Operational immune tolerance towards transplanted allogeneic pancreatic islets in mice and a non-human primate

Abstract

Aims/hypothesis: Patients with autoimmune type 1 diabetes transplanted with pancreatic islets to their liver experience significant improvement in quality of life through better control of blood sugar and enhanced awareness of hypoglycaemia. However, long-term survival and efficacy of the intrahepatic islet transplant are limited owing to liver-specific complications, such as immediate blood-mediated immune reaction, hypoxia, a highly enzymatic and inflammatory environment and locally elevated levels of drugs including immunosuppressive agents, all of which are injurious to islets. This has spurred a search for new islet transplant sites and for innovative ways to achieve long-term graft survival and efficacy without life-long systemic immunosuppression and its complications.

Methods: We used our previously established approach of islet transplant in the anterior chamber of the eye in allogeneic recipient mouse models and a baboon model of diabetes, which were treated transiently with anti-CD154/CD40L blocking antibody in the peri-transplant period. Survival of the intraocular islet allografts was assessed by direct visualisation in the eye and metabolic variables (blood glucose and C-peptide measurements). We evaluated longitudinally the cytokine profile in the local microenvironment of the intraocular islet allografts, represented in aqueous humour, under conditions of immune rejection vs tolerance. We also evaluated the recall response in the periphery of the baboon recipient using delayed-type hypersensitivity (DTH) assay, and in mice after repeat transplant in the kidney following initial transplant with allogeneic islets in the eye or kidney.

Results: Results in mice showed >300 days immunosuppression-free survival of allogeneic islets transplanted in the eye or kidney. Notably, >70% of tolerant mice, initially transplanted in the eye, exhibited >400 days of graft survival after re-transplant in the kidney without immunosuppression compared with ~30% in mice that were initially transplanted in the kidney. Cytokine and DTH data provided evidence of T helper 2-driven local and peripheral immune regulatory mechanisms in support of operational immune tolerance towards the islet allografts in both models.

Conclusions/interpretation: We are currently evaluating the safety and efficacy of intraocular islet transplantation in a phase 1 clinical trial. In this study, we demonstrate immunosuppression-free long-term survival of intraocular islet allografts in mice and in a baboon using transient peri-transplant immune intervention. These results highlight the potential for inducing islet transplant immune tolerance through the intraocular route. Therefore, the current findings are conceptually significant and may impact markedly on clinical islet transplantation in the treatment of diabetes.

Keywords: Allogeneic rejection; Anterior chamber of the eye; Immune tolerance induction and maintenance; Immunosuppression-free; Intraocular transplantation; Long-term graft survival; Non-invasive longitudinal intravital imaging; Pancreatic islet transplant; Th2 cytokines.

Fig. 1

Transient peri-transplantation anti-CD154 antibody treatment leads to long-term survival of intraocular islet allografts. (a, b) Representative longitudinal images of B6 mouse eyes transplanted with allogeneic DBA/2 islets in the anterior chamber of the eye while treated transiently with isotype Ig control/PBS (a) or anti-CD154 antibody (MR-1) (b). Images on POD 7 show the transplanted islets engrafted on top of the iris that were rejected by POD 24 in mice treated with PBS/Ig control (a) but were still clearly visible on POD 347 in the anti-CD154-treated mice, long after stopping treatment on POD 7 (b). (c) Non-fasting blood glucose in STZ-induced diabetic B6 mice before and after transplantation of 250–300 IEQs (DBA/2) in the eye anterior chamber with MR-1 (n=7) or Ig control (n=5) treatments. Grey area indicates duration of the treatment. Normoglycaemia is defined as <11.11 mmol/l (dotted horizontal line) and diabetes/hyperglycaemia as >16.66 mmol/l (see also Methods). (d, e) Kaplan–Meier survival curves of islet allografts in diabetic B6 mice treated transiently (grey shaded areas) with MR-1/Ig control/PBS and transplanted initially (first transplant) either in the anterior chamber of the eye (d) or under the kidney capsule (e). For mice transplanted in the eye: MR-1 n=13, Ig control n=6 and PBS n=17; For mice transplanted in the kidney: MR-1 n=19 and Ig control n=5. (f) Survival of repeat transplant (second transplant) of islet allografts in the kidney following initial islet transplantation (first transplant) either in the anterior chamber of the eye or in the kidney. In MR-1-treated mice, median survival time was 70 days in mice initially transplanted in the kidney and remained undefined in those initially transplanted in the eye (p=0.012 by logrank Mantel–Cox test; also see ESM Fig. 1 for corresponding Ig controls). ACE, anterior chamber of the eye; BG, blood glucose; D-B6, diabetic B6; Ig Ctrl, Ig control; KDN, kidney; TX, transplant

