice compared with that in other groups (p,0.05,
Figure 2B). These results suggest that Treg cells could be induced
and played suppressive function by expressing IL-10 in B9-23/
DEX treated diabetic mice.
Treg cell expansion is known to be linked to the function of
immature DC [19,21], and DEX was previously reported to
prevent DC maturation in vitro . To analyze the maturation
of DCs in treated diabetic mice, the splenocytes of treated mice
were prepared and stained with anti-CD11c-FITC,
anti-MHCIIPE or CD80-PE. For IL-10 expression in DCs, the samples were
intracellularly stained with anti-CD11c-FITC and anti-IL-10-PE
mAbs and analyzed by flow cytometry (Figure 3A). As shown in
Figure 3B, the expression of MHCII or CD80 on DCs were
decreased significantly in B9-23/DEX treated diabetic mice
compared with that in B9-23 treated mice (p,0.05). The
expression of CD40 or CD86 on DCs were also decreased
significantly in B9-23/DEX treated diabetic mice compared with
that in B9-23 treated mice (Data not shown). The percentage of
Figure 2. Tolerance was restored in treated diabetic mice. A. On day 7 after the second immunization, splenocytes were intracellularly stained
with anti-CD4-APC, anti-CD25-PECy5 and anti-Foxp3-PE mAbs for Treg analysis. Gating on CD4+ T cells (R1), Treg cells (CD4+CD25+Foxp3+) were
quantified relatively to total CD4+ T cells. The numbers of Treg cells were counted in all groups by flow cytometry. For statistical analysis, mice treated
with B9-23/DEX were compared with other control groups and ANOVA were used; *p,0.05. B. On day 7 after the second immunization, splenocytes
were re-stimulated with B9-23, and then intracellularly stained with anti-CD4-FITC, anti-CD25-PECy5, anti-Foxp3-PE and anti-IL-10-APC mAbs for IL-10
expression in Treg cells analysis. Gating on CD4+ CD25+ T cells (R1), Treg cells expressed IL-10 (CD4+CD25+Foxp3+ IL-10+) were quantified relatively to
total Treg cells. Data shown are representative of 3 independent experiments. Bar, mean and SD from 2-4 independent experiments, each using at
least three mice per group (n = 3). For statistical analysis, mice treated with B9-23/DEX were compared with other control groups and ANOVA were
IL-10+ DCs was increased significantly in B9-23/DEX treated
diabetic mice compared with that in other control groups
(p,0.05). These results indicate that DCs maturation was blocked
in B9-23/DEX treated diabetic mice.
Reduction of Pancreatic CD4 T Cells in Treated Diabetic
Cell mediated immunity plays a central role in autoimmune
responses and also contributes to the destruction of insulin
producing b cells in NOD mice and T1D patients . To
test the subpopulations of T cells in B9-23/DEX treated mice, the
blood, spleen and pancreas were prepared for flow cytometry
analysis. As shown in Figure 4A, the percentage of CD4 T cells in
blood was lowered significantly in B9-23/DEX treated diabetic
mice compared with that in B9-23 treated mice (p,0.05), but
there were no differences of CD4 T cells in blood between B9-23/
DEX and DEX treated mice. In spleens, there were no differences
of CD4 T cells among all groups. Remarkably, the percentage of
infiltrated CD4+ T cells in pancreas was decreased significantly in
B9-23/DEX treated diabetic mice compared with other groups
(p,0.05) (Figure 4B). Since DEX has immunosuppressive
function, the percentage of CD4+ T cells in blood and spleen of DEX
treated mice was decreased compared with T1D control or B9-23
groups. The numbers of infiltrated pancreatic CD4 T cells in T1D
control and B9-23 treated mice were higher than that in DEX and
B9-23/DEX treated groups. In B9-23/DEX treated group, there
was the least number of infiltrated CD4 T cells in the pancreas
than that in other groups except nave mice (data not shown).
