Critical Review
Islet Inflammation in Human Type 1 Diabetes
Mellitus
1
Institute of Biomedical and Clinical Sciences, University of Exeter Medical
School, Exeter, UK
2
GG&C Pathology Department, Southern General Hospital, Glasgow, UK
Abstract
Type 1 diabetes mellitus (T1DM) is caused by the selective
deletion of pancreatic b-cells in response to an assault
mounted within the pancreas by infiltrating immune cells.
However, this apparently clear and focussed annunciation
conceals a stark reality in which the cellular and molecular
events leading to b-cell loss remain poorly understood in
humans. This reflects the difficulty of studying these proc-
esses in living individuals and the fact that, using pathological
specimens, islet inflammation has been analysed in fewer
than 200 recent-onset cases of T1DM worldwide, over the
past century. Nevertheless, insights have been gained and the
composition of the islet infiltrate is being disclosed. This is
shown to be primarily lymphocytic in nature, with populations
of both CD81 and CD41 T cells displaying an autoreactivity
Keywords: cell death; pancreatic islet cells; b-cell; islets of Langerhans;
Insulin; Insulitis
Introduction
Type 1 diabetes mellitus (T1DM) in humans is considered to
have an autoimmune aetiology in which the insulin-secreting
b-cells of the pancreatic islets are destroyed selectively by
influent immune cells responding to the aberrant presentation
of b-cell antigens (1–7). However, this seemingly straightfor-
ward summary conceals a considerable degree of ambiguity
that still surrounds almost every stage of the process. In this
C 2014 International Union of Biochemistry and Molecular Biology
V
Volume 66, Number 11, November 2014, Pages 723–734
Address correspondence to: Noel G. Morgan, Institute of Biomedical and
Clinical Sciences, University of Exeter Medical School, Barrack Road,
Exeter EX2 5DW, UK. Tel: +01392 408300.
E-mail: n.g.morgan@exeter.ac.uk
Received 27 October 2014; Accepted 17 November 2014
DOI 10.1002/iub.1330
Published online 7 November 2014 in Wiley Online Library
(wileyonlinelibrary.com)
IUBMB Life
Noel G. Morgan1*
Pia Leete1
Alan K. Foulis2
Sarah J. Richardson1
against specific islet antigenic peptides. The T cells are often
accompanied by influent CD201 B cells, although new data
imply that the proportions of these individual cell types vary
and that patients fall into at least two distinct categories hav-
ing either a hyper-immune (CD20Hi) or a pauci-immune
(CD20Lo) phenotype. The overall rate of b-cell decline appears
to correlate with these two phenotypes such that hyper-
immune patients lose b-cells more quickly and tend to
develop disease at an earlier age than those with the pauci-
immune profile. In this article, we review the evidence which
underpins our current understanding of the aetiology of T1DM
and highlight both the established features as well as areas
of on-going ambiguity and debate. V C 2014 IUBMB Life,
66(11):723–734, 2014
article, we will attempt to highlight these ambiguities and indi-
cate where active research efforts are still required.
There are various reasons why, almost 100 years after the
discovery of insulin, the molecular basis of human T1DM
remains shrouded in mystery but, foremost among these, is
the fact that the pancreas is inaccessible to non-invasive inves-
tigation in humans. Currently there are no reliable ways to
image the organ such that the cellular and molecular events
associated with islet inflammation can be studied in living indi-
viduals, although efforts are continuing towards the achieve-
ment of this goal (8). Thus, the limited information which is
available has been derived from samples recovered post-
mortem from patients with T1DM (9–11), from glands recov-
ered at the time of organ donation (6,12–14) or from surgical
biopsy samples recovered from living patients (15,16). Unfortu-
nately, even among these valuable resources, there are impor-
tant issues which militate against a full understanding of the
disease pathology. First, access to the whole pancreas is only
possible at the end of a patient’s life and this is usually many
years after disease diagnosis. Second, the bulk of the pancreas
723

