Islet transplantation is an emerging therapy for type 1 diabetes and hypoglycemic unawareness. However, a key challenge for islet transplantation is cellular rejection and the requirement for long-term immunosuppression. In this study, we established a diabetic humanized NOD-scidIL2Rγnull (NSG) mouse model of T-cell–mediated human islet allograft rejection and developed a therapeutic regimen of low-dose recombinant human interleukin-2 (IL-2) combined with low-dose rapamycin to prolong graft survival. NSG mice that had received renal subcapsular human islet allografts and were transfused with 1 × 107 of human spleen mononuclear cells reconstituted human CD45+ cells that were predominantly CD3+ T cells and rejected their grafts with a median survival time of 27 days. IL-2 alone (0.3 × 106 IU/m2 or 1 × 106 IU/m2) or rapamycin alone (0.5–1 mg/kg) for 3 weeks did not prolong survival. However, the combination of rapamycin with IL-2 for 3 weeks significantly prolonged human islet allograft survival. Graft survival was associated with expansion of CD4+CD25+FOXP3+ regulatory T cells (Tregs) and enhanced transforming growth factor-β production by CD4+ T cells. CD8+ T cells showed reduced interferon-γ production and reduced expression of perforin-1. The combination of IL-2 and rapamycin has the potential to inhibit human islet allograft rejection by expanding CD4+FOXP3+ Tregs in vivo and suppressing effector cell function and could be the basis of effective tolerance-based regimens.

Clinical islet transplantation is a cutting-edge therapy for the treatment of type 1 diabetes (T1D) and hypoglycemic unawareness (13). However, its success is limited by the need for long-term multiagent immunosuppression and the ensuing side effects, coupled with the progressive loss of graft function over time, most likely from chronic rejection (1,2,4). Clinically, there are competing objectives of reducing immunosuppressive burden while maintaining long-term graft function.

Altering the immunological microenvironment can potentially prolong islet allograft survival while simultaneously reducing the requirement for long-term immunosuppression. This requires the removal of potential and existing alloreactive T cells and, at the same time, expanding regulatory T cells (Tregs). Two such agents with the capacity to achieve these dual objectives are recombinant human interleukin-2 (IL-2) and rapamycin, both of which are used clinically. In the clinic, low-dose IL-2 therapy has been used successfully to treat chronic graft-versus-host disease (GVHD) (5), hepatitis C virus–induced vasculitis (6), systemic lupus erythematosus (SLE) (7), and T1D (8). Rapamycin (sirolimus) is approved for the prevention of rejection in kidney transplantation (9), and animal studies have shown that low-dose rapamycin allows selective expansion of Tregs in vivo (10,11). Furthermore, rapamycin has been used successfully in clinical islet transplantation as part of the Edmonton protocol (12). In animal models, IL-2 treatment prolonged corneal allograft survival (13), and the combination of IL-2 and rapamycin treatment prolonged skin graft survival in minor antigen mismatch and semiallogeneic mouse recipients (14); in many of these studies, selective expansion of FOXP3+ Tregs was found (57,13,14).

Rapamycin through its inhibition of the mammalian target of rapamycin pathway affects Treg homeostasis (9,12) and suppresses the activation and proliferation of immune cells, including T cells (15). It inhibits IL-2–induced phosphorylation and subsequent T-cell proliferation (16) and impairs CD8+ T-cell antitumor immune responses (17). Rapamycin also causes expansion of functional CD4+CD25+FOXP3+ Tregs in both healthy individuals and patients with T1D in vitro (18) and increases Treg-specific suppressive ability in patients with T1D (19). In experimental studies, rapamycin has been shown to be protolerogenic by expanding naturally occurring Tregs while also depleting CD4+ T effector cells (18) by promoting their apoptosis (20). The combination of IL-2/rapamycin can also induce FOXP3+ Tregs from CD4+CD25 conventional T cells (21).

Because of the limited success of translating the findings obtained in naïve mouse models to the clinic, we established a humanized mouse model that would allow us to study the human T-cell response to islet allografts in vivo. Previously, in this model, we showed that infusion of in vitro–expanded human FOXP3+ Tregs prevented porcine neonatal islet cell cluster xenograft rejection by suppressing the T-cell–mediated antigraft response (22). In this study, we aimed to determine whether low-dose IL-2 and/or rapamycin could promote the expansion of CD4+FOXP3+ Tregs and limit effector T cells in vivo to promote islet allograft survival.

Animals

Breeding pairs of NOD-scidIL2Rγnull (NSG) mice were obtained from The Jackson Laboratory and housed under specific pathogen–free conditions. NSG mice also were obtained from the Animal Resource Centre (Murdoch, Western Australia, Australia). Experiments were conducted using established guidelines for animal care and approved by the animal ethics committee of Western Sydney Local Health District.

Human Islet Preparation and Mononuclear Cell Isolation

Human islets were isolated from donor pancreata and cultured for 24 h (2). Human islets that did not reach release criteria for clinical transplantation were used in this study. Human spleen mononuclear cells (hSPMCs) were isolated by Ficoll density gradient centrifugation (Sigma-Aldrich, Castle Hill, New South Wales, Australia). The use of human tissue for research was approved by the human research ethics committee of the Western Sydney Local Health District.

Humanized Mouse Models and Treatment Regimen of IL-2 and Rapamycin

NSG mice were administered hSPMCs (fresh and frozen) (hNSG mice) intraperitoneally. Recombinant human IL-2 (Aldesleuki; Chiron and Novartis) and/or rapamycin (sirolimus, oral solution; Wyeth) treatments were administered intraperitoneally into transplanted NSG mice with hSPMCs (hNSG recipient mice). Treatment groups are outlined in Supplementary Table 1 and included 1) IL-2 at 0.3 × 106 IU/m2 of body surface area/day (IL-20.3) (equivalent to 7.5 IU/g) and 2) 1 × 106 IU/m2 of body surface area/day (IL-21) (equivalent to 24.9 IU/g) for 6 days/week for 3 weeks, 3) IL-20.3 or 4) IL-21 plus rapamycin 1 mg/kg for week 1 and 0.5 mg/kg for weeks 2 and 3 (rapamycin 0.5/1 mg/kg), and 5) rapamycin (0.5/1 mg/kg) for 3 weeks. The doses of rapamycin were chosen on the basis of studies that showed that a dose of 1 mg/kg/day corresponded to therapeutic doses for solid organ transplantation (23).

Humanized Diabetic Human Islet Transplant Mouse Model

NSG mice were injected with streptozotocin at doses of 170–180 mg/kg to induce diabetes, which was defined as blood glucose level (BSL) ≥20 mmol/L (24). Diabetic mice were transplanted with 2,000 human islets/mouse under their left renal capsule. After successful transplantation, mice received one of two doses of IL-2 as above for 1 day (day −1) intraperitoneally. The following day, mice were transfused with allogenic 1 × 107 hSPMCs (day 0). At the same time, untreated mice were divided into rapamycin treatment or no treatment as above, and IL-2–treated mice were continually treated with IL-2 or IL-2/rapamycin as described above. Successful islet function was defined as random BSL of ≤12 mmol/L after islet transplantation before transfusion and ≤10 mmol/L after IL-2 and/or rapamycin treatment. Nephrectomy was performed on control NSG mice after transplantation and before reconstitution.

Flow Cytometric Analysis

Flow cytometry was performed on an LSR II cytometer, and data were analyzed using FACSDiva (BD Biosciences) and FlowJo version 10 (25) software. Peripheral blood (PB) and/or spleen samples of the hNSG mice were collected and stained at weeks 3, 4, 5, 7, and/or 9 after transfusion with hSPMCs. Antibodies comprised allophycocyanin-conjugated anti-human CD3, CD4, CD25; fluorescein isothiocyanate–conjugated anti-human CD4, CD19; Pacific blue–conjugated anti-human CD3, CD4; V450-conjugated anti-mouse CD45; phycoerythrin-conjugated anti-human CD3, CD8, CD56; peridinin chlorophyll–conjugated and BUV395-conjugated anti-human CD45; BV421-conjugated anti-human perforin; and PE-CF594–conjugated anti-T-bet (BD Biosciences) and phycoerythrin-conjugated anti-human FOXP3 (eBioscience). LIVE/DEAD Fixable Near-IR stain (Thermo Fisher Scientific) was used before surface and intracellular staining (FOXP3, perforin, and T-bet).

