Low energy X-ray (grenz ray) treatment of purified islets prior to allotransplant markedly decreases passenger leukocyte populations

ABSTRACT

Grenz rays, or minimally penetrating X-rays, are known to be an effective treatment of certain recalcitrant immune-mediated skin diseases, but their use in modulating allograft rejection has not been tested. We examined the capacity of grenz ray treatment to minimize islet immunogenicity and extend allograft survival in a mouse model. In a preliminary experiment, 1 of 3 immunologically intact animals demonstrated long-term acceptance of their grenz ray treated islet allograft. Further experiments revealed that 28.6% (2 of 7) grenz ray treated islet allografts survived >60 d. A low dose of 20Gy, was important; a 4-fold increase in radiation resulted in rapid graft failure, and transplanting a higher islet mass did not alter this outcome. To determine whether increased islet allograft survival after grenz treatment would be masked by immunosuppression, we treated the recipients with CTLA-4 Ig, and found an additive effect, whereby 17.5% more animals accepted the graft long-term versus those with CTLA-4 Ig alone. Cell viability assays verified that islet integrity was maintained after treatment with 20Gy. As well, through splenocyte infiltration analysis, donor CD4+ T cell populations 24-hours after transplant were decreased by more than16-fold in recipients receiving irradiated islets compared with control. Donor CD8+ T cell populations, although less prevalent, decreased in all treatment groups compared with control. Our results suggest that brief treatment of isolated islets with low energy grenz rays before allotransplantation can significantly reduce passenger leukocytes and promote graft survival, possibly by inducing donor dendritic cells to differentiate toward a tolerogenic phenotype.

Introduction

In 2000, the use of a glucocorticoid-free immunosuppression protocol pushed the feasibility of islet transplantation forward for patients with Type 1 Diabetes. Rejection of islets, however, is still a major obstacle in this transplant setting. Emphasis on immunosuppressive therapy and treatment in islet transplantation is predominantly recipient-based, with little emphasis on donor tissue. Given the prevalence of antigen presenting cells (APC), dendritic cells, macrophages and passenger leukocytes bearing MHC II antigens in donor tissue and their suspected importance as stimulators of the immune response, treatment of donor tissue may be warranted to minimize these effects in allotransplantation.¹ Pre-treatment of donor islets before transplant to reduce their immunogenicity could reduce the amount of immunotherapy required by the recipient, affecting the direct and indirect pathways of rejection. Previous work has been performed trying to decrease, eliminate or inactivate the passenger leukocyte population in islet preparations using irradiation, long-term culture, low temperature culture, and adding antibodies to block receptors.^1-8 Ablamunits and Baranova et al. decreased leukocyte populations by dissociating islets followed by re-aggregation.³ In 1989, using an allogeneic islet transplant rat model, James and colleagues found that low dose gamma irradiation (2.5Gy) was insufficient in prolonging graft survival. However, in combination with irradiation, the addition of cyclosporine depleted the passenger leukocyte population prolonging graft survival in 100% of cases for >100 days.⁴ Similarly, it has been demonstrated that direct irradiation of recipient lymphatic organs followed by treatment with anti-CD4 led to prolonged cardiac allograft survival, as compared with individual therapies alone.⁹ In contrast, Shizuru and colleagues prolonged islet allograft survival with anti-CD4 treatment alone and in combination with depletion of APCs by low temperature culture, between highly divergent species.^9,10

At high doses, radiation causes an activated inflammatory response, however when administered at a low dose, it can modulate inflammation by affecting leukocyte populations, cytokine production (increasing TGF-β and IL-6) and by reducing reactive oxygen species.^11,12 Grenz rays, or very low energy, minimally penetrating X-rays, have been used historically to successfully treat a variety of benign immune-mediated skin disorders such as chronic eczema and psoriasis. Although no longer commonly used in dermatology, grenz ray treatment has been shown to be safe at cumulative doses <100 Gy per field and lifetime, and this therapeutic modality is known to be remarkably effective for certain recalcitrant dermatosis (nail psoriasis, hyperkeratotic frictional hand dermatitis, etc.). Because grenz rays have low penetration (all energy is dissipated within the first ≈2 mm of the skin), we reasoned that this form of radiation might have a significant immunomodulatory effect on isolated islets, which because of their small dimensions (unlike larger solid organs) would be able to absorb most or all of the energy delivered by grenz rays.

