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Insulitis and β Cell Mass in the Natural History of Type 1 Diabetes
Short title: Insulitis and Type 1 Diabetes
Martha Campbell Thompson1, Ann Fu1, John S. Kaddis2, Clive Wasserfall1, Desmond A.
Schatz3, Alberto Pugliese4, and Mark A. Atkinson1,3
From the 1Department of Pathology, Immunology, & Laboratory Medicine, College of Medicine,
University of Florida, Gainesville, FL; 2City of Hope, Los Angeles, CA, 3Department of
Pediatrics, University of Florida, Gainesville, FL; 4Diabetes Research Institute and Departments
of Medicine, Microbiology and Immunology, University of Miami Miller School of Medicine,
Miami, FL.
Correspondence to: Martha Campbell Thompson, Pathology, Immunology and Laboratory
Medicine, 1395 Center Drive, University of Florida, Gainesville, FL 32610 0275; Phone: (352)
273 6129; Fax: (352) 273 7753; Email: [email protected]
Word count: Abstract (229); Manuscript (4979); Tables (2); Online Supplementary Tables (5);
Figures (6); Online Supplementary Figures (6).
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Descriptions of insulitis in human islets throughout the natural history of type 1 diabetes
are limited. We determined insulitis frequency (proportion of islets displaying insulitis to
total islets, %), infiltrating leukocyte subtypes, and β cell and α cell mass in pancreata recovered from organ donors with type 1 diabetes (n=80), as well as donors without diabetes, both with (AAb+; n=18) and without (AAb ; n=61) islet autoantibodies. Insulitis was observed in 4/4 (100%) donors with type 1 diabetes duration ≤1 year and two AAb+ donors (2/18; 11%). Insulitis frequency showed a significant but limited inverse correlation with diabetes duration (r= 0.58; P=0.01) but not with age at onset. Residual β cells were observed in all type 1 diabetees donors with insulitis, while β cell area and mass were significantly higher in type 1 diabetes donors with insulitis compared to those without insulitis. Insulitis affected 33% of insulin+ islets compared to 2% of insulin islets in donors with type 1 diabetes. A significant correlation was observed between insulitis frequency and CD45+, CD3+, CD4+, CD8+, and CD20+ cell numbers within the insulitis (r= 0.53
0.73, p=0.004 0.04) but not CD68+ or CD11c+ cells. The presence of β cells as well as insulitis several years after diagnosis in children and young adults suggest that the chronicity of islet autoimmunity extends well into the post–diagnosis period. This information should aid considerations of therapeutic strategies seeking type 1 diabetes prevention and reversal.
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Abbreviations:
AAb /AAb+ Autoantibody negative/positive donors without diabetes
DKA Diabetic ketoacidosis
GADA Glutamic acid decarboxylase 65 autoantibodies
IA 2A Insulinoma associated protein 2 autoantibodies
IAA Insulin autoantibodies
IHC Immunohistochemistry
INS/Ins+/Ins Insulin/insulin positive/insulin negative
GCG Glucagon
nPOD Network for Pancreatic Organ donors with Diabetes
T1D Type 1 diabetes
ZnT8A Zinc transporter 8 autoantibodies
Key words: β cell, α cell, autoantibody, autoimmunity, pancreas, insulin, glucagon, DKA, HLA
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Type 1 diabetes is a chronic autoimmune disorder resulting from poorly understood
combinations of immunologic, genetic, and environmental factors that drive immune responses
against multiple β cell antigens, resulting in the irreversible loss of functional pancreatic β cells
(1). These destructive processes are thought to begin months to years before the clinical
symptoms of type 1 diabetes occur. Ongoing autoimmunity and β cell destruction are
asymptomatic during this pre diabetic period but can be identified serologically by the presence
of autoantibodies against one or more of several β cell autoantigens including glutamic acid
decarboxylase 65 (GADA), insulinoma associated protein 2 (IA 2A), insulin (IAA), and zinc
transporter 8 (ZnT8A) (2). The number, rather than titer, of these so called “islet autoantibodies”
can be used to determine risk for type 1 diabetes development (reviewed in (3)).
While the initial description for inflammation of pancreatic islet cells (i.e., insulitis) in
type 1 diabetes occurred over a century ago, a limited number of studies have characterized this
lesion in patients with the disease or during the preclinical phase (4). Certain exceptions exist,
yet a meta analysis of the literature would suggest that insulitis is present in young donors (<14
years) within 1 year of type 1 diabetes diagnosis as well as in donors with multiple islet
autoantibodies but having no diabetes (5 9). Difficulties in studying human islets/β cells in vivo can be ascribed to several factors including their relative scarcity within the pancreas (1 2%), anatomical inaccessibility, declining patient autopsy rates, and inherent risks associated with pancreatic biopsy (reviewed in (10)). This inability to perform pathological evaluations is unfortunate as it holds the potential to help explain, in part, multiple facets of disease heterogeneity including age variation at diagnosis and disease progression including rate of C peptide decline after onset or with experimental therapy (11; 12). In an attempt to overcome these limitations, organized efforts have been developed to obtain high quality pancreas
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biospecimens from organ donors to study mechanisms of β cell loss in type 1 diabetes (e.g.,
PanFinn, Belgian Diabetes Registry, JDRF Network for Pancreatic Organ donors with Diabetes
(nPOD)) (7; 13; 14).
In this current study, our major objective was to screen pancreata from nPOD donors
with and without type 1 diabetes, as well as donors with and without islet autoantibodies but no
diabetes, for islets with insulitis followed by leukocyte subtyping of infiltrating cells. Insulitis
frequency and leukocyte subtypes, in islets still expressing insulin as well as insulin negative
islets, were correlated with donor clinical attributes (age at onset or demise, diabetes duration,
autoantibody numbers, HLA, diabetic ketoacidosis (DKA)). The β cell and α cell area and mass
were also determined for each donor group and tested for correlations to insulitis frequency and
diabetes duration.
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RESEARCH DESIGN AND METHODS
Study Design
Pancreata were recovered from organ donors during a seven year period through the JDRF nPOD program according to procedures previously described (14 16). This report provides results from
donors with: 1) no history of diabetes or other pancreatic disease and negative for islet cell
autoantibodies (AAb , n=61); 2) no history of diabetes or other pancreatic disease but positive
for islet autoantibodies (AAb+, n=18); or 3) type 1 diabetes (n=80). The lower age limit in this
study was 4 years as the youngest donor with type 1 diabetes was 4.4 years old. Clinical and
demographic data are summarized (Suppl. Table 1) and include proportions of donors by
numbers of islet autoantibodies and those with DKA in relation to cause of death. Diabetes
durations were known for 79/80 donors with type 1 diabetes; one donor with unknown diabetes
duration was a 34 year old male Caucasian donor with clinical history of insulin dependent
diabetes, undetectable C peptide levels, and no insulin+ islets by histopathology (nPOD case
6032). Ethical permission was obtained from the Institutional Review Board at the University of
Florida, and informed consent was obtained from a legal representative of each donor.
Laboratory Assessments
Islet autoantibody assays, serum C peptide levels, and high resolution HLA genotyping were performed as previously described (14 16). Islet autoantibodies were measured by radioimmunoassay against all four major type 1 diabetes associated autoantigens (GADA, IA2
A, ZnT8 A, IAA) in all but one donor who was diagnosed with type 1 diabetes at 10.7 years old and had diabetes for 6 years (nPOD 6062). Of note, after institution of insulin therapy for type 1 diabetes, IAA could not be distinguished from antibodies induced by insulin injections, so IAA
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were excluded from autoantibody counts for the donors with type 1 diabetes subgroup in Table 1
and Suppl. Tables 1 and 3 (19).
