Regulatory T cells from patients with end-stage organ disease can be isolated, expanded and cryopreserved according good manufacturing practice improving their function

Background Here, we isolated, expanded and functionally characterized regulatory T cells (Tregs) from patients with end stage kidney and liver disease, waiting for kidney/liver transplantation (KT/LT), with the aim to establish a suitable method to obtain large numbers of immunomodulatory cells for adoptive immunotherapy post-transplantation. Methods We first established a preclinical protocol for expansion/isolation of Tregs from peripheral blood of LT/KT patients. We then scaled up and optimized such protocol according to good manufacturing practice (GMP) to obtain high numbers of purified Tregs which were phenotypically and functionally characterized in vitro and in vivo in a xenogeneic acute graft-versus-host disease (aGVHD) mouse model. Specifically, immunodepressed mice (NOD-SCID-gamma KO mice) received human effector T cells with or without GMP-produced Tregs to prevent the onset of xenogeneic GVHD. Results Our small scale Treg isolation/expansion protocol generated functional Tregs. Interestingly, cryopreservation/thawing did not impair phenotype/function and DNA methylation pattern of FOXP3 gene of the expanded Tregs. Fully functional Tregs were also isolated/expanded from KT and LT patients according to GMP. In the mouse model, GMP Tregs from LT or KT patient proved to be safe and show a trend toward reduced lethality of acute GVHD. Conclusions These data demonstrate that expanded/thawed GMP-Tregs from patients with end-stage organ disease are fully functional in vitro. Moreover, their infusion is safe and results in a trend toward reduced lethality of acute GVHD in vivo, further supporting Tregs-based adoptive immunotherapy in solid organ transplantation

Rapamycin Combined with Anti-CD45RB mAb and IL-10 or with G-CSF Induces Tolerance in a Stringent Mouse Model of Islet Transplantation

Background: A large pool of preexisting alloreactive effector T cells can cause allogeneic graft rejection following transplantation. However, it is possible to induce transplant tolerance by altering the balance between effector and regulatory T (Treg) cells. Among the various Treg-cell types, Foxp3 +Treg and IL-10-producing T regulatory type 1 (Tr1) cells have frequently been associated with tolerance following transplantation in both mice and humans. Previously, we demonstrated that rapamycin+IL-10 promotes Tr1-cell-associated tolerance in Balb/c mice transplanted with C57BL/6 pancreatic islets. However, this same treatment was unsuccessful in C57BL/6 mice transplanted with Balb/c islets (classified as a stringent transplant model). We accordingly designed a protocol that would be effective in the latter transplant model by simultaneously depleting effector T cells and fostering production of Treg cells. We additionally developed and tested a clinically translatable protocol that used no depleting agent. Methodology/Principal Findings: Diabetic C57BL/6 mice were transplanted with Balb/c pancreatic islets. Recipient mice transiently treated with anti-CD45RB mAb+rapamycin+IL-10 developed antigen-specific tolerance. During treatment, Foxp3 +Treg cells were momentarily enriched in the blood, followed by accumulation in the graft and draining lymph node, whereas CD4 +IL-10 +IL-4 - T (i.e., Tr1) cells localized in the spleen. In long-term tolerant mice, only CD4 +IL-10 +IL-4 - T cells remained enriched in the spleen and IL-10 was key in the maintenance of tolerance. Alternatively, recipient mice were treated with two compounds routinely used in the clinic (namely, rapamycin and G-CSF); this drug combination promoted tolerance associated with CD4 +IL-10 +IL-4 - T cells. Conclusions/Significance: The anti-CD45RB mAb+rapamycin+IL-10 combined protocol promotes a state of tolerance that is IL-10 dependent. Moreover, the combination of rapamycin+G-CSF induces tolerance and such treatment could be readily translatable into the clinic. © 2011 Gagliani et al

Murine Pancreatic Islets Transplantation under the Kidney Capsule

Type 1 diabetes (T1D) is an autoimmune disease caused by the lack of insulin-producing pancreatic beta cells leading to systemic hyperglycemia. Pancreatic islet transplantation is a valid therapeutic approach to restore insulin loss and to promote adequate glycemic control. Pancreatic islet transplantation in mice is an optimal preclinical model to identify new therapeutic strategies aiming at preventing rejection and optimizing post-transplant immuno-suppressive/-tolerogenic therapies. Islet transplantation in preclinical animal models can be performed in different sites such the kidney capsule, spleen, bone marrow and pancreas. This protocol describes murine islet transplantation under the kidney capsule. This is a widely accepted procedure for research purposes. Stress caused in the animals is minimal and it leads to reliable and reproducible results

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

Immune Depletion in Combination with Allogeneic Islets Permanently Restores Tolerance to Self-Antigens in Diabetic NOD Mice.

The destruction of beta cells in type 1 diabetes (T1D) results in loss of insulin production and glucose homeostasis. Treatment of non-obese diabetic (NOD) mice with immune-depleting/modulating agents (e.g., anti-CD3, murine anti-thymocyte-globulin (mATG)) can lead to diabetes reversal. However, for preclinical studies with these and other agents seeking to reverse disease at onset, the necessity for exogenous insulin administration is debated. Spontaneously diabetic NOD mice were treated with a short-course of mATG and insulin provided as drug therapy or by way of allogeneic islet implants. Herein we demonstrate that exogenous insulin administration is required to achieve disease reversal with mATG in NOD mice. Unexpectedly, we also observed that provision of insulin by way of allogeneic islet implantation in combination with mATG leads to a pronounced reversal of diabetes as well as restoration of tolerance to self-islets. Expansion/induction of regulatory cells was observed in NOD mice stably cured with mATG and allogeneic islets. These data suggest that transient provision of allogeneic insulin-producing islets might provide a temporary window for immune depletion to be more effective and instilling stable tolerance to endogenous beta cells. These findings support the use of a never before explored approach for preserving beta cell function in patients with recent onset T1D

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