Fig. 2

Intraocular islet allografts survived and remained functional long after stopping anti-CD154 monotherapy in a diabetic baboon. (a) Longitudinal images of the baboon eye before (POD 56 and POD 154) and after (POD 429 and POD 728) stopping anti-CD154 (5C8) antibody treatment on POD 248. Inset shows intact islets on POD 728, which was 480 days after stopping immunosuppression. (b) Fluorescence micrographs showing positive insulin and glucagon immunostaining in a frozen eye section obtained after necropsy of the baboon on POD 728. (c) C-peptide levels in aqueous humour of the recipient baboon measured by electrochemiluminescence immunoassay. Aqueous humour samples were collected from the diabetic baboon islet-transplanted right eye (OD) and non-transplanted left eye (OS) before (POD 31; n=1) and after stopping anti-CD154 antibody treatment (POD 255 and POD 429; n=1 each). (d) C-peptide levels in plasma of recipient baboon before/after islet transplantation and before/after stopping immunosuppression (5C8). The box and whisker plot showss the median values (horizontal black lines), the interquartile range, and the minimum and maximum values in each dataset (individual data points shown as white circles). (e) Blood glucose and (f) change in plasma C-peptide levels during 60 min IVGTTs performed before intraocular islet transplantation on POD −48 and after transplantation on POD 73, POD 128 and POD 204. C-peptide levels (shown as Δ C-peptide) were normalised to the mean (i.e. ratio) of values measured at −10 and −5, and 0 min (0 min = time of injection of glucose bolus; 0.5 g/kg) during the IVGTTs. Aq., aqueous; BG, blood glucose; ND, not detected; TX, transplant

Fig. 3

Cytokine profiles within the local islet environment varied significantly between rejection vs tolerance of intraocular islet allografts. (a–f) Cytokine levels measured by Bio-Plex assay in aqueous humour samples collected from B6 mice exhibiting long-term survival (tolerant; n=13 mice) or ongoing rejection (rejecting; n=9), or from mice that had completely rejected (>20 days post rejection onset; n=10) their intraocular islet allografts, as well as from non-transplanted B6 control mice (No TX Ctrl; n=8). Results are shown as means ± SEM. *p<0.05 (by ANOVA) . (g–j) Cytokine levels measured by Bio-Plex assay in aqueous humour samples collected from the right (OD) and left (OS) eyes of the transplanted baboon during 5C8 treatment (POD 31; OD only; n=1) and after stopping 5C8 (anti-CD154) treatment on POD 429 (n=1 each). ND, not detected; TX, transplant

Fig. 4

Operational immune tolerance of intraocular islet allografts associated with donor-specific peripheral immune regulation (bystander pression) in trans vivo DTH assay. (a–c) Net swelling in trans vivo DTH by the recipient baboon on POD 629 (a) and POD 728 (b) and an untreated control (Ctrl) baboon (non-transplanted) (c) in response to challenge by recall antigen TT/D alone (positive control), donor antigen (Donor Ag) and a mixture of both (n=1 in each condition). (d) Per cent inhibition of the recall response to TT/D by the recipient baboon (black bars) and non-transplanted untreated control (Ctrl) baboon (hatched bars) in the presence of soluble antigens from the specific donor baboon from which islets were isolated (Donor Ag) and naive third-party control baboons Ctrl Ag (1) and Ctrl Ag (2); n=1 each). Swelling data in the different conditions were normalised to response to TT/D alone and pooled from repeat trans vivo DTH assays (n=3 for recipient baboon on POD 629, POD 665 and POD 728; n=2 for untreated control) and presented as means ± SD (see also Methods). *p<0.05 (by unpaired Student’s t test) vs control antigens. (e) Cytokine dependence of donor-specific linked immune regulation in the recipient baboon. DTH recall response (shown as net swelling) to TT/D by the recipient baboon in the presence of the donor baboon antigens (Donor Ag) without and with blocking antibodies against IL-10, IL-35 (anti-IL-12[P35] + anti-Ebi3) and TGF-β, or Ig isotype control (Ctrl Ab). Data shown as means ± SD