Histological Examination

The left kidney containing the human islet grafts, right kidney, skin, intestine, liver, and spleen of the NSG mice, hNSG mice, and hNSG recipient mice were fixed in 10% formalin or frozen in optimal cutting temperature compound (Miles Scientific) for hematoxylin-eosin (H-E) staining and immunohistochemistry (IHC). Polyclonal guinea pig anti-insulin (Dako) with the R.T.U. VECTASTAIN Universal ABC Kit (Vector Laboratories) were used for detection of insulin secretion by human islet allografts as described previously (26). Primary anti-human CD4 (monoclonal, RPA-T4) and anti-human CD8 (monoclonal, RPA-T8) (eBioscience) antibodies were used for detection of infiltrating T cells within islet allograft (IHC). Immunofluorescence staining of human CD4+FOXP3+ (hCD4+FOXP3+) Tregs by primary rabbit anti-human CD4 (clone: EPR6855) and mouse anti-human FOXP3 (clone: 236A/E7) (Abcam) with secondary goat anti-mouse IgG (Alexa Fluor 488) and donkey anti-rabbit IgG (TRITC; Jackson ImmunoResearch) antibodies was performed on kidney human islet allograft paraffin sections that were counterstained with VECTASHIELD Antifade Mounting Medium with DAPI (Jackson ImmunoResearch) (26).

IHC images stained for human CD4+ (hCD4+) and CD8+ (hCD8+) T cells and insulin were blindly analyzed using Aperio ImageScope version 12.4.0.7018 software (Leica Biosystems). Fields used for statistical analysis were restricted to islet allograft sites, and staining was quantified using Aperio Positive Pixel Count Algorithm version 9 software (Leica Biosystems) according to the manufacturer’s instructions and as reported by previous studies (27,28). Quantitative analysis of immunofluorescence-positive cells was reported in a similar way to previous studies (29).

The histology of skin, intestine, liver, and right kidney from experimental and control NSG mice was used for xeno-GVHD evaluation and for evaluating the alloimmune response in the islet allografts of left kidney. H-E images of skin, intestine, liver, and right kidney (Aperio ImageScope version 12.4.0.7018 software) were blindly scored as grade 0–4 to assess degree of organ and tissue damage. The scoring criteria are listed in Supplementary Table 2.

Cell Sorting

The hCD4+ and hCD8+ T cells were sorted from spleens of hNSG recipient mice and donor hSPMCs on a FACSAria III Cell Sorter (BD Biosciences). Allophycocyanin-conjugated anti-human CD3, phycoerythrin-conjugated anti-human CD4, and fluorescein isothiocyanate–conjugated anti-human CD8 (BD Biosciences) and DAPI (Invitrogen) were used for staining.

Real-time RT-PCR

Real-time RT-PCR was used for analysis of human IL-2, IL-4, IL-6, IL-10, interferon-γ (IFN-γ), and transforming growth factor-β (TGF-β) in sorted hCD4+ and hCD8+ T cells; perforin-1 on sorted hCD8+ T cells; and IL-17A and B-lymphocyte–induced maturation protein-1 (Blimp-1), T-bet, GATA3, and RORγt in sorted hCD4+ T cells from the spleens of hNSG recipient mice and donor hSPMCs. TaqMan gene expression assays were used with GAPDH (Thermo Fisher Scientific) for normalized gene expression.

Cytokine Cytometric Bead Array

Human IFN-γ, tumor necrosis factor (TNF), IL-2, IL-4, IL-6, IL-10, and IL-17A in mouse serum were detected on BD FACSCanto II (BD Biosciences) by BD cytometric bead array human T helper 1 (Th1)/Th2/Th17 cytokine kit. Data were analyzed by FCAP Array software (BD Biosciences) (26).

Statistical Analysis

GraphPad Prism 7.0 (GraphPad Software, San Diego, CA) was used for statistical analysis. The log-rank (Mantel-Cox) test was used for comparison of survival data between groups. Differences between two groups were evaluated using unpaired t test or Mann-Whitney test (nonparametric test), while three or more groups were compared using a one-way ANOVA followed by Tukey multiple comparison test and Kruskal-Wallis test (nonparametric) followed by Dunn multiple comparison test. P < 0.05 was considered statistically significant. Data are expressed as mean ± SEM.

Data and Resource Availability

The data sets generated and/or analyzed during the current study are available from the corresponding author upon reasonable request.

Optimization of hSPMC Transfusion in NSG Mice

To establish a humanized mouse model for studies of human islet transplantation, the relative importance of cell number and time from transfusion in NSG mice was evaluated. Initially, 5 × 106 and 1 × 107 of hSPMCs were both assessed. The proportion of human CD45+ (hCD45+) cells after transfusion in NSG mice was found to be time dependent (Supplementary Fig. 1A and B). A greater proportion of hCD45+ cells was found at week 9 compared with weeks 4 and 5, regardless of whether mice were transfused with 5 × 106 fresh hSPMCs or 1 × 107 fresh or frozen hSPMCs (Supplementary Fig. 1B). In the doses used, the number of cells used (5 × 106 and 1 × 107) had no significant impact on the degree of hCD45+ cell reconstitution in NSG mice (Supplementary Fig. 1B). hCD45+ cells in PB and spleen of hNSG mice were predominantly CD3+ T cells (Fig. 1A and B), comprising hCD4+ and hCD8+ subsets (CD4+ T cells > CD8+ T cells) (Fig. 1C and D) with low proportions of B cells or natural killer cells (Supplementary Fig. 1C–E).

Figure 1

Human CD3+ (hCD3+) T cells in NSG mice after transfusion of hSPMCs. A: The representative pseudocolor plot of hCD45 vs. hCD3 (gating on hCD45+ cells) in PB of NSG mice (M) transfused with 1 × 107 hSPMCs (M/PB/hSPMC) at week 5. B: The proportion of hCD3+ T cells in reconstituted hCD45+ cells in PB (n = 14, including 5 × 106 [n = 4] and 1 × 107 fresh [n = 5] and 1 × 107 frozen [n = 5]) (M/PB) and spleen of NSG mice transfused with hSPMCs (n = 7, including 5 × 106 [n = 2] and 1 × 107 [n = 3] fresh hSPMCs or 1 × 107 frozen hSPMCs [n = 2]) (M/Spleen) at week 5. C: The representative pseudocolor plot of hCD4 vs. hCD8 (gating on hCD3+ T cells) in PB of NSG mice transfused with 1 × 107 of hSPMCs at week 5. D: The proportion of hCD4+ and hCD8+ T cells in reconstituted hCD3+ T cells of PB and spleens from NSG mice transfused with hSPMCs at week 5 (n = 7, including 5 × 106 [n = 1] and 1 × 107 fresh [n = 3] and 10 × 106 frozen hSPMCs [n = 3]). Data were from five independent experiments. Nonparametric Mann-Whitney test and unpaired t test were used for the comparisons of proportion of hCD3+ T cells in reconstituted hCD45+ cells (B) and proportion of hCD4+ and hCD8+ T cells in reconstituted hCD3+ T cells in hNSG mice (D), respectively. Error bars indicate mean ± SEM. ***P < 0.001.

Figure 1

Human CD3+ (hCD3+) T cells in NSG mice after transfusion of hSPMCs. A: The representative pseudocolor plot of hCD45 vs. hCD3 (gating on hCD45+ cells) in PB of NSG mice (M) transfused with 1 × 107 hSPMCs (M/PB/hSPMC) at week 5. B: The proportion of hCD3+ T cells in reconstituted hCD45+ cells in PB (n = 14, including 5 × 106 [n = 4] and 1 × 107 fresh [n = 5] and 1 × 107 frozen [n = 5]) (M/PB) and spleen of NSG mice transfused with hSPMCs (n = 7, including 5 × 106 [n = 2] and 1 × 107 [n = 3] fresh hSPMCs or 1 × 107 frozen hSPMCs [n = 2]) (M/Spleen) at week 5. C: The representative pseudocolor plot of hCD4 vs. hCD8 (gating on hCD3+ T cells) in PB of NSG mice transfused with 1 × 107 of hSPMCs at week 5. D: The proportion of hCD4+ and hCD8+ T cells in reconstituted hCD3+ T cells of PB and spleens from NSG mice transfused with hSPMCs at week 5 (n = 7, including 5 × 106 [n = 1] and 1 × 107 fresh [n = 3] and 10 × 106 frozen hSPMCs [n = 3]). Data were from five independent experiments. Nonparametric Mann-Whitney test and unpaired t test were used for the comparisons of proportion of hCD3+ T cells in reconstituted hCD45+ cells (B) and proportion of hCD4+ and hCD8+ T cells in reconstituted hCD3+ T cells in hNSG mice (D), respectively. Error bars indicate mean ± SEM. ***P < 0.001.

