Download Clinical application of regulatory T cells intype 1 diabetes

© 2013 John Wiley & Sons A/S.
Published by John Wiley & Sons Ltd.
Pediatric Diabetes 2013
doi: 10.1111/pedi.12029
All rights reserved
Pediatric Diabetes
Review Article
Clinical application of regulatory T cells
in type 1 diabetes
Marek-Trzonkowska N, Myśliwec M, Siebert J, Trzonkowski P. Clinical
application of regulatory T cells in type 1 diabetes.
Pediatric Diabetes 2013.
Regulatory T cells (Tregs) are responsible for the maintenance of peripheral
tolerance. Animal studies have shown that administration of Tregs can
prevent type 1 diabetes (DM1).
Several clinical trials attempted to induce Tregs with various agents, and thus
provide long-term tolerance of β cells in DM1. Nevertheless, most of these
studies have focused on clinical parameters (e.g. C-peptide) and not Treg
numbers nor their function after treatment. Therefore, it is not possible to
conclude if the majority of these therapies failed because the drugs did not
induce Tregs, or if they failed despite Treg expansion.
The current knowledge regarding Tregs, along with our experience in Treg
therapy of patients with graft versus host disease, prompted us to use ex vivo
expanded Tregs in 10 children with recent-onset DM1. No adverse effects in
the treated individuals were observed. There was a significant increase in Treg
number in peripheral blood immediately after the treatment administration,
while the first clinical differences between treated and control patients were
observed 4 months after Treg injection. Treated individuals had higher
C-peptide levels and lower insulin requirements than non-treated children.
Eleven months after diagnosis of DM1, there are still 2 individuals who are
independent of exogenous insulin.
These results indicate that autologous Tregs are a safe and well-tolerated
therapy in children with DM1, which can inhibit or delay the destruction of
pancreatic β cells. Additionally, Tregs can be a useful tool for local protection
of transplanted pancreatic islets. Isolation and expansion of antigen-specific
Tregs is one of the directions for future studies on cellular therapy of DM1.
Natalia
Marek-Trzonkowskaa ,
Małgorzata Myśliwecb ,
Janusz Sieberta and Piotr
Trzonkowskic
a Department of Family Medicine,
Medical University of Gdańsk, Gdańsk,
Poland; b Department of Pediatrics,
Diabetology and Endocrinology,
Medical University of Gdańsk, Gdańsk,
Poland; and c Department of Clinical
Immunology and Transplantology,
Medical University of Gdańsk, Gdańsk,
Poland
Key words: cellular therapy –
immunotherapy – Tregs – β cell
preservation – DM1
Corresponding author: Natalia
Marek-Trzonkowska, VMD, PhD,
Department of Family Medicine,
Medical University of Gdańsk,
ul. Dȩbinki 2,
80-210 Gdańsk,
Poland.
Tel: (48) 058-349-15-92;
fax: (48) 058-349-15-91;
e-mail: [email protected]
Submitted 1 January 2013.
Accepted for publication 31 January
2013
Regulatory T cells
Regulatory T cells (Tregs) are a specific lymphocyte
subset that, contrary to conventional T cells, do not
actively fight infectious microorganisms, but suppress
excessive responses of other immune cells (1–12)
(Fig. 1).
The presence of such a cell population in the immune
system was hypothesized in the 1970s by Gershon (13,
14). However, the evidence supporting the concept was
provided 25 yr later in 1995, when Sakaguchi showed
that a lack of cells with a CD4+ CD25+ phenotype led
to autoimmune-mediated multiple organ dysfunction
(15).
Currently, it is known that Tregs regulate cellular
activity of the innate and adaptive immune system.
Tregs can inhibit FcεRI-dependent degranulation of
mast cells (Mcs) (Fig. 1A) in a cell contact-dependent
manner, and thus can prevent/abrogate anaphylaxis.
It has been reported that the pathway is mediated via
interactions between the Treg surface molecule, OX40,
belonging to the tumor necrosis factor receptor family,
and its ligand, OX40L, expressed on Mcs (3).
Tregs are also effective inhibitors of antigen
presenting cells (APC), thus, they not only suppress
activated effector T cells (Teff), but also prevent
their activation. It is well established that direct
1
Marek-Trzonkowska et al.
Fig. 1. Selected, basic mechanisms used by Tregs. Tregs act on both innate and adaptive immune response. (A) Tregs inhibit degranulation
of mast cells (Mc) and thus dampen anaphylaxis in an OX40-OX40L-dependent manner. (B) Tregs inhibit maturation of dendritic cells
(DC); immature DCs are weak stimulators of T cells; Tregs induce expression of indoleamine 2,3-dioxygenase (IDO) in DCs via the CTLA-4
(cytotoxic T lymphocyte antigen-4)-CD80/CD86 pathway; IDO inhibits T cell function. (C) Tregs inhibit activation of both helper (Th) and
cytotoxic (Tc) effector T cells via cyclic adenosine monophosphate (cAMP) transferred through gap junctions. (D) Tregs induce apoptosis
of monocytes/macrophages (Mono/Mac) and induce alternative activation of Mono/Mac; alternatively, activated Mono/Mac have a strong
anti-inflammatory potential. (E) Tregs inhibit B cell proliferation and induce their apoptosis via a PD-1 (programmed death-1 receptor) PD-L1
(programmed death-1 ligand)-dependent manner; Tregs block plasma cell differentiation and affinity maturation of antibodies (Abs). (F) Tregs
induce apoptosis of neutrophils (Neu) in a granzyme A- or B-dependent fashion; Tregs inhibit production of reactive oxygen intermediates
(ROI) and cytokines by Neu. (G) Tregs inhibit proliferation and function (production of interferon gamma; IFN-γ and cytotoxicity) of natural
killer (NK) cells via membrane-bound transforming growth factor beta (mTGF-β).
cell-to-cell contact between Tregs and dendritic cells
(DCs) suppresses DCs maturation. Consequently, DCs
express low levels of costimulatory molecules (CD80
and CD86) required for the activation of naïve T
cells. Additionally, in a mechanism dependent on
CTLA-4-CD80/CD86 interactions, Tregs induce the
expression of indoleamine 2,3-dioxygenase (IDO) by
DCs. IDO is a potent regulatory molecule that converts
tryptophan into proapoptotic metabolites, resulting in
the suppression of both helper (Th) and cytotoxic (Tc)
Teffs (Fig. 1C) (2). Another strategy used by Tregs
to suppress Teffs is the transfer of cyclic adenosine
monophosphate (cAMP), a potent inhibitory second
messenger, via membrane gap junctions (Fig. 1C).
