Journal of Trace Elements in Medicine and Biology 73 (2022) 127040

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Journal of Trace Elements in Medicine and Biology

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Review

Antigen-specific immunotherapies in type 1 diabetes

Xuejiao Zhang a, Ying Dong b, Dianyuan Liu a, Liu Yang a, Jiayi Xu c, Qing Wang a, *
a
Department of Endocrinology, China-Japan Union Hospital of Jilin University, Changchun 130000, China
b
Department of Radiation Oncology, Jilin Cancer Hospital, Changchun 130000, China
c
School of Public Health, Jilin University, Changchun 130000, China

A R T I C L E I N F O A B S T R A C T

Keywords: Type 1 diabetes mellitus (T1DM) is an autoimmune disease caused by the destruction of pancreatic beta cells, in
Type 1 diabetes mellitus which immune system disorder plays an important role. Finding a cure for T1DM and restoring beta cell function
Antigen-specific immunotherapies has been a long-standing goal. Research has shown that immune regulation with pancreatic islet auto-antigens
Immune tolerance
may be the most specific and safe treatment for T1DM. Immunological intervention using diabetogenic auto-
ZnT8
antigens as a target can help identify T1DM in high-risk individuals by early screening of autoantibodies
Prevention
(AAbs) before the loss of pancreatic islet function and thus achieve primary prevention of T1DM. However,
induction of self-tolerance in patients with pre-diabetes can also slow down the attack of autoimmunity, and
achieve secondary prevention. Antigen-based immune therapy opens up new avenues for the prevention and
treatment of T1DM. The zinc transporter 8 (ZnT8) protein, presents in the serum of pre-diabetic and diabetic
patients, is immunogenic and can cause T1D autoimmune responses. ZnT8 has become a potential target of
humoral autoimmunity; it is of great significance for the early diagnosis of T1D. ZnT8-specific CD8+ T cells can
be detected in most T1DM patients, and play a key role in the progression of T1D. As an immunotherapy target, it
can improve the dysfunction of beta cells in T1DM and provide new ideas for the treatment of T1D. In this
review, we summarize research surrounding antigen-specific immunotherapies (ASI) over the past 10 years and
the ZnT8 antigen as an autoimmune target to induce self-tolerance for T1DM.

1. Introduction
Type 1 diabetes mellitus (T1DM) is an autoimmune metabolic dis­
ease characterized by the damage of beta cells [1]. The physical, social,
and economic costs of T1DM are huge; according to statistics late
diagnosis and lack of access to insulin are considered to be the main
causes of death from T1DM [2]. At present, the most effective treatment
is lifelong exogenous insulin replacement therapy, but this inevitably
leads to diabetic complications and cannot prevent the autoimmune
destruction of beta cells [3]. Allogeneic islet transplantation was suc­
cessful, but because of severe immune rejection this method was not
widely used [4]. The development of antigen-based immunotherapy
targeting the antigen-specific immune tolerance pathways is expected to
prevent or reverse T1DM [5]. It is aimed at regulating the potential
defects of the immune system that cause the destruction of beta cells by
auto-antigens to alleviate T1DM [6]. Furthermore, ZnT8 antibodies are
newly discovered independent biomarkers for the diagnosis of T1DM in
addition to glutamic acid decarboxylase AAbs (GADA), insulinoma
associated protein tyrosine phosphatase 2 AAbs (IA-2A), and insulin
AAbs (IAA) [7,8]. In this paper, we summarize the development and use
of ASIs over the past 10 years and the ZnT8 auto-antigen as a target to
induce immune tolerance for T1DM.
1.1. T1DM and autoimmune pathogenesis
Although the pathogenesis of T1DM is not yet clear, it is certain that
a disorder of the immune system plays an essential role in beta cell
destruction. One consequence of autoimmune attack and loss of beta cell
function is a decrease or even lack of insulin secretion, which is manifest
as hyperglycemia and gradually progresses to diabetes [9,10]. An im­
mediate need for exogenous insulin and lifelong replacement therapy
are important features of T1DM. Insulin is the only hormone in the body
that lowers blood glucose, and beta cells are responsible for sensing and
releasing insulin [11]. T1DM mostly occurs in children and adolescents,
but age is not a limiting factor [12]. Autoimmune-mediated diabetes can
be divided into two types according to different ages: the classic T1DM,
and the latent type autoimmune diabetes in adults (LADA); the latter
exhibits slow progression in adults, with the rate of beta cell failure

* Correspondence to: Department of Endocrinology, China-Japan Union Hospital of Jilin University, 126 Xiantai St, Changchun 130033, China.
E-mail address: wang_qing@jlu.edu.cn (Q. Wang).

https://doi.org/10.1016/j.jtemb.2022.127040
Received 10 January 2022; Received in revised form 18 June 2022; Accepted 14 July 2022
Available online 15 July 2022
0946-672X/© 2022 The Authors. Published by Elsevier GmbH. This is an open access article under the CC BY-NC-ND license
(http://creativecommons.org/licenses/by-nc-nd/4.0/).
X. Zhang et al. Journal of Trace Elements in Medicine and Biology 73 (2022) 127040

