ECDI-fixed allogeneic splenocytes induce donor-specific tolerance for long-term survival of islet transplants via two distinct mechanisms

Abstract

A major challenge for human allogeneic islet transplantation is the development of effective methods to induce donor-specific tolerance to obviate the need for life-long immunosuppression that is toxic to the insulin-producing beta cells and detrimental to the host. We developed an efficient donor-specific tolerance therapy that utilizes infusions of ethylene carbodiimide (ECDI)-treated donor splenic antigen-presenting cells that results in indefinite survival of allogeneic islet grafts in the absence of immunosuppression. Furthermore, we show that induction of tolerance is critically dependent on synergistic effects between an intact programmed death 1 receptor-programmed death ligand 1 signaling pathway and CD4(+)CD25(+)Foxp3(+) regulatory T cells. This highly efficient antigen-specific therapy with a complete avoidance of immunosuppression has significant therapeutic potential in human islet cell transplantation.

Fig. 1.

Repeated infusions of ECDI-treated donor splenocytes induce significant graft protection in allogeneic islet cell transplantation. (A) Time line of the treatment protocols. (B) Graft survival. Day 0 indicates the day of islet transplantation. ECDI-treated BALB/c cells vs. control, P = 0.0036; ECDI-treated SJL cells vs. control, P = 0.2178; untreated BALB/c cells vs. control, P < 0.0001.

Fig. 2.

Graft histology. Protected grafts at day 14 (A) and day 70 (B) after transplantation were stained with H&E, anti-insulin (red), CD4 (green), +Foxp3 (red), and CD8 (red). DAPI staining is shown in blue. Graphs are representatives of at least four sectioned and stained grafts of each group. Magnification × 100. Asterisks indicate intact islets.

Fig. 3.

Long-term tolerized recipients show spontaneous acceptance of a second same-donor islet graft without further intervention. (A) Long-term tolerized B6 recipients (60–90 days after the first transplantation) were nephrectomized to remove the first graft and were transplanted with a second same-donor (BALB/c, n = 3) or third-party (SJL, n = 3) graft. Day 0 indicates the day of the second islet graft transplantation. (B) Graft histology of the second islet graft. Upper panels: a protected same-donor BALB/c graft; Lower panels: a rejected third-party SJL graft. Insulin is shown in red; DAPI is shown in blue. Magnification × 100.

Fig. 4.

Diminished alloantigen-specific T-cell and antibody responses are associated with protection of allogeneic islet grafts. (A) DTH responses. P = 0.0018, rejecting vs. tolerized recipients. (B) Specific anti-donor antibodies were measured for IgG1, IgG2a, IgG2b, and IgG3. The top two rows of histograms are results from two control (rejected) recipients. The bottom two rows of histograms are results from two long-term tolerized recipients. Data are representative of two individual experiments. Shaded histogram indicates syngeneic cells. (C) Mixed lymphocyte reaction and IFN-γ production. Thy1.2⁺ T cells from the spleens and peripheral lymph nodes from control and tolerized recipients were used. P values are indicated in the graphs. Data are representative of three separate experiments.

Fig. 5.

CD4⁺CD25⁺ Tregs are required for tolerance induction by i.v. treatment with ECDI-treated donor splenocytes but not for tolerance maintenance. (A) PC61 treatment at the time of tolerance induction abrogated graft protection in recipients receiving ECDI-treated donor cell infusions. Day 0 indicates the day of islet transplantation. The dotted line indicates the blood glucose level of 250 mg/dl, which was used to diagnose graft rejection. (B) Quantification of the CD4⁺CD25⁺Foxp3⁺ T-cell population in peripheral lymphoid organs from rejecting, tolerized, or PC61-treated recipients on day 15 after transplantation. Data were expressed as the percentage of total CD4⁺ T cells that were Foxp3⁺ cells. dLNs = draining lymph nodes; pLNs = peripheral lymph nodes. *Rejecting vs. tolerized, P = 0.0076; **, PC61-treated vs. tolerized, P = 0.0013; ^#PC61-treated vs. tolerized, P = 0.0086; ^&PC61-treated vs. tolerized, P = 0.026. (C) Anti-donor IFN-γ production by rejecting, tolerized, or PC61-treated recipients. *Rejecting vs. tolerized, P = 0.0009; **rejecting vs. PC61-treated, P = 0.0012. (D) PC61 treatment in long-term tolerized recipients (n = 3). Treatment was given on day 118 and day 120 after islet transplantation as indicated by the arrows. Blood glucose levels were followed for an additional 50 days after PC61 treatment. (E) Anti-TGF-β treatment at the time of tolerance induction abrogated graft protection in recipients receiving infusions of ECDI-treated donor cell.

Fig. 6.

Absence of PD-L1 signaling impairs tolerance induction mediated by the infusions of ECDI-treated donor cells. (A) Islet graft survival in PD-L1^−/− recipients with or without ECDI treatment. Day 0 indicates the day of islet transplantation. (B) Anti-donor IFN-γ production by PD-L1^−/− vs. wild-type recipients receiving ECDI treatment. *, PD-L1^−/− ECDI-treated vs. wild-type ECDI-treated; P = 0.0005. (C) Quantification of the CD4⁺CD25⁺Foxp3⁺ T-cell population in peripheral lymphoid organs from PD-L1^−/− vs. wild-type recipients receiving ECDI-treated cells.