Pathogenic CD8 T cells can recognize b cell autoantigens and
play an important role in destruction of islet in T1D patients or
mice [26,27]. To test the role of CD8 T cell population in
tolerogenic treatment, the blood, splenic and pancreatic samples
independent experiments. Bar, mean and SD from 3 independent experiments, each using at least three mice per group (n = 3). For statistical analysis,
mice treated with B9-23/DEX were compared with that in mice treated with B9-23 group and student-t test were used between the indicated pair;
**p,0.01. C. On day 7 after the second immunization, the pancreatic samples were immunostained with anti-CD8-APTC mAb and analyzed by flow
cytometry. The infiltrated lymphocytes (R1) were gated for CD8 T cells analysis. The CD8 T cells (CD8+, R2) were counted relatively to the infiltrated
were stained for flow cytometry analysis. There were no
differences of CD8 T cells in blood and spleen among all groups
(data not shown), and the same as the pancreatic CD8 T cells
Decrease of Autoreactive CD4 Effector Tm Cells in
Treated Diabetic Mice
When naive T cells are activated with antigen, the expression of
several adhesion and homing molecules can be changed for
effector Tm phenotype of CD44high, CD62Llow . In T1D mice
or patients, autoantigen-specific T cells have been shown to be
antigen specific Tm cells whereas in healthy individuals . To
test whether the tolerogenic vaccination influence Tm cells in
treated diabetic mice, the blood, splenic and pancreatic samples
were prepared and stained for Tm cells on day 45 after the second
treatment. Gating on CD4+ T cells (R1 in Figure 5A), effector
memory CD4+CD44+CD62L- T cells or central memory
CD4+CD44+CD62L+ T cells were counted relatively to total
CD4 cells. As shown in Figure 5B, the effector memory
CD4+CD44+CD62L- T cells in blood (Upper) were lowered
significantly in B9-23/DEX treated mice compared with that in
other groups (p,0.05) while there were no differences of
CD4+CD44+CD62L- T cells in spleen of all groups (Middle).
Remarkably, the infiltrated CD4+CD44+CD62L- T cells in
pancreas (Lower) were lowered significantly in B9-23/DEX
treated mice compared with that in other groups (p,0.05).
Consistently, the number of central memory
CD4+CD44+CD62LT cells was lowered significantly in blood and pancreas of B9-23/
DEX treated mice compared with that in other groups (Figure 5C,
p,0.05). The percentage of central memory CD4+CD44+
CD62L+ T cells was increased significantly in blood of B9-23
treated mice compared with that in other groups (Figure 5D,
p,0.05). These results suggest that autoreactive effector memory
CD4+CD44+ CD62L- T cells were reduced in blood and pancreas
of treated diabetic mice.
Induction of Functional Effector Memory Treg Cells in
Treated Diabetic Mice
Naive Treg cells might be activated in the periphery by
selfantigen and subsequently converted to mTreg cells in T1D mice
or patients [28,29]. The mTreg cells induced in vitro were capable
of persisting as effector memory cells after transfer and were
protective against the development of T1D [28,30]. Several studies
have reported the existence of a small population of Tregs and also
mTreg cells in the peripheral blood of healthy adult individuals
and preferentially activated Tm cells in diabetic patients [4,29].
Since effector Tm cells appear phenotype of CD44highCD62Llow,
the CD4+Foxp3+ CD44+CD62L- Treg cells were analyzed as
effector memory Treg cells. On day 45 after the second treatment,
the splenocytes of mice were prepared and immunostained for
effector mTreg cells analysis by flow cytometry. Gating on Treg
cells (CD4+Foxp3+, R1 in Figure 6A), the effector mTreg cells
(CD4+Foxp3+ CD44+CD62L-) were counted relatively to total
Treg cells. As shown in Figure 6A, the induced CD4+Foxp3+
CD44+CD62L- effector mTreg cells were increased significantly in
B9-23/DEX treated mice compared with that in other groups
(p,0.05). The number of Treg cell in B9-23/DEX treated mice
was higher than that in other groups, while the number of Treg
cells in DEX treated mice was higher than that in T1D control
and B9-23 treated groups (data not shown). This result suggests
tolerogenic treatment stimulated the induction of CD4+Foxp3+
CD44+CD62L- effector mTreg cells in treated diabetic mice.
Additional experiments showed that the Treg cells from B9-23/
DEX treated mice were functional and B9-23 specific, as they
effectively inhibited the proliferation of B9-23 specific Teff and did
not suppressed the proliferation of MOG35-55 specific Teff in
coculture (p,0.05) (Figure 6B). These data established the
capability of B9-23/DEX for induction of self-antigen specific
CD4+Foxp3+ CD44+CD62L- effector mTreg cells in treated
T1D results from autoimmune destruction of insulin-producing
b cells in the pancreatic islets. Once autoimmune responses are
established, interventions are less effective on activated T cells,
including Tm cells. Immunosuppressives that block activation and
expansion of T cells have been used for T1D therapy . Treg
cells have been applied as strategies for prevention or therapy of
autoimmune disease [2,11,28]. We previously demonstrated that
DEX as adjuvant of B9-23 could induced and expanded Treg cells
for T1D prevention . The immune regulation of Treg cells has
been studied, while their potential for developing immunological
memory has received little attention. Here, we reveal that
tolerogenic vaccination with B9-23/DEX reduced effector CD4
Tm cells and induced functional and specific effector mTreg cells
for restoring immune tolerance for T1D treatment.