Close modal

Human CD3+ T Cells Were Capable of Rejecting Allogeneic Human Islet Allografts

To determine whether hNSG mice were capable of rejecting human islet allografts, human islets were transplanted under the kidney capsule of diabetic NSG mice, and graft function was confirmed by BSL monitoring, histology, and/or nephrectomy (Fig. 2, Supplementary Fig. 2, and data not shown). NSG mice with functioning islet grafts were transfused with 1 × 107 hSPMCs. Functioning islet allografts were rejected with a mean survival time (MST) of 27 days (Fig. 2A and B). Histological evaluation of rejecting human islet allografts showed marked cellular infiltration, predominantly composed of hCD4+ and hCD8+ T cells with no significant difference in the numbers of each subset and no intact islets (detected by insulin stain), while control islet grafts from NSG mice without transfusion had intact islets with significantly stronger insulin staining by IHC (Fig. 2C–F).

Figure 2

Human islet allografts were rejected in the diabetic hNSG recipient mice. A: The BSL (dashed lines) and weight (solid lines) profiles of the NSG recipient mice that had successful islet transplantation following transfusion of hSPMCs (n = 8). B: Survival curves of human islet allografts in the hNSG recipient mice after transfusion of hSPMCs (n = 8, MST = 27 days). C: The representative micrographs of H-E and IHC for CD4+ and CD8+ cells (brown) and insulin staining (brown) in rejected human islet allografts within the left kidneys from hNSG recipient mice at day 35 after transfusion of 1 × 107 hSPMCs (original magnification ×10). D: Representative micrograph of H-E and IHC of insulin-positive staining (brown) human islet grafts in the diabetic NSG recipient mice after successful transplantation without transfusion (original magnification ×10). E: Quantification of IHC staining of hCD4+ and hCD8+ T-cell infiltrations in rejected islet allografts (n = 5). F: Insulin in rejected islet allografts (n = 7) and control islet grafts (n = 5). Pixel Count Algorithm is based on staining intensity and expressed as positive pixel count/mm2. The ratio of strong-positive pixel count over total pixel number, per graft site, was quantified per sample and normalized to total positive pixels/mm2. Strong-positive pixel intensity was chosen to exclude false-positive and weak-positive pixel counts within fields. Unpaired t test and Mann-Whitney test were used for the comparisons of CD4+ and CD8+ T cells (E) and positive insulin in islet grafts (F), respectively. Error bars indicate mean ± SEM. **P < 0.01.

Figure 2

Human islet allografts were rejected in the diabetic hNSG recipient mice. A: The BSL (dashed lines) and weight (solid lines) profiles of the NSG recipient mice that had successful islet transplantation following transfusion of hSPMCs (n = 8). B: Survival curves of human islet allografts in the hNSG recipient mice after transfusion of hSPMCs (n = 8, MST = 27 days). C: The representative micrographs of H-E and IHC for CD4+ and CD8+ cells (brown) and insulin staining (brown) in rejected human islet allografts within the left kidneys from hNSG recipient mice at day 35 after transfusion of 1 × 107 hSPMCs (original magnification ×10). D: Representative micrograph of H-E and IHC of insulin-positive staining (brown) human islet grafts in the diabetic NSG recipient mice after successful transplantation without transfusion (original magnification ×10). E: Quantification of IHC staining of hCD4+ and hCD8+ T-cell infiltrations in rejected islet allografts (n = 5). F: Insulin in rejected islet allografts (n = 7) and control islet grafts (n = 5). Pixel Count Algorithm is based on staining intensity and expressed as positive pixel count/mm2. The ratio of strong-positive pixel count over total pixel number, per graft site, was quantified per sample and normalized to total positive pixels/mm2. Strong-positive pixel intensity was chosen to exclude false-positive and weak-positive pixel counts within fields. Unpaired t test and Mann-Whitney test were used for the comparisons of CD4+ and CD8+ T cells (E) and positive insulin in islet grafts (F), respectively. Error bars indicate mean ± SEM. **P < 0.01.

Close modal

Graft loss was considered to be due to the islet alloimmune T-cell response and not as a result of xeno-GVHD because there was no clinical evidence of xeno-GVHD at the time of graft failure. Only one mouse had weight loss of >10% (Fig. 2A). The skin, intestine, liver, and contralateral kidney without the islet graft were examined histologically, and no xeno-GVHD was seen at the time of rejection (Fig. 3). The only abnormality in the nongrafted tissues was the presence of a scattered inflammatory infiltrate surrounding the branches of the portal vein of the liver at day 35 after transfusion (Fig. 3). In untransplanted NSG mice transfused with hSPMCs, evidence of xeno-GVHD was seen at week 9 after transfusion, with histology demonstrating loss of hair follicles in the skin and massive cellular infiltration in the liver with significant increases in histopathological GVHD grades compared with normal controls. Structural changes were also present in the spleen and minimal cellular infiltrate within the kidneys (Fig. 3 and Supplementary Fig. 3).

Figure 3

Histological evaluation of GVHD in hNSG mice and transplanted hNSG recipient mice. A: Representative H-E micrographs of skin and intestine (original magnification ×10) and liver and right kidney (original magnification ×20) in the NSG mice without transfusion of hSPMCs (normal control), NSG recipient mice with 1 × 107 hSPMCs at day 35 (Tx/hSPMC/d35), and NSG mice with 1 × 107 hSPMCs at week 9 (hSPMC/w9) after transfusion. B: Histological scores of skin, intestine, liver, and kidney damage grades (0–4) for normal control (n = 9), Tx/hSPMC/d35 (n = 8), and hSPMC/w9 groups (n = 9). Kruskal-Wallis test followed by Dunn multiple comparison test was used for comparisons. Error bars indicate mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.01.

Figure 3

Histological evaluation of GVHD in hNSG mice and transplanted hNSG recipient mice. A: Representative H-E micrographs of skin and intestine (original magnification ×10) and liver and right kidney (original magnification ×20) in the NSG mice without transfusion of hSPMCs (normal control), NSG recipient mice with 1 × 107 hSPMCs at day 35 (Tx/hSPMC/d35), and NSG mice with 1 × 107 hSPMCs at week 9 (hSPMC/w9) after transfusion. B: Histological scores of skin, intestine, liver, and kidney damage grades (0–4) for normal control (n = 9), Tx/hSPMC/d35 (n = 8), and hSPMC/w9 groups (n = 9). Kruskal-Wallis test followed by Dunn multiple comparison test was used for comparisons. Error bars indicate mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.01.

Close modal

Proportion of hCD4+FOXP3+ Tregs Was Reduced Over Time After Engraftment

hCD4+FOXP3+ Tregs, including CD25+Foxp3+ and CD25Foxp3+ Tregs, were assessed in PB and spleen of hNSG mice. The proportion of CD4+FOXP3+ Tregs was reduced in the PB and spleens of hNSG mice following transfusion alone (Fig. 4A and B) and subsequent to transplantation (Fig. 4C). At week 5, there was no significant difference in the proportion of CD4+FOXP3+ Tregs in the PB of hNSG mice with and without transplantation (Fig. 4C). These CD4+FOXP3+ Tregs were predominantly CD25+FOXP3+ (Fig. 4A). However, by week 9 after transfusion, the proportion of CD4+FOXP3+ Tregs had fallen in both PB and spleen in hNSG mice compared with the proportion found at week 5 (Fig. 4B).

Figure 4

Reduction of hCD4+CD25+FOXP3+ Tregs over time in hNSG mice that were transfused with 1 × 107 hSPMCs. A: The representative pseudocolor plots (smooth) of hCD4 vs. hFOXP3 and hFOXP3 vs. hCD25 (gating on hCD4+ T cells) in spleens of the hNSG mice at week 5 and week 9. B: The proportion of hCD4+FOXP3+ Tregs in PB (M/PB) and spleens (M/Spleen) of the hNSG mice at weeks 4, 5, and 9 of reconstitution (n = 11–16). Control is donor hSPMCs before transplantation (green circles, n = 11). C: The proportion of hCD4+FOXP3+ Tregs in PB of the hNSG recipient mice at week 3 (n = 9) and week 5 (n = 8) after hSPMC transfusion compared with donor hSPMCs before transplantation (green circles, n = 11). Data were from five independent experiments. Kruskal-Wallis test followed by Dunn multiple comparison test and Mann-Whitney test was used for comparisons. Error bars indicate the mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 4

Reduction of hCD4+CD25+FOXP3+ Tregs over time in hNSG mice that were transfused with 1 × 107 hSPMCs. A: The representative pseudocolor plots (smooth) of hCD4 vs. hFOXP3 and hFOXP3 vs. hCD25 (gating on hCD4+ T cells) in spleens of the hNSG mice at week 5 and week 9. B: The proportion of hCD4+FOXP3+ Tregs in PB (M/PB) and spleens (M/Spleen) of the hNSG mice at weeks 4, 5, and 9 of reconstitution (n = 11–16). Control is donor hSPMCs before transplantation (green circles, n = 11). C: The proportion of hCD4+FOXP3+ Tregs in PB of the hNSG recipient mice at week 3 (n = 9) and week 5 (n = 8) after hSPMC transfusion compared with donor hSPMCs before transplantation (green circles, n = 11). Data were from five independent experiments. Kruskal-Wallis test followed by Dunn multiple comparison test and Mann-Whitney test was used for comparisons. Error bars indicate the mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001.

