Inflammatory biomarkers in the blood and pancreatic tissue of organ donors that predict human islet isolation success and function

ABSTRACT

The pancreas of brain-dead donors is the primary source of islets for transplantation. However, brain death mediates systemic inflammation, which may affect the quantity and quality of isolated islets. Our aim was to identify inflammatory biomarkers in donor blood and/or pancreatic tissue capable of predicting islet isolation success. Blood samples were collected from 21 pancreas donors and 14 healthy volunteers. Pancreatic tissue samples were also collected from the corresponding donor during organ procurement. Six serum cytokines were measured by a fluorescent bead-based immunoassay, and the expression of fifteen inflammatory target genes was quantified by quantitative reverse transcription polymerase chain reaction (RT-qPCR). There was no correlation between serum inflammatory cytokines and mRNA expression of the corresponding genes in peripheral blood mononuclear cells (PBMCs) or pancreatic tissue. The IL6 expression in pancreatic tissue correlated negatively with post-isolation islet yield. Islets isolated from donors highly expressing IFNG in PBMCs and MAC1 in pancreatic tissue functioned poorly in vivo when transplanted in diabetic NODscid mice. Furthermore, the increased MAC1 in pancreatic tissue was positively correlated with donor hospitalization time. Brain death duration positively correlated with higher expression of IL1B in PBMCs and TNF in both PBMCs and pancreatic tissue but failed to show a significant correlation with islet yield and in vivo function. The study indicates that the increased inflammatory genes in donor pancreatic tissues may be considered as biomarkers associated with poor islet isolation outcome.

Introduction

Pancreatic islet transplantation has been established as a viable option for the treatment of type 1 diabetes (T1D).^1-3 However, apart from immune rejection, transplanted islets face many challenges, and a significant number fail to engraft. Consequently, transplantation from multiple donors is often required to provide a sufficient number of islets that can effectively function for a prolonged period, leading to a shortage of donor organs and critically limits the potential of islet transplantation.

The pancreas of brain-dead donors is the primary source of islets for transplantation. The condition of the donor at the time of procurement influences organ quality and greatly impacts the outcome of islet isolation. For example, donor-specific factors, such as low body mass index (BMI), high hemoglobin A1c (HbA1c) level, high serum sodium concentration, and prolonged cold ischemia of the pancreas have been shown to correlate with low islet yield and quality.^4-6 In whole organ transplantation, cerebral injury, and brain death (BD) of the donor significantly reduce the graft function, decrease the organ engraftment, and increase the rejection potential.⁷ The mechanisms underlying donor organ damage have not been fully elucidated. Brain death is known to be followed by various reactions, including hemodynamic, inflammatory, neuro-hormonal, metabolic, and blood coagulation responses, resulted in detrimental effects on cells throughout the body.^8,9 Brain death induces systemic inflammation through several mechanisms: for example, circulating inflammatory mediators released from the ischemic brain induce systemic inflammatory responses and up-regulate the expression of cellular adhesion molecules in donor organs,⁷ leading to the increased immunogenicity of donor tissues. Furthermore, inflammatory cytokines released by the donor immune system are toxic and damage cells in donor organs.⁷ Islets are found especially susceptible to inflammatory cytokines, such as tumor necrosis factor (TNF)-α, interleukin (IL)-1β, and interferon (IFN)-γ,^10,11 which are further elevated during the islet isolation process and subsequent transplantation.^12-14 Consistent with these findings, Jung et al. showed that the pancreas of living donors provides greater islet yields than that of brain-dead donors.¹⁵

Our hypothesis set forth to conduct this study was that proinflammatory biomarkers increase in donor blood and/or pancreatic tissue from the time of BD and negatively affect islet isolation outcome. To test this hypothesis, we analyzed data collected at our islet center and measured the expression of proinflammatory biomarkers in serum and peripheral blood mononuclear cells (PBMCs) of pancreas donors and evaluated the correlation between their expression in serum, PBMCs and the pancreatic tissue of the same donor. We further evaluated the correlation of donor proinflammatory biomarkers expression with specific donor factors and islet isolation outcomes.

Results

Comparison of inflammatory biomarkers expressed in the blood of brain-dead pancreas donors vs. healthy controls

Inflammatory biomarkers detected in the blood of pancreas donors were compared to those of healthy controls by measuring cytokine levels in sera and mRNA expression of inflammatory genes in PBMCs.