Insulitis Screening and Insulitic Islet Subtyping for Insulitis Frequencies
Pancreata were processed to formalin fixed paraffin blocks for each pancreas region (head, body,
tail) as previously described (20). For each donor, serial sections (average 2 blocks/region) were
stained by hematoxylin and eosin and two double immunohistochemistry (IHC) stains (Ki67 and
insulin, CD3 and glucagon) (Suppl. Table 2) (21). When insulitic islets were found in a given
donor, additional blocks were screened (as detailed below). Stained sections were scanned at 20x
magnification using an Aperio CS scanner (Leica/Aperio, Vista, CA) and all images stored in an
online pathology database (eSLIDE, Leica/Aperio, Vista, CA).
Screening for insulitic islets was performed on CD3+ glucagon stained sections. An islet
was defined as ≥10 α cells. Insulitis was defined as an islet with ≥6 CD3+ cells immediately
adjacent to or within the islet with ≥3 islets per pancreas section according to recent criteria (4).
Islets with insulitis were marked in an image layer using ImageScope software (Leica/Aperio,
Vista, CA). The two IHC serial images were aligned using the synchronization tool, and insulin+
islets were also marked on image layer. All islets/section from donors with insulitis were
subsequently subtyped as follows: 1) insulin+ CD3 ; 2) insulin+ CD3+ 3) insulin CD3+; and 4)
insulin CD3 (see Table 1 for numbers of islets analyzed). Then, all islets were counted by
subtype. The process was reversed for AAb+ donors (i.e., islet subtypes were counted using the
Ki67 insulin image after markup for CD3+ insulitic islets and Ins islets using the CD3 glucagon
image). The number of pancreas sections subtyped for insulitis ranged from 2 16/donor (8.1±4.1
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sections/donor, n=162 sections) (Table 1). The lowest number of available sections was due to partial pancreas recovery (tail only, 6198).
Insulitis frequency (%) was calculated as total insulitic islets (sum of insulin+ CD3+ and insulin CD3+ islets) divided by total number of islets (sum of 4 subtypes). Frequency of insulin+ insulitic islets to total insulin+ islets was determined by the ratio of (insulin+ CD3+ islets)/(sum of insulin+ CD3 and insulin+ CD3+ islets) with similar calculations for frequency of insulin insulitic islets (insulin CD3+/(sum of insulin CD3+ and insulin CD3 )).
Insulitis Leukocyte Phenotyping
Paraffin sections from blocks having the maximum insulitis frequency for each donor were stained and positive leukocytes/insulitic islet (≥6 CD3+ cells) counted using multi immunofluorescence. Serial sections (4 m) were dewaxed and rehydrated with Tris buffer. Heat induced antigen retrieval was performed using Triology (Cell Marque, Rocklin, CA) at 95⁰ for
20 minutes followed by rinsing in water for 20 minutes. The staining series was designed to phenotype leukocytes [total leukocytes (CD45), T (CD3) and B (CD20) lymphocytes, T lymphocyte subsets (CD4, CD8), and monocytes (dendritic cells (CD11c) and macrophages
(CD68)) (21)] in conjunction with subtyping islets for insulin immunopositivity. The staining series was as follows: 1) CD45+glucagon+insulin; 2) CD20+CD3+glucagon; 3)
CD8+CD4+glucagon; and 4) CD11c+CD68+insulin. Chromogranin A staining was also used to delineate endocrine cells. Antigens are listed in order of primary antibody incubation and corresponding secondary antibody and conjugated fluorochrome (AF488 AF555 AF647) (Suppl.
Table 2). After blocking, sections were sequentially incubated with the primary antibody followed by the appropriate secondary antibody. For anti CD4, a Cy3 Tyramide signal
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amplification kit (PerkinElmer, Waltham, MA) was used according to the manufacturer’s
instructions. All sections were mounted with ProLong Gold Anti fade mounting media
containing DAPI (Life Technologies, Grand Island, NY). Positive controls included human
spleen, tonsil, and donor intra pancreatic lymph nodes, and negative controls included omission
of the primary antibody.
Numbers of leukocytes/insulitic islet were determined using multi channel image
acquisition software on a Zeiss Axiophot microscope (AxioVision, Carl Zeiss Inc., Thornwood,
NY). Fluorescent channels were viewed in combination with DAPI to count positive
leukocytes/islet.
β cell and α cell Area and Mass
Insulin and glucagon immunopositive areas were determined using the IHC sections to estimate
β cell and α cell areas, respectively, in relation to total tissue area using a single Aperio
colocalization algorithm (22). An average of 6 sections was used per donor (2 sections/head,
body, and tail regions). The β cell and α cell areas were expressed as a ratio (%) to the total
sectional area, including acinar and interstitial regions, to permit use of pancreas weights.
Average β cell and α cell area/pancreas was calculated from regional area averages. The β cell
or α cell mass (mg) was calculated by multiplication of the respective average area and pancreas
weight (g).
Statistical Analysis
Number and percent values were reported for all categorical factors, mean (± SD, n=number of
donors unless noted in figure legend) for normally distributed continuous variables, and median
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(minimum, maximum) for continuous variables with a skewed distribution. Donor characteristics
were analyzed using Satterthwaite corrected t test if continuous and parametric, the Kruskal
Wallis test if continuous and non parametric, Pearsons’s chi square if categorical values in all
cells exceeded 5, or by Fisher’s exact test if categorical values in one or more cells was <5. P
values were not calculated if values from 2 or more cells were equal to 0. Donor characteristics
were correlated to insulitis frequency and leukocyte subtypes/islet to other subtypes by Spearman
correlation and plotted with linear regression lines. Data were analyzed with exclusion of the
AAb+ donors with insulitis due to small sample size (n=2).
To obtain a subset of type 1 diabetes donors and compare clinical characteristics in the presence or absence of insulitis, selection of controls for all 18 type 1 diabetes insulitis cases was
attempted using a 1:1 ratio to identify the single best match. A matching method was used to
minimize bias lesion effect estimates from key covariates (i.e., age at diabetes onset, sex,
ethnicity, and BMI). A propensity score, given the selected covariates, was calculated for all type
1 diabetes donors (23). The logit of the propensity score was used to match donors without
insulitis to those with insulitis. A randomly sorted nearest available neighbor matching method
without replacement was used with an optimal caliper width equal to 0.20 SD of the logit value
(0.15), as has been shown to be optimal (24), along with a SAS macro (25; 26). All reported P values are two tailed and significant if <0.05. All analyses were performed using GraphPad
Prism version 5.00 for Windows (GraphPad Software, San Diego, California CA, USA) or SAS software version 9.3 (SAS Institute, Cary, NC).
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RESULTS
Insulitis Screening in Donors With Type 1 Diabetes and AAb+ Donors With No Diabetes
A total of 159 pancreata were reviewed for insulitis (no diabetes (n=61 AAb ; n=18 AAb+);
n=80 type 1 diabetes) (Suppl. Table 1). Insulitis was observed in 18 type 1 diabetes donors
(18/80; 23%) and 2 AAb+ donors (2/18; 11%). Variability in the lobular distribution of insulin+
islets and insulitic islets was high in donors with type 1 diabetes (Fig. 1A-D). In AAb+ donors
with insulitis, insulitis was primarily observed in insulin+ islets (Suppl. Fig. 1). Numbers of
CD3+ cells/islet varied in a given donor irrespective of islet insulin immunopositivity or size,
and CD3+ cells were observed diffusely infiltrating islets, in various sized aggregates, or both
(Suppl. Fig. 2).
All 18 donors with type 1 diabetes and insulitis had insulin+ islets detected in at least one
section while only 5/62 (8%) donors with type 1 diabetes and no insulitis had insulin+ islets.
Insulitis was found in 509/1525 (33%) insulin+ islets and in 379/17,718 (2%) insulin islets in
donors with type 1 diabetes (Table 1). In AAb+ donors with insulitis, the opposite was found;
insulitis affected 167/4297 (4%) of insulin+ islets and 8/30 (27%) of those that were insulin .