Relatively little is known about the mechanisms
which Tregs use to inhibit monocytes/macrophages. It
was observed that, after coculture with Tregs, monocytes/macrophages differentiate towards alternatively
activated macrophages (AAMs), and this process is
partially dependent on interleukin 10 (IL)-10, IL-4, and
IL-13. Nevertheless, the mechanism is not fully elucidated. AAMs are characterized by anti-inflammatory
2
potential and immunoregulatory activity (Fig. 1D).
Additionally, AAMs downregulate human leukocyte antigen (HLA)-DR, thus becoming weak antigen presenters (4). Tregs can also efficiently inhibit
autoreactive B cells in an antigen-specific manner and
are able to prevent production of autoantibodies. These
mechanisms are primarily mediated through interactions between the surface molecules programmed
death-1 (PD-1; expressed by B cells) and PD-1 ligand (PD-L1; on Tregs). In addition, Tregs can induce
apoptosis of B cells and suppress their proliferation, as
well as suppress plasma cell differentiation and affinity
maturation of antibodies (Fig. 1E) (6, 10).
Tregs are also efficient suppressors of granulocytes.
It was observed that Tregs effectively inhibit the
production of reactive oxygen intermediates (ROI)
and cytokines by neutrophils. These effects were
partially dependent on IL-10, TGF-β, and direct
contact. In addition, Tregs were found to induce
neutrophil apoptosis in a granzyme-dependent manner
(Fig. 1F) (8, 9). There are also multiple studies
on Treg and natural killer (NK) cell interactions.
Pediatric Diabetes 2013
Therapeutic potential of Tregs in type 1 diabetes
Currently, it is well established that Tregs are efficient
inhibitors of interferon gamma (IFN-γ) production,
proliferation, and cytotoxicity of NK cells. The main
mechanism responsible for these effects seems to be
signal transduction via membrane-bound TGF-β upon
direct cell-to-cell contact (Fig. 1G) (11, 12).
The wide repertoire of mechanisms utilized by Tregs,
and the variety of cell types that are their targets,
serve the main goal of Tregs, which is maintenance of
peripheral tolerance and prevention of autoimmune
diseases. In addition, Tregs modulate immune
responses induced by pathogens and environmental
factors; thus, they do not become life-threatening
reactions (2). This cell subset also plays a crucial role in
the induction of tolerance to transplanted solid organs
and controls allergic reactions (16–18). Numerous
studies suggest that certain immunosuppressive
drugs exert their therapeutic effects, at least
partially, via stimulation of Treg activity and their
generation/proliferation (19–22).
Tregs are generally divided into two groups: natural
(nTregs) and inducible/adaptive (iTregs/aTregs). The
first subset derives from the thymus, constitutively
expresses high levels of CD25, is FoxP3+ (forkhead box
P3 transcription factor), and mediates its suppressive
function predominantly in a cell contact-dependent
manner. The second group of Tregs is comprised of Tr1
and Th3 cells, which derive from conventional CD4+ T
cells and act predominantly via IL-10 and transforming
growth factor β (TGF-β) secretion, respectively
(23, 24).
Regulatory T cells in type 1 diabetes
Studies of animals and humans lacking a certain
immune cell subset are a valuable source of knowledge,
as they show the real function and significance of the
lacking cell population in the body. Owing to these
types of observations, it is known that the lack of
Tregs and/or impairment of their function lead to
autoimmune diseases, including type 1 diabetes (DM1)
(25–27).
One of the best-defined examples of this type of
abnormality is IPEX syndrome (immune dysregulation, polyendocrinopathy, enteropathy, X-linked),
resulting from a mutation in the foxp3 gene, which
encodes the transcription factor FoxP3, crucial for the
activity of nTregs (25, 28). The characteristic symptoms of IPEX in neonatal age are atopic dermatitis,
watery diarrhea, and DM1 (29).
In addition, it was observed that early intervention
with ex vivo expanded Tregs can prevent the onset of
DM1 in genetically predisposed animals, while Treg
administration at the onset of DM1 induces regression
of the pathological lesions in the pancreas (26, 30).
Numerous studies on Treg function established the
Pediatric Diabetes 2013
opinion that these cells could provide an opportunity
for effective and targeted immunosuppression (31).
Currently, there are two main points of view on
Tregs in DM1. On the one hand, there are reports that
the number and function of Tregs in DM1 patients
is decreased (32, 33). On the other hand, there is
data which indicate that the main problem in DM1
is extensively activated autoreactive T cells that are
resistant to physiologically acting Tregs (34–36). In
our studies, we did not find differences in Treg function
and number between healthy individuals and children
with recent-onset DM1 (37). Nevertheless, we cannot
rule out that some changes in Treg effectiveness appear
as the disease progresses (38). This may result from
long-term activation of Tregs upon the autoimmune
reaction and the chronic inflammatory process which
accompanies DM1 (39–41). Therefore, initiation of
Treg therapy at the time of DM1 onset, when the
function of these cells is still unchanged, is reasonable.
Approaches to induce Tregs in diabetic
patients
Since the protective role of Tregs in DM1 has
been suggested, several clinical studies attempted to
potentiate their action and/or expand them in vivo with
an aim to preserve β cells in recent-onset DM1 (42–53).
One of the very first approaches was based on
oral insulin administration (42). It is well-known that
immune response in the gut is precisely regulated;
thus, pathogenic microorganisms are recognized and
destroyed, while at the same time, physiological flora
and dietary antigens are tolerated. About 20 yr ago,
it was shown that oral antigen administration can
suppress various experimental autoimmune diseases
(54–57). In prediabetic mice, orally administered
insulin was found to inhibit DM1 onset (42), and
even reverse hyperglycemia (58). The effect resulted
from induction of antigen-specific Tregs, which downregulated autoimmunity via the inhibitory cytokines
IL-10 and TGF-β (42, 55, 57, 58). As observations
made in animal models suggested that oral insulin can
attenuate anti-β cell response and thus delay onset of
the disease, a study in humans was launched.
The trial recruited relatives of DM1 patients positive
for anti-islet cell antibodies (ICA), with a low firstphase insulin response and a high 5-yr projection
risk of DM1 onset (42). However, satisfactory results
from animal models were not replicated in humans
(42, 59). Further analysis revealed that individuals
with high levels of anti-insulin autoantibodies (IAA;
≥80 nU/mL) at the time of study entry benefited from
the treatment. Oral insulin administration delayed
onset of DM1 in these patients for nearly 5 yr. At
the same time, the therapy was found to accelerate
development of DM1 in patients with lower baseline
3
Marek-Trzonkowska et al.
IAA levels (<80 nU/mL). Nevertheless, after the
discontinuation of therapy, the risk of DM1 onset
in the treated patients and those receiving placebo
equalized (42, 59). Thus, oral insulin administration
did not induce the expected tolerance.