being much slower and the residual ability of beta cell function much
stronger than that of classic T1DM [13].
How is the autoimmune process in beta cells implemented in the
pancreatic islets? Islet-specific cytotoxic T cells cloned from a patient
with T1DM leads to beta cell destruction after transplantation into HLA-
A2 transgenic non obese diabetic (NOD) -scid IL2Rγnull mice, indicating
that autoreactive CD8+ T cells are involved in T1DM initiation and beta
cell destruction [14]. This showed that the immune process of T1DM is
mediated by auto-reactive T lymphocytes. Therefore, persistent
auto-reactive T-cells in diabetic patients are the key to the onset of
T1DM [1]. Why do T cells target the destruction of beta cells? It may
depend on the recognition of diabetogenic antigen epitopes produced in
beta cells by anti-islet T-cells [15]. Another study also demonstrated this
point, showing that auto-reactive T-cells in the pancreatic islets are
auto-antigen specific [16]. So, the second characteristic feature of T1DM
is the existence of persistent beta cell specific auto-reactive CD8+ T cells
in the circulation [17,18], which play a leading role in the pathogenesis
of T1DM [19]. The destruction of beta cells leads to the release of
auto-antigens and expansion of AAb-producing B lymphocytes, so the
third distinguishing feature of T1DM is the presence of pancreatic
islet-specific AAbs against beta cell auto-antigens in the serum, which
can be used as a reliable biological marker for the diagnosis of T1DM
[20]. AAbs are key to distinguishing T1DM from type 2 diabetes mellitus
(T2DM), about 96 % of patients are detected to be positive for at least
one type of AAb, including GADA, IAA, or IA-2A [21,22], and the ZnT8A
[7,23]. However, why do T cells not produce abnormal immune
responses to auto-antigens in normal individuals? That is because of the
existence of central immune tolerance and peripheral immune tolerance
to ensure that the body responds correctly to self-antigens [24]. But in
some cases, when immune tolerance is lost, auto-reactive T cells are
activated and proliferate after encountering auto-antigens and then
secrete and release inflammatory factors to induce insulitis, resulting in
the destruction and loss of beta cells [25,26]. Therefore, the fourth
typical feature of T1DM is insulitis [27,28]. Autoantigen-specific path­
ogenic T cells and insulitis are considered to be immune factors that
mediate the destruction of pancreatic beta cells in T1DM [29,30].
Taken together, a plausible way to treat T1DM based on the etiology
and pathogenesis is to combat the destructive pathogenic T cells and
restore the function of pancreatic islet beta cells at the same time.
Therefore, rebuilding the immune tolerance of beta cells has become the
target of treatment for T1DM (Fig. 1).
1.2. The development of T1DM treatment
T1DM has been proven to be an autoimmune disease that damages
beta cells due to over activation of effector T cells. A humanized Fc re­
ceptor nonbinding anti-CD3 monoclonal antibody (mAb), teplizumab,
was developed based on this strategy for the treatment of patients with
stage 2 and 3 T1D. Clinical trials have confirmed that teplizumab can
improve the stimulated C-peptide responses [31], and can significantly
delay the onset of diabetes in high-risk groups [32]. Based on these
clinical trial results, the US Food and Drug Administration (FDA) is

Fig. 1. Mechanism of T cell-mediated beta cell destruction in T1D. T cells are enabled to identify self and non-self through positive selection, those with strong
binding affinity to self-peptides are removed by negative selection. Some T cells that escape the central tolerance require action from peripheral mechanisms. APCs
migrate from the pancreas take up and present diabetogenic auto-antigens and activate T cells; activated pathogenic T cells migrate to pancreatic islets and begin the
process of beta cell destruction, eventually leading to T1D. Tregs attempt to prevent beta cell damage by secreting cytokines. Autoreactive B cells are also involved in
beta cell damage. They activate autoreactive T cells, which act on islets by secreting inflammatory cytokines (IFN-γ, TNF, IL-17) and CTL-mediated killing; in turn,
auto-reactive T cells provide help signals to auto-reactive B cells, which further exacerbate T1D by promoting the differentiation of auto-reactive B cells into plasma
cells and the production of islet-specific AAbs. Bregs inhibit beta cell damage by secreting the cytokines IL-10 and TGF-γ. ZnT8 is the main autoantigen in T1D. The
secretion of insulin granules can increase the exposure of ZnT8 autoantigen to the cell surface and initiate ZnT8 epitope-specific T cell-mediated beta cell destruction,
resulting in T1D. As beta cells are destroyed, autoantigens release increases, amplifying the T cell-mediated immune response.Abbreviations: T1D: type 1 diabetes;
APCs: antigen presenting cells; Tregs: regulatory T cells; CTL: cytotoxic T lymphocytes; IFN-γ: interferon-γ; TNF: tumor necrosis factor; IL-17: interleukin-17; AAbs:
autoantibodies; Bregs: regulatory B cells; IL-10: interleukin − 10; TGF-γ: transforming growth factor-γ; ZnT8: zinc transporter 8.