Insulitis patterns do not differ between autopsy or organ donor cases. Photomicrographs of pancreas sections from patients
FIG 1 with type 1 diabetes mellitus collected either at the time of autopsy (A) or upon organ donation (B). Sections were immuno-
stained for insulin (brown) and glucagon (red), and each image shows an individual islet. Evidence of immune cell infiltration
(seen as small nucleated cells; black arrows) is observed around each of the islets. [Color figure can be viewed in the online
issue, which is available at wileyonlinelibrary.com.]
plays a role in digestion, and during the post-mortem phase,
the organ is often subject to autolysis such that the state of
preservation of the tissue may be less than optimal when it is
recovered. This may be offset by the rapid processing of an
organ at the time of donation in heart-beating donors; how-
ever, such glands are rarely available from patients with
recent-onset T1DM. Figure 1 illustrates the process of insulitis
in individual islets from pancreases collected either at autopsy
or organ donation. The biopsy approach could, in principle,
provide a route by which many of these problems are over-
come; however, in practice, this also presents difficulties.
Some of these are technical given that the surgical or post-
operative procedures can lead to unexpected complications, as
has happened recently in the DiViD study from Norway (16).
In addition, this approach relies on the assumption that the
disease process will inevitably be captured in the biopsied
region; however, this does not necessarily hold true as there is
strong evidence that T1DM progresses in a focal manner. As
such, both temporal and spatial differences exist in the distri-
bution of inflamed islets and, collectively, these limit the
amount of useful information that may be available from
within the biopsied area.
Based on this finding, it will already be clear that a
detailed study of the cellular aetiology of T1DM in humans is
not a trivial matter and that the quest to provide a more com-
plete picture continues. In particular, consortia around the
world are collaborating to develop new collections of samples
which build on the extant historical collections such that the
process can be studied in molecular detail (6,12). Nevertheless,
we should emphasise that conclusions about the underlying
pathology of human T1DM are drawn from the study of fewer
than 200 cases worldwide. These cases and their historical
context have been summarised very comprehensively in recent
reviews by In’t Veld (17,18) and will not be rehearsed here,
although we will draw on the available information to present
a contemporary view of the process of islet inflammation in
human T1DM. In stating this objective, we should also empha-
724
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sise that it is not our purpose to discuss in detail the data
which have arisen from the study of the various animal models
of T1DM. This is not because we consider that these cannot
provide important insights (they clearly do!) but, rather,
because the available evidence implies that the human disease
is not recapitulated fully in any of the available animal models.
b-Cell Loss in Type 1 Diabetes Mellitus
Early studies of the pancreatic gland recovered from patients
with T1DM identified the presence of small nucleated cells
around the periphery of (and sometimes within) the islets of
Langerhans (19–21). These were correctly considered to be
immune cells, and it is now clear that such cells congregate in
the vicinity of the islets during disease progression. However,
islets do not become inflamed in synchrony; rather, the pro-
cess follows a focal course. Thus, in patients studied soon after
diagnosis, regions of the pancreas can be identified in which
normal, apparently healthy, islets are found, whereas other
regions exist (often located in close proximity) containing islets
in which b-cell destruction is complete (4,9,11,22). Such islets
are sometimes referred to as ‘pseudoatrophic’ (18,23) to
denote the fact that although b-cells are absent, they retain a
normal complement of the other islet endocrine cells. This
raises an important point in that immunological approaches
have been (and remain) the mainstay of pathological investiga-
tion and all conclusions about the loss of b-cells depend on an
analysis of the presence (or absence) of immunoreactive insu-
lin as a means to identify these cells. In studies of T1DM pan-
creas, the loss of insulin immunoreactivity has almost univer-
sally been interpreted as synonymous with b-cell ablation;
however, it should be understood that this has rarely been
proven conclusively. This is mainly because there are no other
known immunological markers that define human b-cells
uniquely. Thus, it is theoretically possible that degranulation
of b-cells could be misinterpreted as cell ablation during
Process of Islet Inflammation in Human T1DM
 Islet architecture is altered during b-cell destruction. Individual islets from a single nPOD case (6228) were immunostained for
FIG 2 insulin (brown) or glucagon (red). Islets in the initial phase of peri-insulitis tend to have a normal architecture with both a- and
b-cells present (A). Islets in which most of the b-cells have been destroyed (B) are usually smaller and adopt a more condensed
appearance. Occasionally, insulin-deficient islets (IDIs) adopt a more diffuse appearance (C), with loss of their typical cellular
organisation. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