It is worth noting that, even though the aim of
the study was to induce immune tolerance dependent
on IL-10 and TGF-β producing Tregs, neither the
Treg number nor IL-10/TGF-β levels were measured
in the patients. Therefore, the study did not reveal
if the treatment was ineffective because oral insulin
administration did not induce antigen-specific Tregs,
as was expected, or if it was ineffective despite the
generation of iTregs.
The next approach that aimed to inhibit the
progression of DM1 and gain long-term tolerance via
iTregs was a therapy with anti-CD3 antibody. There
were two anti-CD3 antibodies used in the clinical trials:
hOKT3γ1 Ala-Ala (teplizumab) and ChAglyCD3
(otelixizumab). These humanized antibodies had
mutated Fc fragments that prevented them from
binding to Fc receptors (FcRs). This modification
eliminated the risk of adverse effects resulting from
cell activation via FcR stimulation. In addition, it has
been suggested that these antibodies act in a biphasic
mode. In phase 1, the antibody binds to the CD3
molecule and induces anergy/apoptosis of activated
T cells. In phase 2, the drug increases the number
of iTregs that produce anti-inflammatory IL-10 and
TGF-β, inducing long-term tolerance (60, 61).
In the study of Herold et al., teplizumab was
administered to 21 individuals with recent-onset DM1,
while the control group comprised of 21 non-treated
diabetic patients. After 6- and 12-month observation,
the treated patients had higher levels of stimulated
C-peptide than the control individuals. Moreover, the
effect persisted 2 yr after the treatment, and the patients
who received the drug had lower insulin requirements
and lower levels of glycated hemoglobin (HbA1c).
Depletion of T cells after teplizumab therapy was
transient. The T cell number recovered around 1 month
after the treatment. Clinical responders at months 1
and 3 after the therapy were characterized by increased
numbers of CD8+ T cells as compared to the baseline
values. No significant differences were found between
the responders and non-responders in terms of CD4+
T cell numbers on day 90 as compared to the baseline
values at the time of study entry. No results regarding
Treg subsets were shown (43).
As these initial results were very promising, phase III
of the trial was launched, consisting of 516 individuals
from 83 clinical centers from Europe, North America,
Israel, and India. Patients were divided into four groups
(groups 1–3 received different doses of the drug, group
4 was treated with placebo). However, after a 15-month
observation, the study did not reach its primary and
4
secondary end-points. There was no decrease in HbA1c
levels from baseline and no improvement in C-peptide
response after stimulation in the treated individuals.
Nevertheless, the study suggested that teplizumab may
be more effective in: (i) younger individuals, (ii) patients
with high baseline C-peptide levels at the time of study
entry, and (iii) those diagnosed with DM1 <6 wk before
starting the therapy (44).
The studies with otelixizumab brought similar
observations. Despite the initially promising results,
higher C-peptide concentrations and lower insulin
requirements 18 months after the treatment (45), the
beneficial effect waned with time. Only the youngest
patients benefited from the treatment up to 36 months
after otelixizumab administration (46).
Adverse effects related to the treatment with
anti-CD3 antibodies were usually mild and easily
controlled with non-steroidal anti-inflammatory drugs
and antihistamines. The most frequent side effects were
headache, fever, rash, nausea, diarrhea, and vomiting.
An increased frequency of infections was not observed
in teplizumab studies. However, in otelixizumabtreated patients, transient reactivation of Epstein–Barr
virus was reported. Neither of the antibodies caused
sustained T cell depletion. The patients were fully
immunocompetent as early as 1 month after the last
injection of the drug. In approximately 40–50% of
the patients, anti-idiotypic antibodies appeared that
could potentially neutralize the effect of the therapy.
Nevertheless, the phenomenon was observed after the
last dose of the antibody and was not considered
a problem because repeated treatments were not
anticipated (43–46).
The next treatment that was suggested to induce
Tregs in DM1 patients was glutamic acid decarboxylase
(GAD)-alum (Diamyd; Diamyd Medical, Stockholm,
Sweden), an autoantigen-based vaccine. The drug was
composed of adjuvant (alum) and recombinant human
glutamic acid decaboxylase 65 (GAD65), a specific
isoform of GAD expressed in human pancreatic β
cells. The aim of the therapy was to interfere with the
process of GAD65 recognition by autoreactive T cells
using the recombinant peptide and to induce long-term
tolerance.
Results of the first studies with GAD-alum in
patients with latent autoimmune diabetes of adults
were very promising. The phase II dose-escalation
clinical trial showed that a 20-μg dose of the vaccine
was the most effective and preserved fasting and
stimulated C-peptide levels for a 24-wk period. In
addition, the patients who received 20-μg dose of
GAD-alum had higher numbers of CD4+ CD25+ T
cells, which was interpreted as induction of Tregs
(48). However, no additional markers of these T
cells were analyzed. Therefore, it is not possible to
determine if GAD-alum induced natural or adaptive
Pediatric Diabetes 2013
Therapeutic potential of Tregs in type 1 diabetes
Tregs. We also have to remember that CD25, as an α
chain of IL-2 receptor, appears and is upregulated on
conventional T cells upon activation; thus, the molecule
cannot be used to unquestionably determine Tregs.
Furthermore, it has been shown that autoreactive
T cells, including GAD-65-specific lymphocytes, are
CD25+ (62). Thus, there is uncertainty as to which cell
subset was induced by GAD-alum, Tregs, conventional
activated T cells, or autoreactive T cells. In addition,
Agardh et al. reported no changes in serum levels
of the most relevant regulatory and proinflammatory
cytokines in the patients treated with various doses of
GAD-alum (48).
As GAD-alum preserved β cell function in the
initial studies, a multicenter study was launched.
However, 15-month follow-up showed no differences
in stimulated C-peptide levels between the treated and
placebo groups. In addition, patients who received
GAD-alum had significantly higher levels of antiGAD65 antibodies than individuals who were treated
with placebo (61).
The next antigen-based vaccine used in DM1 that
was suggested to induce Tregs was DiaPep277. The
vaccine consisted of the dominant epitope of heat shock
protein 60 (HSP-60), present in secretory granules
of β cells. It has been previously reported that the
peptide is a Treg-inducing autoantigen that enhances
the regulatory function of Tregs (63). Studies in nonobese diabetic mice showed that DiaPep277 can inhibit
β cell destruction and improve insulin secretion in
recent onset DM1. The phase II clinical trial revealed
that administration of DiaPep277 increased Cpeptide levels and lowered insulin requirements, which
suggested β cell protection (49–51). However, the next
study, which recruited only children, did not confirm
the vaccine’s effectiveness (52). It has been suggested
recently that the best responders to DiaPep277 are
adults with low/moderate risk HLA genotypes (53).