Insulitis was found in type 1 diabetes donors ranging in age from 3 26.2 years (13.3±6.5) at
disease onset and with disease durations of 0 12 years (4.6±3.4) (Table 1). The highest insulitis
frequency (10.6%) was observed in a 13 year old who presented with DKA at disease onset
(nPOD 6228). The lowest insulitis frequency (0.3%) was observed in a 12 year old with type 1
diabetes for 9 years (nPOD 6264). The two AAb+ donors with insulitis had multiple
autoantibodies and were 22 and 23 years old (Table 1). The AAb+ donors had insulitis
frequencies (1.4%, 6.4%) within the range observed for donors with type 1 diabetes; few insulin
islets, with or without insulitis, were observed in these donors.
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The percent of islets with insulitis (insulitis frequency) was tested for a correlation to
diabetes duration. Insulitis frequency showed a low, yet significant, inverse relationship to
diabetes duration while age at onset or demise did not (Fig. 3).
Correlation of Insulitis Prevalence With Diabetes Duration
As past studies reporting insulitis primarily examined young donors with type 1 diabetes within a
year of onset, the prevalence of donors with insulitis were analyzed in this study using similar
criteria (6). All four donors with recent onset type 1 diabetes (duration ≤1 year) had insulitis; 3/4
were <15 years old at disease onset (Table 2). In donors with disease durations >1 year, insulitis
was found in 7/51 (14%) donors who were <15 years of age at disease onset and in 7/24 (29%)
donors who were ≥15 years at disease onset. Using 12 years diabetes duration as the cut off, the prevalence of insulitis was 45% (18/40) of donors with type 1 diabetes.
To further define other potential clinical variables as they relate to insulitis, 15 type 1
diabetes donors with insulitis were matched for age at onset, ethnicity, sex, and BMI with 15
type 1 diabetes donors without insulitis (Suppl. Table 3; In 3 cases, no matches were found and
thus excluded). Diabetes duration was significantly shorter in type 1 diabetes donors with
insulitis vs. those without insulitis (5 versus 14.5 years, respectively). Correspondingly, donors
with insulitis were significantly younger at demise compared to matched donors without insulitis
(20 versus 27.6 years, respectively). Interestingly, islet autoantibody numbers were not different between the matched type 1 diabetes donors. Hospitalization durations were not significantly
different in this matched subset and ranged from a mean of 3.2 3.7 days across all donor groups
(Suppl. Tables 1 and 3). Correlation of DKA during the hospitalization showed a significantly
higher proportion of donors with (7/15, 47%) versus without (1/15, 7%) insulitis (p=0.04).
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Infiltrating Leukocytes Have Similar Proportions of B and T Cells and T Cell Subsets and
Numbers were Proportional to Insulitis Frequency
Infiltrating leukocytes in insulitic islets were examined by multi immunofluorescence on serial
sections (Fig. 4). Mean numbers of CD45+ cells/islet in each donor were significantly correlated
to mean numbers of both T (CD3+) and B (CD20+) cells/islet (Fig. 5). Absolute numbers of
CD3+ and CD20+ cells varied between islets in a given donor depending on the nature of the
infiltrate (e.g., diffusely associated with islet, aggregate, mixture). Numbers of both CD3+ and
CD20+ cells appeared independent of islet insulin immunopositivity and diabetes duration
(Suppl. Fig. 2 3). Numbers of CD3+ cells were significantly correlated with CD8+ and CD4+
cells/islet (Fig. 5B). Leukocyte numbers/islet were also plotted for both donor groups with
insulitis, and high variability was observed particularly for CD45+, CD3+ and CD20+ (Fig. 5C).
This variability was also seen in the AAb+ donors without diabetes (Fig. 5C). Five donors had
insulitis aggregates with >50 CD3+ cells/islet; four were donors with type 1 diabetes who were
all less than 12 years of age but had widely varying diabetes durations (nPOD 6209, 6243, 6052,
6070) and one was an AAb+ donor without diabetes (nPOD 6267).
We also tested whether numbers of leukocytes/islet were correlated with insulitis
frequency and found significant correlations with CD45+, CD3+, CD20+, CD8+, and CD4+
cells/islet but not for CD68+ and CD11c+ cells/islet (Suppl. Fig. 4, Suppl. Table 4). Because of
the variability between islets in numbers of leukocytes/insulitic islet, we also tested whether a
ratio of markers (CD3+ cells / sum of CD3+ and CD20+ cells) would normalize inter islet
variability but found no significant correlation to insulitis frequency (Suppl. Fig. 4).
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β cell and α cell Mass and Insulitis
Pancreas weights were variable in donors without diabetes: 71.1±24.2g (n=49) in AAb donors,
70.7±28.0g (n=15) in AAb+ donors without insulitis, and 60.7±17.9 in two AAb+ donors with insulitis (Fig. 6A). As anticipated, both donor groups without diabetes (AAb and AAb+) showed increasing pancreas weights after age 4 up to ~30 years (Suppl. Fig. 5A-B). Donors with type 1 diabetes had significantly lower pancreas weights (39.51±17.9g no insulitis, n=60;
33.8±12.9g with insulitis, n=16) compared to donors without diabetes (Fig. 6A). Pancreas weights were decreased at all ages in donors with type 1 diabetes compared to donors without diabetes (Suppl. Fig. 5C).
β cell area was also variable in donors without diabetes: 1.18±0.71% for AAb donors
(n=57) and 0.67±0.38% for AAb+ donors without insulitis (n=16) (Fig. 6B). The two AAb+ donors with insulitis had β cell area of 2.26±0.4%. As expected, significant reductions in β cell area was observed for donors with type 1 diabetes compared to nondiabetic donors (Fig. 6B). β cell area was also significantly different between the two donor groups with type 1 diabetes
(0.02±0.05% no insulitis, n=54; 0.15±0.17% with insulitis, n=18) (Fig. 6B).
β cell mass was 797±523mg for AAb donors (n=45) and 448±268mg for AAb+ donors without insulitis (n=15) (Fig. 6C). β cell mass was 1407± 649mg in the two AAb+ donors with insulitis. β cell mass was decreased in donors with type 1 diabetes regardless of insulitis status compared to those without type 1 diabetes; however, β cell mass was significantly higher in type
1 diabetes donors with insulitis (47.9±53.4mg, n=17; P<0.05) compared to those without insulitis (5.5±10.1mg, n=53) (Fig. 6C). β cell mass from five donors without diabetes (3 AAb ,
2AAb+) overlapped with the highest values for β cell mass in donors with T1D and insulitis due to low pancreas weight (3) or low β cell area (2). Estimated β cell mass was further tested for
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correlation with insulitis frequency, but no association was found (Suppl. Fig. 6A, P>0.05).
Similarly, we compared β cell mass to type 1 diabetes duration and age at onset to determine if
insulin mass was greater in donors with shorter diabetes durations or younger donors; again, no
significant associations were found (Suppl. Fig. 6B-C, P>0.05).
In marked contrast to differences in β cell area and mass, α cell area and mass were not
significantly different between groups (Fig. 6D-E). The α cell area was 0.73±0.45% in AAb
donors (n=57), 0.62±0.44% in AAb+ donors without insulitis (n=16), 1.37±0.04% in AAb+
donors with insulitis (n=2), 1.04±0.84% in donors with type 1 diabetes and no insulitis (n=57),
and 1.37±0.62% in donors with type 1 diabetes and insulitis (n=17). The α cell mass was
495±320mg in AAb donors, 402±428mg in AAb+ donors without insulitis, 827±224mg in
AAb+ donors with insulitis, 396±377mg in donors with type 1 diabetes and no insulitis, and
513±375mg in donors with type 1 diabetes and insulitis.
