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Concise Review: Cell-Based Therapies and Other
Non-Traditional Approaches for Type 1 Diabetes
REMI J. CREUSOT,
a
MANUELA BATTAGLIA,
b
MARIA-GRAZIA RONCAROLO,
c
C. GARRISON FATHMAN
d
Key Words. T cells •Cell therapy •Autoimmunity •Prevention •Type 1 diabetes •
Immunoregulation •Antigen-specific
ABSTRACT
The evolution of Type 1 diabetes (T1D) therapy has been marked by consecutive shifts, from
insulin replacement to immunosuppressive drugs and targeted biologics (following the under-
standing that T1D is an autoimmune disease), and to more disease-specific or patient-oriented
approaches such as antigen-specific and cell-based therapies, with a goal to provide efficacy,
safety, and long-term protection. At the same time, another important paradigm shift from
treatment of new onset T1D patients to prevention in high-risk individuals has taken place,
based on the hypothesis that therapeutic approaches deemed sufficiently safe may show better
efficacy if applied early enough to maintain endogenous bcell function, a concept supported by
many preclinical studies. This new strategy has been made possible by capitalizing on a variety
of biomarkers that can more reliably estimate the risk and rate of progression of the disease.
More advanced (“omic”-based) biomarkers that also shed light on the underlying contributors
of disease for each individual will be helpful to guide the choice of the most appropriate thera-
pies, or combinations thereof. In this review, we present current efforts to stratify patients
according to biomarkers and current alternatives to conventional drug-based therapies for T1D,
with a special emphasis on cell-based therapies, their status in the clinic and potential for treat-
ment and/or prevention. STEM CELLS 2016; 00:000—000
SIGNIFICANCE STATEMENT
This article summarizes the significance of the paradigm shift in the thinking about the treat-
ment of Type 1 diabetes. Current treatment strategies are now directed toward prevention of
disease progression to maintain endogenous beta cell function. The use of immunomodulating
strategies including antigen- and cell-based therapies as well as the need to identify new bio-
markers that allow a measure of disease stage and time to onset of hyperglycemia for selection
of appropriate patients to enroll in prevention trials are discussed.
INTRODUCTION
Type 1 diabetes (T1D), like its more common
Type 2 counterpart, has been rising in preva-
lence and incidence primarily in Western coun-
tries [1, 2] (Fig. 1). Insulin replacement
therapy has been the primary treatment of all
forms of diabetes for almost 100 years, but
inadequate control of its delivery has allowed
a number of complications to markedly dimin-
ish the quality of life of affected individuals,
and contributed to an increasingly intolerable
financial burden. The realization that a subset
of patients presents with an autoimmune form
of insulin-dependent diabetes was made in the
1970s [3]. The initial model suggesting the
potential pathogenesis of this disorder as a
chronic autoimmune disease directed against b
cells was proposed by George Eisenbarth in
the mid-1980s [4]. This understanding led to
subsequent attempts to develop more specific
treatments for this autoimmune form of diabe-
tes, initially with immunosuppressive therapies
that had proven effective in other chronic
autoimmune diseases, including cyclosporine A
(CsA) or anti-thymocyte globulin and predni-
sone [5–7]. Despite initial suggestion of effi-
cacy of CsA, no subsequent study has been
able to confirm these initial results. In addi-
tion, the lack of lasting effects once CsA was
withdrawn and the serious renal toxicity of
the drug severely limited enthusiasm for this
approach.
Subsequent natural history studies have
made the approach to treatment more com-
plex as these studies have demonstrated that
underlying autoimmune responses are present
for varying periods of time, usually years, in
genetically predisposed individuals before the
a
Department of Medicine,
Columbia Center for
Translational Immunology
and Naomi Berrie Diabetes
Center, Columbia University
Medical Center, New York,
USA;
b
Diabetes Research
Institute, IRCCS San Raffaele
Scientific Institute, Milan,
Italy;
c
Division of Stem Cell
Transplantation and
Regenerative Medicine,
Department of Pediatrics,
Stanford University School of
Medicine Stanford, CA, USA;
d
Division of Immunology and
Rheumatology, Department
of Medicine, Stanford
University School of
Medicine Stanford, CA, USA
Correspondence: C. Garrison
Fathman MD, Division of
Rheumatology and Immunology,
Department of Medicine,
Stanford University, 269 Campus
Drive West, Stanford, California
94305, USA. Telephone: 650-
723-7887; Fax: 650-725-1958; e-
mail: cfathman@stanford.edu
Received July 7, 2015; accepted
for publication December 7,
2015; first published online in
STEM CELLS EXPRESS February 3,
2016.
V
CAlphaMed Press
1066-5099/2016/$30.00/0
http://dx.doi.org/
10.1002/stem.2290
This is an open access article
under the terms of the Creative
Commons Attribution-NonCom-
mercial-NoDerivs License, which
permits use and distribution in
any medium, provided the origi-
nal work is properly cited, the
use is non-commercial and no
modifications or adaptations are
made.