Lymphocytes and dendritic leukocytes present in allografts have been shown to migrate from the graft into the recipient lymphoid tissue (e.g. spleen), in the first 4–5 d after transplant, rather than remaining within the graft itself. Such donor cells can persist in the recipient long after the transplant procedure, and they can localize near CD4 T cell populations and act as stimulators toward allorejection.^13,14

Radiation therapy has been used as antiproliferative therapy in cancer patients, and in many forms and doses radiation has been shown to be immunosuppressive.^12,15,16 Grenz ray therapy, or ultra-soft X-ray, has been replaced with immunosuppressive medications as a treatment modality for a variety of skin disorders.^11,17-19 Grenz rays represent very low energy radiation, which typically penetrates skin only to a depth of about 2 mm, and these rays can be absorbed by an individual cell.^11,18-21 Most studies in transplantation with low dose radiation therapy involved whole donor irradiation, while studies using Grenz ray therapy historically involved treatment of localized skin conditions with an emphasis on patient results.^9,17-19,22 Given the potential benefit of low dose radiation, we used a mouse allograft model to investigate if pretreatment of isolated donor islets with grenz rays would prolong their survival posttransplant, and in animals with or without additional immunosuppression.

Results

Cytological evaluation of Islets after low dose irradiation

After isolation islet membrane integrity was examined. Administration of low dose, 20Gy (2000rad), irradiation was followed by 1-hour culture, islet membrane integrity was maintained at 84.6% ± 7.8 and was not significantly different than islets tested immediately after isolation, 87.8 ± 1.4, or after 1-hour culture with no irradiation treatment at 88.4% ± 1.5, n = 4 (Fig. 1A and C). Increasing the irradiation dose to 80Gy, a 4-fold increase, affected islet integrity where percentage viability dropped to 66.0%, n = 2 (data not shown). Apoptosis was also assessed by TUNEL after irradiation treatment, with no significant difference observed between treated (9.3% ± 3.5) and non-treated groups (24.3% ± 8.3) when compared with islets assayed immediately after isolation (13.4 ± 1.3, Fig. 1B and D).

Figure 1.

Cytological evaluation of mouse islets after low dose irradiation. BALB/c mouse islets were isolated and analyzed immediately post-isolation, with irradiation + 1 hour incubation and without irradiation + 1 hour incubation, n = 4. Incubations were at 37°C and 5% CO₂. Islets treated with SYTO® 13 Green-Fluorescent nucleic acid stain which permeates live cells with intact membranes (green) were not significantly different where 87.8% ± 1.4 of the islets post-isolation stained green compared with those cultured for 1 hour without irradiation at 88.4% ± 1.5 and those irradiated at 84.6% ± 7.8 (n = 4, A and C). Ethidium bromide stains for dead cells, where membrane integrity has been compromised (red) (A and C). Apoptosis was measured by TUNEL staining and there was no significant difference between the percentage of TUNEL+ cells in the post-isolation group at 13.4% (n = 2), the islets cultured for 1 hour with no irradiation at 24.3% ± 8.3 (n = 4) and after culture 1 hour with irradiation treatment 9.3 ± 3.5 (n = 5) (B and D). Apoptotic TUNEL-positive cells (yellow/green) were identified within islets stained for insulin (red) and counterstained with DAPI (blue) (D). Bar represents 100 μm.