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X. Zhang et al.
currently considering approval of teplizumab as a treatment for T1D.
Other immunotherapy drugs targeting T cells in clinical trials include
anti-thymocyte globulin (ATG) [33]. An animal randomized controlled
study showed that Low-dose ATG slowed decline of C- peptide and
reduced HbA1c in new-onset T1D [34]; Abatacept, inhibits the activa­
tion of B cells and other antigen-presenting cells by acting on follicular
helper T cells [35]; Alefacept, a second costimulatory receptor CD2 In­
hibitor [36]. These drugs have a significant effect on delaying the pro­
gression of the disease, but these effects are sustainable for not all
patients. Therefore, the treatment time window is particularly
important.
Increasing evidence showed that B lymphocytes are important not
only for autoantibody-mediated diseases, but also for those that are ul­
timately T cell-mediated, such as T1DM. There is a collaborative inter­
action between autoreactive B and T lymphocytes. B lymphocytes
contribute to islet-specific autoimmunity by presenting antigens to
autoreactive T cells and providing costimulatory signals, acting on the
activation of pathogenic T cells [37]. In turn, activated T cells provide
help signals to autoreactive B cells to further aggravate impaired B cell
tolerance and promote the production of autoantibodies, promoting the
occurrence and development of T1D [38,39]; in addition, regulatory B
cells (Bregs) play an immunoregulatory role by secreting cytokines IL-10
and transforming growth factor-β (TGF-β) [40]. Immunotherapy to
antibody B cell against new-onset T1DM was initially performed mainly
by pan-B-cell-depletion by targeting surface antigens with monoclonal
antibodies. rituximab (RTX), a monoclonal antibody specific to B-cell
surface protein CD20. Although RTX treatment of T1DM caused signif­
icant B cell depletion, the long-term efficacy has not been established yet
[41]. One study showed efficacy in preserving beta cell function for 1
year; Extended follow-up of participants, whereas such therapy showed
limited effect after 2 years, the improvement in C-peptide preservation
was limited to the early period after RTX and disappeared 30 months
later [42]. Thus, pan-B-cell-depletion is not very effective as used and
frequent depletion might be required to minimize the presence of
autoreactive B cells.
Then it leads to antigen-specific treatment using micro- and nano­
particles for treatment of autoimmune disease. It includes micro- and
nanoparticle-based therapy, such as antigen- or peptide-conjugated
nanoparticles, antigen- or peptide-conjugated microparticles and
pMHC-coated nanoparticles [43–46]; cell-based immunotherapy such as
antigen-coupled syngeneic lymphoid cells and autoantigen-loaded
erythrocytes [47,48]. Conjugate antigens or polypeptides to the sur­
face of microparticles or nanoparticles or encapsulate antigens inside
microparticles or nanoparticles, transport them to designated sites, and
release them through conformational changes when blood glucose levels
fluctuate. Its tolerance effect may involve multiple mechanisms,
including clonal anergy, clonal deletion, immune deviation, production
of regulatory cells and the release of autoantigen-loaded apoptotic
bodies [49]. The Antigen-polymer conjugate polymeric modular nano­
particles (acNPs) platform can precisely control the size of antigens,
precisely control over their size, Antigen loading, Antigen release and
deliver single or multiple pathogenic antigenic epitopes with loading of
single or multiple Antigens [50].
In T1DM, antigen-specific immunotherapy (ASI) offers a good and
safe therapy to induce immune tolerance. Although islet antigen mon­
otherapy has shown promise in animal models, prevention or reversal
has not been achieved in human clinical trials. So there is still an urgent
need to optimize those immunotherapies, and combination therapy may
be needed to further improve the therapeutic efficacy. Anti-CD20
combined with insulin has a moderate protective effect in prevention
of T1D onset, but no therapeutic efficacy in NOD mice [51]. B cell
depletion might be combined with T cell-based therapies to restore
immune tolerance. A study reported that the combined treatment of
intravenous anti-CD20 mAb and oral anti-CD3 mAb was more effective
than anti-CD20 monotherapy in NOD mice [52]. In human, an increase
in T cell frequency and activity was observed in T1D patients receiving
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Journal of Trace Elements in Medicine and Biology 73 (2022) 127040
RTX treatment [53]. In line with this notion, a 2-year clinical trial also
confirmed that the combined therapy with regulatory T cells (Tregs) and
rituximab was generally consistently superior to monotherapy with
Tregs in controlling recent-onset T1DM in paediatric patients [54],
which also reminds us that it can be personalized at least according to
the age of the patient.
Pancreatic islet transplantation has brought great hope to T1DM
patients. However, due to the shortage of donors, poor revasculariza­
tion, immunosuppression and many other serious problems, the failure
of islet transplantation has greatly affected the development of islet
transplantation. To address these issues, biomaterial-based strategies
are being explored. The islet encapsulation strategy encapsulates
xenogeneic islets by semi permeable membrane encapsulate, creating a
protective environment between the graft and recipient, allowing oxy­
gen and nutrients to enter the encapsulated cells, insulin and metabolic
waste products to be excreted while preventing immune cells [55]. It can
promote islet transplantation without using toxic immunosuppressants
to prevent the host immune response. In theory, it can effectively protect
the survival of transplanted islets and improve insulin secretion.
Therefore, this strategy has high therapeutic potential. Current Islet
encapsule approaches include macroencapsulation, microencapsulation
and nanoencapsulation. Researchers have studied from the choose of
islet encapsulation biomaterials and coencapsulation or modification of
the islet material [56], the application in islet transplantation [55],
merits and demerits of different approaches to the encapsulation tech­
nology [55,57], immune modulation [58], alternative islet sources and
implantation sites [59], and the major challenge faced by islet trans­
plantation [57,59,60] and other perspectives. The goal is to minimize
the immune system’s attack on the graft and to improve islet secretion.
Relevant clinical studies have confirmed that encapsulated islets can
effectively reduce the demand for exogenous insulin in patients with
T1D [61,62]. Although this strategy yielded positive results over time in
the experiments, all patients returned exogenous insulin requirements to
pre-transplant levels within a 7-year follow-up period [61], and most
patients cannot achieve insulin independence from this treatment
strategy [62]. Further studies have found that the fibrotic response of
the graft is significantly associated with treatment failure [63], and
interleukin-1 (IL-1) and tumor necrosis factor (TNF-α) is also contribute
to graft damage [64]. In addition, the oxygen and nutrient delivery of
the encapsulated islets are also key factors that determine the success or
failure of transplantation. Therefore, an ideal islet encapsulation envi­
ronment should have the following points: able to provide islet struc­
tural support, able to ensure a good blood supply, biocompatible [65],
and minimize the level of fibrosis and inflammation to achieve optimal
efficacy. More research is still needed to optimize this treatment
strategy.
In general, immunotherapy for T1D is in a stage of rapid develop­
ment, and the above treatments have been proven to be effective in
relieving disease progression in T1D patients. However, there are still
many challenges, such as unsustainable immune tolerance and it is
necessary to accurately grasp the treatment time window, which brings
great difficulties to clinical application. Based on the above consider­
ations, ASI induces self-tolerance to β-cell self-antigens, selectively
eliminates self-reactive T cells without affecting overall immunity, and
can delay the time of immune tolerance to a certain extent. Currently,
relevant research is still in progress (Table 1). ASI may be important
contributors to future therapeutic prospects, and their specificity may be
beneficial from a safety perspective.
1.3. T1DM and immune tolerance
The immune system is one of the most complex networks in humans;
its main function is to recognize and protect the body from foreign
pathogens, while maintaining self-tolerance to avoid abnormal immu­
nity caused by its own tissues [66]. Self-tolerance maintenance and
immune regulation are the two important functions of the immune
X. Zhang et al. Journal of Trace Elements in Medicine and Biology 73 (2022) 127040