STEM CELLS 2016;00:00–00 www.StemCells.com V
C2016 The Authors STEM CELLS published by
Wiley Periodicals, Inc. on behalf of AlphaMed Press
CONCISE REVIEWS
appearance of overt hyperglycemia. Particular major histo-
compatibility class II haplotypes (HLA-DR3/4, HLA-DQ8) confer
the greatest risk as genetic factors [8]. In addition to serving
as a diagnosis tool for T1D in new onset diabetic patients, cir-
culating autoantibodies against bcell proteins (specificity and
quantity) in at-risk individuals, as well as abnormalities in the
oral glucose tolerance test, can generally help predict the risk
of, and time remaining, before the onset of hyperglycemia
[9]. These predictive data have raised the possibility of
attempting therapeutic intervention before the onset of
hyperglycemia in high-risk individuals identified based on bio-
markers like those mentioned above. Prevention of T1D may
represent a viable alternative to an actual cure by perma-
nently blocking the autoimmune response while there are suf-
ficient bcells remaining, and may offer a more cost-effective
approach in the short-term to deal with the alarming rise in
the incidence of disease (Fig. 1). To make the matter even
more complex, many patients may present a spontaneous but
temporary remission after onset, known as the honeymoon
period, possibly reflecting reduced stress on residual bcells
after initial insulin treatment. This honeymoon period may
perhaps represent a sweet window of opportunity (pun
intended) to exploit for the use of intervention therapy.
In this review, we will discuss how our therapeutic arsenal
to fend off this autoimmune disease has greatly diversified
beyond traditional drugs and biologicals to include various
forms of cell therapies, as well as other less conventional
approaches. The field is witnessing a paradigm shift from
immunosuppressive therapies applied after the onset of
hyperglycemia (a time at which bcell function has generally
been irreversibly lost) to prevention strategies attempting to
shut off the autoimmune response and preserve bcell func-
tion in high-risk individuals. This of course entails the use of
reliable biomarkers to identify the most appropriate at-risk
subjects for such intervention trials, and perhaps also guide
the type of therapy that should be employed, paving the way
to more personalized therapies. Current studies using new
techniques of transcriptomics and proteomics [10–13] are
attempting to more precisely stratify those at risk by identify-
ing novel biomarkers that may be superior to those currently
used to define the stage or rate of progression of disease,
and thus help select appropriate subjects to enter into pre-
vention trials. Although the strategies described in this review
have all shown remarkable efficacy in preclinical models, it
should be noted that little or no clinical efficacy data is avail-
able for most of them, whether they are evaluated in the
treatment of recent onset patients after safety has been dem-
onstrated or in prevention studies following safe but ineffec-
tive use in recent onset patients.
ASHIFT FROM TREATMENT TO PREVENTION ISDRIVEN BY
BIOMARKERS GUIDING WHEN AND HOW TO INTERVENE
The progress in identifying the patients who are at “high-risk”
and should be entered into prevention trials has been sup-
ported by an improved understanding of T1D disease proc-
esses that allows screening for at-risk individuals and
stratification of the individual’s risk and time of progression
to the development of hyperglycemia. The first level of
screening is comprised of family history (number of relatives
with T1D and degree of relationship) and HLA haplotype
(HLA-DR3/4 heterozygosy combined with HLA-DQ8 conferring
the highest known risk) [9]. Although these risk factors are
fixed from birth, new relatives may become diagnosed later
and the relative risk re-evaluated. These parameters have
served to enroll young subjects into studies on how environ-
mental factors influence disease progression (e.g., primary
prevention studies examining diet alterations in genetically at-
risk babies with no evidence of autoimmunity, Table 1). These
individuals can be closely and regularly monitored and
undergo a second level of screening consisting of well-
established biomarkers such as circulating autoantibodies to b
cell antigens insulin, GAD65, IA-2, ZnT8, and IGRP [9, 14],
which have served as good predictive tools [15–18] and
enrollment criteria for prevention studies. In vitro immunoas-
says performed on peripheral blood cells including T cell
responses to bcell antigens or identification of diabetogenic
T cells by tetramer staining complete this assessment of the
breadth (how many autoantigens targeted) and amplitude
(antibody titers or frequency of tetramer-positive T cells) of
the autoimmune response [14]. More recently, biomarkers
based on epigenetic changes have been discovered, such as
circulating demethylated insulin DNA [19, 20] and differences
in methylation level at specific CpG sites in immune cells [21].
Increased levels of demethylated insulin DNA in the blood
correlates with the extent of bcell damage [19], while the
extent of insulitis may now be evaluated by refined imaging
techniques [22].
These biomarkers of prediction are important to evaluate
disease risk and rate of progression (indicating with a high
degree of confidence if and approximately when a patient will
progress to overt hyperglycemia unless the course of progres-
sion is altered by treatment), and, therefore, to determine
when to treat. Individuals with a comparable risk level may
experience a different rate of progression, according to their
genetic makeup (other genes besides HLA) and their environ-
ment, which differentially affect mechanisms of immune toler-
ance and pathogenesis. As environmental factors change, the
rate of progression may increase or decrease, placing the sub-
ject at greater risk or slowing down the development of the
Figure 1. T1D incidence has doubled every 20 years. Data for
Finland are from the Finnish National Public Health Institute; data
for Sweden are from the Swedish Childhood Diabetes Registry;
data for Colorado are from the Colorado IDDM Registry, the Bar-
bara Davis Center for Childhood Diabetes, and SEARCH for Diabe-
tes in Youth; data for Germany are a compilation of two reports;
and data from Poland are from Diabetologia 2010;54:508-515.
Reprinted with permission from the Ann N Y Acad Sci
2008;1150:1-13, with additional modifications and permission
from Marian Rewers and Jay Skyler.
2Unconventional approaches to treat Type 1 diabetes
V
C2016 The Authors STEM CELLS published by Wiley Periodicals, Inc. on behalf of AlphaMed Press STEM CELLS
disease (Fig. 2). As a result of multiple etiological factors act-
ing in concert, the disease can progress from very fast in the
case of fulminant T1D [23] to very slow in the case of latent
autoimmune diabetes in adults (LADA) [24]. However, the
most aggressive forms of disease, like fulminant T1D, might
not benefit from prevention unless the trigger becomes well
understood.