Close modal

Combination Low-Dose IL-2 and Rapamycin Prolonged Human Islet Allograft Survival in NSG Recipient Mice Reconstituted With hSPMCs

To determine the importance of IL-2 dose for expansion of hCD4+FOXP3+ Tregs and whether expansion of hCD4+FOXP3+ Tregs could prolong human islet allograft survival, recipient mice of human islet allografts were treated with one of two doses of IL-2 alone (IL-20.3 or IL-21) or combination of IL-2 with rapamycin for 3 weeks. Treatment with either dose of IL-2 alone and rapamycin alone did not prolong islet allograft survival (MST 35, 26, and 32 days, respectively) (Fig. 5A). However, the combination of either dose of IL-2 with rapamycin significantly prolonged human islet allograft survival (MST 60 and 42 days, respectively) (Fig. 5A). hNSG recipient mice that did not receive treatment had an MST of 27 days (Figs. 2 and 5A).

Figure 5

Combination low-dose IL-2 and rapamycin treatment for 3 weeks led to prolongation of human islet allograft survival. A: Survival curves of human islet allografts in the diabetic NSG mice that had successful islet transplantation following transfusion of 1 × 107 hSPMCs only (MST = 27) (Tx/hSPMC) (Tx/Without) and plus treatment with IL-20.3 (MST = 35) (Tx/hSPMC/IL-20.3), IL-21 (MST = 26) (Tx/hSPMC/IL-21), rapamycin (MST = 32) (Tx/hPBMC/Rapamycin), IL-20.3/rapamycin combination treatment (MST = 60) (Tx/hSPMC/IL-20.3/Rap), and IL-21/rapamycin combination treatment (MST = 42) (Tx/hSPMC/IL-21/Rap). **P = 0.0026, ***P < 0.001. B and C: The representative H-E micrographs of human islet allograft and insulin IHC staining (brown for positive staining) from reconstituted hNSG recipient mice with IL-21 [Tx/IL-2(1)], rapamycin (Tx/Rapamycin), and IL-20.3/rapamycin [Tx/IL-2(0.3)/Rap] groups at day 35 (d35) and the IL-21/rapamycin [Tx/IL-2(1)/Rap] group at d44 after hSPMC transfusion (original magnification ×10). D and E: Quantification of IHC insulin staining of human islet allografts from Tx/IL-21 (n = 5), Tx/rapamycin (n = 5), and Tx/IL-20.3/Rap (n = 7) groups at d35 and Tx/IL-21/Rap group at d44 after hSPMC transfusion (n = 7) compared with human islet grafts in the diabetic NSG mice after successful transplantation without transfusion (n = 5) (Tx/Control) and NSG recipient mice with hSPMCs and without treatment (n = 7) [Tx/hSPMC (Tx/Without)]. Pixel Count Algorithm is based on staining intensity and expressed as positive pixel count/mm2. The log-rank (Mantel-Cox) test was used for comparison of survival data. Kruskal-Wallis test followed by Dunn multiple comparison test was used for comparisons for quantification of IHC insulin. Data were from nine independent experiments. Error bars indicate the mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 5

Combination low-dose IL-2 and rapamycin treatment for 3 weeks led to prolongation of human islet allograft survival. A: Survival curves of human islet allografts in the diabetic NSG mice that had successful islet transplantation following transfusion of 1 × 107 hSPMCs only (MST = 27) (Tx/hSPMC) (Tx/Without) and plus treatment with IL-20.3 (MST = 35) (Tx/hSPMC/IL-20.3), IL-21 (MST = 26) (Tx/hSPMC/IL-21), rapamycin (MST = 32) (Tx/hPBMC/Rapamycin), IL-20.3/rapamycin combination treatment (MST = 60) (Tx/hSPMC/IL-20.3/Rap), and IL-21/rapamycin combination treatment (MST = 42) (Tx/hSPMC/IL-21/Rap). **P = 0.0026, ***P < 0.001. B and C: The representative H-E micrographs of human islet allograft and insulin IHC staining (brown for positive staining) from reconstituted hNSG recipient mice with IL-21 [Tx/IL-2(1)], rapamycin (Tx/Rapamycin), and IL-20.3/rapamycin [Tx/IL-2(0.3)/Rap] groups at day 35 (d35) and the IL-21/rapamycin [Tx/IL-2(1)/Rap] group at d44 after hSPMC transfusion (original magnification ×10). D and E: Quantification of IHC insulin staining of human islet allografts from Tx/IL-21 (n = 5), Tx/rapamycin (n = 5), and Tx/IL-20.3/Rap (n = 7) groups at d35 and Tx/IL-21/Rap group at d44 after hSPMC transfusion (n = 7) compared with human islet grafts in the diabetic NSG mice after successful transplantation without transfusion (n = 5) (Tx/Control) and NSG recipient mice with hSPMCs and without treatment (n = 7) [Tx/hSPMC (Tx/Without)]. Pixel Count Algorithm is based on staining intensity and expressed as positive pixel count/mm2. The log-rank (Mantel-Cox) test was used for comparison of survival data. Kruskal-Wallis test followed by Dunn multiple comparison test was used for comparisons for quantification of IHC insulin. Data were from nine independent experiments. Error bars indicate the mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001.

Close modal

Rejection and survival were monitored by BSL and confirmed by histology of islet allografts. At day 35, islet allografts from hNSG recipient mice receiving either IL-2 alone or rapamycin alone showed a heavy infiltrate of mononuclear cells and no intact islets (Fig. 5B–E). Histological findings were similar to those observed in rejected allografts in the hNSG recipient mice without treatment (Fig. 2C). In contrast, hNSG recipient mice treated with combined IL-2/rapamycin had intact islets with positive insulin staining and were surrounded by a smaller cellular infiltrate at the same time point (Fig. 5B–E). By day 44, intact islets remained with very few infiltrating lymphocytes (Fig. 5B–E). However, this increased to a larger mononuclear cell infiltrate on day 62, containing hCD4+ and hCD8+ T cells (Supplementary Fig. 4). There was no histological evidence of xeno-GVHD in skin, intestine, or right kidney at days 35–44 after reconstitution, and less liver damage was observed (Fig. 6A–C). However, signs of chronic xeno-GVHD with limited skin and liver involvement was observed at day 62 despite functional islet allografts (Fig. 6A–D), with no significant histological evidence of GVHD in the contralateral right kidney (Supplementary Fig. 4).

Figure 6

Histological evaluation of GVHD in transplanted and hNSG mice after treatment with IL-2 and/or rapamycin. A: Representative H-E micrographs of skin, intestine, and liver (original magnification ×10) from the transplanted NSG mice transfused with 1 × 107 hSPMCs and treated with IL-21, rapamycin, and IL-20.3/rapamycin at day 35 (d35) and/or d62 after transfusion, and IL-21/rapamycin at d44 after transfusion. Histological scores of skin, intestine, and liver damage grades (0–4) from hNSG recipient mice with IL-21 (Tx/IL-21/d35), rapamycin (Tx/rapamycin/d35), IL-20.3/rapamycin (Tx/IL-20.3/Rap/d35), and IL-21/rapamycin (Tx/IL-21/Rap/d44) at d35–d44 after transfusion and IL-20.3/rapamycin at 62 days after transfusion (Tx/IL-20.3/Rap/d62) (n = 5 for every group) compared with NSG mice without transplantation and transfusion (n = 9) (normal control) (B), NSG recipient mice without treatment at d35 after hSPMCs transfusion (n = 8) (Tx/hSPMC/d35) (C), and hNSG mice at week 9 (n = 9) (hSPMC/w9) (D). Kruskal-Wallis test followed by Dunn multiple comparison test was used for comparisons. Error bars indicate mean ± SEM. *P < 0.05, ***P < 0.001.

Figure 6

Histological evaluation of GVHD in transplanted and hNSG mice after treatment with IL-2 and/or rapamycin. A: Representative H-E micrographs of skin, intestine, and liver (original magnification ×10) from the transplanted NSG mice transfused with 1 × 107 hSPMCs and treated with IL-21, rapamycin, and IL-20.3/rapamycin at day 35 (d35) and/or d62 after transfusion, and IL-21/rapamycin at d44 after transfusion. Histological scores of skin, intestine, and liver damage grades (0–4) from hNSG recipient mice with IL-21 (Tx/IL-21/d35), rapamycin (Tx/rapamycin/d35), IL-20.3/rapamycin (Tx/IL-20.3/Rap/d35), and IL-21/rapamycin (Tx/IL-21/Rap/d44) at d35–d44 after transfusion and IL-20.3/rapamycin at 62 days after transfusion (Tx/IL-20.3/Rap/d62) (n = 5 for every group) compared with NSG mice without transplantation and transfusion (n = 9) (normal control) (B), NSG recipient mice without treatment at d35 after hSPMCs transfusion (n = 8) (Tx/hSPMC/d35) (C), and hNSG mice at week 9 (n = 9) (hSPMC/w9) (D). Kruskal-Wallis test followed by Dunn multiple comparison test was used for comparisons. Error bars indicate mean ± SEM. *P < 0.05, ***P < 0.001.