Inflammatory cytokine expression in serum: Of the 6 cytokines tested, IL-6, IL-8, and IL-10 showed significantly higher concentrations in donor sera than in controls. TNF-α levels were also higher in donors than in controls, but the difference was not statistically significant (Table 1). IL-1β and IFN-γ were undetectable in donor sera.

Table 1.

Serum cytokine levels in brain dead donors and healthy controls.

Cytokines (pg/mL)	BD-donors	Controls	p value
IL-6	67 ± 56.6	4 ± 5.9	.0002
IL-8	22 ± 23.7	3 ± 2.7	.006
IL-10	60 ± 99.9	3 ± 3.4	.04
TNF-α	8 ± 8.4	4 ± 2.5	.17

Inflammatory mRNA expression in PBMCs: IL10 was expressed high in PBMCs of donors, while consistently low in healthy controls PBMCs (Figure 1A). TLR2 and TLR4 expression were widely variable in both groups, but the mean expression levels of both markers were significantly higher in donors PBMCs than controls (Figure 1B,C). The expression of both ICAM1 and MIF was variable, and the donor’s mean expression value was lower than the controls (Figure 1D,E). Expression of FASLG, TNF, CD40LG, and IFNG in PBMCs was also lower in donors (Figure 1F–I).

Figure 1.

Inflammatory mRNA expression in PBMCs – Comparison between brain-dead donors and normal controls: mRNA expression levels of (A) IL10, (B) TLR2, (C) TLR4, (D) ICAM1, (E) MIF, (F) FASLG, (G) TNF, (H) CD40LG, and (I) IFNG were measured by RT-qPCR in PBMCs of donors (n = 21) and control subjects (n = 14) and presented relative to the expression of housekeeping gene ACTB: *P < .05, **P < .01, ****P < .0001 by unpaired Student’s t-tests or Mann-Whitney tests.

Our results have shown that as compared to normal controls, donor blood contains higher levels of IL-6, IL-8, and IL-10 in serum and expresses higher levels of inflammatory cytokine mRNA, especially IL-10, in their PBMCs.

Expression of inflammatory markers in serum, PBMCs, and pancreatic tissue of brain dead organ donors

The expression level of a specific cytokine in serum was compared to the mRNA level in PBMCs and pancreatic tissue in the same donor. Although four cytokines (TNF-α, IL-6, IL-8, and IL-10) were detected in most of the donor sera, their expression level did not correlate with the corresponding mRNA level in PBMCs or the pancreatic tissue. We also examined whether the level of inflammatory biomarkers in PBMCs reflected their expression in the pancreatic tissue of the same donor. Results of the following biomarkers showed the positive correlation between mRNA expression in PBMCs and pancreatic tissue: IFNG (P = .002, r = 0.66), ICAM1 (P = .006, r = 0.61), and IL6 (P = .03, r = 0.5).

Correlation between inflammatory biomarker expression and donor factors

We also assessed possible correlations between several donor factors and inflammatory biomarker expression in donor serum, PBMCs, and pancreatic tissue. Of all the donor factors analyzed, only the BD duration and hospitalization time significantly correlated with the expression of inflammatory biomarkers, as presented in Figure 2. Brain death duration correlated positively with TNF expression in both PBMCs (Figure 2A) and pancreatic tissue (Figure 2B) and with IL1B expression only in PBMCs (Figure 2C), but not pancreatic tissue. Hospitalization time correlated positively with MAC-1 in pancreatic tissue (Figure 2D) but not in PBMCs.

Figure 2.

mRNA expression of inflammatory biomarkers in PBMCs and pancreatic tissue correlate with brain death (BD) duration and hospitalization time. mRNA expression of inflammatory genes was assessed by RT-qPCR. Of all the inflammatory biomarkers tested, only (A) TNF in PBMCs, (B) TNF in pancreatic tissue, and (C) IL1B in PBMCs correlated positively with BD duration. (D) MAC1 in pancreatic tissue correlated positively with hospitalization time. r: Pearson’s correlation coefficient; r_s: Spearman’s correlation coefficient.

Correlations between inflammatory biomarker expression and isolated islet quantity and quality

The correlations between islet isolation outcome and inflammatory biomarker expression in donor serum, PBMCs, and pancreatic tissue were summarized in Figure 3. The expression of IL6 in pancreatic tissue correlated negatively with post-isolation islet yield. Donors expressing higher levels of IL6 in pancreatic tissue yielded significantly lower islet equivalent (IEQ)/pancreas (g) compared to those expressing lower levels of IL6 (Figure 3A).