Because of the heterogeneity of etiological factors that
may control the rate of progression, it is unlikely that patients
stratified as having a similar risk will be equally responsive to
a particular treatment (Table 2). Treating a high-risk patient
with the wrong drug would cost precious time during which b
cells will continue to be destroyed. In prevention studies, as
opposed to new onset cases, more time will be needed
before it can be determined whether the treatment is effec-
tive. Conversely, treating a low-risk patient, who may never
advance to onset, even with the right prevention therapy,
would involve unnecessary costs and risks. Thus, a third level
of screening that is more sophisticated (using novel bio-
markers featured in larger datasets) will be required to help
determine how best to treat each patient by providing clues
as to the underlying defects that need to be acted upon. A
combination of genetic, transcriptomic, and proteomic tests
performed on blood samples will likely be part of such
screening in the future, and extensive research is being con-
ducted to this end. Furthermore, advances in viromics have
enabled the development of sensitive blood tests that can
detect prior exposure to particular viruses, some of which
have long been suspected to play a role as a trigger for the
disease in some individuals [44]. If these tests help confirm a
link between these pathogens (which are not uncommon, and
therefore would not be sufficient to induce T1D), then the
prospect of vaccinating genetically at-risk individuals becomes
possible.
In recent years, it has become clear that combination
therapies, selected to address multiple underlying defects, will
become more prominent in our effort to tackle T1D heteroge-
neity in both prevention and treatment. Such combination
therapies are expected to be effective in larger cohorts of
patients with overlapping defects. Although many of the
immunosuppressive strategies have not been effective when
administered post-hyperglycemia, they might be appropriate
to use at an earlier stage of disease where they may prove
more efficacious. Besides efficacy and safety, the cost will
Table 1. Main clinical trials focused on the prevention of T1D
Prevention trials Drug Type of study
Diet/supplement-based prevention
NCT01055080 (FINDIA) Baby diet alteration Phase 1, primary prevention
NCT00570102 (MIP) Baby diet alteration Phase 2, primary prevention
NCT01115621 (BABYDIET) Baby diet, delayed gluten Phase 1, primary prevention
NCT00179777 (TRIGR) Controlled diet in infants Phase 2, primary prevention
NCT00333554 (NIDDK) Omega-3-fatty acids Phase 2, primary prevention
NCT00141986 (CDA) Vitamin D Phase 1, primary prevention
bcell antigen-based prevention
NCT00004984 (DPT-1) Parenteral or oral insulin Phase 2, secondary prevention
NCT00419562 (NIDDK) Oral insulin Phase 3, secondary prevention
ISRCTN76104595 (Pre-POINT) Oral insulin Phase 1, primary prevention
NCT00654121 (BDR Trial) Subcut. insulin (Actrapid HM) Phase 2, secondary prevention
NCT00223613 (DIPP) Intranasal insulin Phase 3, secondary prevention
NCT00336674 (INIT-II) Intranasal insulin Phase 2, secondary prevention
NCT01122446 (DIAPREV-IT) Diamyd (GAD-Alum) Phase 2, secondary prevention
Combinations of the above
NCT02387164 (DIAPREV-IT2) Diamyd (GAD-Alum 1Vit. D) Phase 2, secondary prevention
Prevention using biological-based immunotherapy
NCT01773707 (NIDDK, TN18) Abatacept (CTLA4-Ig) Phase 2, secondary prevention
NCT01030861 (NIDDK, TN10) Teplizumab (anti-CD3) Phase 2, secondary prevention
Cell-based prevention
CoRD study (Sydney) Umbilical cord blood Phase 1, secondary prevention
Note: Clinical trials are color-shaded based on whether they are ,,or
Figure 2. Rates of T1D disease progression. In T1D, earlier onset
reflects a faster rate of progression through risk levels (repre-
sented by narrower height). The risk level can be evaluated
according to family history and HLA haplotype, which are fixed at
birth, as well as circulating anti-bcell autoantibodies and certain
metabolic measurements, which are dynamic [9]. The rate of pro-
gression (a) takes into account all causes and factors that may
contribute to pathogenesis, including (but not limited to) defec-
tive deletional tolerance, defective immune regulation, defective/
delayed clearance of damaged bcells, viral infection (high tropism
for bcells and/or molecular mimicry), and bcell intrinsic factors
such as susceptibility to infection, apoptosis or dedifferentiation
(Table 2). Progression through these stages may not be linear for
all individuals, as precipitating events during life (depicted by a
thunderbolt) may accelerate the rate, while other environmental
changes may curb it. Furthermore, accumulation of genetic (pre-
fixed) factors may cause an individual to start at an intermediate
risk, but genetic analysis other than HLA haplotype is not yet rou-
tinely performed. Successful prevention of disease will require
more advanced tools to evaluate the risk level, the rate of pro-
gression, and preferably, the nature of the deficiencies that con-
tribute to the rate of progression (Table 2).
Creusot, Battaglia, Roncarolo et al. 3
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C2016 The Authors STEM CELLS published by Wiley Periodicals, Inc. on behalf of AlphaMed Press
need to be leveraged against the long-term benefits. Expen-
sive therapies that induce durable tolerance and protection
may, however, save money in the long run. It is unusual for
new drugs to be approved for prevention of disease without
prior testing in new-onset patients: the immunomodulatory
drugs and cell-based therapies described below are no excep-
tion to this rule.