Close modal

Combination Low-Dose IL-2 and Rapamycin Therapy Was Associated With Expansion of CD4+CD25+FOXP3+ Tregs

To determine whether prolonged islet allograft survival following IL-2/rapamycin treatment was associated with the expansion of human Tregs (hTregs), the proportion of hCD45+ cells and hCD4+FOXP3+ Tregs was assessed. Compared with the no-treatment group, reconstituted hCD45 cells at week 3 of transfusion was decreased in all treatment groups (except IL-2 only) (Fig. 7A). However, by week 5, a similar proportion of hCD45 cells was seen in all groups (Fig. 7A). In the PB of mice treated with either IL-20.3/rapamycin or the higher dose of IL-21/rapamycin, the proportion of CD4+FOXP3+ Tregs was significantly increased (Fig. 7B). Interestingly, when the IL-21/rapamycin–treated mice were divided into those with a functioning graft and those with a rejected graft, the proportion of splenic CD25+FOXP3+ Tregs was markedly increased in the subset with functioning islet allografts (Fig. 7C). Furthermore, these findings, in PB and spleen, were replicated in the islet allograft, where a reduction in infiltrating CD4+ T cells and an increased proportion of CD4+FOXP3+ Tregs were seen in hNSG recipient mice following IL-2/rapamycin treatment (Fig. 8A and B). Taken together, these data support the proposal that IL-2/rapamycin therapy led to prolonged human islet allograft survival that was closely associated with the expansion of CD4+CD25+FOXP3+ Tregs in the graft, spleen, and PB.

Figure 7

Human islet allograft survival was associated with expansion of CD4+CD25+FOXP3+ Tregs. A: At weeks 3 and 5 of transfusion, the proportion of reconstituted hCD45 in PB of NSG mice transfused with 1 × 107 hSPMCs without transplantation (n = 16) (Without) and transplanted NSG recipient mice transfused with 1 × 107 hSPMCs and no treatment (n = 8) (Tx/Without) plus treated with IL-20.3 (n = 6) (Tx/IL-20.3), IL-21 (n = 6) (Tx/IL-21), rapamycin (n = 6) (Tx/rapamycin), IL-20.3/rapamycin (n = 9) (Tx/IL-20.3/Rap), and IL-21/rapamycin (n = 7) (Tx/IL-21/Rap). B: The representative pseudocolor plot of hCD4 vs. hFOXP3 (gating on hCD4+ T cells) and the proportion of hCD4+Foxp3+ Tregs in PB of the seven groups of hNSG mice and/or transplanted hNSG recipient mice at week 5. C: Representative pseudocolor plots (smooth) of hCD4 vs. hFOXP3 and hCD25 vs. hFOXP3 (gating on hCD4+ T cells) in spleen of IL-21/rapamycin-treated NSG recipient mice that rejected human islet allografts or had functional human islet allografts at day 44 after transfusion. Kruskal-Wallis test followed by Dunn multiple comparison test was used for the comparisons. Error bars indicate the mean ± SEM. **P < 0.01, ***P < 0.001.

Figure 7

Human islet allograft survival was associated with expansion of CD4+CD25+FOXP3+ Tregs. A: At weeks 3 and 5 of transfusion, the proportion of reconstituted hCD45 in PB of NSG mice transfused with 1 × 107 hSPMCs without transplantation (n = 16) (Without) and transplanted NSG recipient mice transfused with 1 × 107 hSPMCs and no treatment (n = 8) (Tx/Without) plus treated with IL-20.3 (n = 6) (Tx/IL-20.3), IL-21 (n = 6) (Tx/IL-21), rapamycin (n = 6) (Tx/rapamycin), IL-20.3/rapamycin (n = 9) (Tx/IL-20.3/Rap), and IL-21/rapamycin (n = 7) (Tx/IL-21/Rap). B: The representative pseudocolor plot of hCD4 vs. hFOXP3 (gating on hCD4+ T cells) and the proportion of hCD4+Foxp3+ Tregs in PB of the seven groups of hNSG mice and/or transplanted hNSG recipient mice at week 5. C: Representative pseudocolor plots (smooth) of hCD4 vs. hFOXP3 and hCD25 vs. hFOXP3 (gating on hCD4+ T cells) in spleen of IL-21/rapamycin-treated NSG recipient mice that rejected human islet allografts or had functional human islet allografts at day 44 after transfusion. Kruskal-Wallis test followed by Dunn multiple comparison test was used for the comparisons. Error bars indicate the mean ± SEM. **P < 0.01, ***P < 0.001.

Close modal
Figure 8

Low-dose IL-2 combined with rapamycin inhibits human islet allograft rejection by expanding hCD4+FOXP3+ Tregs and suppressing effector cell function. A: hCD4+FOXP3+ Tregs within human islet allografts from hNSG recipient mice. The representative fluorescence micrographs of hCD4, hFOXP3, and DAPI nuclear stain for the human islet allografts within the left kidneys of the hNSG recipient mice without treatment (n = 5) (Tx/Without), IL-2–only treatment (n = 5) (Tx/IL-2), and the combination of IL-2/rapamycin treatment (n = 5) (Tx/IL-2/Rap) at day 35 after transfusion of 1 × 107 hSPMCs. B: The percentages of hCD4+ T cells in total counted cells (DAPI) (the numbers of CD4+ cells divided by total cell counts) and hCD4+FOXP3+ Tregs in hCD4+ cells (the numbers of hCD4+FOXP3+ Tregs divided by the number of hCD4+ T cells). Total cell number, hCD4+, and hCD4+FOXP3+ cells were counted on high-resolution micrographs using multipoint cell counter. C: The level of human IFN-γ in the serum of hNSG mice (n = 5) and hNSG recipient mice with IL-21 (n = 3) or rapamycin (n = 4) only and IL-20.3/rapamycin (n = 4) and IL-21/rapamycin (n = 5) at 35 days after reconstitution. D: Representative pseudocolor plots of hCD8 vs. perforin (gating on hCD3+CD8+ T cells) and hCD4 vs. T-bet (gating on hCD3+CD4+ T cells) of flow cytometric analysis by intracellular staining and the proportions of hCD8+perforin+ in hCD3+CD8+ T cells and hCD4+T-bet+ in CD3+CD4+ T cells of spleens from the NSG recipient mice without treatment (n = 6), with IL-2 treatment (n = 6), and with the combination of IL-2 and rapamycin treatment (n = 7) at day 35 after transfusion of hSPMCs. One-way ANOVA followed by Tukey multiple comparison test was used for the comparisons of the proportion of CD4+ T cells, hCD4+FOXP3+ Tregs, hCD8+perforin+, and hCD4+T-bet+. Kruskal-Wallis test followed by Dunn multiple comparison test was used to compare serum levels of hIFN-γ used. Error bars indicate the mean ± SEM. *P < 0.05, **P < 0.01. Mag, magnitude.

Figure 8

Low-dose IL-2 combined with rapamycin inhibits human islet allograft rejection by expanding hCD4+FOXP3+ Tregs and suppressing effector cell function. A: hCD4+FOXP3+ Tregs within human islet allografts from hNSG recipient mice. The representative fluorescence micrographs of hCD4, hFOXP3, and DAPI nuclear stain for the human islet allografts within the left kidneys of the hNSG recipient mice without treatment (n = 5) (Tx/Without), IL-2–only treatment (n = 5) (Tx/IL-2), and the combination of IL-2/rapamycin treatment (n = 5) (Tx/IL-2/Rap) at day 35 after transfusion of 1 × 107 hSPMCs. B: The percentages of hCD4+ T cells in total counted cells (DAPI) (the numbers of CD4+ cells divided by total cell counts) and hCD4+FOXP3+ Tregs in hCD4+ cells (the numbers of hCD4+FOXP3+ Tregs divided by the number of hCD4+ T cells). Total cell number, hCD4+, and hCD4+FOXP3+ cells were counted on high-resolution micrographs using multipoint cell counter. C: The level of human IFN-γ in the serum of hNSG mice (n = 5) and hNSG recipient mice with IL-21 (n = 3) or rapamycin (n = 4) only and IL-20.3/rapamycin (n = 4) and IL-21/rapamycin (n = 5) at 35 days after reconstitution. D: Representative pseudocolor plots of hCD8 vs. perforin (gating on hCD3+CD8+ T cells) and hCD4 vs. T-bet (gating on hCD3+CD4+ T cells) of flow cytometric analysis by intracellular staining and the proportions of hCD8+perforin+ in hCD3+CD8+ T cells and hCD4+T-bet+ in CD3+CD4+ T cells of spleens from the NSG recipient mice without treatment (n = 6), with IL-2 treatment (n = 6), and with the combination of IL-2 and rapamycin treatment (n = 7) at day 35 after transfusion of hSPMCs. One-way ANOVA followed by Tukey multiple comparison test was used for the comparisons of the proportion of CD4+ T cells, hCD4+FOXP3+ Tregs, hCD8+perforin+, and hCD4+T-bet+. Kruskal-Wallis test followed by Dunn multiple comparison test was used to compare serum levels of hIFN-γ used. Error bars indicate the mean ± SEM. *P < 0.05, **P < 0.01. Mag, magnitude.