Yet another agent with Treg-inducing potential and
a suggested beneficial role in DM1 that was clinically
tested was vitamin D3 . Bock et al. analyzed the effect
of short-term, high-dose vitamin D3 administration
on pancreatic β cell function in healthy, non-diabetic
individuals. It was observed that the percentage of
Tregs increased significantly in the treated group, while
no changes were found in the placebo subjects. Despite
the increase in Treg numbers, there were no differences
in fasting C-peptide levels and area under the curve for
C-peptide after 3 months in all examined individuals
(64). However, we have to keep in mind that the
study recruited healthy individuals with normal insulin
secretion, which may explain the lack of effect of
vitamin D3 on C-peptide levels. Therefore, there is a
need for further studies to elucidate if Tregs induced
with vitamin D3 can prevent β cell destruction in DM1
patients.
Pediatric Diabetes 2013
One of the most recent approaches to induce Tregs
in vivo in DM1 patients was presented by Long
et al. The trial recruited 10 individuals with DM1
4–48 months after diagnosis. The patients were treated
with rapamycin and IL-2. Rapamycin is known to
preferentially inhibit proliferation of Th1 and Th17
effector cells. Some studies also suggested that it can
potentiate Treg function, while IL-2 is known to be a
crucial cytokine for nTreg survival and proliferation
(65). In addition, it has recently been reported that
low-doses of IL-2 increase Treg numbers and exert
beneficial effects on clinical outcome in some patients
with autoimmune vasculitis (66) and graft vs. host
disease (GVHD) (67). However, IL-2 and rapamycin
co-administration did not induce clinical improvement
in DM1 patients. Despite the increase in nTreg number,
the therapy resulted in transient β cell dysfunction (65).
Isolation and ex vivo expansion of nTregs
for clinical administration
As the knowledge regarding Tregs has been increasing
rapidly, and multiple animal studies have confirmed the
therapeutic potential of this cell subset, in 2007, nTregs
was considered as a method for so-called ‘intelligent
immunosuppression’. Autologous nTregs seemed to
provide all the benefits of standard immunosuppressive
drugs, without their adverse effects (31).
However, as nTregs are present in peripheral blood
in very low numbers, there was a need for a clinical
protocol for the expansion of human nTregs ex vivo.
The main obstacles were weak proliferative potential
of nTregs and decreasing immunosuppressive activity
during culture in vitro.
In June 2008, after multiple in vitro and animal
studies, we administered, for the first time in humans,
ex vivo expanded nTregs to a patient with chronic
GVHD, resistant to standard immunosuppression.
nTreg infusion resulted in the regression of symptoms,
which enabled the tapering of medication doses and
improved the patient’s quality of life (68). Our team
continues studies with nTreg therapy in GVHD.
Owing to the positive results of this trial, lack of
adverse effects directly after nTreg administration
and at each time point after the treatment (no
increased frequency of infections, no primary disease
reactivation, etc.), we decided to use nTregs to preserve
β cell function in children with recent-onset DM1
(clinical trial reg. no. NKEBN/8/2010; phase I) (Fig. 2).
As nTregs are a rare cell population, according to our
protocol, we needed approximately 250 mL of blood
to separate and expand them to the therapeutically
relevant numbers. Therefore, appropriate venous
access and the required blood volume are the first
limiting factors for the therapy, and only children
above 5 yr can be recruited to the study. nTreg yield
5
Marek-Trzonkowska et al.
Fig. 2. Therapy with natural regulatory T cells (nTregs): outline of the procedure. (A) Patient is admitted to the pediatric ward for drawing
of blood. (B) Blood is processed in blood bank into Buffy coat and plasma. (C) Buffy coat and plasma are sent to the good manufacturing
practice (GMP) facility for nTreg isolation, expansion, and certification (GMP facility of Medical University of Gdansk in Poland is shown).
(D) nTregs are expanded under GMP conditions in presence of artificial antigen presenting cells (APC), which are beads coated with anti-CD3
and anti-CD28 antibodies, autologous serum, and interleukin-2 (IL-2) (schematic view). Under the described conditions, nTregs proliferate
vigorously. Just after the nTreg isolation, their number is very low and cells occupy just few wells on 96-well plate (day ‘0’). Cells are cultured
up to 14 d. Within this period, they proliferate to the clinically relevant numbers (day ‘10’ - 10-d culture is shown). (E) When the required nTreg
number is reached, cells are collected, counted, and washed to remove the whole culture medium. (F) At this time, the patient is admitted to
the hospital again and receives nTregs in a slow intravenous infusion (nTregs are suspended in 250 mL of 0.9% NaCl).
immediately after the sorting is very low and, before
administration to the patient, their number must be
multiplied. For example, if the total postsort number
of nTregs is 1 × 106 , they must be multiplied by a
1000 to few 1000-fold to obtain the dose of 20 × 106
nTregs/kg of body weight (kg b.w.). Multiplication
of nTregs can be performed in a so-called expansion
culture, which lasts for up to 2 wk. Within this period,
nTregs are incubated with IL-2 and beads coated
with anti-CD3 and anti-CD28 antibodies, which are
extremely strong activators of nTreg proliferation (37)
(Fig. 2). Nevertheless, the expansion is challenging, as
only a very pure population of sorted nTregs can be
successfully multiplied. Thus, purity is also one of the
most crucial factors affecting the clinical results of the
therapy. We confirmed in our preclinical in vitro studies
that Teff proliferate more vigorously than nTregs (69).
Therefore, even small initial impurities (low percentage
of Teff within the isolated nTregs), may become a
6
serious problem, leading to the expansion of potentially
harmful Teff, but not therapeutic nTregs. For this
reason, we chose fluorescence-activated cell sorting
(FACS) for the final step of nTreg isolation. This
method guarantees the highest purity of cell isolation
(approximately 100% of nTregs after sorting), which
can be sustained through the entire expansion, and
thus, the final cell product still keeps the characteristics
of nTregs (37).
There are several other safety issues that must be
addressed during the production of nTregs for clinical
applications. Isolation and expansion of nTregs for
therapy must be performed at a facility complying
with the requirements of good manufacturing practice
(GMP) (Fig. 2). The reagents used for the cell
culture should also be kept to these standards. In
our laboratory, all reagents used for expansion,
polystyrene beads coated with anti-CD3 and antiCD28 antibodies (Invitrogen, Carlsbad, CA, USA),
Pediatric Diabetes 2013
Therapeutic potential of Tregs in type 1 diabetes
IL-2 (1000 units/mL; Proleukin; Chiron, San Diego,
CA, USA), and culture medium (CellGro, CellGenix,
Breisgau, Germany) are GMP approved. The cell
sorter that we use for nTreg isolation (Influx; BD
Biosciences, San Jose, CA, USA) currently has a master
file with the Food and Drug Administration (FDA) for
cellular therapy applications. The biggest advantage
of this device is a fully replaceable sample line that
can be exchanged between patients (Fig. 2). Thus,
the risk of cross-contamination, potentially associated
with sorting with the previously available machines, is
eliminated.