Influence of HLA on Insulitis
Donors with type 1 diabetes had a higher frequency of DRB1*03:01 DQB1*02:01 or DRB1*04
DQB1*03:02 genotypes (DR3/DR4) compared to AAb or AAb+ donors (35.9% vs. 0%,
P<0.0001 and P=0.0012, respectively). HLA DR3/DR4 frequencies were not different when
comparing the matched donors within the type 1 diabetes group, with and without insulitis
(Suppl. Table 3). The DRB1*1501 DQB1*0602 haplotype was very rare and considered
protective for type 1 diabetes. The overall frequency of this haplotype in AAb donors (25%)
was similar to the general U.S. Caucasian population (22%) (27). This haplotype was found in
four AAb+ donors with single autoantibodies and in none of those with multiple autoantibodies.
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In the type 1 diabetes donors, only one had the DRB1*1501 DQB1*0602 haplotype, and quite interestingly, this donor also had insulitis (nPOD 6195).
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DISCUSSION
The concept of insulitis has been the subject of previous reports; however, this study is the first
to report insulitis frequency, leukocyte subtypes, pancreatic weights, and β cell and α cell mass
in organ donors encompassing the entire natural history of type 1 diabetes [i.e., preclinical phase
(no diabetes, islet autoantibody positive) as well as acute and chronic phases]. Beyond this, the
current study is large, utilized standard operating procedure driven protocols, and utilized only
high quality tissues. As a result, we believe the results noted herein provide much in the way of
new information as well as validating, or refuting, past observations.
Insulitis was defined by the presence of ≥6 CD3+ cells/islet and 3 insulitic islets/section
(4). Overall, insulitis was identified in 18/80 (23%) donors with type 1 diabetes and 2/18 (11%)
AAb+ donors without diabetes. Notably, numbers of donors with insulitis (insulitis prevalence)
was higher for older donors and for longer disease durations in both young and older patients
than reported using meta analysis of existing studies (6). Historically, insulitis prevalence has
been reported in 51% (41/81) of patients with disease duration ≤1 year compared to only 3%
(4/132) in patients with durations >1 year (6). As shown in Table 2, we observed an insulitis
prevalence of 100% (4/4) in donors with type 1 diabetes durations ≤1 year and 19% (14/75) in
donors with durations >1 year. We also observed a high proportion of donors with insulitis for
diabetes durations up to 12 years (18/40 (45%)). At the same time, our overall insulitis
prevalence of 23% (18/79) in donors with type 1 diabetes compares well with the overall insulitis
prevalence rate of 21% (45/213) from former studies (6). Heterogeneity in the distribution of
insulitic islets could partly explain the lower detection rates from former studies using autopsy
collections or biopsies as fewer blocks and pancreas regions could be tested per patient (5; 7).
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Studies by Oram et al. and several others support the persistence of β cells in patients with long standing type 1 diabetes (11; 28 30). Our data also show the persistence of β cells in donors with type 1 diabetes, with and without insulitis, up to 12 years post onset. While other investigators have also reported insulin+ islets without insulitis in patients with type 1 diabetes, few have emphasized that insulin islets are also involved in the insulitic process (reviewed in
(31)). The proportions of insulin+ and insulin insulitic islets in our study (33% insulin+, 2% insulin ) were similar to those reported by Foulis and others (28 35% insulin+, 1 5% insulin ) (5;
32). Persistent insulitis after loss of β cells could imply the presence of degranulated and/or dedifferentiated β cells, undetected by these methods, or alternatively, spread of autoimmunity or
“innocent bystander” effects on other endocrine cells. Prospective studies demonstrated that appearance of multiple islet autoantibodies is usually sequential rather than simultaneous, and that their number (≥2) is a strong predictor of progression to type 1 diabetes (1). Herein, insulitis was observed in 2/5 (40%) donors with multiple autoantibodies, a finding similar to In’t Veld et al. where insulitis was detected in 2/7 (29%) donors with multiple autoantibodies (7). Of note, the AAb+ donors with insulitis in our study were younger (21 and 22 years compared to 46 and
52.5 years) and the entire pancreas was available for study compared to the former study of biopsy material. Interestingly, all four donors showed similar insulitis frequencies (1.4 6.4%; 3
9%) and normal β cell mass, and all four donors had the autoantibody combination of GADA+ and IA 2A+.
Another novel aspect of our study is in showing similarities between the preclinical and clinical phases of type 1 diabetes in the leukocyte subtypes in insulitic islets and independent of the presence of residual β cells. Both phases were characterized by diffuse and/or focal aggregates and varying numbers of leukocytes/islet within and between donors. Several studies
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show a predominance of CD8+ T lymphocytes and macrophages in insulitic islets (7; 30; 32 38).
The defining study was considered that by Willcox et al. using 29 patients from the Foulis
collection, and re examination of 21 patients from the Foulis collection with two nPOD donors
(6052, 6113) was recently reported by Arif et al. (32; 39). In the former study, insulitic islets
were ranked by both insulin percentage and immune cell numbers (n= 279 insulitic islets), while
only insulin+ islets were evaluated in the latter study. All leukocytes were significantly reduced
in insulin islets (32). In marked contrast, our study found that CD45+, CD3+, CD20+, and T cell
subsets increased linearly with numbers of insulitic islets (insulitis frequency) irrespective of
islet β cells. Potential differences between studies may result from differences in age or disease
duration or technical differences in methods.
An intriguing hypothesis by Skog et al. proposed that ascending infection through the
pancreatic ducts could result in insulitis due to greater susceptibility of β cells compared to other
endocrine cells (40). Though insulitic islets were frequently observed near pancreatic ducts in
this study, no consistent pattern was observed for the presence of peri ductal infiltrates and
insulitic islets. The heterogeneous leukocyte composition of insulitis might support this
hypothesis if the offending agent (virus or bacterium) or their toxic by products had minimal
proclivity for ductular epithelial cells.
In the present study, donors with type 1 diabetes had small pancreata regardless of age or
disease duration. Pancreas weights were not different between AAb or AAb+ donor groups
without diabetes and are similar to the age related changes reported in normal pancreas volume
by Saisho et al. (41). Our current findings contrast with our previous report using a subset (n=8)
of the 18 AAb+ donors in this study (42). In our first study, all donors were >18 years and
excluded if pancreatitis was noted in the medical chart or by histopathology. Age or pancreatitis
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exclusion criteria were not utilized in the current study. As it is difficult to predict when organ
donors with multiple AAb+ could progress to type 1 diabetes, pancreas size loss in the natural
history of type 1 diabetes might be best studied in longitudinal clinical trials of AAb+ subjects by noninvasive radiography (43; 44). Beyond this, the notion of pancreatitis in type 1 diabetes is
of evolving interest, given histopathological findings of multifocal CD3+ infiltrates in acinar
regions of donors with type 1 diabetes. Indeed, Rodriguez Calvo et al. reported an increased
number of exocrine CD8+ T cells in nPOD donors with type 1 and type 2 diabetes (45).
Reports indicate marked variability in β cell area or mass in normal adults with adult
levels reached by 5 years (17; 18; 46; 47). Indeed, some donors without diabetes (both AAb+ and
AAb ) exhibit β cell mass within the range observed for type 1 diabetes donors. Reasons for this
heterogeneity are unknown but could result from host genetic and environmental factors
including in utero and neonatal periods when β cell mass is increasing. Rising rates of obesity
could also account for some variability. Our β cell area and mass data are in agreement with past
literature also reporting high variability in both measures of islet β cell content. For instance, our
mean control β cell area (1.2%) was similar to that reported in children and young adults by
Meier et. al. [1.7% (5.7 15 years of age), 1.3% (18.5 21.5 years of age)] (18) as well as In’t
Veld’s report of AAb+ donors without diabetes (mean relative β cell area of 1.2% (0.51–2.61)
(7). In the report by In’t Veld et al., comparison of β cell areas and mass in AAb+ and AAb
donors did not show a significant difference (7). Two other groups have also reported normal β
cell mass in patients with autoantibodies (48; 49). Interestingly, β cell mass in five donors
without diabetes was in the upper range found for donors with T1D and insulitis and was related
to young donor age, low pancreas weights, and/or low β cell area.
