Combination of an Antigen-Specific Therapy and an Immunomodulatory Treatment to Simultaneous Block Recurrent Autoimmunity and Alloreactivity in Non-Obese Diabetic Mice

Abstract

Restoration of endogenous insulin production by islet transplantation is considered a curative option for patients with type 1 diabetes. However, recurrent autoimmunity and alloreactivity cause graft rejection hindering successful transplantation. Here we tested whether transplant tolerance to allogeneic islets could be achieved in non-obese diabetic (NOD) mice by simultaneously tackling autoimmunity via antigen-specific immunization, and alloreactivity via granulocyte colony stimulating factor (G-CSF) and rapamycin (RAPA) treatment. Immunization with insB9-23 peptide alone or in combination with two islet peptides (IGRP206-214 and GAD524-543) in incomplete Freund's adjuvant (IFA) were tested for promoting syngeneic pancreatic islet engraftment in spontaneously diabetic NOD mice. Treatment with G-CSF/RAPA alone or in combination with insB9-23/IFA was examined for promoting allogeneic islet engraftment in the same mouse model. InsB9-23/IFA immunization significantly prolonged syngeneic pancreatic islet survival in NOD mice by a mechanism that necessitated the presence of CD4+CD25+ T regulatory (Treg) cells, while combination of three islet epitopes was less efficacious in controlling recurrent autoimmunity. G-CSF/RAPA treatment was unable to reverse T1D or control recurrent autoimmunity but significantly prolonged islet allograft survival in NOD mice. Blockade of interleukin-10 (IL-10) during G-CSF/RAPA treatment resulted in allograft rejection suggesting that IL-10-producing cells were fundamental to achieve transplant tolerance. G-CSF/RAPA treatment combined with insB9-23/IFA did not further increase the survival of allogeneic islets. Thus, insB9-23/IFA immunization controls recurrent autoimmunity and G-CSF/RAPA treatment limits alloreactivity, however their combination does not further promote allogeneic pancreatic islet engraftment in NOD mice.

Fig 1. InsB_9–23/IFA immunization temporarily controls recurrent autoimmunity in NOD mice.

A, diabetic NOD mice were transplanted with islets from NOD donors (syngeneic) and treated with insB_9–23/IFA or PBS/IFA. Percentage of islet graft survival after transplantation is shown. B, correlation between blood glucose levels at the time of transplant and the number of days of syngeneic islet engraftment for all mice. Each symbol represents one mouse. Survival curves were compared with the log-rank test. Correlation was done with Pearson coefficient. P values and R² values are indicated in the graphs.

Fig 2. InsB_9–23/IFA treatment efficacy is dependent on CD4⁺CD25⁺ Treg cells.

Spontaneously diabetic female NOD mice were transplanted with syngeneic islets and with insB_9–23/IFA. A, some mice were received anti-CD25 mAb administration injected at the same time of transplantation or B, 10 days after transplantation. Arrows indicate the time when anti-CD25 mAb treatments initiated. Overall graft survival is shown. Survival curves were compared with the log-rank test. P values are indicated in the graphs.

Fig 3. Combination therapy with InsB_9–23, IGRP_206–214 and GAD_524–543 is less efficacious in promoting syngeneic islet transplant tolerance in NOD mice.

A, diabetic NOD mice were transplanted with syngeneic islets. Recipients were treated with a mix of 3 islet peptides, insB_9–23, IGRP_206–214 and GAD_524–543/IFA and monitored for graft engraftment. Graph shows the percentage of islet graft survival after transplantation. Control, PBS/IFA-treated, mice were pooled from different experiments and used as reference in all experiments (see Materials and Methods). B, correlation between blood glucose levels at the time of transplant and the number of days of syngeneic islet engraftment for all mice. Survival curves were compared with the log-rank test. Correlation was done with Pearson coefficient. P and R² values are indicated in the graphs.

Fig 4. G-CSF/RAPA treatment does not reverse diabetes in NOD mice and does not control the recurrence of autoimmunity.

A, diabetic NOD mice were treated with G-CSF/RAPA and monitored for diabetes progression. Graph shows the blood glucose values of mice prior and after treatment over time. B, diabetic NOD mice were transplanted with islets from NOD donors and treated with G-CSF/RAPA. Graph shows the percentage of islet graft survival after transplantation. Differences between treated and untreated mice are not statistically significant (ns).

Fig 5. G-CSF/RAPA treatment induces transplant tolerance to allogeneic islets in NOD mice that depends on IL-10 production.

A, spontaneously diabetic female NOD mice were transplanted with allogeneic islets from BALB/c donors. Recipients were treated with G-CSF/RAPA and monitored for graft survival. Anti-IL-10 was administered in NOD mice transplanted and treated with G-CSF/RAPA. Arrow indicates the time when anti-IL-10 mAb treatments initiated. Overall graft survival is shown. Graph shows the percentage of islet graft survival after transplantation. B, correlation between blood glucose levels at the time of transplant and the number of days of syngeneic islet engraftment. C, graph shows the percentage of islet graft survival in G-CSF/RAPA vs. G-CSF/RAPA/insB_9–23/IFA-treated recipients. Survival curves were compared with the log-rank test. P values are indicated in the graphs.