Table 1
Reports of antigen-specific immunotherapy for type 1 diabetes mellitus (T1DM) in the last decade.
Author Year Subjects Cases Autoantigen Administration route Pathogenesis Clinical Efficacy Evaluation
(n)

Casas [156] 2022 individuals aged 12 GAD-alum Intralymphatic induced secretion of IL-13 C-peptide secretion increased
12–24 y with T1DM almost 4 years after the initial
< 6 months from immune intervention and insulin
diagnosis, requirement remained stable
Assfalg 2021 islet autoantibody- 44 Insulin Oral Associated with the T cell No differences in insulin and c-
[157] negative children responses to insulin. peptide in serum
aged
6 months to
2.99 years who had
a first-degree
relative with type 1
diabetes
Larsson et al. 2018 Patients aged 4–17.9 50 GAD-Alum – Retain residual beta cell No rapid decrease in C-peptide
[158] y function and increase
regulatory T cells.
Tavira et al. 2018 Recent-onset 12 GAD-Alum Intralymphatic+subcutaneous Depend on Th2-associate C-peptide remained stable at 180
[159] patients cytokine IL-13, IL-13 days while insulin dose
suppresses Teff decreased in 24 h in Lymph
proliferation and IL-2 nodes group; An increase in
secretion. insulin requirement with a
greater loss of C-peptide in
subcutaneous group.
Krischer 2017 Nondiabetic 560 Insulin Oral – Improve short-term c-peptide
[160] relatives of probands secretion
with T1DM
Alhadj 2017 < 100 d from 27 Proinsulin C19- Intradermal injection Induce IL-10 responses and Insulin use and stimulated c-
[161] diagnosis of T1DM A3 adaptive Tregs arising peptide remained stable
peptide after treatment; and retain
residual beta cell function
and increase regulatory T
cells.
Bonifacio 2015 Children 25 Insulin Oral Induce insulin-responsive –
[162] 2–7 y and proinsulin-responsive
regulatory T cells.
Roep [19] 2013 < 5 y from diagnosis 80 An engineered Intramuscular injections Deletion of CD8 + T cells C-peptide increases
of T1DM and > 18 y DNA plasmid reactive to proinsulin.
encoding
proinsulin (BHT-
3021).
Ludvigsson 2012 Recent-onset T1DM 334 GAD-Alum Subcutaneous injections – No significant increase in c-
[163] 10–20 y peptide secretion
Wherrett[ 2011 < 100 d from 145 GAD-Alum Subcutaneous injection – No significant increase in c-
164] diagnosis of T1DM peptide secretion
Vehik [165] 2011 Subjects with a 275 Insulin Oral Induce adaptive Tregs –
projected 5 y risk of from cohorts of preexisting
diabetes of 26–50 % insulin-specific T cells.
Fourlanos 2011 Recent-onset T1DM 52 Insulin Intranasal Induce immune tolerance –
[166] to insulin.

GAD-Alum: Glutamic acid decarboxylase formulated with aluminum hydroxide; alum has been the adjuvant of choice to minimize the possibility of promoting beta-
cell-mediated destruction together with GAD65.

system [66]. When the immune system fails in certain individuals, and
causes an attack on its own components resulting in the overexpression
of AAbs, this can lead to disease, which is termed as autoimmune dis­
ease, characterized by the production of AAbs and activation of
auto-reactive T lymphocytes [67]. When an autoimmune disease occurs,
auto-reactive T cells become resistant to suppression during the active
phase; but if immunity is normal, the underlying auto-reactive T cells
will not cause autoimmune diseases owing to the regulation of a variety
of inhibitory mechanisms, collectively known as “immune tolerance”
[68]. Immune tolerance refers to the states of nonresponse in immunity
to foreign antigens and self-tissues [69]. It is an essential mechanism
used to balance physiological responses and constantly fights off
harmful molecules in the human body and eliminates or inactivates
auto-reactive T lymphocytes that recognize self-antigens, to prevent the
immune system from destroying itself and maintain the immune balance
[70].
Immune tolerance is divided into central tolerance and peripheral
tolerance [24]. The former occurs in the thymus, passing through
positive selection makes T cells able to recognize a peptide/major his­
tocompatibility complex (pMHC), and the interaction between T cell
receptor (TCR) and MHC/self-antigen complex. T cells that do not pass
the central tolerance test undergo an intrinsic suicide program called
negative selection to remove them and prevent auto-reactivity [24].
There is evidence that defects in thymic T cell negative selection related
to insulin reactivity is a key factor in the genetic predisposition in T1DM
[71]. The purpose of central selection is to ensure that T cells entering
the periphery do not respond to their own antigens [24]. However, many
self-reactive T cells still escape from thymic negative selection, which
leads to the need for peripheral mechanisms to ensure that self-tolerance
is maintained to prevent autoimmune diseases [72].
Peripheral immune tolerance may delete those escaped T cells in the
peripheral lymphoid organs through various mechanisms including
clone deletion, clone anergy, and immune suppression of regulatory T
cells [73]. The most important mechanism is the presence of specific cell
populations to suppress abnormal immune responses that unintention­
ally target their own tissues; Tregs develop in the thymus and periphery