Close modal

Serum Human IFN-γ Is Reduced in Mice Receiving Combination Low-Dose IL-2 and Rapamycin Therapy

Serum from hNSG recipient mice 35 days after transfusion was examined for human IL-2, IL-4, IL-6, IFN-γ, IL-10, TNF, and IL-17A by cytometric bead array. Other than human IFN-γ, the remainder of the cytokines were absent or low in the serum, with no significant difference observed between groups (Fig. 8C and data not shown). While hIFN-γ was detectable in hNSG recipient mice receiving no treatment or receiving rapamycin alone, in those receiving IL-20.3/rapamycin, the serum concentration of hIFN-γ was significantly reduced compared with NSG mice transfused with hSPMCs only (Fig. 8C).

Prolongation of Human Islet Allograft Survival Was Associated With Reduced CD8+ Effector T Cells and Expanded Regulatory CD4+ T Cells in Mice Receiving Combination Low-Dose IL-2 and Rapamycin Therapy

Spleens were removed from hNSG recipient mice with and without IL-2 treatment alone at the time of rejection or following IL-2/rapamycin treatment (day 35). Splenocytes were analyzed by flow cytometry for T-bet and perforin, respectively (Fig. 8D). Intracellular staining of splenocytes from hNSG recipient mice confirmed a significant decrease in perforin expression in reconstituted hCD8+ T cells following IL-2/rapamycin treatment compared with no treatment or IL-2 only (Fig. 8D). These findings were supported by gene expression analysis by RT-PCR on sorted hCD4+ and hCD8+ T cells where there was a significant reduction in IFN-γ gene expression and a trend to lower perforin-1 expression in hCD8+ T cells in hNSG recipient mice receiving IL-2/rapamycin treatment, with no difference in IL-10, TGF-β, or IL-2 gene expression (Supplementary Fig. 5A).

In splenic hCD4+ T cells, there was a significant increase in the expression of T-bet, a key regulator of Th1 responses, in hNSG recipient mice receiving IL-2 alone or IL-2/rapamycin treatment compared with the no-treatment group (Fig. 8D). This finding was supported by gene expression data that showed a trend toward increased expression of Blimp-1 (30), T-bet, and TGF-β and no differences in expression of GATA3 and RORγt, which are transcriptional factors for Th2 and Th17 T-cell subsets, respectively (Supplementary Fig. 5B).

Our study shows that low-dose IL-2 in combination with low-dose rapamycin leads to an increased proportion of CD4+FOXP3+ Tregs, which was associated with prolonged (human) islet allograft survival in hNSG recipient mice reconstituted with hSPMCs. In this model, hSPMC transfusion led to hCD45+ cell reconstitution predominantly with CD4+ and CD8+ T cells and at early time points after reconstitution. CD4+FOXP3+Tregs were present in similar proportions to that found in human blood or spleen. As shown previously, this is an excellent model of human T-cell–driven allograft rejection (31). While several studies have shown that human T cells expand rapidly, express an activated immune phenotype, and cause xeno-GVHD after transfer to NSG and other immunodeficient mouse models, the time frame for GVHD exceeds that of allograft rejection, allowing the rejection response to be studied (31,32). It was hypothesized that in vivo expansion of hTregs in the presence of antigen would be more efficient at preventing rejection than infusion of hTregs that were expanded in vitro. To achieve in vivo hTreg expansion, a combination of IL-2 and rapamycin was used. IL-2 is an essential growth factor for Tregs, which constitutively express the high-affinity IL-2R (33). Clinically, low-dose IL-2 has been used in several auto- and alloimmune diseases, including SLE and chronic GVHD (5,7). Low-dose IL-2 treatment leads to a selective expansion of Tregs and clinical improvement in patients with GVHD (5,34). Similarly, in patients with SLE, low-dose IL-2 selectively modulates CD4+ T-cell subsets, with an increase in Treg number and function, and decreases follicular helper T and Th17 cells but not Th1 and Th2 cells (7). The combination of IL-2 and rapamycin has been used in a phase 1 trial of early-onset T1D leading to increased Tregs but a transient impairment of β-cell function (35). This highlights the complexities of clinical translation. In T1D, an autoimmune memory T-cell response is well established that is not able to be reversed, whereas islet transplantation has the advantage of initiating therapy at the time of transplantation when effector memory cells have not yet developed and where their development can be suppressed. Rapamycin has also been reported in some studies to be toxic to islets; however, the extent of this remains controversial, and rapamycin remains the main immunosuppressive for preventing clinical islet allograft rejection (36,37).

On the basis of the complementary role of IL-2 and rapamycin on Treg homeostasis, we proposed that these agents could be used to selectively expand Tregs in vivo and limit effector T-cell expansion. In the humanized mouse model of human islet allograft rejection and at the doses used in this study, the addition of IL-2 alone did not prolong islet allograft survival, although it expanded Tregs in vivo. Likewise, the use of rapamycin in the doses used here led to neither Treg expansion in vivo nor prolongation of graft survival. However, the combination of low-dose IL-2 and rapamycin for 3 weeks resulted in the expansion of CD25+FOXP3+ Tregs and prolonged islet allograft survival. In hNSG recipient mice with long-term surviving islet allografts, the proportion of splenic CD25+FOXP3+ Tregs was enhanced, suggesting that CD25+FOXP3+ Tregs were responsible for promoting graft survival. Interestingly, the IL-2/rapamycin treatment groups showed a significant decrease in the proportion of hCD45+ cells within the PB of the transplanted mice, suggesting that rapamycin either delayed hCD45+ cell reconstitution or suppressed effector T-cell proliferation after hSPMC transfusion. Taken together, these results suggest that IL-2 was an important driver of Treg expansion and that rapamycin played an important role in selectively suppressing effector T-cell expansion and T-cell–mediated islet alloimmunity.

IFN-γ is associated with rejection of solid organ and islet allografts in both experimental mouse models (38,39) and human biopsy specimens (40). IFN-γ was essential for rejection in a CD8+ T-cell–mediated rejection model of islet allotransplantation (38) as well as a CD4+ T-cell–mediated model of cardiac allograft rejection (39). Although IFN-γ is crucial for Th1 responses, it is secreted by natural killer T cells (41), CD8+ T cells (38), and CD4+CD25+ Tregs (42). It is also associated with maintenance of transplant tolerance (42,43) and plays a presumed role in rejection (3840). In the findings presented here, serum and gene expression levels of IFN-γ were associated with increased T-bet gene expression, a Th1-specific transcription factor. When evaluated in the context of the study’s graft survival data, the findings were consistent with Treg-mediated suppression of the Th1 alloimmune response. Increased T-bet expression may reflect expression in Th1-specific Tregs. Graft survival was associated with reduced perforin-1 in CD8+T cells, suggesting an associated reduction in CD8+ T-cell–mediated effector responses (44).

CD4+FOXP3+ Tregs have been shown to be potent suppressors in experimental models of allo- and autoimmunity (45,46). Infusions of FOXP3+ Tregs expanded in vitro have been evaluated in phase 1/2 clinical trials of renal transplantation and GVHD as well as in autoimmune conditions such as T1D (4750) (www.onestudy.org). Major limitations of this approach include the large numbers of Tregs that lack antigen specificity, the requirement for multiple infusions, and the cost of regulatory requirements and production. The ability to expand autologous Tregs in the presence of antigen is an attractive alternative to in vitro manipulation and expansion. We have reported here that the combination of low-dose IL-2 and rapamycin can successfully lead to in vivo expansion of Tregs, suppression of effector T-cell proliferation, and prolongation of islet allograft survival without the requirement for in vitro cell manipulation. It has the added advantage that both IL-2 and rapamycin are approved for clinical use in humans with an acceptable safety profile.

