Insulitis Leukocyte PhenotypingParaffin sections from blocks having the maximum insulitis frequency for each donor were stained, and positive leukocytes/insulitic islets (six or more CD3+ cells) were counted using multi-immunofluorescence. Serial sections (4 μm) were dewaxed and rehydrated with Tris buffer. Heat-induced antigen retrieval was performed using Trilogy (Cell Marque, Rocklin, CA) at 95° for 20 min followed by rinsing in water for 20 min. The staining series was designed to phenotype leukocytes (total leukocytes [CD45], T [CD3] and B [CD20] lymphocytes, T-lymphocyte subsets [CD4 and 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 the corresponding secondary antibody and conjugated fluorochrome (AF488-AF555-AF647) (Supplementary 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 Amplification Kit (PerkinElmer, Waltham, MA) was used according to the manufacturer's instructions. All sections were mounted with ProLong Gold Antifade mounting media containing DAPI (Life Technologies, Grand Island, NY). Positive controls included human spleen, tonsil, and donor intrapancreatic lymph nodes, and negative controls included omission of the primary antibody.The numbers of leukocytes/insulitic islets were determined using multichannel image acquisition software on a Zeiss Axiophot microscope (AxioVision; Carl Zeiss Inc., Thornwood, NY). Fluorescent channels were viewed in combination with DAPI to count the number of positive leukocytes/islet.

β-Cell and α-Cell Area and MassInsulin- 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 six sections was used per donor (two sections/head, body, and tail regions). The β-cell and α-cell areas were expressed as a ratio (percent) to the total sectional area, including acinar and interstitial regions, to permit the use of pancreata weights. The average β-cell and α-cell area per pancreas was calculated from regional area averages. The β-cell or α-cell mass (in milligrams) was calculated by multiplication of the respective average area and pancreas weight (in grams).

Insulitis Screening and Insulitic Islet Subtyping for Insulitis FrequenciesPancreata were processed to formalin-fixed paraffin blocks for each pancreas region (head, body, and tail) as previously described (20). For each donor, serial sections (average two blocks per region) were stained by hematoxylin-eosin and two double-immunohistochemistry (IHC) stains (Ki67 and insulin, CD3 and glucagon) (Supplementary Table 2) (21). When insulitic islets were found in a given donor, additional blocks were screened (as detailed below). Stained sections were scanned at ×20 magnification using an Aperio CS scanner (Leica/Aperio, Vista, CA), and all images were stored in an online pathology database (eSLIDE; Leica/Aperio).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 six or more CD3+ cells immediately adjacent to or within the islet with three or more islets per pancreas section, according to recent criteria (4). Islets with insulitis were marked in an image layer using ImageScope software (Leica/Aperio). The two IHC serial images were aligned using the synchronization tool, and insulin+ islets were also marked on the image layer. All islets/sections 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 insulin− islets using the CD3-glucagon image). The number of pancreas sections subtyped for insulitis ranged from 2 to 16 sections/donor (8.1 ± 4.1 sections/donor, n = 162 sections) (Table 1). The lowest number of available sections was due to partial pancreas recovery (tail only in nPOD 6198).Insulitis frequency (percent) was calculated as the total number of insulitic islets (sum of insulin+ CD3+ and insulin− CD3+ islets) divided by the total number of islets (sum of four subtypes). The frequency of insulin+ insulitic islets in relation to the total number of insulin+ islets was determined by the ratio of (insulin+ CD3+ islets)/(sum of insulin+ CD3− and insulin+ CD3+ islets) with similar calculations for the frequency of insulin− insulitic islets (insulin− CD3+)/(sum of insulin− CD3+ and insulin− CD3−).