In addition, each seventh day of the expansion and
immediately before nTreg administration, we check
the cells for bacterial contamination and presence of
genetic material of hepatitis B virus (HBV), hepatitis
C virus (HCV) and human immunodeficiency virus
(HIV). At the same time points, phenotype and
immunosuppressive activity (functional test of IFN-γ
suppression) of nTregs are analyzed. The release
criteria for nTregs are FoxP3 expression >90% (in our
studies it is 93% on average), passed IFN-γ suppression
assay, and negative microbiological tests (37).
and administered in a slow infusion to the patient
(Fig. 2).
No adverse reactions related to the treatment
were observed. In all the patients, a significant
increase in nTreg frequency in peripheral blood was
observed immediately after the injection. The first
clinical differences between the treated and control
patients were observed 6 months after DM1 diagnosis
(4 months since administration of nTregs). After this
period, children who received nTregs had nearly
twofold higher levels of fasting C-peptide than nontreated individuals. The results were clinically relevant,
as treated patients required twofold lower doses of
exogenous insulin than the control individuals, while
HbA1c and glucose levels were comparable in both
groups (37). Among patients treated with nTregs, there
are still two individuals who do not require exogenous
insulin 11 months after DM1 diagnosis.
There is one similar trial recruiting adults with DM1,
currently underway in USA. However, the results are
not yet available. We are looking forward to the
completion of this trial, which hopefully will further
encourage nTreg-based therapies in DM1.
Therapy of type 1 diabetic children with
nTregs
Perspectives for the future clinical use
of nTregs
The main inclusion criterion that we used to recruit
children with DM1 for nTreg therapy was a fasting
C-peptide level of >0.4 ng/mL in two consecutive
measurements. C-peptide levels reflect the production
of endogenous insulin and, indirectly, β cell mass.
Therefore, relatively high C-peptide levels indicate that
there are still some active β cells that can be spared
from the autoimmune attack, which is the main idea of
the therapy with nTregs. Other inclusion criteria were
age 5–18 yr, up to 2 months since DM1 diagnosis,
presence of at least one type of anti-islet antibody in
high titer (GAD65, ICA, IAA, and IA2), body mass
index range of 25th–75th percentiles for a particular
age, and adequate venous access. The exclusion criteria
were any cytopenia or low hemoglobin levels, presence
of the HLA-DQB1*0602 allele, positive test for HBV,
HCV, HIV, Treponema pallidum, or other active
infections, history of neoplasm, excessive anxiety
related to the procedure, and other chronic diseases
requiring pharmacological treatment. The control
group comprised of 10 children with DM1 fulfilling
the same inclusion criteria as the research group, but
not treated with nTregs.
Patients from the treated group received either
10 × 106 /kg b.w. or 20 × 106 /kg b.w. of expanded
nTregs. Technically, the transfer was performed after
complete washout of the culture media and reagents.
Subsequently, nTregs were suspended in 250 mL
of 0.9%NaCl (Polfa, Starogard Gdanski, Poland),
Most of the experimental therapies of DM1 focused
on patients with preserved C-peptide. While for the
patients with long-lasting DM1 and undetectable Cpeptide, the only chance for insulin independence is
pancreas or pancreatic islet transplantation. However,
pancreas transplantation is associated with a high
risk of severe complications (70, 71), while islet
survival after transplantation is still a big challenge
(72–78). Main limitations of a positive outcome in
islet recipients include not only immune rejection,
but also reactivation of the autoreactive response
toward transplanted islets (79–81). Furthermore, even
immunosuppression used for islet protection is toxic
for the graft (80, 82). Currently, no treatment exists
that could provide permanent β cell tolerance and islet
protection in DM1 recipients (83–85).
As nTregs are able to inhibit both auto- and
alloreactivity (2, 86–88), we found these cells a
perfect tool to provide protection for transplanted
islets from rejection and anti-β cell response
that reactivates in DM1 recipients. Therefore, we
coated human pancreatic islets with live nTregs
using a polymer of ethylene glycol (poly(ethylene
glycol)-N-hydroxysuccinimide ester; biotin-PEGNHS) and streptavidin as a binding bridge.
Fluorescence and confocal microscopy confirmed
the feasibility of the approach (nTregs covered the
islets with a uniform layer). In addition, functional
tests in vitro showed that the coating procedure does not
Pediatric Diabetes 2013
7
Marek-Trzonkowska et al.
affect islet and nTreg viability and function. Further
experiments showed that surface-bound nTregs were
able to protect the islets against allogeneic effector
cells, and one of the mechanisms was associated with
the inhibition of IFN-γ production.
These results show that Tregs may be used
successfully as a fully biocompatible protection for
pancreatic islets. However, there is a need for animal
studies to confirm safety and efficacy of this technique
in vivo before any clinical trial in humans would be
launched.
Summary
The results of available studies strongly indicate that
success of the treatment in DM1 correlates with the
level of nTregs. The agents used to increase the number
of these cells, as well as the studies on adoptive transfer
of expanded nTregs, are currently a main interest of
the research groups working on therapies for DM1.
Our results indicate that autologous nTregs are a safe
and well-tolerated therapy of DM1 in children. Higher
C-peptide concentrations in the treated individuals as
compared to the control group indicate that nTregs
inhibit the autoimmune process of pancreatic β cell
destruction. This is the first study of this type in diabetic patients; therefore, it is hard to predict how long
the beneficial effect of nTreg administration will last.
For the same reason, it is difficult to define the factors
necessary for the treatment success. According to our
experience, it seems that one of the most important
criteria for future recruitment is short disease duration/prediabetes and high C-peptide level. The number
of nTregs administered and infusion of the additional
doses of nTregs are other important issues that need
to be addressed in future studies. Finally, one of the
directions for future therapies with nTregs is isolation
and expansion of antigen-specific cells. Elaboration of
the technique that would enable this approach could
result in the higher efficacy of cellular therapy of DM1
and persistent tolerance in the treated patients.
Acknowledgements
The presented studies were supported by the Polish Ministry
of Science and Higher Education, grant no. IP2011 033771
and the National Center of Science, grant no. UMO
2011/01/D/NZ3/00262.
References
1. Shevach EM, DiPaolo RA, Andersson J, Zhao DM,
Stephens GL, Thornton AM. The lifestyle of naturally
occurring CD4+ CD25+ Foxp3+ regulatory T cells.
Immunol Rev 2006: 212: 60–73.
2. Vignali DA, Collison LW, Workman CJ. How
regulatory T cells work. Nat Rev Immunol 2008: 8:
523–532.