Efficacy and glucose tolerance after allotransplant with low dose irradiated Islets and immunosuppression

Islets were isolated, treated and cultured for 1-hour before transplant under the kidney capsule. Graft survival, measured by percentage of euglycemic animals in non-treated control groups did not extend past 22 d (Fig. 2). In contrast, 2 out of 7 (28.6%) recipient mice that received irradiated donor islets with no subsequent immunosuppression showed prolonged graft survival up to 60 d posttransplant (*p<0.05). The same dose was previously tested with one of 3 mice accepting the graft long-term (unpublished). Given immunosuppression, CTLA-4 Ig, alone, 62.5% of animals (5 of 8) had prolonged graft survival, >60 days, and this was increased to 80.0% when the donor islets were irradiated before transplant (8 out of 10, with both irradiation and CTLA-4 Ig, ns p>0.05) (Fig. 2). Increasing the radiation dose 4-fold resulted in graft failure by 21 d (Figure S1). Increasing the number of islets transplanted from 500 to 700 islets per transplant produced similar graft survival proportions irrespective of dose (2 of 7 at 50 days) (Figure S1, *p<0.05 compared with control).

Figure 2.

Allograft survival in mice after donor islet treatment with low dose irradiation with or without immunosuppression. BALB/c mouse islets were isolated and exposed to 20Gy low dose irradiation and incubated 1 hour before transplant into diabetic C57BL/6 recipient mice. Treated mice were administered intraperitoneal CTLA-4 Ig at 10 mg/kg one hour before transplant (day = 0) and day = 2, 4 and 6 posttransplant. Control and CTLA-4 Ig alone groups did not receive irradiation exposure. Graft survival measured by percent euglycemic, in non-treated control animals did not extend 22 d. Two of 7 (28.6% survival proportion) recipient mice that received irradiated donor islets without subsequent immunosuppression exhibited prolonged graft survival up to 60 d posttransplant (*p < 0.05 at 60 days). Given immunosuppression alone, 62.5% (5 of 8) of animals had prolonged graft survival, with a 17.5% increase in recipients receiving irradiated donor islets followed by CTLA4-Ig (8 of 10, 80%) (ns).

To assess graft function, glucose tolerance was tested approximately 50 d after transplant on normoglycemic mice (Fig. 3). Mice that received irradiated islets with no immunosuppression after transplant exhibited a slight delay in response to glucose challenge, which was non-significant compared with the naïve animals tested. There was no delay between naïve animals and those that received irradiated islets followed by immunosuppressive therapy. There was a significant difference between irradiated islets and those that received immunosuppression, CTLA-4 Ig (*p < 0.05).

Figure 3.

Intraperitoneal glucose tolerance test on euglycemic mice transplanted with donor islets treated with low dose irradiation followed by recipient immunosuppression. BALB/c mouse islets were isolated and exposed to 20Gy low dose irradiation and incubated 1 hour before transplant into diabetic C57BL/6 recipient mice. Treated mice were administered intraperitoneal CTLA-4 Ig at 10 mg/kg one hour before transplant (day = 0) and day = 2, 4 and 6 posttransplant. Euglycemic animals were administered 3 g/kg of 25% dextrose and blood glucose was monitored at baseline (time = 0), 15, 30, 60, 90 and 120 minutes (A). There was a delayed glucose response in the irradiated treatment group, although not significant in comparison to the naïve animals but different from those that received CTLA-4 Ig alone (*p < 0.05). Data represented as Area Under the Curve (B).

Immune response 24 hours after allotransplant with low dose irradiated Islets

A sub-set of mice was killed 24 hours after irradiated islets were transplanted to determine the population of T cells present in the spleen. Recipient (H-2kb+) CD4+ populations were not significantly different between groups regardless of treatment (Fig. 4A). Donor (H-2kd+) CD4+ populations within the recipients treated with irradiation decreased by 16.5 times compared with the control (0.004% vs. 0.067%, n = 5, Fig. 4B and E). There was no significant difference between the mouse splenocytes removed from animals that were transplanted with irradiated islets and administered CTLA-4 Ig immunosuppression therapy, although they did decrease by 10 times when compared with the control (0.007% vs. 0.067%, n = 5). Recipient levels of CD8+ cells were not significantly different between groups (Fig. 4C). Few donor CD8+ T cells were detected in the recipients 24 hours after transplant (Fig. 4D). However, there were lower CD8+ populations in all treatment groups (irradiated 0.004%, combination treatment 0.002%) compared with control (0.038%, n = 5, Fig. 4D and E). There were no CD8+ donor cells detected in the mice treated with only CTLA-4 Ig (n = 5, Fig. 4D). Although donor CD4+ and CD8+ cell populations were very low, they were all higher in the islet transplant recipient mice than in the mice that received a sham operation where there were no detectable donor cells (data not shown).