OVERVIEW OF THE CURRENT LANDSCAPE OF NON-CELL
THERAPIES
Cell- or Pathway-Neutralizing Biologics
Regulatory authorities have historically prioritized treatment of
new onset patients because these cases are more pressing, the
risk tolerance greater, and the studies shorter, smaller, and less
expensive. In an attempt to replace the use of globally immuno-
suppressive drugs, a number of promising biologics have been
evaluated in new onset patients, such as anti-CD3 mAb [49–51],
anti-CD20 mAb [52], CTLA4-Ig [53]. These drugs showed signifi-
cant but limited (transient) efficacy in new-onset patients. These
drugs are now being tested in high-risk normoglycemic patients
where they might have a more pronounced and durable effect in
sustaining euglycemia (Table 1). These drugs were deemed safe
enough by the US Food and Drug Administration to be used pro-
phylactically at a dose unlikely to cause serious adverse events.
Current studies using such biologics to prevent disease in high-
risk patients include two major TrialNet studies: TN10 (Clinical-
Trial.gov NCT01030861) using Teplizumab (anti-CD3 mAb) and
TN18 (ClinicalTrial.gov NCT01773707) using Abatacept (CTLA4-Ig).
Many other biologics used to treat other autoimmune diseases
are also being evaluated for T1D. However, these drugs remain
relatively nonspecific and may still carry accrued risks of infections
or malignancies in susceptible subjects.
Low Dose IL-2: A Safer and More Selective Approach?
Because of different sensitivities, it was found that regulatory
T cells (Tregs), a subset of T cells that protects from autoim-
munity, are selectively stimulated by low doses of IL-2, a T
cell growth factor [54]. This new approach is particularly note-
worthy because of its safety profile, based on several pub-
lished studies on the use of low dose IL-2 to treat
inflammatory diseases including chronic graft-versus-host dis-
ease (GvHD) [55–57] and hepatitis C virus-induced vasculitis
[58, 59], and preliminary studies on its use as a potential
treatment of T1D [60]. In addition, preclinical studies in non-
obese diabetic (NOD) mice showed that low dose IL-2 admin-
istered after onset of hyperglycemia restored euglycemia in a
majority of the treated mice [61]. In each instance, low dose
IL-2 therapy was associated with a dose-dependent increase
in the number of circulating Tregs and a marked diminution
of inflammatory cytokine expression in the serum of the
Table 2. Factors contributing to the rate of disease progression
Possible mechanisms
(not mutually exclusive)
Possible causes or
predisposing genes Possible biomarkers
Possible therapeutic
approach to prevent
(and reverse) disease References
Defective deletional tolerance:
higher frequency of
bcell-reactive T cells
(and B cells)
HLA-DR, INS, PTPN22
polymorphism
HLA-DR, selected SNP analysis,
MHC tetramer analysis/ELISPOT
Antigen-specific therapies [8, 25–29]
Defective immune regulation:
reduced number,
responsiveness and/or
function of regulatory
T cells
IL2RA, IL2, CTLA4, IL10,
PTPN22 polymorphism
Vitamin D and/or fiber
deficiency Foxp3
promoter methylation
Treg suppression, TSDR assay,
STAT5 responsiveness
Expanding Tregs and/or
boosting their function
(in vivo or ex vivo);
antigen-specific therapies
[8, 25, 30–36]
Antigen-presenting cells:
hyperactivity under
inflammatory conditions
or defective tolerogenic
properties
Genetic predispositions?
Environmental factors?
Functional characterization
of certain blood cells?
Blockade of specific
costimulatory pathways
or cytokines
[37, 38]
Response of bcells: apoptosis,
stress ( 6generation of
neo-antigens),
de-differentiation or
trans-differentiation
IFIH1 polymorphism,
inflammation, some
unknown genetic
determinants, inability
to cope with excessive
stress
Selected SNPs, circulating
demethylated insulin DNA
(also reflects bcell immune
destruction); impaired
glucose tolerance
Drugs increasing bcell
replication, reducing
bcell stress (imatinib?),
or stabilizing bcell
phenotype
Anti-inflammatory drugs?
[20, 39–41]
Defective/delayed clearance
of damaged bcells / bcell
antigens (disease initiation)
Genetic predispositions? None (too early to detect)? None at the moment [42, 43]
Interaction with microbes:
molecular mimicry
(cross-reactivity with
bcell antigens), immune
deviation or dysregulation
HLA-DR, IFIH1, TLR7/8
polymorphism; Infection
(e.g., enterovirus);
Dysbiosis (imbalanced
microbiome)
Infection history (difficult)
Microbiome profiling
Probiotics? Vaccines?
Anti-inflammatory drugs?
[8, 35, 44–48]
Combinations of genetic and environmental factors contribute to the initiation and progression of T1D disease, leading to various deficiencies at
the level of both immune cells and bcells. Gene-wide association studies have identified some 40 gene polymorphisms each contributing a
small risk to the disease, some of which are listed here. The more deficiencies that exist in a particular individual, the faster the progression is
expected to be. However, different patients are characterized by different combinations of such deficiencies, leading to substantial heterogeneity
in how they progress and how rapidly. Biomarkers that assess not only the risk but also the underlying deficiencies will help inform the choice
of prevention therapies to be applied to more homogeneous cohorts of patients for better efficacy. References are only provided as examples
to illustrate the concepts.
4Unconventional approaches to treat Type 1 diabetes
V
C2016 The Authors STEM CELLS published by Wiley Periodicals, Inc. on behalf of AlphaMed Press STEM CELLS
treated mice or patients in the initial short-term safety trial
[60]. An additional dose finding study to determine the optimal
dose of IL-2 required to increase the number and response of
Tregs has been completed in T1D patients (ClinicalTrial.gov
NCT01827735). Subsequently, an efficacy trial has begun in
patients with recent onset T1D (ClinicalTrial.gov NCT01862120).