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In terms of potential limitations, it must be emphasized that these data were obtained
from pancreata of multi organ donors with brain death who were subjected to intensive care
procedures. Interpretations of data must be considered, as posed by In’t Veld et al., in light of
medications received during critical care and duration of intensive care (reviewed in (50)). The
majority of donors in this study had short hospitalization durations, yet further studies are needed
to both validate and expand these findings. The computer assisted image analysis algorithm used
in this study has inherent limitations with single minimum and maximum intensity thresholds
applied to all sections. Alternatively, its unbiased approach utilizing the entire section cross
sectional areas could add greater consistency compared to manual point counting methods.
When studying organ donors, there is the possibility that the referring/initial diagnosis provided
to nPOD may be of questionable nature. To address this, full medical histories are carefully
reviewed by Board Certified Endocrinologists and Pathologists. Even when histories are limited,
we carefully consider age at diagnosis, BMI, time to insulin dependence, history of insulin
dependence, treatment with oral agents, c peptide, HLA, presence of DKA, ketosis, and more.
In sum, the proportion of insulitic islets (insulitis frequency) in donors with type 1
diabetes was inversely associated with disease duration and not age at onset, number of
autoantibodies, or HLA genotype. Numbers of donors with insulitis (insulitis prevalence) was
higher in older patients and for longer disease durations in both young and older patients than
previously reported. In donors with type 1 diabetes, proportions of insulin+ islets with insulitis
was higher than in insulin islets, yet insulin islets were also affected by insulitis, particularly in
donors with multiple AAb+. Insulitic lesions were composed of a mixture of B and T
lymphocytes whose numbers increased with insulitis frequency. The β cell mass was
significantly higher in donors with type 1 diabetes and insulitis compared to donors without
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insulitis while β cell mass was similar between AAb+ donors and AAb donors. These data
underscore the chronic and heterogeneous nature of insulitis in type 1 diabetes. The presence of
insulitis as well as insulin+ islets several years after diagnosis, and the limited correlation between insulitis frequency and disease duration suggest that the chronicity of islet
autoimmunity extends well into the post–diagnosis period. Together with growing evidence for
the chronicity of autoimmune responses and for the persistence of C peptide secretion in patients, even decades after onset, our findings support the existence of a potential long window of therapeutic opportunity (51; 52).
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Acknowledgments. The authors acknowledge Dr. Alvin Powers (Vanderbilt University,
Memphis, TN) and Dr. Jake Kushner (Baylor College of Medicine, Houston, TX) for nPOD
donor referrals and Robert Pietras for image analysis software design. The authors thank Dr.
Amanda Posgai (University of Florida) for editorial assistance. All nPOD staff members are
thanked for donor recovery, data collection, and helpful discussions. Organ Procurement
Organizations partnering with nPOD are listed at http://www.jdrfnpod.org/for partners/npod
partners. Immunohistochemistry slides used in this study are available for viewing through the
nPOD Online Pathology database (21). This study used data from the Organ Procurement and
Transplantation Network (OPTN) supplied by the United Network for Organ Sharing as the
contractor for the OPTN. The interpretation and reporting of these data are the responsibility of
the author(s) and in no way should be seen as an official policy of or interpretation by the OPTN
or the U.S. Government.
Funding. This work was supported by the JDRF (25 2013 268, 17 2012 3, and 25 2012 516
M.C T., M.A.A., A.P., J.S.K.) and NIDDK (UC4 DK 104155 01, 1DP3DK101120 01 M.C T.,
M.A.A., D.A.S.).
Duality of interest. No potential conflicts of interest relevant to this article were reported.
Author Contributions. M.C.T. designed the experiments, performed pathology reviews, image
analyses, and statistical calculations, and wrote the manuscript. A.F. performed experiments and
edited the manuscript. C.W. designed the autoantibody studies, performed experiments, and
edited the manuscript. J.S.K. and A.P. researched data, performed statistical calculations and
contributed to discussion and editing of the manuscript. D.A.S and M.A.A. contributed to study
design and discussion and edited the manuscript. M.C.T. is the guarantor of this work and, as
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Diabetes Page 24 of 58
such, had full access to all the data in the study and takes responsibility for the integrity of the
data and the accuracy of the data analysis.
Prior Presentation. Preliminary immunotyping results were presented at the Immunology of
Diabetes Society 12th International Congress, Vancouver Island, Canada, June 15 19, 2012 and
13th International Congress, Lorne, Australia, December 7 11, 2013. Pancreas weights were
reported on a subset of these donors (53). Of the 159 nPOD cases presented herein, 43 were previously examined by Coppieters et al (Suppl. Table 5).
Page 24 of 40
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Table 1. Insulitis frequency in donors. Multiple slide sections were reviewed from each donor pancreas and islets were counted by insulin immunopositivity (Ins+, Ins ) and insulitis status (CD3 (negative), CD3+ (positive)). the total number of insulitic islets divided by total islet numbers was used to calculate insulitis frequency (%) for each donor. Donors were listed in order of increasing insulitis frequency (%).Demographic information for ages at demise and diabetes onset, diabetes duration, and autoantibody types were also shown.
Insulitis Case No. Ins+ Ins+ Ins Ins Total Age Diabetes Age Frequency § || ID Sections CD3 CD3+ CD3+ CD3 Islets* Onset Duration Demise Autoantibody Results (%) † (yrs) (yrs) (yrs) 6264 6 7 3 0 1017 1027 0.3% 3.00 9.00 12.00 Negative 6212 12 51 13 10 1601 1675 1.4% 15.00 5.00 20.00 6268 5 0 1 13 808 822 1.7% 9.00 3.00 12.00 6265 6 8 6 5 508 527 2.1% 3.00 8.00 11.00 GADA+ 6039 4 0 2 5 324 331 2.1% 16.70 12.00 28.70 GADA+ IA 2A+ ZnT8A+ 6046 5 92 2 6 268 368 2.2% 10.80 8.00 18.80 IA 2A+ ZnT8A+ 6113 14 21 28 9 1640 1698 2.2% 11.52 1.58 13.10 6224 8 30 24 12 1486 1552 2.3% 19.50 1.50 21.00 Negative 6088 7 73 7 6 433 519 2.5% 26.20 5.00 31.20 GADA+ IA 2A+ ZnT8A+
T1D 6245 6 131 7 28 1021 1187 2.9% 15.00 7.00 22.00 GADA+ IA 2A+ 6247 6 77 20 25 1068 1190 3.8% 23.40 0.60 24.00 6195 16 0 2 53 1385 1440 3.8% 14.20 5.00 19.20 GADA+ IA 2A+ ZnT8A+ 6070 4 56 15 8 333 412 5.6% 15.60 7.00 22.60 IA 2A+ 6198 2 16 9 5 181 211 6.6% 19.00 3.00 22.00 GADA+ IA 2A+ ZnT8A+ 6052 9 10 27 41 921 999 6.8% 11.00 1.00 12.00 IA 2A+ 6209 11 32 42 54 1247 1375 7.0% 4.75 0.25 5.00 IA 2A+ ZnT8A+ 6243 8 162 55 32 859 1108 7.9% 8.00 5.00 13.00 6228 15 250 246 74 2447 3017 10.6% 13.00 0.00 13.00 GADA+ IA 2A+ ZnT8A+ Total 144 1,016 509 386 17,547 19,458 6197 5 1981 28 0 4 2013 1.4% 22.00 GADA+ IA 2A+ AAb+¶ 6267 13 2149 139 8 18 2314 6.4% 23.00 GADA+ IA 2A+
Total 18 4,130 167 8 22 4,327
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* Total Islets calculated as sum of 4 islet subtypes. †Insulitis frequency (%) calculated as sum insulitic islets (Ins+ CD3+ and Ins CD3+)/Total Islets. ‡ Calculated as age at demise minus diabetes duration. § All donors with insulitis had autoantibody testing by RIA. RIA data values were converted to NIDDK units and defined as positive if one or more of the following applied: GADA if >20, IA2A if >5, or ZnT8A if >0.020. || IAA were not listed for donors with T1D as they could not be distinguished as autoantibodies versus antibodies to parenteral insulin as described in Methods. An empty field indicates that only insulin autoantibodies were detected by RIA. ¶ AAb+ donors without diabetes.