Figure 4.

CD4+ and CD8+ (T)cell expression levels of recipient and donor populations in splenocytes from low dose irradiation islet recipients 24 hours posttransplant. Isolated BALB/c mouse islets were exposed to low dose irradiation and incubated 1 hour before transplant into diabetic C57BL/6 recipient mice. Treated mice were administered intraperitoneal CTLA-4 Ig at 10 mg/kg one hour before transplant. A sham operation was tested to determine the effect of the operation, itself, on T cell populations and there were no detectable donor cells present (data not shown). Twenty-four hours posttransplant recipient splenocytes were sorted by flow cytometry into C57BL/6 recipient mouse b haplotype cells (H-2Kb⁺), T cell receptor⁺ cells (TCRβ⁺) and CD4+ or CD8+ T cell populations (A, C, E); and compared with BALB/c donor mouse d haplotype cells (H-2Kd⁺), T cell receptor⁺ cells (TCRβ⁺) and CD4+ or CD8+ T cell populations (B, D, E). Recipient CD4 populations were similar between all groups regardless of treatment (A). Regardless of treatment group, donor CD4+ and CD8+ populations were very low in the recipient. Donor CD4 populations within the recipient decreased by 16.5 times with irradiation treatment alone when compared with control (n = 5) and by 10 times with the combination treatment (B, E; n = 5). Recipient populations of CD8+ cells remained unchanged (C). Donor CD8+ T cells were decreased in all treatment groups 24 hours after transplant; no CD8+ T cells were detected in the CTLA-4 Ig alone treatment group (D, E).

Discussion

Grenz rays were first described by Bucky et al. in 1923 when he noted that the rays were able to deliver an effective dose of irradiation with a minimal amount of damage to the treatment tissue or to surrounding tissues and organs.²⁰ The therapeutic effects of the rays were considered optimal for dermatitis and certain skin conditions because of their low penetrating depth of 1–2 mm.^18-20 Taking this into consideration, we hypothesized that the effect could be beneficial when exposing islets to these rays before transplant. We did not see any signs of membrane integrity damage or increased apoptosis in islets after exposure to 20Gy of grenz ray.

Using an allotransplant model, we initially transplanted the grenz ray treated islets without subsequent immunosuppression and surprisingly found that 28.6% of the mouse islet allografts survived long-term and became tolerized. Although the increase seems marginal, it is important to note that the cells are treated after donor isolation and before patient recipient transplant. The recipient, in this case, is not subjected to additional drug therapy to achieve increased transplant graft survival. This effect was mirrored in other studies where very pure islets, devoid of acinar and ductal tissue, and containing potentially a decreased number of passenger leukocytes, decreased immunogenicity and resulted in long-term survival in a small fraction of recipients without additional immunosuppression of the recipient.^7,23 When islets were subjected to irradiation and immunosuppression therapy was subsequently administered to the recipient, a similar trend was observed where there was a cumulative 17.5% increase in graft survival in the combination treatment vs. immunosuppressive therapy alone.

Glucose homeostasis was normal when islets were pre-treated with irradiation followed by immunosuppression therapy. In contrast, we observed an impaired glucose response in animals transplanted with irradiated islets without subsequent immunosuppression therapy. It would be impractical to think that irradiation treatment alone would be sufficient to completely prevent rejection. However, it would also be negligent to discredit the observed increase in islet survival in irradiated donor islet recipients, which we accredit, at least in part, to a decrease in donor passenger leukocytes.

