Tolerance of Vascularized Islet-Kidney Transplants in Rhesus Monkeys

Abstract

We previously reported that transplantation (Tx) of prevascularized donor islets as composite islet-kidneys (IK) reversed diabetic hyperglycemia in both miniature swine and baboons. In order to enhance this strategy's potential clinical applicability, we have now combined this approach with hematopoietic stem cell (HSC) Tx in an attempt to induce tolerance in nonhuman primates. IKs were prepared by isolating islets from 70% partial pancreatectomies and injecting them beneath the autologous renal capsule of five rhesus monkey donors at least 3 months before allogeneic IK Tx. HSC Tx was performed after mobilization and leukapheresis of the donors and conditioning of the recipients with total body irradiation, T cell depletion, and cyclosporine. One IK was harvested for histologic analysis and four were transplanted into diabetic recipients. IK Tx was performed either 20-22 (n = 3) or 208 (n = 1) days after HSC Tx. All animals accepted IKs without rejection. All recipients required >20 U/day insulin before IK Tx to maintain <200 mg/dL, whereas after IK Tx, three animals required minimal doses of insulin (1-3 U/day) and one animal was insulin free. These results constitute a proof-of-principle that this IK tolerance strategy may provide a cure for both end-stage renal disease and diabetes without the need for immunosuppression.

Keywords: islet isolation; islet transplantation; kidney transplantation/nephrology; tolerance: chimerism; tolerance: experimental; translational research/science.

Fig. 1

a) Number of CD3+ T-cells Pre and post HSC transplantation in all the HSC transplanted recipients. b) Macrochimerism among total leukocytes in the peripheral blood: S0209, S0610 and S1010 had transient macrochimerism which was determined by the presence of H38+ cells in peripheral blood lymphocytes following HSC Tx. This macrochimerism was below the level of detection (<0.5%) at the time of a primary kidney (S0610) or an IK Tx (S1010). HSC, hematopoietic stem cell; Tx, transplantation.

Fig. 2

Assessment of anti-donor responses at the T or B cell levels following HSC Tx, but prior to kidney Tx, in S0409. A) MLR showed that the animal developed hyporesponsiveness as compared to prior to HSC Tx, but this was not donor specific. B) Neither anti-donor IgM nor IgG developed following HSC Tx. HSC, hematopoietic stem cell; MLR, mixed lymphocyte reaction; Tx, transplantation.

Fig. 3

Serum creatinine levels (mg/dl) following kidney transplantation in S0409, the animal that received allogeneic kidney from the HSC donor on day 105 following HSC Tx without immunosuppression. Two transient rises in serum creatinine levels were caused by either dehydration or ureteral stent occlusion by debris, and were resolved by hydration and/or replacement of the ureteral stent. HSC, hematopoietic stem cell; Tx, transplantation.

Fig. 4

A) Anti-donor and third party MLR responses at 50 days and 105 days after kidney transplantation in S0409. Anti-donor responses decreased over time while anti-third party responses were maintained after kidney transplantation. B) Immunofluoresence specimens showed IgM deposits as well as C3 and C4d, but no IgG deposits were observed (Fig. 4B-c, d, e and f), indicating class switch from IgM to IgG did not occur.Gross findings (a) and histology of the kidney graft (b, c, d, e and f) on day 75 following kidney Tx in S0409. a) Grossly the kidney was well vascularized without edema or spot hemorrhages. b) Minimal lymphoid aggregation was seen around vessels with interstitial cell infiltrates (H&E ×200). Immunofluorescence staining showed IgM (c), C3 (e) and C4d deposits (linear pattern (f)) but no IgG deposits were observed (d). H&E: hematoxylin and eosin staining. MLR, mixed lymphocyte reaction; Tx, transplantation.

Fig. 5

BG levels after IK preparation (partial pancreatectomy). Donor BG levels did not increased after removal of IKs. BG, blood glucose; IK, islet-kidney.

Fig. 6

BG levels prior and post IK Tx in S0610. S0610 received an IK graft from the actual HSC and primary KTx donor on day 208, without immunosuppression. STZ was administrated intravenously on day 201. BG, blood glucose; IK, islet-kidney; STZ, streptozotocin; Tx, transplantation.

Fig. 7

Histological findings from S0610 IK graft, excised at day 242. Islets (indicated by black arrows) were observed under renal capsule of the IK (H&E, ×100) (a), and no cell infiltrate was observed in the kidney section of the IK (H&E, ×200) (b). Immunofluorescence staining of the IK graft showed neither IgM (c), IgG (d), C3 (e) nor C4d (f). H&E: hematoxylin and eosin staining. IK, islet-kidney.

Fig. 8

Schematic diagram of IK Tx in the induction period of HSC Tx with a non-myeloablative conditioning regimen. Recipients received bi-daily doses of anti-monkey CD3 immunotoxin (CD3 IT) (25 µg/kg) on days −4, −3, −2 and −1 prior to HSC Tx. 100 cGy of total body irradiation (TBI) was given on day −2. Cyclosporine was administered from day 0 to day 45 following HSC Tx (target blood nadir levels 400–600 ng/ml). CyA: cyclosporine A; TBI: total-body irradiation. HSC, hematopoietic stem cell; IK, islet-kidney; Tx, transplantation.

Fig. 9

Blood sugar levels (mg/dl) and requirement of rapid insulin (units) prior and after IK Tx in S8710 (A), S1010 (B), and S9110 (C). These animals were transplanted with an IK on days 20, 22, and 22 post HSC Tx respectively. S8710 was euthanized on day 69 due to GI complications. S1010 received an additional 2,000 IE/kg from the original islet donor, which was administered in the mesenteric vein on day 112. S1010 and S 9110 had their IKs removed on days 342 and 242 after HSC Tx respectively. In both cases a rapid rise in BG was observed, confirming that stable BG levels were solely maintained by the IK grafts. BG, blood glucose; HSC, hematopoietic stem cell; IK, islet-kidney; Tx, transplantation.

Fig. 10

A) Insulin staining of excised IKs, demonstrating numerous insulin positive islets remaining under renal capsule of the IKs (a: S8710; b: S1010). No remaining islets were seen in native pancreata by insulin staining (c: S8710; d: S1010). B) Immunofluorescence staining of the excised IK (S9110 was negative for IgM (a) and IgG (b). IK, islet-kidney.

Fig. 11

MLR data from S0610, S8710, S9110 and S1010 pre, 7 weeks post, and 15 weeks post IK Txs. These animals were relatively hyporesponsive against donors, but donor specific unresponsiveness was not induced. MLR, mixed lymphocyte reaction; Tx, transplantation.