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X. Zhang et al.
are critical for maintaining tolerance [74]. Tregs include naturally
arising Treg cells (Foxp3+ CD25+ CD4+ Tregs), induced Treg cells
(iTreg), type 1 regulatory T cells (Tr1) that produce the immunosup­
pressive cytokine interleukin-10 (IL-10), and Type 3 helper T cells (Th3)
that produce transforming growth factor beta (TGF-beta) [75]. Tregs are
essential suppressors of unwanted autoimmune responses [76]. The
mechanism of Tregs includes contact-dependent suppression and
contact-independent suppression mechanisms [77]. The former can
inhibit costimulatory signals and cytokine secretion by connecting
Cytotoxic T lymphocyte antigen 4 (CTLA-4) on Tregs to CD80/CD86 on
APCs. The latter contact-independent mechanisms disrupt the metabolic
state of T effector cells via the production of the immunosuppressive
cytokines IL-10 and TGF-beta [77,78]. Among them, the most typical
mechanism is the natural CD25+ CD4+ Tregs that can specifically ex­
press Foxp3 transcription, which plays an extremely critical role in
maintaining peripheral tolerance and preventing autoimmune diseases
[79]. Animal experiments have shown that a deficiency of Foxp3+ Tregs
can activate even weak or rare autoimmune T-cell clones, and subse­
quently cause an autoimmune disease [80]. Tregs deficiency in humans
has also been confirmed to be associated with autoimmune diseases such
as T1DM [81,82]. In the absence of specific antigen activation, Tregs
have no effect on regulating disease [83]. Therefore, Foxp3+ CD25+
CD4+ natural Tregs actively participate in the maintenance of
self-tolerance, and their abnormality is one of the causes of autoimmune
disease. A balance is formed between autoimmunity and self-tolerance,
similar to a scale. The dynamic balance between Tregs and auto-reactive
T cells determines the risk, timing, and progression of disease.
The onset and development of T1DM is also a process of dynamic
evolution. Although the specific pathogenesis is not yet clear, it can be
determined that disorder of the immune system plays an extremely
important role in the loss of immune tolerance and the subsequent
destruction of beta cells [84]. Naive T cells first encounter
diabetes-related auto-antigens presented by APCs in lymph nodes, the
result of which is a tolerance in healthy humans; but in T1DM, naïve T
cells are activated as pathogenic auto-reactive T cells and then migrate
to the pancreatic islets and begin to damage beta cells [85]. Only when a
new balance is reached can autoimmune diseases be prevented, in which
Tregs, that are responsible for peripheral tolerance and attempt to pre­
vent unwanted autoimmune responses, recognize self-polypeptides and
inhibit auto-reactive pathogenic T cells.
Rebuilding immune tolerance refers to inducing the body to actively
suppress the immune response to a specific antigen [86]. If tolerance to
diabetogenic auto-antigens can be effectively reinstated then the beta
cell attack is prevented. Rebuilding immune tolerance can be roughly
divided into two parts, one is by decreasing the number of pathogenic T
cells by inducing apoptosis and functional inactivation of highly acti­
vated effector cells [87], or inducing the deletion of pathogenic T cells
[88,89]. The other is through promoting immune regulation and
increasing the number of Tregs and enhancing the function of Tregs [90,
91]. This may be a future strategy for the treatment and prevention of
autoimmune disease such as T1DM through reestablishing
self-tolerance.
1.4. A new strategy for inducing immune tolerance: Evaluation of ZnT8-
antigen specific immunotherapy
ZnT8 is a product encoded by the SLC30A8 gene located on chro­
mosome 8, and is specifically expressed on the insulin granular mem­
brane of pancreatic beta cells [92,93]. The protective effects of ZnT8
loss-of-function mutations are either different between mouse or
human [94]. The polymorphic variant rs13266634 (R325W) in the
human ZnT8 gene SLC30A8 is strongly associated with diabetes risk
[94]. In NOD mice, ZnT8 is a minor diabetogenic antigen that can
participate in diabetes in conditions in which the islet is first made
receptive to immunological insults [95]. It is the zinc transporter with
the highest known expression levels and unique tissue specificity in
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Journal of Trace Elements in Medicine and Biology 73 (2022) 127040
pancreatic tissues [96] where it maintains the balance of zinc in beta
cells by importing and exporting zinc ions [97]. Pancreatic islet beta
cells are the only source of insulin in the human body, and insufficient
insulin secretion is the main pathogenic factor of T1DM [98]. Zinc is an
important ion in humans, which is involved in the synthesis, storage,
and secretion of insulin, and plays an important role in the regulation of
insulin signaling and glucose homeostasis [99]. ZnT8 is mainly
responsible for zinc enrichment in insulin secretory granules, and the
increase of zinc accumulation in beta cells is the result of overexpression
of ZnT8 [100].
Peptides derived from ZnT8 have been identified as biomarkers of
diabetogenic self-antigens independent of other auto-antigens that can
be recognized by auto-reactive CD8+ T cells; hence, it is a major CD8+ T
cell target of autoimmunity [101]. Therefore, the autoimmune process
of ZnT8 is being studied at the level of both AAbs and T-cells. T-cell
reactivity of ZnT8 antigen has been shown to exist in both rodent models
and T1DM patients [102]. The proper application of ZnT8 antibody