Allorejection, to some extent, involves the donor dendritic cell population and passenger lymphocyte population.^13,14,24,25 In a study where splenectomies were performed in rodents with renal and cardiac allografts, prolonged graft survival rates were observed in animals with reduced intra spleen graft-derived dendritic cells.^13,26 Building on this finding, we removed the spleens from recipient mice 24 hours post-islet transplant to determine if the donor T cell population was reduced after irradiation treatment. The recipient population of self-T cells remained consistent regardless of the treatment conditions, with a slight increase in T cells in the irradiated islet treatment group receiving immunosuppression, although non-significantly. We also found CD4/CD8 double-positive cells present in both the control and treated samples, with an occurrence of < 1% of the total T cell population, a commonly found frequency in mice.^27,28

It was observed, as expected, that donor CD4+ T cells were indeed decreased in the irradiated group and in the irradiated CTLA-4 Ig combination treatment group when compared with the controls; albeit not statistically significant. The donor CD8+ population likewise was decreased, in all treatment groups when compared with control; no CD8+ T cells were found in the CTLA-4 Ig treatment groups.

To decrease immunogenicity, it is currently well practiced to culture islets in lower temperatures (22–24°C). Moreover, islets are maintained in culture, rather than transplanted immediately after isolation, reducing the acinar, ductal and endothelial tissue components of the islet preparation, thus decreasing antigen presentation to the host, prospectively extending islet graft survival. There is always variability between donors and between recipients and this heterogenicity affects the transplant outcome. It would be naïve, for us to believe that all the passenger leukocytes are affected by the irradiation of the donor islets. There may be some islets or portions, thereof, that were not penetrated by the grenz rays and may therefore elicit an immune response from the recipient. The procedure would require further refinements and optimization to move forward. Even though both the recipient and donor are in-bred strains in this study, there is still variability between individuals and their individual cell types i.e. islets and immune population, and the transplant recipient immune reaction and profile would also affect outcomes and graft rejection. Numerous treatments and procedures are cumulatively beneficial and not mutually exclusive. Here, we used neither lower temperature nor long-term culture conditions to delineate the benefit of low dose irradiation on the islets. Indeed, there are pros and cons to treating islets in culture, which may impact islet yield, viability or sterility. All will play a major role in determining the optimum treatment strategy that will provide the most favorable outcome. Grenz ray low dose irradiation treatment does not appear to significantly affect islet yield or viability, is short in duration thereby limiting potential culture contamination, and has demonstrated the ability to prolong graft survival; therefore, its utility in islet transplantation may be worth investigating further.

Materials and methods

Mice

BALB/c (H-2kd) male donor mice, greater than 10 weeks of age, and 10–12 week age-matched male C57BL/6 (H-2kb) recipient mice were purchased through Jackson Laboratories (Bar Harbor, ME, USA). Animals were housed in the Health Sciences Laboratory Animal Services vivarium at the University of Alberta and treated in accordance to the guidelines established by the Canadian Council on Animal Care.

Islet isolation and irradiation

Islets were isolated as described previously.²⁹ Three separate islet isolations were assayed and transplanted. Immediately post-isolation the same islet pool was divided into groups: irradiated treatment islets and non-irradiated control islets. These 2 groups were transplanted into recipient animals with the following immunosuppression: irradiated with no immunosuppression, irradiated with CTLA-4 Ig, non-irradiated with no immunosuppression and non-irradiated with CTLA-4 Ig (CTLA-4 Ig monotherapy). To perform irradiation we used a model 3320 grenz ray source (Universal X-Ray Products, Chicago, IL) fitted with the largest size conical shield. The X-ray output at the bottom of the cone was measured using a Model 9015S-P Radiation Monitor Controller attached to a Model 9060A Electrometer/Ion Chamber equipped with Model 10 × 5–6M dedicated mammo chamber (Radcal Corp., Monrovia, CA). The grenz ray control box output was adjusted to 14 kV, 6 mAmp, and at these settings we determined that 2000 rad (i.e., 20Gy) was reproducibly delivered to the bottom of the cone in 5 minutes. Plastic lids were removed from petri culture plates containing the purified islets in a minimum volume of tissue culture medium, and the open plates placed under the cone for the predetermined time. In the initial experiments, islets received a total dose of 20Gy administered over 5 minutes, and in a subsequent experiment, 80Gy administered over 20 minutes. During the irradiation treatment, both control and treatment islet preparations were aliquoted into 1 mL volumes (200–300 islets/mL) dispersed evenly as a droplet to give a maximum thickness of 2 mm over a 100 mm x 15 mm sterile, non-treated Petri dish and lightly agitated and rotated at 2.5 minute intervals. Note that both control and irradiated islets were treated equally, but with one dish under the grenz source, and the other at the same temperature on the bench. After irradiation of the experimental plate and simultaneous incubation of the control plate, additional tissue culture medium was added to each plate and the islets incubated at 37°C in 5% CO₂ for 1 hour before transplant.