Once these studies (in Table 3) are completed and the safety
profile confirmed, a move to recruit high-risk patients in low
dose IL-2 studies will be expected.
However, the potential success of low dose IL-2 therapy in
T1D patients rests on two assumptions: (i) Tregs are function-
ally defective and (ii) IL-2 production is impaired. Studies on
whether CD4
1
CD25
1
Tregs are defective in T1D have yielded
conflicting results (decreased frequency [62], decreased func-
tion [63], or normal frequency and function [64]), which may
reflect inadequate identification of Tregs by available markers;
recruitment of patients different in age and disease progres-
sion and differences in experimental conditions. Follow-up
studies using more specific markers (FOXP3
1
CD127
low
and
demethylation of regulatory elements of the FOXP3 gene)
showed that both the frequency and function of Tregs are
normal in the blood of T1D patients, even though a transient
decrease of suppressor activity may occur early after diagnosis
[65], and in a subset of T1D patients [30]. Studies from the
Battaglia lab showed that reduced suppressive function of
Tregs may be restricted to the pancreatic lymph nodes in
patients with long lasting T1D [31]. A defect in IL-2 produc-
tion by total peripheral blood mononuclear cells of patients
with new onset T1D was reported several years ago [66] but
never confirmed as a key immunological feature of T1D
patients. A recent study showed that the T1D-susceptibility
IL2RA haplotype identified by rs12722495 is associated with
decreased signaling via the IL-2 pathway in both memory T
cells and Tregs and that this is linked to diminished Treg func-
tion [32]. However, this phenotype is limited to carriers of
this single nucleotide polymorphism (SNP) and not to all indi-
viduals. Thus, it is likely that this treatment may benefit some
patients more than others, again based on their underlying
defects that contribute to disease.
A Wide Array of Approaches to Reestablish Antigen-
Specific Tolerance
The overall objective of this strategy is to deliver bcell anti-
gens in particular ways such that their presentation in vivo
results in elimination or inactivation of antigen-specific diabe-
togenic T cells, or induction of antigen-specific immunoregula-
tory populations, to confer durable protection from
autoimmunity without compromising the general immunosur-
veillance for infectious agents and malignant cells. The tradi-
tional method has been to administer protein antigens via
tolerogenic routes (mainly oral or intranasal insulin and
GAD65/Alum), but this approach has not produced significant
clinical benefit in recent onset patients [67]. Because of lack
of adverse side effects, these therapies are now being tested
in secondary prevention trials (i.e., in patients with ongoing
autoimmunity evidenced by circulating autoantibodies) (Table
1). It is worth pointing out that oral insulin has also been
tested in a primary prevention trial (in young subject with no
evidence of autoimmunity, Pre-POINT trial, Table 1) and data
suggest that insulin-specific Tregs were induced at the highest
dose [68]. Antigens coupled with apoptotic cells have been
known for several decades to be very tolerogenic and showed
efficacy in preclinical models of T1D [69]. This strategy has
now been tested in patients with multiple sclerosis and was
well tolerated [70]. Massive apoptosis resulting from deple-
tion of B cells and CD8
1
T cells (using a short course of bio-
logics) is accompanied by release of TGF-b, which combined
with exogenous antigens such as GAD65 peptides, supports
the generation of protective Tregs, because CD4
1
T cells are
left untouched and available for conversion [71]. This promis-
ing approach validated in mouse models of T1D and multiple
sclerosis remains to be tested for safety in humans.
A less conventional alternative to protein antigen delivery
lets the body produce specific antigens in cells or sites
Table 3. Main clinical trials using low-dose IL-2 or cell-based therapies in recent onset T1D patients
New onset trials Drug Type of study
Low-dose IL-2
NCT01827735 (DILT1D) Proleukin (IL-2) Phase 1/2, onset <24 months
NCT02265809 (DILfrequency) Aldesleukin (IL-2) Phase 1/2, onset <60 months
NCT01353833 (DF-IL2) Aldesleukin (IL-2) Phase 1/2, onset <24 months
NCT01862120 (DFIL2-Child) IL-2 Phase 2, recent onset
NCT02411253 (DIABIL-2) rhIL-2 Phase 2, recent onset
Cell-based therapies
ISRCTN06128462 (Gdansk) Polyclonal Tregs Phase 1, onset <2 months
NCT01210664 (UCSF) Polyclonal Tregs Phase 1, onset 3-24 months
NCT00445913 (Pittsburgh) Autologous DCs Phase 1, long-term T1D (5y1)
NCT02354911 (Pittsburgh) Autologous DCs Phase 2, new onset <100d
NCT01068951 (Uppsala) MSCs Phase 1, new onset
NCT00690066 (Mesoblast) Prochymal (MSCs) Phase 2, onset 2-20 wks
NCT02057211 (Uppsala) MSCs Phase 2, new onset <3 weeks
NCT01322789 (Sao Paulo) MSCs Phase 1/2, new onset <6 weeks
NCT00305344 (Florida) Umbilical cord blood (UCB) Phase 1/2, post-onset
NCT00989547 (Munich) Umbilical cord blood (UCB) Phase 1, post-onset
NCT01350219 (Tianhe) UCB-derived stem cells Phase 2, post-onset
NCT01996228 (Tianhe) UCB-derived stem cells Phase 1/2, post-onset
NCT00315133 (Sao Paulo) Autologous HSCs Phase 1/2, onset <12 weeks
NCT01285934 (Northwestern) Autologous HSCs Phase 1/2, onset <5 months
Note: Clinical trials are color-shaded based on whether they are ,,or Stem cells used for the generation of new b
cells are not covered here. DCs: dendritic cells; HSCs: hematopoietic stem cells; MSCs: mesenchymal stem/stromal cells; Tregs: regulatory T cells.