Vaccination with self-antigen or peptides induced immune
tolerance by generating Treg cells for T1D prevention or therapy.
Injections of GAD65p217 or GAD65p290 had no effect on T1D
development in NOD mice . This self antigen-based
immunotherapy provides an approach to selectively tolerate self
antigenspecific T cells, while keeping the remainder of the immune system
intact . When insulin protein was co-immunized with specific
DNA plasmid, CD4+CD25 islet-specific Treg cells were induced
and effectively prevented T1D . Otherwise, adjuvants as well as
different routes of antigen administration can be used to manipulate
the nature of the T cell response. Co-administration of IFA with
insulin peptides subcutaneously protected T1D while
intraperitoneal vaccination did not prevented T1D in NOD mice . DEX
can induce antigen-specific tolerance by influencing DC
maturation, suppressing Th1 immune response, and promoting
development of Treg cell . Intraperitoneal injection of DEX into
BALB/c mice for 1, 3, or 5 days enhanced the proportion of Treg
cells in lymphoid organs, especially in the thymus . Our
previous study demonstrated B9-23/DEX vaccination could
suppress established T cell responses by inducing Treg cells and
expanded antigen-specific Treg for T1D prevention . In this
study, the proportion and number of Treg cells were consistently
increased in B9-23/DEX treated diabetic mice which indicated this
vaccination could induce antigen-specific Treg cells for T1D
treatment. The B9-23 treatment could not induce Treg cells for
T1D therapy which was similar with that in T1D prevention
(Figure 2). Furthermore, the maturation of DCs was blocked in
B9compared with that in other groups and ANOVA were used; *p,0.05. B. Summary of effector CD4 Tm cells in blood, in spleen or infiltrated in
pancreases. Data shown are representative of 3 independent experiments. For statistical analysis, mice treated with B9-23/DEX were compared with
other control groups and ANOVA were used; *p,0.05. C. The numbers of effector CD4 Tm cells in blood, in spleen or infiltrated in pancreases were
counted by flow cytometry. Data shown are representative of 3 independent experiments. For statistical analysis, mice treated with B9-23/DEX were
compared with other control groups and ANOVA were used; *p,0.05. D. The central memory CD4+CD62L+CD44+ T cells were counted relatively to
total CD4 cells in PBMC (Upper), spleen (Middle) and pancreas (Lower). Shown in each panel is 1 of at least 3 experiments with similar results. For
statistical analysis, mice treated with B9-23 were compared with that in other groups and ANOVA were used; *p,0.05.
23/DEX treated diabetic mice (Figure 3) which was consistent with
that in tolerogenic vaccination for preventing T1D.
Many studies have demonstrated that CD4+ T cells play a
predominant role in the development of insulitis, while CD8+ T
cells migrate into the islets later and differentiate into killer cells
with the aid of CD4+ T cells . CD8 T cells from young NOD
diabetic islets were able to transfer rapid onset of diabetes in NOD
mice . Others have demonstrated autoreactive T cells were
preferentially activated in T1D patients . Here, we also found
autoreactive CD4 and CD8 T cell subsets in the pancreas of T1D
mice. The CD4 T cells were decreased in the blood and pancreas
of B9-23/DEX treated diabetic mice while there were no changes
of CD8 T cells (Figure 4). Since the immunosuppressive function
of DEX acts on T cells, the percentage of CD4 T cells in the
blood, spleen and pancreas of DEX treated mice were decrease
compared with that in T1D control group (Figure 4). In T1D mice
and patients, the islet antigen specific T cells have already
encountered and responded to the islet antigen, so it can convert
to Tm cells and show memory phenotype . Many studies have
shown that CD44HighCD62LLow T cells appeared to be effector
memory cells in T1D mice [6,7], so the CD4+CD44+CD62L- cells
were used as effector Tm cells of mice on day 45 after treatment in
this study. Here, the percentage and number of effector CD4 Tm
cells were decreased significantly in blood and pancreas of B9-23/
DEX treated diabetic mice compared with that in other control
groups (Figure 5B) which indicate tolerogenic vaccination could
function on effector memory cells. However, the phenotype and
function of autoreactive Tm cells remains a challenge.