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Table 2. Table of insulitis prevalence in nPOD donors by age of onset and diabetes duration.
Diabetes Duration ≤ 1 year Diabetes Duration > 1 year Totals*
Age at Number Total Proportion Number Total Proportion Number Total Proportion
Onset Donors Number with Donors Number with Donors Number with
(Years) Insulitis+ Donors Insulitis% Insulitis+ Donors Insulitis% Insulitis+ Donors Insulitis%
1.4 14.2 3 3 100% 7 51 14% 10 54 19%
15 36 1 1 100% 7 24 29% 8 25 32%
Total 4 4 100% 14 75 19% 18 79 23%
Age groups and diabetes durations adapted from In’t Veld (6).
*One donor with T1D had unknown duration of disease (Methods).
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Figure Legends
Figure 1. Lobular variability in insulin+ islets and insulitis. Islets were imaged from a 13 year
old patient with T1D for 5 years (6243). Serial paraffin sections were stained for Ki67+INS (A,
C, E) and CD3+GCG (B, D,F) and islets were subtyped as described in Methods. Five insulin+
islets (A) are seen in a lobule adjacent to a lobule with two insulin islets in the patient with T1D
(A, B, blue arrows). Three insulin+ islets had insulitis (B, black arrows) and both insulin islets
did not have insulitis. One of the insulin+ islet with insulitis is shown at higher magnification
((C, D) as well as an insulin insulitis islet (E, F). Few islet cells were Ki67+ (A, C) indicating
no effect of insulitis on proliferating cell numbers. Scale bars: A, B 500 m; C-F 50µm.
Figure 2 Heterogeneity of islet β cells and numbers of CD3+ cells in insulitic islets in young
donors at diabetes onset and after 5 years duration. Representative islets were imaged for a 13
year old at diabetes onset (6228; A-F) and another 13 year old with T1D for 5 years (6243; G-L).
Serial paraffin sections were stained using dual immunohistochemistry (Ki67+Insulin,
CD3+Glucagon) as described in Methods. Decreasing proportions of β cells are shown in
different islets from both donors (A-C, G-I) as well as heterogeneity in numbers of CD3+
cells/islet (D-F, J-L). Heterogeneity in CD3+ counts between two insulin islets of similar sizes
is also shown for one donor (K, L). Scale bars: A-F, H, K: 50 m; G, I, J, L: 100µm.
Figure 3 Insulitis frequency in relation to diabetes duration and donor ages at demise or disease
onset. The insulitis frequency (%, total insulitic islets/total islets) is shown on the Y axis in
comparison to years for diabetes duration (A), age at T1D onset (B), and age at demise (C). Data
were displayed (pink) for the two AAb+ donors with insulitis for age at demise but were
excluded from statistical analyses due to small sample size. Insulitis frequency (%) had a low but
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significant correlation to diabetes duration but not to donor age at demise or disease onset. Linear
regression line with Spearmen r and p values are shown.
Figure 4. Insulitis leukocyte subtyping by multiple immunofluorescence. Serial sections of an islet with insulitic aggregate were imaged from a 5 year old donor with T1D for 4 months
(6209). Numbers of leukocytes/islet were counted for total leukocytes (CD45, A), B and T
lymphocytes (CD20, CD3, B), T lymphocyte subsets (CD8, CD4, C), dendritic cells and
macrophages (CD11c, CD68, D) as described in Methods. Islet endocrine cells were identified
using glucagon (GCG) and insulin (INS) stains. The merged images (A-D) include DAPI stained
DNA and the individual fluorescence channels are shown below the merged images. Scale bars:
50µm
Figure 5 Leukocyte numbers increase in parallel with CD45+ and CD3+ numbers but are heterogeneous between islets. Numbers of CD45+, CD3+, CD20+, CD8+ and CD4+ cells were counted for each insulitic islet and averaged per donor (n=13 donors with T1D, 2 10 islets/donor). Data were also displayed (pink) for each leukocyte marker for the two AAb+ donors with insulitis for comparison purposes but were excluded from statistical analyses due to small sample size. Correlations between numbers of leukocytes/islet were performed by
Spearman analysis with linear regression lines shown. Numbers of CD45+ cells/islet were significantly correlated with numbers of CD3+ and CD20+cells/islet (A). Numbers of CD3+ cells/islet were also significantly correlated with numbers of CD8+ and CD4+ cells/islets (B).
Numbers of leukocyte subtypes/islet were also analyzed to examine variability in cell numbers between all islets for donors with T1D and two AAb+ donors with insulitis with means depicted
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by box and whisker plots with Tukey error bars (nonparametric) (C). Numbers in parentheses
following each marker indicate the total numbers of individual islets analyzed for each leukocyte
marker (CD45) or set of markers. Variability in overall numbers of leukocytes/islet was
observed. Numbers of leukocytes /islet had similar trends in AAb+ donors without diabetes.
Figure 6 Pancreas weights and β cell area and mass are decreased in donors with T1D. Group
means were depicted by box and whisker plots with Tukey error bars (nonparametric). Data
were displayed (pink) for the two AAb+ donors with insulitis and were excluded from statistical
analyses due to small sample size (n=2). Pancreas weights were significantly lower in donors
with T1D compared to AAb and AAb+ donors without diabetes and no difference was detected
between donors with T1D based on insulitis status (A). The β cell area was significantly lower in
both donor groups with T1D compared to nondiabetic AAb and AAb+ donors (B). T1D donors
with insulitis had a significantly higher β cell area compared to T1D donors without insulitis (B).
The β cell mass was similar between AAb and AAb+ donors and was significantly different
from donor groups with T1D (C). T1D donors with insulitis had significantly higher insulin+ β
cell mass compared to T1D donors without insulitis (C). The α cell area (D) and mass (E) were
not significantly different between groups. *Significantly different from AAb and AAb+ donors
(P<0.001), **Significantly different from AAb and AAb+ donors (P<0.001) and T1D donors
without insulitis (P<0.05).
Supplementary Figure 1. Insulitic islets in AAb+ donors without diabetes. Islets were imaged
from a 22 year old donor (6197; A-B) and a 23 year old donor (6267; C-D). Cells in the insulitic
islets were immunotyped using multiple immunofluorescence as described in Methods. Islet
endocrine cells were identified using glucagon (GCG) and insulin (INS) stains. Heterogeneous
Page 37 of 40
Diabetes Page 38 of 58
distribution of Ins+ and Ins islets was observed between lobules (A). An insulitic islet is shown with diffuse infiltration by CD45+ cells (B). A large insulitic aggregate is shown composed of similar numbers of CD20+ and CD3+ lymphocytes (C). Another large islet with fewer CD20+ and CD3+ cells demonstrates the wide variability between leukocyte numbers/islet within a given donor (D). Scale bars: 200µm A, 50 µm B, D, 100µm C.
Supplementary Figure 2. B and T lymphocytes in insulitic islets from donors with long standing T1D. Focal aggregates in islets were imaged from a 12 year old donor with T1D for 3 years (6268) (A-D) and a 22.6 year old patient with T1D for 7 years (6070) (E-I). Sections were stained for CD20 (B, F), CD3 (C, G), CD8 and CD4 (I), glucagon (GCG, D), insulin (INS, H), or chromogranin A (CGA, I) as described in Methods. Both CD20+ and CD3+ lymphocytes were found in insulitic aggregates though in varying numbers. DAPI stained DNA to delineate nuclei is shown in merged images (A, E, I). Scale bars: 50µm.