Creusot, Battaglia, Roncarolo et al. 5
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amenable for tolerance induction following gene transfer [72].
Plasmid DNA encoding autoantigens such as insulin or its
InsB
9-23
immunodominant peptide prevented disease in NOD
mice [73–75] and was given to recent-onset T1D patients in a
phase 1 trial [76]. Data from this trial demonstrated both
safety and diminution of insulin-reactive CD8
1
T cells, thus
tolerogenic DNA vaccines merit consideration for prevention
trials. Delivery of autoantigens by viral vectors used for gene
therapy has also been explored [77, 78]. One legitimate con-
cern when using viral components is the inadvertent activa-
tion of antigen-specific effector T cells that could exacerbate
bcell autoimmunity, especially if expression with ubiquitous
promoters is allowed in professional antigen-presenting cells
(APCs) than can mature and become immunogenic [79]. The
insertion of a microRNA-142 target sequence to abrogate
transgene expression in professional APCs and other hemato-
poietic lineage cells, together with the use of a liver-specific
promoter resulted in a lentiviral vector, which specifically tar-
gets the antigens to hepatocytes [79, 80]. Treatment with
such a vector encoding the InsB
9-23
peptide-induced some
antigen-specific effector T cells but also antigen-specific CD4
1
FoxP3
1
Tregs, which halted islet immune cell infiltration, and
protected mice from T1D [79]. When combined with a single
suboptimal dose of anti-CD3 mAb, it was effective in reversing
hyperglycemia after onset in a Treg-dependent manner [79].
The use of nonintegrating forms of lentiviral vectors will offer
an additional level of safety when implementing such an
approach clinically [81].
So far, antigen-specific therapies for T1D have been
proved efficient in mouse models and to be among the safest
in patients, but evidence for clinical efficacy is lacking. One
possible reason may have to do with the choice of bcell anti-
gens used, which is limited in two aspects: (1) only a single
antigen (mostly insulin or GAD65) is used despite the evi-
dence of epitope spreading reflected by different types of
autoantibodies, and (2) only native antigens are used while it
has become increasingly clear that many diabetogenic T cells
respond to post-translationally modified or processed neo
antigens. Accomplishing long-term antigen-specific tolerance,
whether it is with Tregs or other regulatory cells will require
these issues to be addressed.
CELL-BASED THERAPIES:CURRENT AND FUTURE APPLICATIONS
Cell-based therapies are individualized approaches that cur-
rently involve the transfer of autologous cells that have
immunoregulatory properties and can provide a counterbal-
ance for effector T cells that mediate bcell destruction. While
certain drugs aim at expanding and potentiating Tregs in vivo
(see low dose IL-2 above), cell-based therapies generally
involve Tregs or cells that have the ability to induce or poten-
tiate such immunoregulatory populations in vivo. We will also
discuss the use of different types of stem cells as part of cell-
based therapies to block autoimmune responses, but we will
not cover the generation of new bcells from stem cells for
transplantation, which is reviewed elsewhere [82].
Regulatory T Cells (Tregs)
Several preclinical animal studies have established that the
adoptive transfer of Tregs can prevent various autoimmune
diseases, T1D included. However, only a few studies showed
that Treg cell transfer is efficacious in reverting active dis-
ease and, when it occurred, the transferred cells needed to
be antigen-specific [83–85]. Based on this evidence, Tregs
are now being used in phase 1/2 studies in patients with
autoimmune diseases [86]. Increasing doses of autologous
ex vivo–expanded polyclonal CD4
1
CD25
1
Tregs have been
used safely in newly diagnosed T1D patients [87]. These
studies were instrumental in demonstrating the safety and
feasibility of such a complex approach of personalized medi-
cine, but efficacy has still to be demonstrated in larger
trials.
Based on the data in animal models, to be of any thera-
peutic use, Tregs have to be transferred prior to overt hyper-
glycemia or have to be antigen-specific. It has been, up to
now, difficult to obtain sufficient antigen-specific CD4
1
FoxP3
1
Tregs, but this might be more feasible by inducing
Tregs de novo in vitro. Such an approach has been used in
the context of allogeneic hematopoietic stem cell transplanta-
tion to prevent GvHD where host-specific Tr1 cells were gen-
erated in vitro from donor peripheral blood and transferred
to transplanted hosts [88]. The induction of self-specific Tr1
cells in NOD mice in vivo is feasible and they protect from
diabetes development [89]. However, the generation of
human diabetes-related antigen-specific Tregs in vitro has yet
to be achieved. Studies on adoptive T cell therapy have dem-
onstrated the possibility of engineering T cells using lentivirus,
either by expressing a relevant (bcell antigen-specific) T cell
receptor into polyclonal Tregs [90] or by overexpressing FoxP3
in T cells [91], potentially in antigen-specific T cells that have
been enriched and ex vivo expanded.
Regulatory B Cells (Bregs)
Recently, the IL-10–producing regulatory Bregs have attracted
attention as being altered in autoimmune diseases and thus
represent another potential tool for cell therapy [92]. As with
Tregs, their numbers and function might be compromised in
T1D patients [93], indicating the possibility of using Breg ther-
apy, alone or in concert with Tregs. However, the importance
of antigen-specificity in this case is not clear, and considering
that we are still at the beginning of cell therapy with Tregs,
using Bregs is even more futuristic.
