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

<p>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. <i>P</i> values are indicated in the graphs.</p

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

<div><p>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 <i>via</i> antigen-specific immunization, and alloreactivity <i>via</i> granulocyte colony stimulating factor (G-CSF) and rapamycin (RAPA) treatment. Immunization with insB_9-23 peptide alone or in combination with two islet peptides (IGRP_206-214and GAD_524-543) 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 insB_9-23/IFA was examined for promoting allogeneic islet engraftment in the same mouse model. InsB_9-23/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 insB_9-23/IFA did not further increase the survival of allogeneic islets. Thus, insB_9-23/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.</p></div

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.

<p>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 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0127631#sec002" target="_blank">Materials and Methods</a>). 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. <i>P</i> and R² values are indicated in the graphs.</p

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

<p>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. <i>P</i> values are indicated in the graphs.</p

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

<p>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. <i>P</i> values and R² values are indicated in the graphs.</p

(A) NOD mice with two consecutive blood glucose screenings > 250 mg/dl were treated with mATG + insulin pellet (filled circles, n = 10), or mATG (empty squares, n = 9), or left untreated (asterisks, n = 10). Kaplan-Meyer curves where the endpoint was stable normal glycemia are shown (log-rank test, P = 0.047). (B) Blood glucose levels at study entry (i.e., the day the first mATG dose was administered) are shown per each treated mouse and are divided based on endpoint reached (i.e., success–stable normal glycemia achieved) or not reached (i.e., failed—stable normal glycemia not achieved) (*unpaired t-test, <i>P</i> = 0.034).

<p>None of the animals used in these experiments died spontaneously.</p

Spontaneously diabetic NOD mice were treated with mATG + allogeneic islets (filled circles, n = 7), or allogeneic islets only (asterisks, n = 8).

<p>Kaplan-Meyer curves where the endpoint was death/sacrifice are shown (log-rank test, P = 0.006). Mice died spontaneously of hyperglycemia (n = 3) or were sacrificed when weight loss was higher than 20% (n = 6).</p

Blood glucose levels of diabetic NOD mice shown in Fig 2 are presented grouped (islets, n = 8—upper left) or individually (mATG+islets mouse 001→007).

<p>The age (in weeks, wk) at which the mice entered the study is also reported as median±SD for islets treated animals and individually for mATG+islets treated animals. Horizontal dashed lines indicate blood glucose level above which mice are considered diabetic (i.e., 250 mg/dl). Vertical dashed lines filled with grey area indicate the time of transient hyperglycemia post-treatment. Arrows indicate the day of nephrectomy. Blood glucose levels are reported from the day of study entry until sacrifice (when weight loss was more than 20%) or upon spontaneous death for hyperglycemia.</p

NOD mice shown in Fig 3 (mATG+islets 002, 005, 007) were sacrificed at the end of the study and pancreatic lymph-nodes collected (black bars, n = 3).

<p>In parallel, normoglycemic NOD mice (10 week olds, grey bars, n = 2) and diabetic NOD mice (20 week olds, squared bars, n = 3) were sacrificed and used as controls. The frequency of CD4⁺Foxp3⁺ T cells was tested by flow cytometry (upper panel) and the amount of IL-10 released by total pancreatic lymph-node cells upon polyclonal activation was measured by ELISA (lower panel) (mean±SEM are shown and One-way ANOVA, *** P≤0.0005).</p