histopathological analysis. One extension of this idea (which is
increasingly being mooted as a possible cause of b-cell decline
in T2D; refs. 24–26) is that b-cells might undergo a process of
de-differentiation. In this view, the cells would not necessarily
be lost in significant numbers but, rather, they would lose
insulin immunopositivity and acquire a more ‘mesenchymal’
phenotype associated with the expression of stem cell markers
such as Oct4, Ngn3 and vimentin. Although this finding has
not yet been evaluated in a fully systematic way in the pan-
creases of patients with T1DM (or, arguably, in human sub-
jects with T2D), our view from the examination of many pan-
creatic glands recovered from a wide variety of patients with
T1DM is that the vast majority of islet cells stain positively for
one of the relevant hormones (glucagon, somatostatin, pancre-
atic polypeptide or ghrelin). Hence, we do not find islets con-
taining large numbers of hormone-depleted cells. This implies
that neither complete b-cell degranulation nor b-cell de-differ-
entiation is a frequent occurrence in human T1DM, and it
seems reasonable to conclude that immune-mediated b-cell
ablation remains the primary mechanism of loss. This does
not mean, of course, that processes such as de-differentiation
or selective degranulation might not occur in some b-cells;
however, we are not persuaded that they could account for the
bulk of the apparent cell loss in T1DM.
A further feature which suggests that the major mecha-
nism of insulin depletion in T1DM occurs by b-cell loss is that
the architecture is progressively altered in those islets display-
ing reduced (or absent) b-cells. Islets frequently adopt a more
condensed appearance upon light microscopic examination as
they lose b-cells and they are often smaller and less ovoid in
appearance (as judged on sections of pancreas; Fig. 2). This
feature is characteristic of the pseudoatrophic islets mentioned
above, and these are taken to represent islets in which b-cell
loss is complete and in which the remaining cells have adopted
a reorganised orientation to occupy the newly available space.
Occasionally, insulin-deficient islets may also adopt a more dif-
fuse appearance suggesting that the demise of the b-cells can
also lead to a more generalised loss of cellular organisation
(Fig. 2C).
These considerations raise a further important matter
which also remains largely unresolved. This relates to the
mechanism by which b-cell loss occurs in T1DM and what sub-
sequently happens to the associated debris. It has been widely
supposed that the primary mechanism of b-cell death is by
apoptosis (27–29) and that residual b-cell components are
cleared rapidly by macrophages which normally patrol and
monitor the islet milieu (30). Such hypotheses are based
mainly on evidence arising from in vitro studies which show
that pro-inflammatory cytokines (e.g., interleukin-1b, inter-
feron-c and tumour necrosis factor-a) can promote b-cell apo-
ptosis (31–35). Thus, it is concluded that if similar cytokines
bathe the endocrine cells in inflamed islets, then enhanced b-
cell apoptosis is likely to ensue. Increased apoptosis might also
derive from Fas-mediated b-cell toxicity (35–38) or from
enhanced endoplasmic reticulum stress (39) as has been sug-
gested in some studies. The concept of an apoptotic mode of b-
cell death has received limited support in that although apo-
ptotic b-cells have occasionally been identified in inflamed
islets (40), they are not usually present in large numbers, even
in those islets which appear to be entering the most active
phase of destruction. This may simply reflect the efficiency of
the phagocytic clearance mechanisms and the fact that in any

Morgan et al. 725

 IUBMB LIFE

Representative staining of immune cell subtypes in an islet of a patient with type 1 diabetes mellitus. Serial sections of the
FIG 3 same islet from nPOD case 6052 were stained to reveal the presence of the different immune cell subtypes and endocrine cells
using either immunofluorescence (A–C) or immunohistochemistry (D). Panel A: Insulin1 b-cells (blue), CD451 lymphocytes
(green); panel B: Glucagon1 a-cells (red), CD201 B cells (green); and panel C: CD81 cytotoxic T cells (red), CD681 macrophages
(green). Nuclei were visualised with either ToPro3 (A) or DAPI (B and C). Panel D: CD41 T-helper cells were labelled immuno-
histochemically (brown) and their nuclei counterstained with haematoxylin (blue).

pancreas sample, only a single point in time is being surveyed.
Alternatively, it might mean that non-apoptotic mechanisms
also operate. This could be the case if, for example, the
release of granzymes and/or perforin from influent immune
cells contributes to b-cell loss (41–44). However extensive islet
cell necrosis might be expected to provoke a more intense
immune response (as cellular contents will more readily enter
the extracellular space) and lead to widespread, less targeted
damage. This is rarely seen upon histological examination of
T1DM pancreas and it remains unclear exactly how b-cells die
in human T1DM.
Islet Autoantibodies and Type 1
Diabetes Mellitus
The definition of T1DM as an autoimmune disease has been
driven by the observation that many (although not all) patients
develop circulating antibodies to particular b-cell proteins
(5,45–49). These include insulin itself (which is frequently the
earliest antigen to elicit an antibody response), certain secre-
tory granule proteins (such as a protein phosphatase, IA2, and
a specific zinc transporter, ZnT8) and the cytosolic enzyme
glutamate decarboxylase. Patients often develop antibodies to
multiple autoantigens, and there is an on-going debate as to
whether these antibodies are truly pathogenic or if they are
generated secondarily, as markers of an underlying disease
process. Most evidence favours the latter interpretation,
although it should also be emphasised that individuals can
develop autoantibodies but never progress to clinical T1DM.
Thus, there is no absolute relationship between the two. What
is clear is that the presence of multiple autoantibodies is
strongly predictive of diabetes development in susceptible indi-
viduals (50,51) and that non-diabetic subjects with three or
more circulating autoantibodies have a greatly increased risk
of progressing to T1DM.
The Inflammatory Infiltrate in Type 1
Diabetes Mellitus
Dogma (or, at least, accepted wisdom) states that the islets of
patients with T1DM are inflamed; however, in practice, this