(ZnT8A) complements the use of biomarkers including IAA, GADA, and
IA-2A [9]. Furthermore, ZnT8-specific responses are particularly broad:
GAD triggers responses in 50 % of patients, while other antigens stim­
ulate around 40 %; however, ZnT8 epitopes trigger responses in more
than 64 % of patients [101]. Unlike other pancreatic auto-antigens,
ZnT8 is located on the cell surface; it can be transported to the cell
membrane after insulin secretion, thereby exposing the transmembrane
domain (TMD) and being recognized by serum ZnT8A [103]. This
feature improves the possibility of ZnT8 being directly involved in
antibody-mediated cellular dysfunction and cytotoxicity [104]. After
insulin, ZnT8 is the second antigen that is specifically expressed in the
pancreatic beta-cells and is more restricted in its tissue distribution than
other auto-antigens; there are no other cells destroyed in T1DM [104,
105]. Furthermore, the expression of ZnT8A may not occur until there is
sufficient beta cell damage to make this auto-antigen perceptible to the
immune system [106]. The unique position and tissue specificity of
ZnT8 in pancreatic beta cells makes it an attractive candidate as a
therapeutic target.
1.5. ZnT8 and T1DM
The pathogenesis of T1DM can be divided into three stages: 1) dia­
betogenic AAbs exist, blood glucose is normal and no diabetes-related
symptoms; 2) diabetogenic AAbs continually exist, beginning to
induce impaired glucose tolerance (IGT) but still no symptoms; 3) The
persistence of diabetogenic AAbs leads to T1DM and the occurrence of
related symptoms of diabetes [25,26]. The appearance of AAbs is asso­
ciated with the decline in the number and dysfunction of beta cells [3].
Therefore, autoimmunity persistence in the first stage is owing to the
destruction of beta cells; when the number of beta cells is reduced to a
certain extent, the inability to compensate by the remaining beta cells
leads to hyperglycemia, and then to the second stage. In the asymp­
tomatic first stage, namely pre-diabetes, the emergence of AAbs is the
earliest sign of the establishment of autoimmunity and destruction of
beta cells; about 96 % of patients show at least one AAb positivity at the
onset of T1DM, including GADA, IAA, IA-2A, or the new ZnT8A [7].
ZnT8 is now a confirmed autoimmune target for most patients with
T1DM, and exists in the serum of pre-diabetic and diabetic patients
[107–109]. ZnT8 is a dimer-membrane protein consisted of 369 amino
acids in a form and 320 amino acids in B form [110]. Each monomer
composed of 6 transmembrane domains and a histidine loop, about 50 %
of ZnT8 is located in the endoplasmic reticulum [111]. ZnT8 mainly
maintains the balance of zinc ions in pancreatic beta cells by pumping
zinc ions into intracellular vesicles or extracellular space [97]. Re­
searches have shown, a principal epitope targeted by ZnT8A is affected
by a single amino acid at position 325; This amino acid is encoded by the
SLC30A8 gene as three different variants, ZnT8RA arginine 325 Zinc
transporter 8 autoantibody, ZnT8WA tryptophan 325 Zinc transporter 8
autoantibody and ZnT8QA glutamine Zinc transporter 8 autoantibody
X. Zhang et al.
[112,113]. The COOH-terminal domain of the ZnT8 is the early patho­
genesis of T1DM; therefore, screening these two main ZnT8 variants can
help early prediction of T1DM risk [96]. ZnT8A has been positively
detected in about 60–80 % of newly diagnosed patients with T1DM, of
which 26 % detected no other diabetogenic AAbs, making it a new in­
dependent biological marker for the diagnosis of T1DM [114,115].
Studies have also found that patients who are ZnT8A-positive at an early
stage, are often more likely to develop T1DM than ZnT8A-negative pa­
tients, but there is no correlation between antibody titers and disease
progression [116]. The combined detection of ZnT8A with GADA,
IA-2A, and IAA can improve the sensitivity of detection in T1DM to 98 %
[117]. Andersson et al. reported that the negative rate of AAbs was
reduced from 10 % to 7 % in combination with the detection of ZnT8A
on the basis of the three antigens GADA, IA-2A, and IAA, which
improved the diagnostic sensitivity of T1DM [118]. As mentioned
above, ZnT8-specific responses are also particularly broad: compared
with CD8+ T-cell responses induced by other auto-antigens, ZnT8 epi­
topes elicit responses in more than 64 % of patients [101]. Several
studies have shown that autoimmunity may commence many years
before manifestation of T1DM [28,81,119]. Furthermore, an autopsy
result showed that the number of beta cells in pancreatic islets of pa­
tients with T1DM was about 10 % of that in individuals without T1DM,
which once again demonstrated that the result of autoimmunity is the
destruction of beta cells [120]. T1DM in general has a long prodromal
stage in which only AAbs reactive with diabetogenic antigen can be
detected [81]. Sofla etal followed the fate of beta-cell-specific CD8+ T
cells in non-obese diabetic mice throughout the course of T1DM, showed
that antigen-specific CD8+ T cells can be found in the pancreatic
draining lymph node and pancreas as early as 5 weeks of age. It indi­
cated that autoimmune destruction can be found prior to clinical course,
and there is an asymptomatic period before the typical presentation with
clinical type 1 diabetes, which is associated with beta-cell functional loss
[121]. Thus, the early assessment of ZnT8A can allow identification
from rapid progression to clinical onset of the disease, and subsequent
tolerogenic therapies may be most effective in the earliest disease phase.
The incidence of ZnT8A-positive is correlated with the age of diag­