Cytological evaluation of Islets after low dose irradiation

Aliquots containing approximately 100 islets were removed immediately after isolation, 1 hour after incubation and after irradiation treatment + 1 hour incubation. All incubations were at 37°C and 5% CO₂. For membrane integrity analysis, islet aliquots (100μL) were incubated 5 minutes at room temperature with SYTO® 13 Green-Fluorescent nucleic acid stain which permeates live cells with intact membranes (1:100, Molecular Probes, S7575). The samples were then stained with ethidium bromide to mark for dead cells, where membrane integrity has been compromised and viewed with a Nikon Eclipse TE300 fluorescent microscope (1:100, Sigma-Aldrich, E1510).

Apoptosis of islets after isolation, after 1-hour incubation and following irradiation with 1-hour culture, was quantified using Tdt-mediated dUTP nick-end labeling (TUNEL) staining. The islets were fixed in 4% paraformaldehyde, embedded in agar, processed and embedded in paraffin. Sections were stained for insulin to identify β-cells using guinea pig anti-insulin antibody incubation for 90 minutes (1:100, Dako, A0564) and labeled with Rhodamine (TRITC) conjugated anti-guinea pig IgG with incubation for 20 minutes (1:200, Jackson ImmunoResearch, 106–025–003). The apoptotic nuclei were labeled with fluorescein isothiocyanate-dUTP with TdT enzyme (Deadend Fluorometric TUNEL System; Promega, G3250), counterstained and mounted with DAPI (ProLong Gold DAPI, Invitrogen, P36935) before fluorescence microscopy. Images were processed and analyzed using FIJI ImageJ Software (National Institute of Health, USA).

Islet transplantation and immunosuppression

To induce diabetes, 3 to 5 d before transplantation, C57BL/6 mice were administered an intra peritoneal (i.p.) injection of streptozotocin (STZ) at 175–180mg/kg in acetate phosphate buffer, pH 4.5 (Sigma-Aldrich Canada Co., S0130). The animals were considered diabetic when their blood glucose levels exceeded 18 mmol/L (324 mg/dL) for 2 consecutive daily readings.

After incubation for 1 hour at 37°C and 5% CO₂, islets were transplanted at 500 islets per diabetic recipient. The islets were aspirated into polyethylene (PE-50) tubing using a micro-syringe, and centrifuged into a pellet suitable for transplantation. A left lateral paralumbar subcostal incision was made and the left kidney was delivered into the wound. The renal capsule was perforated and space was made under the capsule to allow transplantation of the islets. Mice were transplanted with a full islet mass, 500 islets ± 10% with purity at 90% ± 5%. A subset of mice was transplanted with 700 islets, to test whether the higher islet mass would affect outcomes. Immunosuppressed animals were treated with CTLA-4 Ig (Fc Conjugated, BioXcell, BE0099), at 10 mg/kg i.p. on d0, 2, 4 and 6. Day 0 injections were administered 1 hour before transplant. Control animals were treated with isotype control Rat IgG1 (anti-horseradish peroxidase, BioXcell, BE0088).

We assessed islet graft function in transplanted mice, 3 times per week using a OneTouch Ultra® Glucometer (Lifescan Canada, Burnaby, BC). Rejection was defined as 2 consecutive non-fasted readings greater than 11.1 mmol/L (200 mg/dL), where the date of rejection was recorded as the time of the first reading.