726 Process of Islet Inflammation in Human T1DM

 Representative photomicrographs of islets from CD20Hi and CD20Lo cases among the nPOD collection. Serial sections of islets
FIG 4 from either an nPOD CD20Hi (A, C and E) or an nPOD CD20Lo (B, D and F) case were stained to identify the presence of CD201
B cells (green; A and B) and insulin1 b-cells (light blue; C and D). Merged images (E and F) also show the cell nuclei (stained
dark blue with DAPI). Increased numbers of CD201 B cells are observed around and within the islets of the CD20Hi cases when
compared with the CD20Lo cases. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

has been observed much less frequently than might be imag-
ined. Indeed, in one recent review, it was noted that human
insulitis is so rare that most pathologists will not witness a sin-
gle case during their entire careers as practitioners (18)!
Nevertheless, there is abundant evidence from the available
pathological collections that insulitis does occur in human
T1DM albeit in a relatively low (10%) proportion of islets.
This is partly because the inflammation dissipates once b-cells
are lost and that most pancreases of patients with T1DM con-
tain a large number of insulin-deficient islets. It may also
reflect the focal nature of the disease and the fact that by con-
trast with animals such as the non-obese diabetic mouse, the
total number of immune cells infiltrating any given islet is
often rather modest. Thus, when islets are observed in pan-
creas sections (i.e., in two dimensions), small numbers of
immune cells may be excluded from view because they lie
either above or below the plane of the section. As a result, an
islet might then be scored by an observer as ‘non-inflamed’,
whereas in reality, it contains a few immune cells elsewhere
within its volume. It is also evident that insulitis is seen most
frequently in patients with recent-onset T1DM and that this
frequency declines with disease duration.
Despite these difficulties, both the number and the pheno-
type of the influent immune cells have been studied in the
inflamed islets of humans with T1DM. Early experiments
showed that the infiltrate is predominantly lymphocytic in