Figure 3.

mRNA expression of inflammatory biomarkers in donor PBMCs and pancreatic tissues correlates with the islet isolation yield and the reversal of diabetes in mice after transplantation: mRNA expression of inflammatory genes was assessed by RT-qPCR. Inflammatory biomarkers that strongly correlated with the outcome of islet isolation and transplantation were further analyzed by separating donors into low- and high-mRNA expression groups based on cutoff values determined by receiver operating characteristic (ROC) analysis. (A) Islet yield per gram pancreas (IEQ/g) for donors expressing low vs. high IL6 mRNA in pancreas tissue; (B and C) Success rate (%) of diabetes reversal after transplanting NODscid mice with islets from donors who expressed low vs high IFNG mRNA in PBMCs (B) and low vs. high MAC1 mRNA expression in pancreatic tissues (C). *P < .05 by unpaired Student’s t-tests and Mann-Whitney tests.

The functional quality of islets, assessed in vivo by the ability to reverse diabetes in NODscid mice, was negatively correlated with mRNA expression of genes associated with inflammation and adhesion molecules. Donors expressing higher levels of IFNG in PBMCs had significantly lower diabetes reversal rates, as compared to those expressing lower levels (Figure 3B). Similarly, donors expressing higher levels of MAC1 had significantly lower diabetes reversal rates compared to those expressing lower levels (Figure 3C). No other inflammatory markers correlated with isolation yield or islet function. Our analysis indicated the negative correlation between diabetes reversal rates with the donor hospitalization period (Figure 4).

Figure 4.

Correlation between donor hospitalization time and success rate (%) of diabetes reversal after transplanting islets into NODscid mice: the success rate to reverse diabetes (%) in NODscid mice showed correlation with the hospitalization time of the donor. r: Pearson’s correlation coefficient.

Discussion

The success of pancreatic islet transplantation relies on both the quality and quantity of the isolated islets. Islets are sensitive to the catastrophic conditions of the brain-dead donor, including inflammation involving inflammatory cell activation, infiltration, and increased their products.^16,17 The aim of this study was to determine if prolonged BD period negatively affects islet isolation outcome, as indicated by increased inflammatory biomarkers in donor blood and pancreatic tissues. The evaluation of the two-layer methods for pancreas cold preservation, even though its benefit was not proven,^18,19 provided us the opportunity to collect donor tissue samples by a single individual, uniformly perform pancreas procurement and preservation, and conduct an assessment of inflammatory markers within appropriate time periods.

We found that serum levels of pro-inflammatory cytokines, IL-6 and IL-8, were higher in BD donors than healthy controls, but no correlation was found between their levels and islet yield or in vivo islet function. In addition, we also found higher levels of IL-10 in sera and IL10 in PBMCs of BD donors than controls, as previously reported the association of brain injury with the increased anti-inflammatory cytokine IL-10.^20,21 However, the elevated IL-10 and IL10 levels again failed to correlate with the islet isolation outcome. These findings indicate that the inflammatory cytokines in the sera of brain dead donors tested in this study do not predict the islet isolation success.

We have found that IFNG is expressed at lower levels in donor PBMCs as compared to normal controls and the IFNG expression level in donor PBMCs negatively correlates with the reversal of diabetes in vivo in immunodeficient mice (Figure 3B), indicative of IFNG as a key biomarker for poor islet quality. IFN-γ induces MAC-1 expression. MAC-1 expressed by immune as well as endothelial cells are responsible, in part, for the adhesion of leukocytes to endothelial cells. As IFNG expression in PBMC, MAC1 expression in pancreatic tissue also associated with the failure to reverse diabetes in NODscid mice (Figure 3C). Furthermore, the MAC1 expression in pancreatic tissue was found to positively correlate with hospitalization time (Figure 2D). Our results have also shown that higher levels of IL6 in the donor pancreatic tissue, but not serum IL-6, are associated with lower islet yield (Figure 3A). IFN-γ regulates IL6 expression in monocytes and plays an important role in IL-6 signaling.²² These results might be attributed to the potential activation of leukocytes and endothelial cells through interactions between inflammatory cytokines and adhesion molecules, therefore influencing islet function. Additional studies would be helpful to determine such a cause/effect relationship.

It is well known that TNF-α, as well as IL-1β alone, cannot induce β cell apoptosis. However, both cytokines increase β cell apoptosis significantly in the presence of IFN-γ.^10,11 Our results have shown that longer BD duration does not clearly influence islet isolation outcome and in vivo function in spite of elevated TNF in donor PBMCs and pancreatic tissue and IL1B in PBMCs (Figure 2A–C). Although BD duration did not correlate with islet function, hospitalization time negatively correlated with the success rate in reversing diabetes (Figure 4).