Dendritic Cells
As the most specialized of APCs, dendritic cells (DCs) have
long been a candidate of choice for their ability to engage T
cells through presentation of bcell antigens, and under a tol-
erogenic phenotype, to achieve deletion or inactivation of dia-
betogenic T cells, converting them into Tregs or restimulating
preexisting Tregs. DC infusions have shown remarkable effi-
cacy in numerous preclinical studies, even in the absence of
exogenously provided antigens [94]. The first clinical trial using
autologous DCs in recent onset T1D patients demonstrated
both safety and the potential to induce Bregs [95]. In this
phase 1 study, the first of its kind for the treatment of auto-
immune diseases, the monocyte-derived DCs were locked in a
nonimmunogenic state by silencing important costimulatory
molecules (CD40, CD80, and CD86), but were not provided
exogenous antigen. Autoantigen expression and maturation
stage are two crucial considerations, because immunogenic
DCs expressing bcell antigens could boost autoimmune T cell
6Unconventional approaches to treat Type 1 diabetes
V
C2016 The Authors STEM CELLS published by Wiley Periodicals, Inc. on behalf of AlphaMed Press STEM CELLS
responses against bcells. The antigen-specific therapies previ-
ously described rely on the acquisition and presentation of
relevant antigens by tolerogenic APCs that are not that well
characterized but possibly comprising different subsets of DCs
and other types of APCs. In the case of the recent DC trial,
the mechanism of action is not completely understood as no
exogenous antigen was provided, and whether these DCs
could pick up and present relevant autoantigens in vivo
remains unclear. Furthermore, there is evidence that DCs
expressing costimulatory molecules but not inflammatory
cytokines (termed semimature DCs) may induce tolerance as
well [96].
Another phase 1 trial employing tolerogenic DCs pulsed
with citrullinated peptides for the treatment of rheumatoid
arthritis has demonstrated safety as well as immunological
responses reflective of regulation [97], suggesting that provi-
sion of autoantigens may not lead to exacerbated responses
as long as the DCs are maintained tolerogenic, which in this
case was achieved by pretreatment with an NF-jB inhibitor.
An alternative or complementary approach to silencing the
expression of costimulatory genes is the overexpression of tol-
erogenic products for which the list is long and includes
immunoregulatory cytokines, inhibitory ligands, and
metabolism-altering enzymes [98]. A safe and clinically viable
way to overexpress genes and achieve a significant therapeu-
tic outcome is by mRNA electroporation, which has been
used widely for autologous DC therapy in cell therapy of can-
cer [99, 100]. This method of modification allows for coex-
pression of multiple products of interest in the same cell and
at the same time, including relevant antigens for added speci-
ficity if desired [101]. Although transient, expression of genes
in DCs by mRNA is sufficient to induce long-lasting responses
resulting in prevention of T1D in mice [102]. A follow-up trial
of the first safety study of autologous DCs in T1D patients is
set to begin in the near future.
Mesenchymal Stem/Stromal Cells
Mesenchymal stem/stromal cells (MSCs) are endowed with
regenerative and immunosuppressive properties that have
fueled their popularity in cell therapy, yet controversies
remain regarding their name and definition [103]. Although
they can generally suppress immune responses on their own
in a nonspecific manner, they have also been shown to induce
or expand Tregs, including in preclinical T1D studies
[104–106]. Because MSCs are nonprofessional APCs, it is
unclear if and how they specifically interact with Tregs and
diabetogenic T cells, and their effect may be indirect through
inflammation relief [107]. Unlike DCs, MSCs are nonimmuno-
genic, and can provide protection even in an allogeneic host,
which makes them attractive for the clinic [108]. They have
been proven to be well-tolerated in T1D patients whether
they were isolated by bone marrow aspiration [109] or from
adipose tissue [110], and associated with improvement of dis-
ease parameters such as C-peptide preservation. Careful char-
acterization of the phenotype and properties of MSCs used in
cell therapy is crucial to demonstrate consistency between
studies and draw meaningful conclusions, regardless of the
source of the cells, isolation, and culture conditions (Table 4).
Two follow-up clinical trials are currently recruiting in Sweden
and Brazil to demonstrate long-term efficacy (Table 3). All tri-
als so far are being conducted in recently diagnosed T1D
patients using MSCs that are either autologous or from first-
degree relatives. Although no serious side effects have been
reported so far, there remains a concern that slow growing
tumors may appear in the long term in some patients receiv-
ing autologous cells, according to some preclinical studies
[108].
Umbilical Cord Blood
Although this approach is limited to a few individuals who
have banked samples, umbilical cord blood (UCB) is a great
source of abundant MSCs and Tregs, which might work in syn-
ergy when infused into patients. As many important self-
reactive Tregs appear to be released early in life [111], these
Tregs may also include more antigen-specific Tregs of rele-
vance. Autologous UCB infusion was found to be safe but did
not have any significant therapeutic effect despite increased
numbers of Tregs [112, 113] and even with oral docosahexae-
noic acid and vitamin D supplementation [114]. Another
phase 1 trial, also in new onset pediatric subjects, is well
under way in Germany (Table 3). As previously mentioned, it
is possible that therapies involving Tregs would be more
effective when applied prior to disease onset. In that model,
the Cord Reinfusion in Diabetes (CoRD) study, enrolling high-
risk children with banked UCB, was recently initiated in Aus-
tralia and represents the first cell-based therapy used for sec-
ondary prevention of diabetes [115]. A distinct process is
being tested in China, whereby lymphocytes are obtained
from the blood by leukapheresis, “reeducated” ex vivo in con-
tact with UCB-derived stem cells, and then reinfused into the
patient. These studies suggest that this treatment improved
preservation of bcell function without notable adverse
effects, but caution must be exercised in the interpretation of
these studies, which were improperly controlled [116].