Morgan et al. 727


nature (Fig. 3A; ref. 52) and that it contains CD81 T cells (Fig.
3C), which are considered to be among the primary cytotoxic
mediators (53). However, additional immune cells are also
present, including CD41 T cells (Fig. 3D), B cells (Fig. 3B) and
macrophages (Fig. 3C; refs. 4 and 53). In addition, the sur-
rounding pancreatic exocrine tissue has recently been
reported to be enriched in both lymphocytes (54) and neutro-
phils (55) in T1DM and the suggestion made that these might
contribute to disease progression. Neutrophils do not appear
to be present within (or near to) inflamed islets in large num-
bers, and hence, their role remains ambiguous. In some stud-
ies, NK cells have also been found in the islet infiltrate (13),
although this is not universally observed (53).
These considerations raise the issue of exactly how islet
inflammation (insulitis) is defined. This is important because,
with attempts to increase the number of available organs for
study, it has been discovered that immune cell infiltration can
occur in the pancreas in association with periods spent by sub-
jects in intensive care units, prior to organ recovery (56). This
occurs independently of the presence or absence of T1DM and
could lead to misdiagnosis of insulitis in pathological specimens.
Fortunately, a comprehensive consensus definition of insulitis
has recently been proposed (23). This deals with various aspects
of the pathological appearance of inflamed islets and concludes
that insulitis can be confirmed in a patient with T1DM if three or
more islets are shown to contain 15 or more lymphocytes in the
peri-islet region and/or within the islet structure. This definition
does not, of course, define insulitis at the level of any given indi-
vidual islet, and this remains a discretionary consideration on
the part of individual investigators. In our hands, we have rarely
seen individual islets which contain more than five immune cells
(lymphocytes or macrophages) on a single cross-section of the
islet in non-diabetic subjects, and therefore, we have used this
as an operational threshold (53).
An analysis of the composition of the immune cell infiltrate
in islets at apparently different stages of b-cell destruction (as
judged by the extent of residual insulin immunopositivity) has
led to the conclusion that CD81 T cells are the predominant
cell type throughout the period of inflammation (4,53,57). This
is consistent with the notion that these cells ultimately mediate
b-cell death. Interestingly, however, the B-cell (CD201) profile
also changes during disease progression and, in an initial
study, was found to align closely with the migration of CD81 T
cells (53). By contrast, the numbers of CD41 T cells and mac-
rophages (defined with anti-CD68) were much less variable
during the period of b-cell decline. This suggests the existence
of a dynamic interplay between some (at least) of the immune
cell subsets during the progression of T1DM and this remains
to be understood. Moreover, very recent data imply that these
initial observations might represent an over-simplification and
that the profile of insulitis is more variable (57). This variabili-
ty is not simply stochastic but, rather, two closely regulated
patterns of insulitis can be defined. The first of these mirrors
the pattern reported by Willcox et al. (53) based on the analy-
sis of insulitis in a cohort of 29 patients with recent-onset
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The percentage of islets which retained insulin
FIG 5 immunopositivity at the time of death in patients
with recent-onset type 1 diabetes mellitus was higher
in the pauci-immune (CD20Lo) cases than in those
with the hyper-immune (CD20 Hi) profile. The per-
centage of residual insulin-containing islets (ICI) was
calculated in pancreas sections from among a cohort
of patients with recent-onset type 1 diabetes mellitus
collected within the United Kingdom. These had
been classified as either CD20Lo (green) or CD20Hi
(red) on the basis of their insulitis profiles. The
pauci-immune (CD20 Lo) cases retained more ICIs
than those with a hyper-immune (CD20Hi) pheno-
type. [Color figure can be viewed in the online issue,
which is available at wileyonlinelibrary.com.]
T1DM, whereas the second is quite different. The main differ-
ence lies in the number of influent CD201 cells which, as
noted above, can be extensive and mapped in parallel with the
influx of CD81 cells or, as revealed in the more detailed recent
study, can be minimal (or absent). As a consequence, the two
distinct profiles have been termed ‘CD20Hi’ or ‘CD20Lo’ to
denote the differential involvement of these cells (Fig. 4). In
fact, even this may be something of a misnomer as the abso-
lute numbers of all immune cell subtypes are reduced among
patients displaying the CD20Lo profile, and in recognition of
this finding, an alternative designation of ‘hyper’- or ‘pauci’-
immune, respectively, has also been suggested.
In both forms of insulitis (CD20Hi and CD20Lo), it is clear
that CD81 T cells predominate and it is likely, then, that these
will mediate the demise of the b-cells in both situations.
Importantly, the two profiles also appear to reflect patient dif-
ferences rather than being islet-specific (or lobular) patterns.
This has not been evaluated completely as it would require the
analysis of all inflamed islets across an entire pancreas and
this would represent a gargantuan task. Nevertheless, based
on the more limited analysis of islets from different regions of
a given pancreas, it appears that patients with T1DM fall into
one of the two categories. The factor that then differentiates
the two is the apparent rate at which the b-cells are killed.
This cannot, of course, be deduced with complete certainty but
the pathological evidence can be reconstructed to make a
strongly supportive case. This is based on several notable fea-
tures. First, the number of residual insulin-containing islets in
patients displaying the CD20Lo profile is greater than in those
who are CD20Hi (Fig. 5). This implies that b-cell killing is less
Process of Islet Inflammation in Human T1DM
efficient in patients with the CD20Lo profile than in those who
are defined as CD20Hi. In support of this, the residual insulin-
containing islets also tend to contain greater numbers of b-
cells in CD20Lo cases. Second (and, perhaps, most strikingly),
when a cohort of patients was studied who were diagnosed
with T1DM within a fixed range of ages (between 0 and 20
years), the group which displayed the CD20Hi profile was, on
average, diagnosed 6 years younger than those designated
CD20Lo. Taken together, these results imply that two differing
profiles of insulitis exist in patients with T1DM and that these
are associated with either a more (CD20Hi) or a less (CD20Lo)
aggressive autoimmune attack on the b-cells.
The factors that determine which profile occurs in any
given patient are entirely obscure (although genetic influences
are an obvious, but untested, possibility); however, these new
data offer an important perspective on the design of clinical
trials intended to slow or halt the progression of autoimmun-
ity. For example, it was previously proposed that the adminis-
tration of an anti-B-cell therapy might be effective in patients
with T1DM and trials with rituximab (a monoclonal anti-CD20
antibody; refs. 58 and 59) have been undertaken. These trials
involved a short-term administration of rituximab followed by
a lengthy period of follow-up, and they were, at best, only par-
tially successful. At first sight, this is disappointing, but the
newly available data on immune cell infiltration suggest that it
might be premature to close the matter without further consid-
eration. In particular, it is noteworthy that the mean age of
disease diagnosis among the patients recruited to the rituxi-
mab trials was 19 years (58,59), and, as such, it seems plausi-
ble that many of them might have displayed the CD20Lo profile
of insulitis (although this prediction cannot be verified since it
is not possible to interrogate their pancreas). If so, then anti-
CD20 antibody is likely to have been minimally effective
(assuming that its efficacy reflects the targeting and deletion of
CD201 cells having access to inflamed islets rather than sim-
ply those within the circulation). By contrast, administration of
rituximab showed more (albeit still limited) protection in the
younger patients. As such patients might be expected to pref-
erentially display the CD20Hi profile of insulitis, this may be
indicative of an altered insulitic profile in response to the anti-
CD20 therapy, at least temporarily. Thus, is it conceivable that
renewed attempts to deplete CD201 cells in a selected sub-
group of (younger-onset) patients might yield more striking
results?
As antibodies are generated from activated B cells and
there is evidence that B cells may comprise a significant pro-
portion of the immune cell infiltrate in certain patients, it is
important to understand whether islet-recruited B cells are
actively secreting antibodies within the vicinity of islets. During
activation and differentiation into plasma cells, B cells usually
down-regulate the expression of CD20 and up-regulate CD138
(60). However, islet-infiltrating B cells retain CD20 and they
are not labelled by antisera directed against CD138, a marker
of terminally differentiated, antibody-secreting plasma cells
(60). Thus, this population appears to play a role that is inde-
Morgan et al.
pendent of antibody synthesis and secretion. One possibility is
that the B cells may be acting as local antigen-presenting cells
within the islet milieu and that they collaborate to promote the
b-cell-directed cytotoxic activity of CD81 T cells (61).
Recent studies have attempted to assess the specificity of
the infiltrating CD81 cells using tetramer staining in which the
target antibodies are loaded with potentially autoreactive pep-
tides derived from islet antigens (62). This has revealed that the
specificity of the total population of CD81 cells is relatively
wide in that specific sub-population and appears to be reactive
against a range of different antigenic peptides. However, it was
also revealed that the majority of such cells within the vicinity
of any given islet display a common autoreactive phenotype,
suggesting that they may be clonal in origin. Moreover, it
appears that the influent cells target defined b-cell antigens,
implying that they are truly autoreactive (62). Whether there
are also additional CD81 cells present which target non-b-cell
antigens (such as virus proteins) remains to be established.
One subtype of immune cells which has not received as
much attention is the population of CD41 T cells present
within insulitic infiltrates. This may be because their numbers
do not alter dramatically during the progress of b-cell destruc-
tion; however, it also remains unclear whether these cells dis-
play a Th1 or Th2 phenotype. In future studies, it will be
important to determine the Th phenotype of these cells and to
examine whether this differs according to the CD20Hi or
CD20Lo status. In an important recent work, CD41 T cells
were grown out from the islets of a patient with T1DM and
were shown to be reactive against epitopes present within
proinsulin in an HLA-restricted manner (63). Thus, these cells
were shown to be truly autoreactive, and it was suggested that
they could be involved in b-cell destruction (63). Few, if any, of
the infiltrating CD41 cells can be classified as Tregs as they
do not stain positively for the presence of FoxP3 (53), a marker
which defines this class.
Recruitment of Immune Cells to
Inflamed Islets
The fact that the number of infiltrating immune cells sur-
rounding (and/or within) inflamed islets is often modest pro-
vokes questions relating to the nature of the attractant that
draws these cells to the site and their origin. It is frequently
assumed that the immune cells migrate to the islets mainly
from influent blood vessels and that they extravasate in the
immediate vicinity of the islets (possibly from efferent capilla-
ries carrying blood to collecting venules leaving the islet
milieu); however, this has not been formally proven. There is
little evidence that the cells reach the islet by egress from ves-
sels which penetrate to the core of the structure as the mor-
phology of the insulitis places the influent cells mainly in the
surrounding pancreatic parenchyma adjacent to islets (often
close to one pole of an islet) rather than within the islet per se.
It is commonly argued that immune cells are drawn to
islets by a chemokine gradient elaborated from the endocrine
729
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Patients with type 1 diabetes mellitus with hyper-immune (CD20Hi) and pauci-immune (CD20Lo) phenotypes may follow differ-
FIG 6 ential paths of disease progression and have different rates of insulin secretion at disease onset. A hypothesis to describe the
progression of b-cell loss in patients with either a hyper-immune (CD20Hi) or pauci-immune (CD20Lo) phenotype of insulitis
(adapted from an original schema proposed by Eisenbarth; ref. 7). The functional b-cell mass of individuals with a genetic sus-
ceptibility to type 1 diabetes mellitus is shown on the y-axis and their relative age on the x-axis. Following an initial trigger, the
decline in b-cell mass occurs more rapidly in those individuals with the hyper-immune phenotype (red line), and overt diabetes
is diagnosed at an earlier age. At this point (A), few residual b-cells are retained within the pancreas, and diabetes results from
the reduced b-cell mass. By contrast, b-cell mass declines more slowly in individuals with the pauci-immune phenotype (yellow
line), and disease onset occurs at an older age. In these individuals, the proportion of islets still containing residual insulin at
the time of diagnosis is increased (compare B with A), suggesting that both b-cell loss and an insulin secretory defect (or a
greater requirement for endogenous insulin) could contribute to disease onset. [Color figure can be viewed in the online issue,
which is available at wileyonlinelibrary.com.]