This study was performed using samples collected and analyzed between 2008 and 2009. During the past decade, the improvements in donor management have continued.^23,24 Among those changes, the use of steroids should have significant potential to affect the inflammatory status of organ donors, which might influence the outcome of islet isolation. In the donors involved in this study, the use of steroids, Solumedrol, was inconsistent and limited to 57% of the donors. We did not notice a clear impact of the presence or absence of steroids on the levels of inflammatory markers and the function of isolated islets. These results were similar to other studies that investigated the effect of steroid treatment on the outcome of the liver and cardiac transplants.^24-26 We believe that our results are highly relevant to current practices. Nevertheless, a large-scale follow-up study is warranted.

Our findings suggest that inflammatory biomarkers expressed in PBMCs and pancreatic tissue may predict islet isolation and transplantation outcomes. However, logistically, it may not be practical to be used as markers determining the acceptance of a donor organ. These inflammatory biomarkers – along with islet quality assessment test results,^27,28 islet number, viability, morphology, functional and metabolic assays,²⁹ and islet gene signatures³⁰ may be useful to determine the suitability of islets for transplantation. Furthermore, the study emphasizes the importance of maintaining proinflammatory gene expressions at low levels, in particular, MAC1^31,32 in pancreatic tissue of the donors with prolonged hospitalization, which may be achieved by an appropriate anti-inflammatory treatment.

Material and methods

Donor sample collection

We had the opportunity to procure the pancreas, peripheral blood, and pancreatic tissue samples from 27 BD-donors using a uniform technique during the period from July 2008 to October 2009. During this period, 57 pancreases were offered by organ procurement organizations (OPOs) to the Southern California Islet Cell Resource Center (SC-ICRC), City of Hope, for islet isolation. Blood and tissue samples were collected from 27 of the 57 pancreas donors offered by a single local OPO, One Legacy (Los Angeles, CA) to minimize the influence of external factors on islet isolation outcome. Blood and pancreas tissue samples were collected by a single SC-ICRC member, and the pancreas was procured and cold preserved by a single team using the two-layer method^18,19 for shipment. Islets isolated from the remaining 30 pancreases were not included in this study as they were procured at distant sites or by other OPOs. Authorization of organ and tissue acquisition for research was obtained by OPO coordinators, either as Document of Gift via the California Department of Motor Vehicles website or written authorization obtained from the next of kin.

Venous blood was collected before aortic cross-clamp and stored in a sterile red-capped tube and a spray-coated K2-EDTA purple-capped tube (BD, NJ, USA). The red-capped tube was kept at room temperature and the K2-EDTA tube was kept on ice until serum and PBMCs separation at the SC-ICRC laboratory, approximately 6 hours after collection. After the removal of excessive adipose tissue, a small specimen was taken from the head of the pancreas, placed in a 1.7-mL microcentrifuge tube, snap-frozen, and kept on dry ice. The organ was placed in the two-layer preservation container for transportation. Upon arrival at the SC-ICRC laboratory, the pancreas was processed for islet isolation, the tissue sample tube was placed in a − 80°C freezer for further RNA extraction, and blood was processed as described below.

Peripheral blood mononuclear cells (PBMCs) isolation, RNA extraction, and RT-qPCR