nosis of T1DM in children [122]. A study showed that among newly
diagnosed T1DM children < 15 y, ZnT8A-positive individuals were
generally older [123]. A longitudinal study of children with genetic risk
after birth also indicated that ZnT8A tends to appear later and is rarely
detected before 6 months [124]. Also in support, a French study
involving 516 children with T1DM showed that children aged 6–17 y
had a higher detection rate of ZnT8A-positive compared with children
< 5 y [125]. Other studies have also reported that the positive rate of
ZnT8A is higher in children aged 5–10 y [126] and that ZnT8A tends to
go undetected before 3 y [127]. The abnormal autoimmunity is mostly
initiated by the two antibodies GADA and IA-2Am with GADA appearing
steadily up to 10–15 y [128,129]. The greater the age at which auto­
immunity begins, the less likely it is to develop into T1DM [130], with
children with negative AAbs < 15 y having a lower risk of developing
T1DM, at around 0.4 % [22]. Therefore, early detection of ZnT8A
combined with GADA and IA-2A is sufficient to predict childhood
T1DM.
However, studies on adult patients with T1DM are few and incon­
sistent. One study reported a negative correlation between the age of
T1DM and the positive rate of ZnT8A, with it decreasing significantly in
elderly T1DM patients [131]; this is contrary to the results of T1DM in
children. In a Chinese population study, it was also found that the pos­
itive rate of ZnT8A decreased with increasing age of diagnosis [117]. In
another Pittsburgh Epidemiology of Diabetes Complications (EDC)
study, there was a positive correlation between the age of patients with
T1DM and positive rate of ZnT8A. The older the patient, the greater the
chance of being ZnT8A-positive; this may be because of a less aggressive
and thus more persistent immune process in those with older age at
onset [132]. It has also been noted that the autoimmune process
observed in T1DM < 35 is more aggressive compared to diabetes
6
Journal of Trace Elements in Medicine and Biology 73 (2022) 127040
recognized after 35 y [106]. In summary, ZnT8 can be used as an
immunobiological target for T1DM in children; early detection is
essential in individuals with a genetic risk for T1DM but without islet
AAbs to achieve the primary prevention [133,134]. Furthermore, in
individuals with multiple islet AAbs but without overt hyperglycemia,
ZnT8 can be used to delay the disease and achieve secondary prevention
[128,129,135]. Detection of pancreatic islet AAbs makes T1DM a pre­
dictable disease. However, the relationship between ZnT8A and adult
patients in T1DM requires further study.
LADA type diabetes is a form of autoimmune-mediated diabetes in
adults where the two vital characteristics are: the rate of beta cell failure
is slower than that of T1DM, and the early symptoms are similar to
T2DM where there is slow progress in insulin therapy and it does not
require insulin replacement for at least 6 months after diagnosis [136].
The former feature indicates that LADA has a wider time window than
T1DM, and timely screening intervention may slow down beta cell
failure. The latter feature suggests that newly diagnosed patients with
LADA are initially insulin-independent and can only be identified by
detecting diabetogenic AAbs. Early research results showed that detec­
tion of IAA, GADA, and IA-2A is currently the only means of dis­
tinguishing LADA from phenotypic T2DM [137,138], and at least one
auto-antigen can be tested in the serum of LADA patients [13]. Huang
et al., in a Chinese study of 3062 individuals, showed that the new ZnT8
auto-antigen was also an independent immune marker for differenti­
ating LADA from phenotypicT2DM; the detection of ZnT8A along with
GADA and IA-2A was shown to improve the diagnostic sensitivity of
Chinese LADA [139,140]. In summary, LADA and phenotypic T2DM can
be distinguished by detection of diabetogenic AAbs, and early identifi­
cation and intervention may better preserve the function of beta cells
and delay disease progression [141–143]. Diabetes-related AAbs
detected from LADA and classic T1DM are similar, so this cannot be used
as a diagnostic criteria.
1.6. ZnT8 and the treatment of T1DM
The mechanism of ZnT8 inducing T1DM autoimmunity may be
related to the structure of ZnT8. ZnT8 is the most abundantly expressed
zinc transporter and is considered to be the regulator of zinc concen­
tration in beta cells, which is critical for insulin biosynthesis and storage
[144]. Studies have shown that proper intracellular zinc concentration
can ensure the function of islet cells, but expressive ZnT8 and zinc
concentrations aggravate islet cell death when exposed to different
cellular stressors, such as hypoxia and cytokines [145]. ZnT8 is a dimer
membrane protein with a Y-shaped molecular architecture similar to
that of immunoglobulin [146]. It readily binds to (Fab)2 and the Fc re­
gion of the bound autoantibody is able to bind immune effector cells to
trigger antibody-dependent cytotoxicity against opsonized beta cells
[147]. Less than half of the protein can be immune surveilled, which
may explain why ZnT8 is a target for autoimmune attack in T1D.
Secondly, the ZnT8 protein is immunogenic and causes T1DM
autoimmune responses, which may be related to the etiology of T1DM.
T1DM is an autoimmune disease characterized by the autoimmune
destruction of pancreatic beta cells mediated by specific T cells, and
CD8+ T cells are the final mediators of islet destruction, CD8+ T-cell
responses against ZnT8 and anti-ZnT8 AAbs were found in T1DM pa­