cells themselves, and there is evidence that these cells can
synthesise and release a range of relevant chemokines such as
CXCL10, MCP-1 and MIP-1a (64–68). If this is the case, then it
seems reasonable to deduce that this may represent one
means by which the b-cells become visible to the immune cells
during an early phase in the development of T1DM. A second
mechanism occurs via the up-regulation of MHC class 1 mole-
cules on the b-cell surface which, although not seen in all
islets, nevertheless represents a striking histological feature in
patients with T1DM (9,69,70). Thus, it seems possible that the
primary ‘defect’ in T1DM may lie at the level of the b-cells
themselves rather than in the immune system, as a dysfunc-
tional immune system which aberrantly targets the b-cells
should be largely independent of chemo-attractants and MHC-
1 hyper-expression.
One obvious possibility that continues to attract attention
as a means to explain why b-cells might become the target for
autoimmunity is that they are mounting an active response to
an environmental insult. This would then represent the pri-
mary ‘triggering’ event which initiates disease progression.
Considerable effort is currently being invested to establish
whether this trigger could be a viral (particularly an enterovi-
ral) infection because such a mechanism would provide an
obvious route by which islet autoimmunity might be initiated
(71). Evidence of enteroviral infection has been reported in
human b-cells from patients with T1DM (13,71–73), and this
can occur in islets which display no signs of inflammation (74).
Thus, it is possible that insulitis follows from the establishment
of an enteroviral infection, and this idea represents a working
hypothesis which is still being pursued by many workers glob-
ally (71). Irrespective of the precise nature of the insult, it
seems probable that the extent to which b-cells become visible
to the immune system is increased as an early event in the
development of T1DM and that insulitis develops as a conse-
quence of this.
As noted above, one of the consistent morphological obser-
vations arising from the study of human insulitis is that the
lesion tends to be diffuse and localised mainly at the periphery
of each islet. There are certainly occasions when immune cells
can be detected among the endocrine cells within the islet
structure; however, this is not seen in the majority of islets.
This arrangement implies that the influent immune cells may
be prevented from accessing the interior of the islet by the
presence of a physical barrier which limits access. In support
of this, it is known that islets are surrounded by a basement
membrane which serves to delineate the islet structure and