Supplementary Figure 3 Leukocytes in insulitic islets from insulin+ and insulin islets. Two islets in a pancreas section were imaged from a 13 year old donor with T1D for 5 years (6243)
(insulin+ islet (A-D); insulin islet (E-H)). Serial sections were stained as described in Methods with endocrine cells identified using glucagon (GCG) and insulin (INS) stains. Aggregates in both islets were comprised of both CD20+ and CD3+ cells (C, G) as well as CD8+ and CD4+ T cells (D, H). Dendritic cells (CD11c+) and CD68+ macrophages were observed adjacent to mononuclear aggregates and endocrine cells (B, F). A large number of macrophages (CD68) are also seen in the adjacent exocrine region in panel B. Scale bars: A-D 50 m; E-H 100 m.
Supplementary Figure 4. Heterogeneity in leukocyte markers in insulitic islets and correlations with insulitis frequency. Mean numbers of leukocyte subtypes / insulitic islets from each donor
Page 38 of 40
Page 39 of 58 Diabetes
with T1D were plotted in comparison to insulitis frequency (%) (A-G). Data were displayed
(pink) for the two AAb+ donors with insulitis and were excluded from statistical analyses due to
small sample size. The ratio of CD3/(CD3+CD20) was also calculated and plotted by insulitis
frequency (H). Numbers of CD45+, CD3+, CD20+, CD8+, and CD4+ cells/islet had variable
significant correlations with insulitis frequency but numbers of CD68+ and CD11c+ cells/islet or
the ratio of CD3+/(CD3+CD20) did not. Linear regression lines with Spearmen r and p values
are shown.
Supplementary Figure 5. Pancreas weight and β cell area and mass by donor age and group.
Data from donors with no diabetes (AAb (A, D, G), AAb+ (B, E, H)) or T1D (C, F, I) were
depicted with data for donors with insulitis indicated in color (AAb+, pink; T1D, blue) within
each respective group. An x axis of 0 80 years was used in all plots; although, some older donors
did not have pancreas weight data due to receipt of partial pancreas sample (n=3) or no recording
(n=13). Smaller Y axis scales were used for donors with T1D in panels for β cell area (F) and
mass (I) to allow better visualization of the significant differences, as shown in Fig. 6, for these
two variables by insulitis status within this group. Pancreas weights were plotted with regression
lines from best fit second order polynomial equations (A-C). Variations in β cell area (%) were
seen by age in donors without diabetes (AAb , AAb+) (D-E) and in donors with T1D (F). The β
cell mass (mg) was calculated from β cell area (%) multiplied by pancreas weight (gram) (G-I)
and similar variations were observed as for β cell areas.
Supplementary Figure 6. β cell mass from donors with T1D and insulitis in comparison to
insulitis frequency, diabetes duration and age at onset. The β cell mass in donors with T1D and
insulitis was not significantly correlated with insulitis frequency (A), diabetes duration (B) or age
Page 39 of 40
Diabetes Page 40 of 58
at onset (C) due to heterogeneity in residual β cell distribution independent of age. Linear regression lines with Spearmen r and p values are shown.
Page 40 of 40
Page 41 of 58 Diabetes
A Ki67 INS B CD3 GCG
E,F C,D
C Ki67 INS D CD3 GCG E Ki67 INS F CD3 GCG
Figure 1. Figure2 .
CD3 GCG Ki67 INS CD3 GCG Ki67 INS G G A D J
K H E B B Diabetes L I C F F Page 42of58 Page 43 of 58 Diabetes
A B C
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Figure 3. Diabetes Page 44 of 58
A B C D Merge Merge
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Figure 4 Page 45 of 58 Diabetes
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Diabetes Page 46 of 58
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Figure 6. Page 47 of 58 Diabetes
A B Insulin+ INS
Insulin- GCG CD45 GCG
C D GCG GCG
CD3
CD20
Suppl. Figure 1 Diabetes Page 48 of 58
A B CD20 C CD3 D GCG
E H INS I CD8 CD4 CGA
F CD20 G CD3
Suppl. Figure 2. Page 49 of 58 Diabetes
E CD45 GCG INS F CD11c CD68 INS
A CD45 GCG INS B CD11c CD68 INS
C CD20 CD3 GCG D CD8 CD4 GCG G CD20 CD3 GCG H CD8 CD4 GCG
Suppl. Figure 3. Diabetes Page 50 of 58 A B
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A B C
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Suppl. Figure 5. Diabetes Page 52 of 58
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Suppl. Figure 6. Page 53 of 58 Diabetes
Supplementary Table 1. Clinical characteristics of 159 organ donors from study. No Diabetes Autoantibody Positive (AAb+) T1D No Diabetes (AAb ) Characteristic No Insulitis Insulitis No Insulitis Insulitis n Value* n Value* n Value* n Value* n Value* Age at Death (yr) 61 28.6 (±16.4) 16 38.8 (±17.8) 2 22.5 (±0.7) 62 30.6 (±11.9) 18 17.8 (±6.8) † Age at Diabetes Onset (yr) 61 11.4 (±8.2) 18 13.3 (±6.5) Diabetes Duration (yr) 61 19.1 (±11.5) 18 4.6 (±3.4) Ethnicity African American 5 8% 1 6% 1 50% 7 11% 1 6% White 49 82% 13 81% 1 50% 53 86% 16 88% Hispanic/Latino 6 10% 2 13% 0 0% 2 3% 1 6% Sex Female 23 38% 7 44% 1 50% 26 42% 9 50% Male 38 62% 9 56% 1 50% 36 58% 9 50% Death‡ or Admit Course§ DKA Related 0 0% 0 0% 0 0% 16 26% 8 44% Other 61 100% 15 100% 2 100% 46 74% 10 56% Whole Body Weight (kg) 61 73.7 (±21.9) 16 75.3 (±27.0) 2 76.5 (±17.7) 62 71.4 (±16.7) 18 59.5 (±19.8) BMI (kg/m2) 61 25.4 (±5.2) 16 25.8 (±6.5) 2 25.9 (±3.3) 62 25.0 (±4.1) 18 22.2 (±4.4) C peptide (ng/mL)|| 61 4.6 (0.4, 22.9) 14 3.9 (0.001, 26.2) 2 17.0 (16.6, 17.5) 61 0.001 (0.001, 0.140) 18 0.001 (0.001, 0.470) HbA1c (% ) 21 5.4 (±1.3) 4 5.8 (±0.8) 2 5.3 (±0.4) 19 9.8 (±2.3) 5 10.3 (±2.9) (mmol/mol) 35.1 (±14.4) 39.6 (±8.4) 33.9 (±3.9) 83.6 (±24.6) 89.1 (±32.0) Autoantibodies Detected¶ 0 61 100% 0 0% 0 0% 30 49% 7 39% 1 0 0% 13 89% 0 0% 23 38% 3 17% ≥2 0 0% 3 19% 2 100% 8 13% 8 44% Hospitalization Stay (days) 57 3.2 (0.3, 23.6) 13 3.2 (1.1, 6.8) 2 3.7 (3.0, 4.5) 60 3.3 (0.9, 11.3) 3.4 (0.9, 21.7) *% reported for all categorical variables; use of mean or median (minimum, maximum) based on evaluation of normal distribution † Calculated as age at death minus diabetes duration ‡ Cause or mechanism of death § Clinical records were examined for mention of DKA || A fill value of 0.001 was used when assay results <0.05 (lower limit of detection) ¶ All RIA data values were converted to NIDDK units and defined as positive if one or more of the following applied: GADA if >20, IA2A if >5, ZnT8A if >0.020, or insulin if >0.010. Insulin autoantibodies excluded from counts for donors with T1D as described in Methods. Three AAb+ donors with no insulitis had positive autoantibody results from the screening serum samples by ELISA and/or RIA in lieu of positive results from the serum sample obtained at pancreas recovery.