Hematopoietic Stem Cells
Perhaps the most drastic of all cell therapies consists of a
major immunological reset with the transient ablation of
Table 4. Phenotype of MSCs used in T1D studies
Source CD11c CD14 CD29 CD31 CD34 CD44 CD45 CD73 CD90 CD105 Reference
Mouse bone marrow 2212 121 1 [104]
Mouse bone marrow 112121[108]
Mouse adipose tissue 2 21211 1 [105]
Human bone marrow 2222111[109]
Human bone marrow 21 21211 1 [*]
Human adipose tissue 211 [110]
Note: Additional characterization may include ability to terminally differentiate (e.g., adipocytes) or to suppress T cell responses. [*] Prochymal
MSCs (NCT00690066).
Creusot, Battaglia, Roncarolo et al. 7
www.StemCells.com V
C2016 The Authors STEM CELLS published by Wiley Periodicals, Inc. on behalf of AlphaMed Press
circulating T cells and their replacement with hematopoietic
stem cells (HSCs). Multiple completed studies have involved
autologous HSCs mobilized from peripheral blood and admin-
istered after a nonmyeloablative regimen consisting of cyclo-
phosphamide and anti-thymocyte globulin [117–119]. This
treatment performed in new-onset T1D patients demon-
strated a remarkable ability to normalize glycemia in a major-
ity of the subjects. Independence from insulin lasted between
several months and several years, up to 3-4 years (as
reported in the last meeting of the Immunology of Diabetes
Society), and a larger trial is now enrolling patients (Clinical-
Trial.gov NCT01285934). However, this therapy is fraught with
considerable side effects associated with the nonmyeloabla-
tive regimen [117–119], which make this approach unattrac-
tive in its current form to many prospective patients and
precludes its use for prevention. Furthermore, the contribu-
tion of HSCs in maintaining normoglycemia is unclear as the
immunosuppressive effect of the regimen may account for
part if not all of the therapeutic benefit. Finally, new T cells
(and other immune cells) generated from autologous HSCs
would still carry any inherent genetic defects that may play a
role in disease etiology. Transplantation of bone marrow-
derived allogeneic HSCs with induction of mixed chimerism
has also been tested in a minority of patients with autoim-
munity, including T1D [120]. The use of HSCs in combination
with islet transplant to induce chimerism and immunological
tolerance has been tested in a recent trial at the University of
Miami based on campath-1H and infusion of donor
CD34
1
HSCs (ClinicalTrials.gov NCT00315614), but did not
show any significant benefit. Although allogeneic HSCs from a
compatible and healthy donor may help correct some genetic
abnormalities, this must be preceded by high doses of chemo-
therapy and radiation to ablate the patient’s bone marrow
and is followed by prolonged immunosuppression to prevent
GvHD. The consequent transplant-related morbidity and mor-
tality limit this approach to patients with concomitant hema-
tological malignancies [121]. It should be noted that
preclinical data with purified allogeneic CD34
1
CD90
1
HSCs
showed complete reversion of T1D in the absence of GvHD
[122]. In addition, novel biologicals are under investigation for
use as safer and less toxic drugs to myeloablate the patient’s
bone marrow [123]. Thus, as safer and more effective HSC
transplantation protocols become available, allogeneic HSCs
might also be indicated in T1D. A more detailed review of the
different applications of stem cells (including MSCs and UCB)
to treat T1D can be found elsewhere [124].
In parallel to efforts in generating insulin-producing cells
from stem cells (embryonic stem cells or induced pluripotent
stem cells) [82], there is an expanded interest in growing tis-
sues specialized in tolerance induction, such as thymic tissue
[125, 126]. Although much can be learned from these studies,
the clinical implementation of such advances is elusive,
including where and how to implant the new tissue.
CONCLUSIONS
A variety of original therapeutic strategies for treating or pre-
venting T1D have emerged in the past decade, with the latest
approaches clearly dominated by cell-based therapies. As the
least expensive and most conventional therapies have failed
to deliver efficient and durable protection from diabetogenic
immune responses, testing of more expensive, and individual-
ized therapies has become justified as long as preclinical stud-
ies indicate a strong prospect of durable efficacy achieved
with a minimal number of treatments. Strategies that are
more antigen-specific and less immunosuppressive tend to
have the best safety profile, and their poor efficacy in new
onset patients should not discourage evaluation in prevention
trials in high-risk patients in which they might perform sur-
prisingly well. The enrollment of subjects for prevention stud-
ies should be guided by more refined biomarkers, which may
help the diabetes community to better understand the under-
lying defects behind the autoimmune response in each
patient, and better tailor the treatment type, dose, and tim-
ing. When appropriate, two or more of these therapies may
be combined in order to address multiple defects and benefit
a larger number of patients. A clear advantage of cell-based
therapies is that they can perform multiple tasks. For exam-
ple, one can envision tolerogenic APCs engineered to express
selected bcell antigens (to specifically engage diabetogenic T
cells), additional immunoregulatory ligands or cytokines (to
potentiate T cell deletion or Treg induction), homing mole-
cules (for targeting to inflamed islets or their draining lymph
nodes), anti-inflammatory cytokines (to quench inflammation),
and even growth factors to promote bcell replication.
ACKNOWLEDGMENTS
RJC is currently funded by the National Institutes of Health
(NIH), the Diabetes & Endocrinology Research Center (Colum-
bia University), and the Irving Institute for Clinical and Trans-
lational Research; MB by the Italian Ministry of Health, the
European Community, the Cariplo Foundation, and the Juve-
nile Diabetes Research Foundation (JDRF); MGR by the Italian
Telethon Foundation and JDRF; and CGF by the NIH and JDRF.
AUTHOR CONTRIBUTIONS
All authors wrote and edited the manuscript
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