8
3. Mekori YA, Hershko AY. T cell-mediated modulation
of mast cell function: heterotypic adhesion-induced
stimulatory or inhibitory effects. Front Immunol 2012:
3: 1–6.
4. Tiemessen MM, Jagger AL, Evans HG, van Herwijnen MJ, John S, Taams LS. CD4+CD25+Foxp3+
regulatory T cells induce alternative activation of human
monocytes/macrophages. Proc Natl Acad Sci USA
2007: 104: 19446–19451.
5. Grossman WJ, Verbsky JW, Tollefsen BL, Kemper
C, Atkinson JP, Ley TJ. Differential expression of
granzymes A and B in human cytotoxic lymphocyte
subsets and T regulatory cells. Blood 2004: 104:
2840–2848.
6. Chung Y, Tanaka S, Chu F et al. Follicular regulatory
T cells expressing Foxp3 and Bcl-6 suppress germinal
center reactions. Nat Med 2011: 17: 983–988.
7. Janssens W, Carlier V, Wu B, VanderElst L,
Jacquemin MG, Saint-Remy JM. CD4+CD25+ T
cells lyse antigen-presenting B cells by Fas–Fas ligand
interaction in an epitope-specific manner. J Immunol
2003: 171: 4604–4612.
8. Lewkowicz P, Lewkowicz N, Sasiak A, Tchórzewski
H. Lipopolysaccharide-activated CD4+CD25+ T
regulatory cells inhibit neutrophil function and promote
their apoptosis and death. J Immunol 2006: 177:
7155–7163.
9. Richards H, Williams A, Jones E et al. Novel role of
regulatory T cells in limiting early neutrophil responses
in skin. Immunology 2010: 131: 583–592.
10. Gotot J, Gottschalk C, Leopold S et al. Regulatory
T cells use programmed death 1 ligands to directly
suppress autoreactive B cells in vivo. Proc Natl Acad
Sci USA 2012: 109: 10468–10473.
11. Trzonkowski P, Szmit E, Myśliwska J, Myśliwski
A. CD4+CD25+ T regulatory cells inhibit cytotoxic
activity of CTL and NK cells in humans-impact of
immunosenescence. Clin Immunol 2006: 119: 307–316.
12. Smyth MJ, Teng MW, Swann J, Kyparissoudis K,
Godfrey DI, Hayakawa Y. CD4+CD25+ T regulatory cells suppress NK cell-mediated immunotherapy of
cancer. J Immunol 2006: 176: 1582–1587.
13. Gershon RK, Kondo K. Cell interactions in the
induction of tolerance: the role of thymic lymphocytes.
Immunology 1970: 18: 723–737.
14. Gershon RK, Eardley DD, Ptak W. Functional
inactivation of suppressor T cells by heat-killed
macrophages. Nature 1976: 262: 216–217.
15. Sakaguchi S, Sakaguchi N, Asano M, Itoh M,
Toda M. Pillars article: immunologic self-tolerance
maintained by activated T cells expressing IL-2
receptor {alpha}-chains (CD25). Breakdown of a single
mechanism of self-tolerance causes various autoimmune
diseases. J Immunol 1995: 155: 1151–1164.
16. Alberu J, Vargas-Rojas MI, Morales-Buenrostro
LE et al. De novo donor-specific HLA antibody
development and peripheral CD4(+)CD25(high) cells
in kidney transplant recipients: a place for interaction?
J Transplant 2012: 2012: 1–8.
17. Ren X, Ye F, Jiang Z, Chu Y, Xiong S, Wang Y.
Involvement of cellular death in TRAIL/DR5dependent suppression induced by CD4(+)CD25(+)
regulatory T cells. Cell Death Differ 2007: 14:
2076–2084.
Pediatric Diabetes 2013
Therapeutic potential of Tregs in type 1 diabetes
18. Sagoo P, Lombardi G, Lechler RI. Relevance of
regulatory T cell promotion of donor-specific tolerance
in solid organ transplantation. Front Immunol 2012: 3:
184.
19. Feng X, Kajigaya S, Solomou EE et al. Rabbit ATG
but not horse ATG promotes expansion of functional
CD4+CD25highFOXP3+ regulatory T cells in vitro.
Blood 2008: 111: 3675–3683.
20. Lopez M, Clarkson MR, Albin M, Sayegh MH,
Najafian N. A novel mechanism of action for
anti-thymocyte globulin: induction of CD4+CD25+
Foxp3+ regulatory T cells. J Am Soc Nephrol 2006: 17:
2844–2853.
21. Suárez A, López P, Gómez J, Gutiérrez C.
Enrichment of CD4+ CD25high T cell population in
patients with systemic lupus erythematosus treated with
glucocorticoids. Ann Rheum Dis 2006: 65: 1512–1517.
22. Watanabe T, Masuyama J, Sohma Y et al. CD52 is
a novel costimulatory molecule for induction of CD4+
regulatory T cells. Clin Immunol 2006: 120: 247–259.
23. Jonuleit H, Schmitt E. The regulatory T cell family:
distinct subsets and their interrelations. J Immunol 2003:
171: 6323–6327.
24. Shevach EM. Mechanisms of foxp3+ T regulatory cellmediated suppression. Immunity 2009: 30: 636–645.
25. Passerini L, Di Nunzio S, Gregori S et al. Functional
type 1 regulatory T cells develop regardless of FOXP3
mutations in patients with IPEX syndrome. Eur J
Immunol 2011: 41: 1120–1131.
26. Salomon B, Lenschow DJ, Rhee L et al. B7/CD28
costimulation is essential for the homeostasis of the
CD4+CD25+ immunoregulatory T cells that control
autoimmune diabetes. Immunity 2000: 12: 431–440.
27. Kukreja A, Cost G, Marker J et al. Multiple immunoregulatory defects in type-1 diabetes. J Clin Invest 2002:
109: 131–140.
28. Bennett CL, Christie J, Ramsdell F et al. The immune
dysregulation,
polyendocrinopathy,
enteropathy,
X-linked syndrome (IPEX) is caused by mutations of
FOXP3. Nat Genet 2001: 27: 20–21.
29. Halabi-Tawil M, Ruemmele FM, Fraitag S et al.
Cutaneous manifestations of immune dysregulation,
polyendocrinopathy, enteropathy, X-linked (IPEX)
syndrome. Br J Dermatol 2008: 160: 645–651.
30. Tang Q, Henriksen KJ, Bi M et al. In vitro-expanded
antigen-specific regulatory T cells suppress autoimmune
diabetes. J Exp Med 2004: 199: 1455–1465.
31. St Clair EW, Turka LA, Saxon A et al. New reagents
on the horizon for immune tolerance. Annu Rev Med
2007: 58: 329–346.