The Histopaque-1077 (Sigma-Aldrich, MO, USA) density gradient centrifugation method was used to isolate PBMCs from the blood sample immediately after arrival at the SC-ICRC, according to the manufacturer’s instruction. Isolated PBMCs were washed 3 times with phosphate-buffered saline (PBS), homogenized in 1 mL Tri Reagent (Molecular Research Center Inc., Ohio, USA) in a 1.7-mL microcentrifuge tube, and stored at −80°C. RNA extraction and RT-qPCR were performed within a few months after sample collection. For RNA extraction from PBMCs and pancreatic tissue, 2 μL Polyacryl carrier (Molecular Research Center Inc., Ohio, USA) was added to 1 mL homogenate and incubated for 5 min at room temperature, followed by the addition of 100 μL 1-bromo-3-chloropropane (BCP) (Molecular Research Center Inc., Ohio, USA) before RNA isolation according to the manufacturer’s instructions. Isolated RNA was stored in RNA storage solution (Thermo Fisher Scientific, Grand Island, NY) at −80°C. RNA samples were quantified spectrophotometrically at 260/280 nm and 2.0 µg RNA from each sample was reverse-transcribed into double-stranded cDNA using the SuperScript III First Strand Synthesis System (Thermo Fisher Scientific). Gene expression was quantified by RT-qPCR using TaqMan Universal PCR Master Mix and TaqMan Gene Expression assay probes (Thermo Fisher Scientific) measured in an Applied Biosystems 7300 Real-Time PCR System (Thermo Fisher Scientific). Thermocycling conditions were as follows: pre-denaturation at 95°C for 20 min, 40 cycles of denaturation at 95°C for 15 seconds, and annealing/extension at 60°C for 60 seconds. mRNA was measured for the following fifteen target genes: IL-1β (IL1B), IL-6 (IL6), IL-8 (IL8), IL-10 (IL10), TNF-α (TNF), IFN-γ (IFNG), macrophage migration inhibitory factor (MIF), macrophage-1 antigen (MAC1), intracellular adhesion molecule 1 (ICAM1), nuclear factor-kappa B subunit 1 (NFKB1), toll-like receptor 2 (TLR2), toll-like receptor 4 (TLR4), TNF superfamily member 10 (TNFSF10), Fas ligand (FASLG), CD40 ligand (CD40LG), and one housekeeping gene, actin beta (ACTB). The results were analyzed using 7300 Sequence Detection Software (version 1.3) and relative gene expression was calculated using the 2^−∆∆CT method.

Serum cytokine measurement

Serum was separated from donor blood collected in a red-capped tube immediately after arrival at the SC-ICRC and stored at −80°C. Cytokine assay was performed within a few months after all test samples were collected. Cytokine measurements were performed by the City of Hope Clinical Immunobiology Correlative Studies Laboratory (CICSL) with a fluorescent bead-based immunoassay using a Human Cytokine/Chemokine Magnetic Bead Panel kit (Millipore, MA). The concentrations of six cytokines, IL-1β, IL-6, IL-8, IL-10, IFN-γ, and TNF-α, were measured in duplicate following the standard operating procedures of the CICSL.

Control blood sample collection

All volunteers (n = 14) who participated in this study were healthy, asymptomatic adults, included eight females and six males, ages ranging 30- to 55-years-old. All volunteers were provided and signed the informed consent to participate in this study, which was approved by the Institutional Review Board at the City of Hope Medical Center and the Beckman Research Institute. Blood samples were collected in a sterile red-capped tube, which was kept at room temperature and in a sterile spray-coated K2-EDTA purple-capped tube, which was kept on ice for approximately 6 hours before serum and PBMCs were isolated. These samples further underwent the same methods as donor samples

Pancreas donor data collection

Donor information was obtained from the chart that accompanied by the pancreas. Based on donor information, five of the 27 donors were excluded from the study for the following reasons: one donor with HbA1c >6.5% had type 2 diabetes for >8 years; two donors had severe polytrauma and received several blood infusions; one donor was positive for multiple narcotics detected by toxicology tests; and one donor had intracranial hemorrhage and a brain tumor. The pancreas of another donor was also excluded because the organ was used to test a new cold preservation solution. After removing the above six donors, the following information was collected from the charts of the 21 remaining donors: age, gender, cause of death, body mass index (BMI), HbA1c (%), cardiac arrest duration, BD duration, hospitalization time (i.e., time between hospital admission to aortic cross-clamp for organ procurement), blood glucose levels (highest, lowest, and at admission), highest amylase and lipase levels, and steroid treatment.

Donor demographics

Donor demographics are shown in Supplemental Table 1. Of the 21 donors, 10 were males and 11 were females. Donor characteristics that may influence islet isolation results were: donor age: 18–72 (median 49); BMI: 22.4–39.1 (median 32.0); HbA1c: 4.7–6.2% (median 5.5%); BD duration: 23–69 hours (median 43 hours) and hospitalization time: 1.8–15.3 days (median 3.5 days).

Human islet isolation and data collection

Islets were isolated from donor pancreas by the SC-ICRC islet manufacturing team in the City of Hope’s current Good Manufacturing Practice (cGMP) facility following the standard operating procedures (SOPs) approved by Food and Drug Administration (FDA). Islet isolation data were collected from islet isolation batch records maintained by the SC-ICRC. Results are summarized in Supplemental Table 2: pancreas weight ranged 47.4–134.5 g with median 96.0 g; cold ischemia time (defined as the time from cross-clamp to enzyme infusion into the pancreatic duct) ranged 4.2–9.2 hours (median: 6.4 hours); the post-isolation islet number ranged 80,425–525,058 IEQ (median: 226,017 IEQ); and the median of post-isolation islet yield per gram of pancreas 2,466 IEQ/g ranging 682–5,621 IEQ/g. One IEQ is considered equivalent to an islet with a diameter of 150 μm.