These findings need to be interpreted within the limitations of the model. NSG mice infused with hSPMCs did not have a completely reconstituted human immune system. The reconstituted cells were predominantly T cells, with limited B-cell repopulation. Previous studies have demonstrated that B cells contribute to allograft tolerance in human and mouse studies, making prolonged islet graft survival in the hNSG mice more remarkable (51). While in vivo–expanded Tregs were associated with prolonged graft survival and an immunoregulatory phenotype, their antigen-specific regulatory function was not confirmed. However, in previous studies, we have shown that in models of islet tolerance, expansion of Foxp3+ Tregs in the presence of antigen was a potent regulator of the graft immune response (26). This model was focused on the strategies that could inhibit the alloimmune response to human islets while limiting the burden of immunosuppression, which remains a major limitation to clinical islet transplantation. It does not address the issue of autoimmune disease recurrence in the host, which may not be amenable to this approach. Despite these limitations, this study demonstrates the potential of low-dose IL-2/rapamycin to limit human islet allograft rejection by expanding Tregs and limiting effector T-cell proliferation. These findings expand on successful results in murine experiments using IL-2 or IL-2/anti–IL-2 antibody complexes and rapamycin as a combination, leading to Treg expansion and effector T-cell suppression (14,52). These studies for the first time show the efficacy of IL-2 and rapamycin as a combination in a human immune model of allorejection of human islet transplants. Many strategies to expand naïve Tregs, ex vivo or in vivo, have failed in primate studies or human trials because of preexisting memory responses or a lack of antigen specificity, despite being successful in naïve mouse models. Our results suggest that it may be possible, using agents already approved for clinical use, to develop a clinical regimen that can minimize the burden of long-term immunosuppression or encouraging operational tolerance through the expansion of CD4+FOXP3+ Tregs in vivo, leading to long-term islet allograft survival in humans. Such a strategy, if successful, would have the potential to expand the indications for islet transplantation in T1D.

This article contains supplementary material online at https://doi.org/10.2337/figshare.12241664.

Acknowledgments. The authors thank the members of the Department of Animal Care at Westmead Hospital. Cell sorting and histological sample processing were performed at the Westmead Scientific Platforms, which are supported by the Westmead Research Hub, the Westmead Institute for Medical Research, the Cancer Institute New South Wales, the National Health and Medical Research Council of Australia (NHMRC), and the Ian Potter Foundation.

Funding. This work was supported by the NHMRC, JDRF/Australian Research Council, and Diabetes Australia and University of Sydney. M.H. was awarded an early career fellowship from the NHMRC (GNT1013185), a Deputy Vice Chancellor University of Sydney research fellowship (IRMA178768) and Sydney Medical School Early Career Researcher Scheme, and Diabetes Australia research program from Diabetes Australia and University of Sydney (Y16G-HUMI). N.R. is a recipient of an NHMRC career development fellowship (GNT1158597). P.J.O. was a recipient of a senior practitioner fellowship from the NMHRC (GNT1125456) and the principal investigator of JDRF/Australian Research Council grant 4-SRA-2016-265-M-B.

Duality of Interest. No potential conflicts of interest relevant to this article were reported.

Author Contributions. M.H. designed and performed experiments, analyzed research data, wrote the manuscript, and finalized the manuscript for publication. W.J.H. performed human islet isolation and islet transplantation. L.N. performed double immunofluorescence staining and histological image analysis. H.B., Y.W.Q., D.L., E.J.V., Y.V.C., and L.W. performed human islet and spleen isolation, histological examination, and/or animal monitoring. S.Y. contributed to the discussion of the research data. K.K. and D.W. contributed to the discussion of the research data and histological examination. N.R. and S.I.A. contributed to the discussion and experimental design and edited the manuscript. P.J.O. proposed the study and designed the experiments, edited and reviewed the manuscript, and gave final approval of the final manuscript for publication. M.H. and P.J.O. are the guarantors of this work and, as such, had full access to all the data in the study and take responsibility for the integrity of the data and the accuracy of the data analysis.

Prior Presentation. Parts of this study were presented in oral abstract form at the 27th International Congress of the Transplantation Society, Madrid, Spain, 30 June–5 July 2018.