Treg cell as a biological therapy to restore self-tolerance may be
a promising immune intervention for T1D . However, this
protective mechanism appears insufficient because of
accumulation of pathogenic T cells over the long disease course . Treg
cells differentiated in vitro had acquired a typical memory
phenotype that was maintained in NOD recipient mice, suggesting
that Treg cells persisted in the hosts as effector memory cells
[28,30]. The mTreg cells could function in the long-term control
of autoimmunity in T1D just as Tm cells have a role in the
prevention of repeated infections and mTreg cells could use
homeostatic mechanisms that are similar to conventional Tm cells
. Since dysregulation of Treg homeostasis appears
characteristic of T1D, mTreg cells must utilize homeostatic mechanisms for
long-term protection , and mTreg cells could be generated in
T1D mice or patients [38,39]. In this study, the percentage of
effector mTreg cells were increased significantly in B9-23/DEX
treated diabetic mice compared with that in other control groups
(Figure 6A) suggesting the induction of effector mTreg cells.
Importantly, these effector mTreg cells specifically suppressed the
proliferation of effector T cells and showed potential to reestablish
immune tolerance in T1D (Figure 6B).
In summary, our results demonstrate that tolerogenic
vaccination effectively reduced effector CD4 Tm cells and induced
effector mTreg cells for T1D treatment. Our findings provide an
effective method for restoring tolerance by induction of effector
mTreg, and may provide an attractive treatment for T1D.
Materials and Methods
Animals and Reagents
Female NOD mice aged at 68 weeks were purchased from
Animal Institute of Chinese Medical Academy (Beijing, China). All
animal protocols [#20120101] were approved by the Animal
Welfare Committee of China Agricultural University and housed
with pathogen-free food and water under 12 h light-cycle
conditions. The B9-23 (SHLVEALYLVCGERG) peptide was
from ChinaPeptides.Co, Ltd. DEX was from Sigma-Aldrich. The
collagenase P was from Worthington. All antibodies for flow
cytometry analysis were from eBioscience.
NOD Mice Treatment and Immunization
The levels of glycemia of female NOD mice were determined
weekly using glucose meter (Beijing Yicheng biological electronic
technology Co., Ltd. JPS-6]. Mice tested positive (Glycemia
level250 mg/dl) twice consecutively were used for treatment
(n = 4). The diabetic mice were treated four times (on days 1, 4, 7,
and 10) with DEX in the two hind footpads (16 mg/mouse). For
the day-7 injection, B9-23 (2 mg/mouse) was coinjected with DEX.
This regimen was given twice in a 2-wk interval. The levels of
glycemia and death rate were checked weekly. Female
nondiabetic NOD mice were immunized with IFA and insulin B9-23
or IFA and myelin oligodendrocyte glycoprotein peptide 35-33
(MOG35-55) twice in a 2-wk interval. On day 4 after the 2nd
immunization, the splenocytes were prepared as responsors for
On day 7 after the second treatment, pancreases of treated
NOD mice (n = 3) were collected and fixed in 4% formaldehyde
for 24 h before being embedded in paraffin. Serial sections of
5 mm thickness were cut and stained with hematoxylin and eosin
(H&E). Pancreatic sections from each group were scored blind for
insulitis and insulitis was graded in at least 10 islets per pancreas:
grade 0 - islet cells had no visible signs of inflammation; grade
1the islets had lymphocytes surrounding the islet margin with little
or no intraislet infiltration; grade 2 - islets were surrounded by
lymphocytes and contained considerable intraislet inflammation;
grade 3 - islets were completely engulfed with lymphocytes .
The mean insulitis score of each pancreas was calculated by
dividing the sum of graded islets by the total number of islets
Immunostaining for Flow Cytometry
The blood and spleens of all groups (n = 3) were prepared and
lysed to blood cells before staining for flow cytometry analysis.
Pancreases of all groups were excised and cut into small pieces.
The samples were digested with collagenase P (1 mg/ml) at 37uC
in water bath and filtered with nylon net, then the samples were
stained with mAbs for flow cytometry analysis.
For Treg cells analysis, the samples were intracellularly stained
with anti-CD4-APC, anti-CD25-PECy5, and anti-Foxp3-PE
mAbs. For IL-10 expression in Treg cells, the samples were
Figure 6. Generation of functional mTreg cells in treated diabetic mice. A. On day 45 after the second immunization, splenocytes were
stained anti-CD4-FITC, anti-CD62L-PECy5, anti-CD44-PE mAbs and intracellularly stained withanti-Foxp3 -APC mAb, then analyzed by flow cytometry.
Gating on Treg cells (CD4+Foxp3+), the effector mTreg cells (CD4+Foxp3+ CD44+CD62L-) were counted relatively to Treg cells. Shown in each panel is
1 of at least 3 experiments with similar results. Bar, mean and SD from 3 independent experiments, each using at least three mice per group (n = 3).
