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

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

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 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

Additional file 1 of Circulating extracellular vesicles are monitoring biomarkers of anti-PD1 response and enhancer of tumor progression and immunosuppression in metastatic melanoma

Additional file 1: Fig. S1. sPD1 and sPD-L1 levels in plasma of MM patients underwent immunotherapy with ICI. Fig. S2. MM patients circulating EVs characterization. Fig. S3. Effect of anti-PD1 treatment on PD-L1+ or uPAR+ EVs release and neoangiogenesis. Fig. S4. Influence of circulating EVs on PD-L1 expression in M0 macrophages. Fig. S5. Modulation of cytokines and chemokines released from M0 macrophages after treatment with circulating EVs of MM patients