1.
Shapiro
AM
,
Ricordi
C
,
Hering
BJ
, et al
.
International trial of the Edmonton protocol for islet transplantation
.
N Engl J Med
2006
;
355
:
1318
1330
2.
O’Connell
PJ
,
Holmes-Walker
DJ
,
Goodman
D
, et al.;
Australian Islet Transplant Consortium
.
Multicenter Australian trial of islet transplantation: improving accessibility and outcomes
.
Am J Transplant
2013
;
13
:
1850
1858
3.
Foster
ED
,
Bridges
ND
,
Feurer
ID
,
Eggerman
TL
,
Hunsicker
LG
,
Alejandro
R
;
Clinical Islet Transplantation Consortium
.
Improved health-related quality of life in a phase 3 islet transplantation trial in type 1 diabetes complicated by severe hypoglycemia
.
Diabetes Care
2018
;
41
:
1001
1008
4.
Pepper
AR
,
Gala-Lopez
B
,
Ziff
O
,
Shapiro
AJ
.
Current status of clinical islet transplantation
.
World J Transplant
2013
;
3
:
48
53
5.
Koreth
J
,
Matsuoka
K
,
Kim
HT
, et al
.
Interleukin-2 and regulatory T cells in graft-versus-host disease
.
N Engl J Med
2011
;
365
:
2055
2066
6.
Saadoun
D
,
Rosenzwajg
M
,
Joly
F
, et al
.
Regulatory T-cell responses to low-dose interleukin-2 in HCV-induced vasculitis
.
N Engl J Med
2011
;
365
:
2067
2077
7.
He
J
,
Zhang
X
,
Wei
Y
, et al
.
Low-dose interleukin-2 treatment selectively modulates CD4(+) T cell subsets in patients with systemic lupus erythematosus
.
Nat Med
2016
;
22
:
991
993
8.
Hartemann
A
,
Bensimon
G
,
Payan
CA
, et al
.
Low-dose interleukin 2 in patients with type 1 diabetes: a phase 1/2 randomised, double-blind, placebo-controlled trial
.
Lancet Diabetes Endocrinol
2013
;
1
:
295
305
9.
Morath
C
,
Arns
W
,
Schwenger
V
, et al
.
Sirolimus in renal transplantation
.
Nephrol Dial Transplant
2007
;
22
(
Suppl. 8
):
viii61
viii65
10.
Hester
J
,
Schiopu
A
,
Nadig
SN
,
Wood
KJ
.
Low-dose rapamycin treatment increases the ability of human regulatory T cells to inhibit transplant arteriosclerosis in vivo
.
Am J Transplant
2012
;
12
:
2008
2016
11.
Battaglia
M
,
Stabilini
A
,
Roncarolo
MG
.
Rapamycin selectively expands CD4+CD25+FoxP3+ regulatory T cells
.
Blood
2005
;
105
:
4743
4748
12.
Shapiro
AM
,
Lakey
JR
,
Ryan
EA
, et al
.
Islet transplantation in seven patients with type 1 diabetes mellitus using a glucocorticoid-free immunosuppressive regimen
.
N Engl J Med
2000
;
343
:
230
238
13.
Tahvildari
M
,
Omoto
M
,
Chen
Y
, et al
.
In vivo expansion of regulatory T cells by low-dose interleukin-2 treatment increases allograft survival in corneal transplantation
.
Transplantation
2016
;
100
:
525
532
14.
Pilon
CB
,
Petillon
S
,
Naserian
S
, et al
.
Administration of low doses of IL-2 combined to rapamycin promotes allogeneic skin graft survival in mice
.
Am J Transplant
2014
;
14
:
2874
2882
15.
Chi
H
.
Regulation and function of mTOR signalling in T cell fate decisions
.
Nat Rev Immunol
2012
;
12
:
325
338
16.
Kuo
CJ
,
Chung
J
,
Fiorentino
DF
,
Flanagan
WM
,
Blenis
J
,
Crabtree
GR
.
Rapamycin selectively inhibits interleukin-2 activation of p70 S6 kinase
.
Nature
1992
;
358
:
70
73
17.
Chaoul
N
,
Fayolle
C
,
Desrues
B
, et al
.
Rapamycin impairs antitumor CD8+ T-cell responses and vaccine-induced tumor eradication
.
Cancer Res
2015
;
75
:
3279
3291
18.
Battaglia
M
,
Stabilini
A
,
Migliavacca
B
,
Horejs-Hoeck
J
,
Kaupper
T
,
Roncarolo
MG
.
Rapamycin promotes expansion of functional CD4+CD25+FOXP3+ regulatory T cells of both healthy subjects and type 1 diabetic patients
.
J Immunol
2006
;
177
:
8338
8347
19.
Monti
P
,
Scirpoli
M
,
Maffi
P
, et al
.
Rapamycin monotherapy in patients with type 1 diabetes modifies CD4+CD25+FOXP3+ regulatory T-cells
.
Diabetes
2008
;
57
:
2341
2347
20.
Wang
Y
,
Camirand
G
,
Lin
Y
, et al
.
Regulatory T cells require mammalian target of rapamycin signaling to maintain both homeostasis and alloantigen-driven proliferation in lymphocyte-replete mice
.
J Immunol
2011
;
186
:
2809
2818
21.
Long
SA
,
Buckner
JH
.
Combination of rapamycin and IL-2 increases de novo induction of human CD4(+)CD25(+)FOXP3(+) T cells
.
J Autoimmun
2008
;
30
:
293
302
22.
Yi
S
,
Ji
M
,
Wu
J
, et al
.
Adoptive transfer with in vitro expanded human regulatory T cells protects against porcine islet xenograft rejection via interleukin-10 in humanized mice
.
Diabetes
2012
;
61
:
1180
1191
23.
Rosborough
BR
,
Raïch-Regué
D
,
Liu
Q
,
Venkataramanan
R
,
Turnquist
HR
,
Thomson
AW
.
Adenosine triphosphate-competitive mTOR inhibitors: a new class of immunosuppressive agents that inhibit allograft rejection
.
Am J Transplant
2014
;
14
:
2173
2180
24.
Stokes
RA
,
Cheng
K
,
Lalwani
A
, et al
.
Transplantation sites for human and murine islets
.
Diabetologia
2017
;
60
:
1961
1971
25.
Hu
M
,
Wang
C
,
Zhang
GY
, et al
.
Infiltrating Foxp3(+) regulatory T cells from spontaneously tolerant kidney allografts demonstrate donor-specific tolerance
.
Am J Transplant
2013
;
13
:
2819
2830
26.
Wu
J
,
Hu
M
,
Qian
YW
, et al
.
In vivo costimulation blockade-induced regulatory T cells demonstrate dominant and specific tolerance to porcine islet xenografts
.
Transplantation
2017
;
101
:
1587
1599
27.
Schwasinger-Schmidt
T
,
Robbins
DC
,
Williams
SJ
,
Novikova
L
,
Stehno-Bittel
L
.
Long-term liraglutide treatment is associated with increased insulin content and secretion in β-cells, and a loss of α-cells in ZDF rats
.
Pharmacol Res
2013
;
76
:
58
66
28.
García-Rojo
M
,
Sánchez
J
,
de la Santa
E
, et al
.
Automated image analysis in the study of lymphocyte subpopulation in eosinophilic oesophagitis
.
Diagn Pathol
2014
;
9
(
Suppl. 1
):
S7
29.
Schneider
CA
,
Rasband
WS
,
Eliceiri
KW
.
NIH Image to ImageJ: 25 years of image analysis
.
Nat Methods
2012
;
9
:
671
675
30.
Cretney
E
,
Xin
A
,
Shi
W
, et al
.
The transcription factors Blimp-1 and IRF4 jointly control the differentiation and function of effector regulatory T cells
.
Nat Immunol
2011
;
12
:
304
311
31.
Kenney
LL
,
Shultz
LD
,
Greiner
DL
,
Brehm
MA
.
Humanized mouse models for transplant immunology
.
Am J Transplant
2016
;
16
:
389
397
32.
King
MA
,
Covassin
L
,
Brehm
MA
, et al
.
Human peripheral blood leucocyte non-obese diabetic-severe combined immunodeficiency interleukin-2 receptor gamma chain gene mouse model of xenogeneic graft-versus-host-like disease and the role of host major histocompatibility complex
.
Clin Exp Immunol
2009
;
157
:
104
118
33.
Boyman
O
,
Sprent
J
.
The role of interleukin-2 during homeostasis and activation of the immune system
.
Nat Rev Immunol
2012
;
12
:
180
190
34.
Matsuoka
K
,
Koreth
J
,
Kim
HT
, et al
.
Low-dose interleukin-2 therapy restores regulatory T cell homeostasis in patients with chronic graft-versus-host disease
.
Sci Transl Med
2013
;
5
:
179ra43
35.
Long
SA
,
Rieck
M
,
Sanda
S
, et al.;
Diabetes TrialNet and the Immune Tolerance Network
.
Rapamycin/IL-2 combination therapy in patients with type 1 diabetes augments Tregs yet transiently impairs β-cell function
.
Diabetes
2012
;
61
:
2340
2348
36.
Hering
BJ
,
Clarke
WR
,
Bridges
ND
, et al.;
Clinical Islet Transplantation Consortium
.
Phase 3 trial of transplantation of human islets in type 1 diabetes complicated by severe hypoglycemia
.
Diabetes Care
2016
;
39
:
1230
1240
37.
Dai
C
,
Walker
JT
,
Shostak
A
, et al
.
Tacrolimus- and sirolimus-induced human β cell dysfunction is reversible and preventable
.
JCI Insight
2020
;
5
:
130770
38.
Diamond
AS
,
Gill
RG
.
An essential contribution by IFN-gamma to CD8+ T cell-mediated rejection of pancreatic islet allografts
.
J Immunol
2000
;
165
:
247
255
39.
Wiseman
AC
,
Pietra
BA
,
Kelly
BP
,
Rayat
GR
,
Rizeq
M
,
Gill
RG
.
Donor IFN-gamma receptors are critical for acute CD4(+) T cell-mediated cardiac allograft rejection
.
J Immunol
2001
;
167
:
5457
5463
40.
Toki
D
,
Zhang
W
,
Hor
KL
, et al
.
The role of macrophages in the development of human renal allograft fibrosis in the first year after transplantation
.
Am J Transplant
2014
;
14
:
2126
2136
41.
Olson
CM
 Jr
.,
Bates
TC
,
Izadi
H
, et al
.
Local production of IFN-gamma by invariant NKT cells modulates acute Lyme carditis
.
J Immunol
2009
;
182
:
3728
3734
42.
Sawitzki
B
,
Kingsley
CI
,
Oliveira
V
,
Karim
M
,
Herber
M
,
Wood
KJ
.
IFN-gamma production by alloantigen-reactive regulatory T cells is important for their regulatory function in vivo
.
J Exp Med
2005
;
201
:
1925
1935
43.
Hassan
AT
,
Dai
Z
,
Konieczny
BT
, et al
.
Regulation of alloantigen-mediated T-cell proliferation by endogenous interferon-gamma: implications for long-term allograft acceptance
.
Transplantation
1999
;
68
:
124
129
44.
Wever
PC
,
Boonstra
JG
,
Laterveer
JC
, et al
.
Mechanisms of lymphocyte-mediated cytotoxicity in acute renal allograft rejection
.
Transplantation
1998
;
66
:
259
264
45.
Webster
KE
,
Walters
S
,
Kohler
RE
, et al
.
In vivo expansion of T reg cells with IL-2-mAb complexes: induction of resistance to EAE and long-term acceptance of islet allografts without immunosuppression
.
J Exp Med
2009
;
206
:
751
760
46.
Hu
M
,
Wang
YM
,
Wang
Y
, et al
.
Regulatory T cells in kidney disease and transplantation
.
Kidney Int
2016
;
90
:
502
514
47.
Trzonkowski
P
,
Bieniaszewska
M
,
Juścińska
J
, et al
.
First-in-man clinical results of the treatment of patients with graft versus host disease with human ex vivo expanded CD4+CD25+CD127- T regulatory cells
.
Clin Immunol
2009
;
133
:
22
26
48.
Di Ianni
M
,
Falzetti
F
,
Carotti
A
, et al
.
Tregs prevent GVHD and promote immune reconstitution in HLA-haploidentical transplantation
.
Blood
2011
;
117
:
3921
3928
49.
Brunstein
CG
,
Miller
JS
,
Cao
Q
, et al
.
Infusion of ex vivo expanded T regulatory cells in adults transplanted with umbilical cord blood: safety profile and detection kinetics
.
Blood
2011
;
117
:
1061
1070
50.
Marek-Trzonkowska
N
,
Mysliwiec
M
,
Dobyszuk
A
, et al
.
Administration of CD4+CD25highCD127- regulatory T cells preserves β-cell function in type 1 diabetes in children
.
Diabetes Care
2012
;
35
:
1817
1820
51.
Karahan
GE
,
Claas
FH
,
Heidt
S
.
B Cell immunity in solid organ transplantation
.
Front Immunol
2017
;
7
:
686
52.
Manirarora
JN
,
Wei
CH
.
Combination therapy using IL-2/IL-2 monoclonal antibody complexes, rapamycin, and islet autoantigen peptides increases regulatory T cell frequency and protects against spontaneous and induced Type 1 diabetes in nonobese diabetic mice
.
J Immunol
2015
;
195
:
5203
5214
Readers may use this article as long as the work is properly cited, the use is educational and not for profit, and the work is not altered. More information is available at https://www.diabetesjournals.org/content/license.