32. Jailwala P, Waukau J, Glisic S et al. Apoptosis of
CD4+ CD25(high) T cells in type 1 diabetes may be
partially mediated by IL-2 deprivation. PLoS One 2009:
4: e6527.
33. Łuczyński W, Wawrusiewicz-Kurylonek N,
Stasiak-Barmuta A et al. Diminished expression of
ICOS, GITR and CTLA-4 at the mRNA level in T
regulatory cells of children with newly diagnosed type 1
diabetes. Acta Biochim Pol 2009: 56: 361–370.
34. Lawson JM, Tremble J, Dayan C et al. Increased
resistance to CD4+CD25hi regulatory T cell-mediated
suppression in patients with type 1 diabetes. Clin Exp
Immunol 2008: 154: 353–359.
Pediatric Diabetes 2013
35. Clough LE, Wang CJ, Schmidt EM et al. Release
from regulatory T cell-mediated suppression during the
onset of tissue-specific autoimmunity is associated with
elevated IL-21. J Immunol 2008: 180: 5393–5401.
36. Walker LS. Regulatory T cells overturned: the effectors
fight back. Immunology 2009: 126: 466–474.
37. Marek-Trzonkowska N, Mysliwiec M, Dobyszuk
A et al. Administration of CD4+CD25highCD127regulatory T cells preserves β-cell function in type 1
diabetes in children. Diabetes Care 2012: 35: 1817–1820.
38. Ryba M, Hak L, Zorena K, Myśliwiec M, Myśliwska
J. CD4+Foxp3+ regulatory T lymphocytes expressing
CD62L in patients with long-standing diabetes type 1.
Centr Eur J Immunol 2009: 34: 90–93.
39. Marek N, Myśliwiec M, Raczyńska K, Zorena K,
Myśliwska J, Trzonkowski P. Increased spontaneous
production of VEGF by CD4+ T cells in type 1 diabetes.
Clin Immunol 2010: 137: 261–270.
40. Kern TS. Contributions of inflammatory processes
to the development of the early stages of diabetic
retinopathy. Exp Diabetes Res 2007: 2007: 95103.
41. Llauradó G, Ceperuelo-Mallafré V, Vilardell C
et al. Arterial stiffness is increased in patients with type
1 diabetes without cardiovascular disease: a potential
role of low-grade inflammation. Diabetes Care 2012:
35: 1083–1089.
42. Skyler JS, Krischer JP, Wolfsdorf J et al. Effects of
oral insulin in relatives of patients with type 1 diabetes:
the Diabetes Prevention Trial-Type 1. Diabetes Care
2005: 28: 1068–1076.
43. Herold KC, Gitelman SE, Masharani U et al. A
single course of anti-CD3 monoclonal antibody hOKT3
gamma1(Ala-Ala) results in improvement in C-peptide
responses and clinical parameters for at least 2 years
after onset of type 1 diabetes. Diabetes 2005: 54:
1763–1769.
44. Sherry N, Hagopian W, Ludvigsson J et al.
Teplizumab for treatment of type 1 diabetes (Protégé
study): 1-year results from a randomised, placebocontrolled trial. Lancet 2011: 378: 487–497.
45. Keymeulen B, Vandemeulebroucke E, Ziegler AG
et al. Insulin needs after CD3-antibody therapy in
new-onset type 1 diabetes. N Engl J Med 2005: 352:
1533–4406.
46. Keymeulen B, Walter M, Mathieu C et al. Four-year
metabolic outcome of a randomised controlled CD3antibody trial in recent-onset type 1 diabetic patients
depends on their age and baseline residual beta cell
mass. Diabetologia 2010: 53: 614–623.
47. Orban T, Bundy B, Becker DJ et al. Co-stimulation
modulation with abatacept in patients with recent-onset
type 1 diabetes: a randomised, double-blind, placebocontrolled trial. Lancet 2012: 378: 412–419.
48. Agardh CD, Cilio CM, Lethagen A et al. Clinical
evidence for the safety of GAD65 immunomodulation in
adult-onset autoimmune diabetes. J Diabetes Complicat
2005: 19: 238–246.
49. Raz I, Avron A, Tamir M et al. Treatment of newonset type 1 diabetes with peptide DiaPep277 is safe and
associated with preserved beta-cell function: extension
of a randomized, double-blind, phase II trial. Diabetes
Metab Res Rev 2007: 23: 292–298.
50. Raz I, Elias D, Avron A, Tamir M, Metzger M,
Cohen IR. Beta-cell function in new-onset type 1
9
Marek-Trzonkowska et al.
51.
52.
53.
54.
55.
56.
57.
58.
59.
60.
61.
62.
63.
64.
65.
10
diabetes and immunomodulation with a heat-shock
protein peptide (DiaPep277): a randomised, doubleblind, phase II trial. Lancet 2001: 358: 1749–1753.
Huurman VA, Decochez K, Mathieu C, Cohen IR,
Roep BO. Therapy with the hsp60 peptide DiaPep277
in C-peptide positive type 1 diabetes patients. Diabetes
Metab Res Rev 2007: 23: 269–275.
Lazar L, Ofan R, Weintrob N et al. Heat-shock
protein peptide DiaPep277 treatment in children with
newly diagnosed type 1 diabetes: a randomised, doubleblind phase II study. Diabetes Metab Res Rev 2007: 23:
286–291.
Buzzetti R, Cernea S, Petrone A et al. C-peptide
response and HLA genotypes in subjects with recentonset type 1 diabetes after immunotherapy with
DiaPep277: an exploratory study. Diabetes 2011: 60:
3067–3072.
Zhang ZY, Lee CS, Lider O, Weiner HL. Suppression
of adjuvant arthritis in Lewis rats by oral administration
of type II collagen. J Immunol 1990: 145: 2489–2493.
Maron R, Blogg NS, Polanski M, Hancock W,
Weiner HL. Oral tolerance to insulin and the insulin
B-chain: cell lines and cytokine patterns. Ann N Y Acad
Sci 1996: 778: 346–357.
Chen Y, Kuchroo VK, Inobe J, Hafler DA, Weiner
HL. Regulatory T cell clones induced by oral tolerance:
suppression of autoimmune encephalomyelitis. Science
1994: 265: 1237–1240.
Weiner HL, Friedman A, Miller A et al. Oral
tolerance: immunologic mechanisms and treatment of
animal and human organ-specific autoimmune diseases
by oral administration of autoantigens. Annu Rev
Immunol 1994: 12: 809–837.
von Herrath MG, Dyrberg T, Oldstone MB. Oral
insulin treatment suppresses virus-induced antigenspecific destruction of beta cells and prevents
autoimmune diabetes in transgenic mice. J Clin Invest
1996: 98: 1324–1331.