Intraperitoneal glucose tolerance in mice after allotransplant with low dose irradiated Islets

At 7-weeks posttransplant, mice were fasted overnight and injected i.p. with 25% dextrose at 3 g/kg body weight to measure glucose tolerance and efficacy. Blood glucose levels were monitored at baseline (time = 0), 15, 30, 60, 90 and 120 minutes post injection.

Nephrectomy to confirm euglycemia

To confirm graft dependent efficacy, islet-bearing kidneys were removed from normoglycemic animals. The animal was placed under anesthesia and a Ligaclip® Extra ligating clip (Johnson & Johnson, Inc., LT200) was used to occlude the renal vessels and ureter under the graft-bearing kidney. The kidney was surgically removed from the animal. Animals were monitored post-nephrectomy for 5 d. A return to hyperglycemia confirmed graft-function over naïve pancreas β-cell regeneration.

Analysis of CD4 and CD8 donor populations in splenocytes removed 24 hours after allotransplant

Single-cell suspensions of splenocytes were isolated from recipient mice 24 hours after transplant. After filtering through a 70 μm cell strainer and red blood cell lysis, cells were surface stained at 4°C for 15 minutes. Splenocytes were stained with H-2kb (0.5 μg/μL, BioLegend, 116505) to distinguish C57BL/6 recipient cells from donor BALB/c H-2kd cells (0.2 μg/μL, BioLegend, 116617). T cells (TCRβ, 0.2 μg/μL, eBioscience, 48–5961–80) were stained and separated into CD4+ (0.2 μg/μL, eBioscience, 17–0041–81) and CD8+ (0.4 μg/μL, eBioscience, 12–0081–83) populations. Data were acquired by LSR II flow cytometry (BD Biosciences) and analyzed with FCS Express 3 software. Assay was adapted from FACS assay analysis using splenocytes.³⁰

Statistical analysis

Data were analyzed using GraphPad Prism (GraphPad Software, La Jolla, CA, USA). Kaplan-Meyer graft survival function curves were compared using the log-rank (Mantel-Cox) statistical method. One-way ANOVA was used to compare AUC for measurement of glucose tolerance over time. One-way ANOVA and t-tests were used to determine significance between CD4+ and CD8+ T cell populations. Statistical significance was considered to be p-values less than 0.05. Graphical representation of data are as mean +/− standard error of the mean (SEM) where *p < 0.05, **p < 0.01 and ***p < 0.001.

Abbreviations

APC

antigen presenting cells

CTLA-4 Ig

cytotoxic T-lymphocyte-associated protein 4 immunoglobulin

Gy

gray (International System of Units delineating absorbed dose of ionizing radiation)

i.p.

intraperitoneal

MHC

Major Histocompatibility Complex

PE-50

polyethylene tubing of outer diameter 0.965mm and inner diameter 0.58mm

rad

absorbed radiation dose

STZ

streptozotocin

TUNEL

Tdt-mediated dUTP nick-end labeling apoptosis assay

Disclosure of potential conflicts of interest

No potential conflicts of interest were disclosed.

Funding

Funding for this study was provided by the Diabetes Research Institute Foundation of Canada (DRIFCan), the Canadian Dermatology Foundation (CDF) and Alberta Diabetes Institute – University Hospital Foundation. N. Abualhassan is supported through the Canadian National Transplantation Research Program and Saudi Arabian Cultural Bureau. A. R. Pepper is supported by the Alberta Innovates - Health Solutions (AIHS) Postdoctoral Fellowship Grant 201400496. A. Bruni is supported by Canadian Institutes of Health Research - Proof of Principle Grant 144255 and Stem Cell Network (SCN). B. Gala-Lopez is supported through an AIHS Clinician Fellowship Grant 201400106 and Izaak Walton Killam Memorial Scholarship. A.M.J. Shapiro is supported through SCN Grant NCESCN CTRA FY17/CT5 and NCESCN DTRA FY17/DT6; AIHS Proof of Principle 144255 and Collaborative Research and Innovation Opportunities (CRIO) Team funding Grant 201201154; the Juvenile Diabetes Research Fund Canadian Clinical Trial Network (JDRF CCTN) and the Diabetes Research Institute Foundation Canada (DRIFCan).