Diabetes Page 54 of 58
Supplementary Table 2. Primary and secondary antibodies used in immunolocalization assays.
Catalogue Antigen Species Clone Company Number CD11c Rabbit EP1347Y Abcam ab52632
CD3 Rabbit Polyclonal DAKO A0452 CD4 Mouse 4B12 DAKO M7310
CD8 Rabbit Sp16 Abcam ab27605 CD20cy Mouse L26 DAKO M0755
CD45 Mouse 2B11 + PD7/26 DAKO M0701 CD68 Mouse PG M1 DAKO M0876
Chromogranin A Rabbit DAK A3 DAKO M0869 Glucagon Mouse K79bB10 Abcam ab10988
Insulin Guinea Pig Polyclonal DAKO A0564 Ki67 Mouse MIB 1 DAKO M7240
Goat AF488 conjugate Invitrogen A 11029 Mouse IgG Rabbit IgG Goat AF555 conjugate Invitrogen A 21429
Guinea pig IgG Goat AF647 conjugate Invitrogen A 21450 Page 55 of 58 Diabetes
Supplementary Table 3. Clinical and analytical characteristics of matched donors with T1D with and without insulitis.
Name (%)*, Mean (± 1 SD)*, or Median (min, max)* P-value n No Insulitis n Insulitis Age at Demise (yrs) 15 27.6 (4.4, 61.0) 15 20 (12.0, 31.2) 0.005 Age at Diabetes Onset (yrs) †, ‡ 14 11.7 (±8.9) 15 14.5 (±6.0) 0.319 Diabetes Duration (yrs) 14 14.5 (3.0, 52.0) 15 5 (0.6, 12.0) <0.001 Ethnicity † 0.791 Caucasian 11 73% 13 86% African American 3 20% 1 7% Hispanic/Latino 1 7% 1 7% Sex † 0.264 Female 4 27% 8 53% Male 11 73% 7 47% Death§ or Admit Course|| 0.035 DKA related 1 7% 7 47% Other 14 93% 8 53% Height (cm) 15 172.7 (81.3, 193.0) 15 165.1 (124.5, 182.8) 0.134 Whole Body Weight (kg) 15 69.9 (±19.8) 15 65.7 (±14.1) 0.508 BMI (kg/m2)† 15 23.9 (±2.5) 15 23.9 (±2.3) 0.984 C peptide (ng/mL) ¶ 15 0.001 (0.001, 0.060) 15 0.001 (0.001, 0.470) 0.142 HbA1c (%) (mmol/mol) 5 11.5 (±1.5) 102.0 (±16.8) 4 9.6 (±2.8) 80.9 (±30.3) 0.223 Autoantibody Results# 0.403 0 8 53% 7 47% 1 4 27% 2 13% >2 3 20% 6 40% Hospitalization Stay (days) 14 3.2 (0.9, 11.3) 14 3.2 (0.9, 21.7) 0.801 Transport Duration (hrs) 13 16.8 (±4.6) 15 14.4 (±5.7) 0.235 Cytomegalovirus Present 0.999 No 8 62% 9 64% Yes 5 38% 5 36% History of Hypertension 0.999 No 8 62% 9 64% Yes 5 38% 5 36% Heavy Alcohol Use No 14 100% 13 100% History of Cigarette Use 0.482 Diabetes Page 56 of 58
No 12 93% 14 100% Yes 1 7% 0 0% Cocaine Use 0.222 No 13 100% 11 79% Yes 0 0% 3 21% Other Drug Use 0.648 No 10 71% 11 85% Yes 4 29% 2 15% HLA Haplotypes DRB1*04 DQB1*03:02 alone 0.390 Absent 13 87% 10 67% Present 2 13% 5 33% DRB1*03:01 DQB1*02:01 alone 0.999 Absent 10 67% 11 73% Present 5 33% 4 27% Both of Above 0.427 Absent 9 60% 12 80% Present 6 40% 3 20% DRB1*15:01 DQB1*06:02 0.999 Absent 15 100% 14 93% Present 0 0% 1 7% Pancreas Weight (grams) 14 39.2 (11.1, 117.4) 14 33.8 (20.4, 59.8) 0.6625 Fractional Insulin Area (%) 14 0.00 (0.00, 0.14) 15 0.08 (0.00, 0.55) 0.0016 Insulin Mass (mg) 13 1.73 (0.11, 30.80) 14 26.77 (1.27, 174.18) 0.0028 * % reported for all categorical variables; use of mean or median based on evaluation of normal distribution. † Factors used for matching (see Table 1 for characteristics of all donors in study). ‡ Calculated as age at death minus diabetes duration. § Cause or mechanism of death. || Clinical records were examined for DKA in relation to cause or mechanism of death or during the terminal admission history. ¶ A fill value of 0.001 was used when assay results <0.05 (lower limit of detection). # All data values were converted to NIDDK units and defined as positive if one or more of the following applied: GADA if >20, IA2A if >5, or ZnT8A if >0.020. Insulin autoantibodies were excluded from counts for donors with T1D as described in Methods.
Page 57 of 58 Diabetes
Supplementary Table 4. Correlation analyses between numbers of leukocytes/islet and insulitis frequency, diabetes duration, and age of onset. Mean numbers of leukocytes/islet for each donor with T1D and insulitis were analyzed for correlations to other CD markers and insulitis frequency (%), diabetes duration, and age of onset. Spearman r values are shown with grey shading indicating no significant correlations. Numbers of CD3+, CD20+, CD8+ and CD4+ cells/islet showed significant correlations to each other, total leukocytes (CD45), and insulitis frequency (%). Mean numbers of CD11c+ or CD68 cells/islet were significantly correlated with each other and numbers of CD3+ cells/islet but not to other leukocytes or other donor variables. * P<0.05 ** P<0.01 *** P <0.001.
Insulitis Diabetes Age CD45 CD3 CD20 CD8 CD4 CD68 CD11c Frequency Duration onset
(%) (Years) (Years) CD45 1.00 0.95*** 0.96*** 0.90*** 0.85*** 0.58 0.51 0.67* 0.42 0.26 CD3 1.00 0.89*** 0.87*** 0.90*** 0.63* 0.68* 0.78*** 0.24 0.38
CD20 1.00 0.78*** 0.78*** 0.33 0.49 0.56* 0.38 0.07
CD8 1.00 0.85 0.22 0.54 0.62* 0.55 0.08
CD4 1.00 0.33 0.55 0.62* 0.42 0.15
CD68 1.00 0.75 0.33 0.32 0.47
CD11c 1.00 0.53 0.55 0.17
Insulitis Frequency (%) 1.00 0.51 0.05
Diabetes Page 58 of 58
Supplementary Table 5. 43 nPOD cases reported herein were previously examined by Coppieters et al. (N = 14 donors without diabetes, N = 4 AAb+ donors without diabetes, and N = 25 donors with T1D). nPOD ID Diabetes Status 6009 No diabetes 6012 No diabetes 6013 No diabetes 6017 No diabetes 6019 No diabetes 6020 No diabetes 6021 No diabetes 6024 No diabetes 6030 No diabetes 6034 No diabetes 6047 No diabetes 6075 No diabetes 6098 No diabetes 6099 No diabetes 6027 AAb+ 6044 AAb+ 6080 AAb+ 6101 AAb+ 6025 T1D 6026 T1D 6031 T1D 6032 T1D 6035 T1D 6039 T1D 6041 T1D 6045 T1D 6049 T1D 6051 T1D 6054 T1D 6061 T1D 6062 T1D 6063 T1D 6064 T1D 6067 T1D 6076 T1D 6077 T1D 6079 T1D 6083 T1D 6087 T1D 6088 T1D 6089 T1D 6113 T1D 6180 T1D