Assessment of in vivo islet function by transplanting in diabetic NODscid mice

Male non-obese diabetic severe combined immunodeficient (NODscid) mice, ages 10–12 weeks, were obtained from Charles River Laboratory (CA) and maintained in the Animal Resources Center of the Beckman Research Institute of the City of Hope. Mice were rendered diabetic by an intraperitoneal injection of 50 mg/kg Streptozotocin (Sigma-Aldrich, St. Louis, MO) on three consecutive days. Mice that exhibited hyperglycemia (blood glucose levels >350 mg/dL for two consecutive days were used as islet recipients. To assess islet function in vivo, islets (1200 IEQ) were transplanted under the kidney capsule of diabetic mice (3–4 mice per each islet donor), and blood glucose levels were measured 2 times weekly after transplantation. This is the standard procedure for assessing islet quality in vivo developed by SC-ICRC and has been used by several other islet transplantation centers.³³ Recipient mice that reduced and maintained blood glucose levels <200 mg/dL were considered to have reversed diabetes, as blood glucose <200 mg/dL is accepted as normoglycemia in normal untreated mice.³⁴ The kidney carrying islets was removed >30 days after transplantation to confirm the recurrence of hyperglycemia and graft-dependent euglycemia. The median success rate of diabetes reversal in NODscid mice was 33% (range: 0–100%) in this study. All animal procedures were approved by the Institutional Animal Care and Use Committee of Beckman Research Institute, City of Hope.

Statistical analysis

Serum cytokine levels and mRNA expression in donors and controls were compared using Student’s t-tests. Correlations between inflammatory biomarkers present in donor serum, pancreatic tissue, and PBMCs and measures of islet transplant success were identified using Pearson’s coefficient for parametric comparisons and Spearman’s coefficient for non-parametric comparisons. Receiver operating characteristic (ROC) analysis of these results (R statistical package v3.4, library ROCR) was then used as an exploratory method to determine the optimal cutoff values with which to classify cytokine biomarker expression. Two groups were then defined as having either “high” or “low” cytokine expression based on whether they fell above or below the cutoff value, respectively. Measures of isolation success, such as islet yield and rate of diabetes reversal in mice, were then compared between groups using Student’s t-tests or Mann-Whitney tests for non-parametric distributions. Two-sided probability values were used in this study, and P < .05 was used to determine statistical significance.

Funding Statement

This work was supported by the National Institute of Diabetes and Digestive and Kidney Diseases [SC-ICRC 5U42RR016607]; Nora Eccles Treadwell Foundation [Cure of Diabetes].

Abbreviations

ACTB	actin beta
BCP	1-bromo-3-chloropropane
BD	brain death
BMI	body mass index
CD40LG	CD40 ligand
cGMP	current Good Manufacturing Practice
CICSL	Clinical Immunobiology Correlative Studies Laboratory
FASLG	Fas ligand
FDA	Food and Drug Administration
HbA1c	hemoglobin A1c
ICAM1	intracellular adhesion molecule 1
IEQ	islet equivalent to a spherical islet 150 μm in diameter
IFN	interferon
IL	interleukin
MAC1	macrophage-1 antigen
MIF	macrophage migration inhibitory factor
NFκB	nuclear factor-kappa B
OPO	organ procurement organization
PBMCs	peripheral blood mononuclear cells
PBS	phosphate buffered saline
RT-qPCR	quantitative reverse transcription polymerase chain reaction
SC-ICRC	Southern California Islet Cell Resource Center
TLR	toll-like receptor
TNF	tumor necrosis factor
TNFSF	TNF superfamily member
T1D	type 1 diabetes

Disclosure of potential conflicts of interest

No potential conflicts of interest were disclosed.

Acknowledgments

We thank the islet manufacturing team at the SC-ICRC for isolating human islets and the CICSL at City of Hope for conducting serum cytokine assessments. We thank Dr. Kerin Higa for her critical reading and editing of the manuscript. We also thank Ms. Miryam Mehra, Organ Acceptance Manager of the SC-ICRC, and Ms. Christina Wheeler, Transplant Administrator at One Legacy, for providing information about organ procurement.

Supplementary Materials

Supplemental data for this article can be accessed on the publisher’s website.