Vehik K, Cuthbertson D, Ruhlig H, Schatz DA,
Peakman M, Krischer JP. DPT-1 and TrialNet Study
Groups. Long-term outcome of individuals treated with
oral insulin: diabetes prevention trial-type 1 (DPT-1)
oral insulin trial. Diabetes Care 2011: 34: 1585–1590.
Chatenoud L, Bluestone JA. CD3-specific antibodies:
a portal to the treatment of autoimmunity. Nat Rev
Immunol 2007: 7: 622–632.
Ludvigsson J, Krisky D, Casas R et al. GAD65 antigen
therapy in recently diagnosed type 1 diabetes mellitus.
N Engl J Med 2012: 366: 433–442.
Endl J, Rosinger S, Schwarz B et al. Coexpression
of CD25 and OX40 (CD134) receptors delineates
autoreactive T-cells in type 1 diabetes. Diabetes 2006:
55: 50–60.
Zanin-Zhorov A, Cahalon L, Tal G, Margalit R,
Lider O, Cohen IR. Heat shock protein 60 enhances
CD4+ CD25+ regulatory T cell function via innate
TLR2 signaling. J Clin Invest 2006: 116: 2022–2032.
Bock G, Prietl B, Mader JK et al. The effect of vitamin
D supplementation on peripheral regulatory T cells
and β cell function in healthy humans: a randomized
controlled trial. Diabetes Metab Res Rev 2011: 27:
942–945.
Long SA, Rieck M, Sanda S et al. Rapamycin/IL-2
combination therapy in patients with type 1 diabetes
66.
67.
68.
69.
70.
71.
72.
73.
74.
75.
76.
77.
78.
79.
80.
81.
augments Tregs yet transiently impairs β-cell function.
Diabetes 2012: 61: 2340–2348.
Saadoun D, Rosenzwajg M, Joly F et al. Regulatory
T-cell responses to low-dose interleukin-2 in HCVinduced vasculitis. N Engl J Med 2011: 365: 2067–2077.
Koreth J, Matsuoka K, Kim HT et al. Interleukin-2
and regulatory T cells in graft-versus-host disease. N
Engl J Med 2011: 365: 2055–2066.
Trzonkowski P, Bieniaszewska M, Juścińska J et al.
First-in-man clinical results of the treatment of patients
with graft versus host disease with human ex vivo
expanded CD4+CD25+CD127- T regulatory cells. Clin
Immunol 2009: 133: 22–26.
Trzonkowski P, Szaryńska M, Myśliwska J,
Myśliwski A. Ex vivo expansion of CD4(+)CD25(+)
T regulatory cells for immunosuppressive therapy.
Cytometry 2009: 75: 175–188.
Manrique A, Jiménez C, López RM et al.
Relaparotomy after pancreas transplantation: causes
and outcomes. Transplant 2009: 41: 2472–2474.
Montiel-Casado MC, Fernández-Burgos I, PérezDaga JA et al. Impact of blood amylase peak over
vascular graft thrombosis in pancreas transplantation.
Transplant Proc 2012: 44: 2627–2630.
Shapiro AM, Lakey JR, Ryan EA et al. Islet
transplantation in seven patients with type 1 diabetes
mellitus using a glucocorticoid-free immunosuppressive
regimen. N Engl J Med 2000: 343: 230–238.
Hering BJ, Kandaswamy R, Harmon JV et al.
Transplantation of cultured islets from two-layer
preserved pancreases in type 1 diabetes with anti-CD3
antibody. Am J Transplant 2004: 4: 390–401.
Sutherland DE, Gruessner R, Kandswamy R,
Humar A, Hering B, Gruessner A. Beta-cell replacement therapy (pancreas and islet transplantation) for
treatment of diabetes mellitus: an integrated approach.
Transplant Proc 2004: 36: 1697–1699.
Hering BJ, Kandaswamy R, Ansite JD et al. Singledonor, marginal-dose islet transplantation in patients
with type 1 diabetes. JAMA 2005: 293: 830–835.
Noguchi H, Matsumoto S, Matsushita M et al.
Immunosuppression for islet transplantation. Acta Med
Okayama 2006: 60: 71–76.
Froud T, Baidal DA, Faradji R et al. Islet transplantation with alemtuzumab induction and calcineurin-free
maintenance immunosuppression results in improved
short- and long-term outcomes. Transplantation 2008:
86: 1695–1701.
Jamiolkowski RM, Guo LY, Li YR, Shaffer SM, Naji
A. Islet transplantation in type I diabetes mellitus. Yale
J Biol Med 2012: 85: 37–43.
Nanji SA, Shapiro AM. Islet transplantation in patients
with diabetes mellitus: choice of immunosuppression.
BioDrugs 2004: 18: 315–328.
Bellin MD, Sutherland DE, Beilman GJ et al. Similar
islet function in islet allotransplant and autotransplant
recipients, despite lower islet mass in autotransplants.
Transplantation 2011: 91: 367–372.
Assalino M, Genevay M, Morel P, DemuylderMischler S, Toso C, Berney T. Recurrence of
type 1 diabetes after simultaneous pancreas-kidney
transplantation in the absence of GAD and IA-2
autoantibodies. Am J Transplant 2012: 12: 492–495.
Pediatric Diabetes 2013
Therapeutic potential of Tregs in type 1 diabetes
82. Posselt AM, Szot GL, Frassetto LA et al. Islet
transplantation in type 1 diabetic patients using
calcineurin inhibitor-free immunosuppressive protocols
based on T-cell adhesion or costimulation blockade.
Transplantation 2010: 90: 1595–1601.
83. Marek N, Bieniaszewska M, Krzystyniak A et al.
The time is crucial for ex vivo expansion of T regulatory
cells for therapy. Cell Transplant 2011: 20: 1747–1758.
84. Lysy PA, Weir GC, Bonner-Weir S. Concise
review: pancreas regeneration: recent advances and
perspectives. Stem Cells Transl Med 2012: 1: 150–159.
85. Barton FB, Rickels MR, Alejandro R et al. Improvement in outcomes of clinical islet transplantation:
1999–2010. Diabetes Care 2012: 35: 1436–1445.
Pediatric Diabetes 2013
86. Marek N, Krzystyniak A, Ergenc I et al. Coating
human pancreatic islets with CD4(+)CD25(high)
CD127(−) regulatory T cells as a novel approach for the
local immunoprotection. Ann Surg 2011: 254: 512–518
discussion 518–519.
87. San
Segundo
D,
Fernández-Fresnedo
G,
Rodrigo E. High regulatory T-cell levels at 1
year posttransplantation predict long-term graft survival among kidney transplant recipients. Transplant
Proc 2012: 44: 2538–2541.
88. Suen JL, Chiang BL. CD4(+)FoxP3(+) regulatory
T-cells in human systemic lupus erythematosus.
J Formos Med Assoc 2012: 111: 465–470.
11