tients [148]. Ehlers [149] found that ZnT8-specific CD8+ T cells were
detectable in the majority of T1DM patients (> 68 %), whereas the
frequency of ZnT8-specific CD8+ T cells was significantly lower in
healthy individuals and patients with T2DM (<8 % and 30 %, respec­
tively). Nayak et al. [95] also found that ZnT8-specific CD8+ T cells
generated after ZnT8 345–359 peptide stimulation could accelerate the
progression of diabetes in irradiated recipient mice (NOD or NOD.
Rag1-/-). Studies have also confirmed the presence of more znt8-specific
CD8+ T cells in the pancreas of patients with T1DM compared with
patients with type 2 diabetes or without diabetes, suggesting that
znt8-specific lymphocytes home to the pancreas [150]. And CD8+ T cells
X. Zhang et al.
isolated from peripheral blood of T1DM patients can secrete more IFN-γ
after stimulation with ZnT8 peptide [150]. In addition, ZnT8 has
become a potential target of humoral autoimmunity [151] and is also
related to the insulin secretory pathway. Glucose-stimulated insulin
secretion (GSIS) promotes the display of ZnT8 on the surface of beta
cells, which may also aggravate beta cell destruction [147]. GSIS is
mainly mediated through a series of events that lead to the ATP-sensitive
potassium channels are inhibited, the consequential decreased efflux of
K+ results in a depolarization of the beta cells plasma membrane, and
voltage-sensitive Ca2+-channels open, facilitating Ca2+ entry, which
triggers insulin secretion [152]. Huang et al. [103] showed that GSIS
increased the expression levels of ZnT8 autoantigen and its exposure on
the beta cell surface. After glucose-stimulated insulin secretion, ZnT8
moves to the cell surface along the insulin secretion pathway, resulting
in an increase in GSIS/ZnT8 exposure. In turn, coupling between the
GSIS and ZnT8 surface display enhanced beta cell damage. The above
results provide a basis for ZnT8 as a surface biomarker.
1.7. Future directions
Great progress has been made in our understanding of T1DM
immunotherapy, but there are still many problems that need to be
solved. First of all, therapies based on auto-antigens have been admin­
istered as native proteins; will the same or better therapeutic effect be
achieved if proteins modified as toleragens are used? Second, pancreatic
AAbs are often triggered years before the symptoms of severe hyper­
glycemia, but the relationship between humoral autoreactivity and islet
pathology remains unclear. Next, as mentioned above, early detection of
ZnT8A combined with GADA and IA-2A can predict T1DM in children at
an early stage, but whether the age of onset will affect the autoimmune
status, severity, and manifestations in adults requires further research to
explore the existence of sensitive markers for the diagnosis of adult
autoimmune diabetes. As an important AAb in T1DM, is ZnT8A also
associated with T2DM? Studies have reported that polymorphism of the
SLC30A8 gene may be a risk factor for T2DM [144,153,154]. Hunt et al.
also collected 800 serum samples from children aged 0–12 y before the
diagnosis of T2DM, and found that in about 3 % of patients a positive
AAb appeared before diagnosis, suggesting that the presence of AAbs as
risk factors may accelerate the development of T2DM [155]. Therefore,
more studies are needed to demonstrate a possible connection between
ZnT8 and T2DM. Moreover, it may not be sufficient to completely
control the self-destruction of beta cells by single-agent immunotherapy;
combination immunotherapies through multiple pathogenic pathways
may be needed to optimize immune intervention, such as by depleting
effector cell populations before induction of antigen-specific immune
tolerance or at the same time. Which drugs can result in lasting relief of
beta cell function? Finally, we should increase our understanding of the
molecular mechanism of T1DM and explore more biomarkers and
therapeutic targets.
2. Conclusions
It is necessary to intervene early in autoimmune progression, prior to
the occurrence of T1DM. Although antigen-specific immunotherapy is
still in its infancy, it may be a potentially powerful weapon in the future
owing to its advantages of safety and effectiveness. Many islet-specific T
cell antigens have been identified that contribute to diabetes develop­
ment. If a solution is developed that can selectively expand antigen-
specific Tregs while reducing the pathogenic responses and reinstating
a homeostatic balance for long term tolerance induction, it will raise
hopes of the treatment for T1DM. Islet-specific expression as well as its
accessibility on the cell surface may provide new ideas for ZnT8 as an
immunotherapy target to improve the dysfunction of beta cells in T1DM.
7
Journal of Trace Elements in Medicine and Biology 73 (2022) 127040
Funding
This work was supported by the National Natural Science Foundation
of China [grant number 82070796].
CRediT authorship contribution statement
QW helped perform the analysis with constructive discussions and
revised the manuscript. XZ contributed to the conception of the review
and wrote the manuscript. YD and LY performed the analysis with
constructive discussions, carried out the study and collected important
background information and references. DL and JX contributed signifi­
cantly to analysis and manuscript preparation. All authors have read and
approved the final content of the manuscript.
Conflict of Interest
The authors declare that the research was conducted in the absence
of any commercial or financial relationships that could be construed as a
potential conflict of interest.
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