730 Process of Islet Inflammation in Human T1DM

which, following their isolation, allows islets to retain their
three-dimensional organisation in vitro (75–78). The composi-
tion of this structure has not been defined systematically but it
is almost certainly composed of a complex mixture of proteins
and proteoglycans, including collagens, hyaluronic acids and
heparan sulphates. Thus, to access the islet interior, it is prob-
able that this physical barrier must be breached, and an
examination of the morphology of inflamed islets implies that
this occurs at specific points around the perimeter of the islet
rather than as a more generalised dissolution of the basement
membrane components. One possibility is that, on arrival
within the peri-islet region, specific immune cells are induced
to secrete relevant proteases and/or other hydrolases that
selectively degrade the basement membrane to facilitate the
entry of the cytotoxic cells (79,80). If this is the case, then it
might offer a therapeutic opportunity as the administration of
inhibitors that attenuate the activity of such enzymes could be
effective in slowing the progression of insulitis. The nature of
the specific immune cell subset responsible for such hydrolase
secretion has not been established and immunolabelling of rel-
evant subsets in human pancreas sections has not, so far,
revealed any unique cell type whose arrival appears to pre-
sage the breaching of the basement membrane. Nevertheless,
this possibility warrants further consideration, especially as
there is increasing evidence that certain proteoglycans are lost
from islets during the progression of T1DM.
Insulin Secretion From Islets Isolated
From Patients with Type 1 Diabetes
Mellitus
It has been implicit in much of the above discussion that
T1DM is caused by net b-cell loss, and many researchers have
extrapolated from empirical observations to argue that the
clinical symptoms arise when the extent of this loss reaches
80% (i.e., when 20% of the original b-cell number remains).
However, this has not been formally proven. Moreover, the
newly developed dual phenotype hypothesis (summarised in
Fig. 6) in which the inflammatory process is envisaged to pro-
gress by one of two distinct mechanisms (the CD20Hi and
CD20Lo phenotypes described above) leads to a situation in
which T1DM occurs when differing proportions of residual
insulin-containing versus insulin-deficient islets remain. The
reasons for this are unknown; however, one possibility is that
T1DM is not solely attributable to the absolute loss of b-cells
(which undoubtedly occurs in all patients) but also as a con-
tributory factor to a decline in the insulin secretory capacity of
any residual b-cells (Fig. 6). This might be particularly true in
patients with a CD20Lo profile of islet inflammation where the
decline in b-cell numbers occurs more slowly and insulin-
containing islets are present in greater proportion (relative to
insulin-deficient islets) at diagnosis (Fig. 5).
There are rather few data available which allow firm con-
clusions to be drawn in relation to these arguments; however,
Morgan et al.
it has been possible to examine insulin secretory responses in
vitro from islets isolated from at least three patients with
T1DM. One of these was a young woman who died 13 years
after an initial diagnosis of diabetes (at the age of 19) (14).
Based on the age profiles of the patients with CD20Hi and Lo,
it might be speculated that this patient would feature among
the CD20Lo group at the time of diagnosis, but this conclusion
is necessarily speculative. Nevertheless, histological analysis
suggested that the majority of islets were insulin deficient at
the time of death with only a small proportion (15%)
retaining insulin immunopositivity. Such islets had reduced
insulin and elevated glucagon contents when compared
with controls, consistent with on-going, although still incom-
plete, b-cell destruction. The islets displayed a strong insulin
secretory response to glucose in vitro, suggesting that, at least
upon isolation, there was no major secretory defect and that
T1DM had resulted from insufficient b-cell mass in this
patient.
A second patient (18 years) who was studied immediately
after disease diagnosis (81) also retained insulin-positive islets
which could be harvested following isolation; however, in this
case, no insulin secretory response to glucose (or forskolin)
was evident, leading the authors to conclude that islet function
was ‘disproportionately impaired’ (81). The third patient was a
girl of 14 years who died 8 months after diagnosis of T1DM
and whose insulin-containing islets were harvested and then
studied after various periods of tissue culture (82). In perifu-
sion studies, these islets initially showed a modest but much
reduced secretory response to glucose when compared with
those isolated from a non-diabetic control subject. If the islets
were maintained in culture for a longer period (17 days), they
recovered secretory function and became indistinguishable
from controls in terms of the magnitude of the response to glu-
cose. Clearly, in total, these data are insufficient to allow firm
conclusions to be drawn, but it may be significant that an insu-
lin secretory defect was observed in the islets of two of three
patients with T1DM, suggesting that this could have contrib-
uted to the disease phenotype in these individuals.
Endocrine Cell Proliferation in Inflamed
Islets
A further area of interest and importance in T1DM is the
extent to which islet cells might attempt to regenerate in the
face of an autoimmune insult. It is widely understood that b-
cell replication occurs at only a very low rate beyond the
immediate post-natal period in humans (83). Indeed, in adults,
the rate of b-cell proliferation is vanishingly small, and there
seems little prospect, therefore, that islet cells could be
replaced at a sufficient rate to offset their decline during the
autoimmune attack leading to T1DM. Nevertheless, it appears
that islet cells are not inert in the face of this assault and that
they can respond by increasing their rate of mitosis. This was
shown in both an older patient (84) and in two studies of
731