For statistical analysis, mice treated with B9-23/DEX were compared with that in mice treated with B9-23 group and ANOVA were used; *p,0.05. B.
On day 45 after the second immunization, Treg cells from diabetic mice treated with DEX/B9-23 were cocultured with Teff (CD4+CD252) from mice
immunized with IFA/B9-23 or IFA/MOG35-55, along with purified CD11c+ cells and B9-23 or IFA/MOG35-55. Proliferation was assessed by MTT
method. Treg cells from mice of each group were purified respectively. Bar, mean and SD from 2-4 independent experiments, each using at least
three mice per group (n = 3); For statistical analysis, mice treated with B9-23/DEX were compared with the indicated group and student-t test were
used between the indicated pair; *p,0.05.
stimulated in culture for 24 h with B9-23 and anti-CD28 mAb
(eBioscience). The samples were treated with monensin (100 mg/
ml) for 2 h and stained with anti-CD4-FITC, anti-CD25-PECy5
mAbs. The cells were fixed with 4% paraformaldehyde,
permeabilized with 0.1% saponin, and then intracellularly stained
with anti-Foxp3-APC mAbs and anti-IL-10-PE mAbs or isotype
control (PE) of IL-10 mAb (eBioscience). Gating on CD4+ CD25+
T cells (R1), IL-10 expressing Treg cells (CD4+CD25+ Foxp3+
IL10+) were quantified relatively to total Treg cells.
For DCs staining, the samples were stained with
anti-CD11cFITC, anti-MHCII-PE or anti-CD80-PE for DCs maturation. To
detect IL-10 expression in DC, the splenocytes were stimulated
with PMA (10 ng/ml), ionomycin (1 mg/ml) and monensin (2 mg/
ml) for 4 h. After stimulation, the samples were washed and
stained with anti-CD11cFITC, fixed (4% paraformaldehyde),
permeabilized (0.1% saponin), and intracellularly stained with
For T cells analysis, the samples were stained with
anti-CD4FITC and anti-CD8-APC mAbs. For effector memory CD4+
CD44+ CD62L- T cell analysis, the samples were stained with
antiCD4-FITC, anti-CD44-PE and CD62L-PECy5 mAbs. For
effector mTreg cells (CD4+Foxp3+CD44+ CD62L-) analysis, the
samples were intracellularly stained with anti-CD4-FITC,
antiFoxp3-APC anti-CD44-PE and anti-CD62L-PECy5 mAbs.
Gating on Treg cells (CD4+Foxp3+), the effector mTreg cells
(CD4+Foxp3+ CD44+CD62L-) and the percentage of central
memory Treg cells (CD4+Foxp3+ CD44+CD62L+) were counted
relatively to Treg cells.
All the samples were analyzed with a FACScalibur and the Cell
Quest Pro Software (BD Bioscience).
CD4+CD25- T effector (Teff) cells from NOD mice immunized
with IFA/B9-23 (n = 3) were enriched via negative selection by
magnetic cell sorting (Miltenyi Biotec, Auburn, CA), as per
manufacturers protocols, and used as responders. Teff cells from
mice immunized with IFA/MOG35-55 (n = 3) were also purified
and used as responders for antigen specific control of Treg cells.
CD4+CD25+ T cells from the spleen of treated diabetic mice
(iTreg) were enriched via positive selection by magnetic cell sorting
and used as suppressors while CD4+CD25+ T cells from the spleen
of nave NOD mice (nTreg) as control. CD11c+ cells were sorted
by magnetic cell sorting (Miltenyi Biotec, Auburn, CA) from the
spleen of nave NOD mice and used as stimulators. The
responders (16105 cells/well) were co-cultured with the
suppressors (0.56105 cells/well), stimulators (16104 cells/well), and B9-23
(10 mg/ml) in U-bottom 96-well plates for 3 days at 37uC.
MOG35-55 specific Teff cells were stimulated with MOG35-55
peptide (10 mg/ml) and co-cultured with the suppressors (0.56105
cells/well), stimulators (16104 cells/well) in U-bottom 96-well
plates for 3 days at 37uC. The proliferation of the responder T
cells was determined by the MTT method described before .
Results are depicted as mean6standard deviation (SD). Pairwise
differences were analyzed by the two-sided Students t test. For
multi-group analysis, ANOVA and the Bonferroni test were used.
Differences are considered significant if p,0.05 and very
significant if p,0.01.
Conceived and designed the experiments: JZ YK. Performed the
experiments: JZ WG XY JK YZ QG YH YK. Analyzed the data: JZ
YK. Contributed reagents/materials/analysis tools: YH GX. Wrote the
paper: JZ YK.
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