pancreas samples harvested post-mortem from patients with
recent-onset T1DM, where it was revealed that islet cell prolif-
eration was increased by as much as 10-fold above that meas-
ured in controls (74,85). More importantly, the increase was
most evident in those islets which were inflamed, implying
that a component associated with the inflammatory process
was responsible for mediating the effect. Interestingly, the pro-
liferative response was not unique to b-cells but a similar
increase was also noted in a-cells, suggesting that the endo-
crine cells were responding to a general mitotic stimulus pres-
ent within the islet milieu. It is reasonable to propose that this
stimulus might have been elaborated from one or more of the
immune cell subtypes present in the inflammatory infiltrate;
however, this factor has not yet been formally identified or
characterised. In support of this conclusion, the rate of endo-
crine cell proliferation has also been examined in the inflamed
islets of two patients who died without a diagnosis of T1DM
but who were immunopositive for multiple islet autoantibodies
(86), suggesting that they might have been in a ‘pre-diabetic’
state. The pancreases of each of these individuals contained
islets which displayed enhanced rates of endocrine cell repli-
cation and, again, this correlated with the presence of inflam-
mation (86).
Clearly, it is of importance to identify the factors responsi-
ble for mediating islet cell proliferation during inflammation.
Once identified, this might offer a novel therapeutic option to
increase endogenous b-cell mass in patients with T1DM if the
relevant factors could be administered in combination with
approaches that limit the autoimmune attack.
Conclusion
Despite the dearth of human samples in which the process of
islet inflammation can be studied, important advances have
been made which have improved our understanding of the
underlying aetiology of human T1DM. These provide clear
cause for continued optimism that the illness will yield its
secrets and that new and improved therapeutic options will
emerge. Arguably, these are most likely to lead to a future in
which T1DM is preventable in susceptible individuals prior to
disease onset rather than curable in those who already have
the disease. Either way, it is important that further studies are
undertaken to address the remaining areas of uncertainty.
As a final statement we also note that in most analyses, it
has been assumed that, once initiated, T1DM progresses in a
broadly uniform manner until b-cell destruction reaches a crit-
ical point at which the clinical diagnosis is made. However, we
have emphasised that the pathology reveals an asynchronous
progression at the islet level. This then raises the alternative
possibility that islet inflammation might wax and wane over
time in a manner which is associated with alternating periods
of relapse and remission as the illness proceeds. Such a mech-
anism would be consistent with the situation in various other
autoimmune conditions. At present, this idea remains only an
interesting hypothesis, but if it proves to have validity, then
732
IUBMB LIFE
further windows of opportunity might be identifiable during
the different phases when therapeutic intervention might be at
its most effective.
Acknowledgements
This work received financial support from the European
Union’s Seventh Framework Programme PEVNET (FP7/2007-
2013) under grant agreement number 261441. Additional sup-
port was received from a Diabetes Research Wellness Founda-
tion Non-Clinical Research Fellowship and, since 2014, a JDRF
Career Development Award (5-CDA-2014-221-A-N) to S.J.R.
The research was also performed with the support of the Net-
work for Pancreatic Organ Donors with Diabetes (nPOD), a col-
laborative type 1 diabetes research project sponsored by the
Juvenile Diabetes Research Foundation International (JDRF)
and with a JDRF research grant awarded to the nPOD-V con-
sortium. Organ Procurement Organizations (OPO) partnering
with nPOD to provide research resources are listed at
www.jdrfnpod.org/our-partners.php.
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Process of Islet Inflammation in Human T1DM