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

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

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

BDC12-4.1 T-Cell Receptor Transgenic Insulin-Specific CD4 T Cells Are Resistant to <i>In Vitro</i> Differentiation into Functional Foxp3⁺ T Regulatory Cells

<div><p>The infusion of ex vivo-expanded autologous T regulatory (Treg) cells is potentially an effective immunotherapeutic strategy against graft-versus-host disease (GvHD) and several autoimmune diseases, such as type 1 diabetes (T1D). However, <i>in vitro</i> differentiation of antigen-specific T cells into functional and stable Treg (iTreg) cells has proved challenging. As insulin is the major autoantigen leading to T1D, we tested the capacity of insulin-specific T-cell receptor (TCR) transgenic CD4⁺ T cells of the BDC12-4.1 clone to convert into Foxp3⁺ iTreg cells. We found that <i>in vitro</i> polarization toward Foxp3⁺ iTreg was effective with a majority (>70%) of expanded cells expressing Foxp3. However, adoptive transfer of Foxp3⁺ BDC12-4.1 cells did not prevent diabetes onset in immunocompetent NOD mice. Thus, <i>in vitro</i> polarization of insulin-specific BDC12-4.1 TCR transgenic CD4⁺ T cells toward Foxp3⁺ cells did not provide dominant tolerance in recipient mice. These results highlight the disconnect between an <i>in vitro</i> acquired Foxp3⁺ cell phenotype and its associated <i>in vivo</i> regulatory potential.</p></div

BDC12-4.1 T cells expand and display memory and Treg cell phenotypes in the periphery.

<p>(A and B) Age-associated increases in total CD4⁺ T cell numbers in the spleen and PLN of BDC12-4.1.RAG^KO mice. Splenocytes and PLN lymphocytes from BDC12-4.1.RAG^KO mice were analyzed at different ages by flow cytometry for the frequency of CD4⁺Vβ2⁺ T cells. The percentage of CD4⁺Vβ2⁺ cells was multiplied with the total number of cells isolated from the spleen or PLN to calculate the total number of CD4⁺ T cells. Trypan blue was used for the exclusion of dead cells prior to counting. (C and D) Representative FACS plots and data for activated T cells, CD44^hiCD62L^low, and Treg cells, CD25⁺Foxp3⁺, from the spleens of donor mice that were used to generate Foxp3⁺ Treg cells in vitro are shown.</p

<i>In vitro</i> polarization induces Foxp3 expression in memory BDC12-4.1 T cells but does not endow <i>in vivo</i> regulatory functions.

<p>CD4⁺CD25⁺ cells from splenocytes of BDC12-4.1 mice polarized into Treg cells (A) <i>in vitro</i> in the presence of a-CD3 (left panel) or InsB:9-23 peptide (right panel). Expanded T cells were gated on CD4⁺CD25⁺ cells (not shown) and expression of CD127 and Foxp3 were determined. (B). 1×10⁶ Tregs polarized with anti-CD3 (left panel) or InsB:9-23 peptide (right panel) were adoptively transferred into prediabetic 8-wk old NOD mice and diabetes development was monitored. Unmanipulated NOD mice monitored on a regular basis in our animal colony served as negative controls with cumulative diabetes incidence of ∼90% by 27 wks of age. Representative results from one of three independent experiments are shown.</p

T cells with InsB:9-23 specific responses increase in frequencies with age.

<p>Total splenocytes from BDC12-4.1.RAG^KO mice were obtained at different ages. 250,000 splenocytes per well were incubated with or without native InsB:9-23 peptide in triplicates. Following 3-day incubation, IFN-γ, IL-2, and IL-4 production were determined by ELISpot assay. Representative images of cytokine production from BDC12-4.1.RAG^KO T-cells with (bottom row) or without antigen (top row) stimulation is shown in A. Background (media) subtracted IFN-γ spot numbers produced in response to antigen stimulation, from BDC12-4.1.RAG^KO mice of different age groups are shown on the Y-axis in B. Representative means ± SEM data from one of two independent experiments with n = 3 per each group with similar results are shown.</p

image_1_Circulating Follicular Helper and Follicular Regulatory T Cells Are Severely Compromised in Human CD40 Deficiency: A Case Report.pdf

<p>Mutations in genes that control class switch recombination and somatic hypermutation during the germinal center (GC) response can cause diverse immune dysfunctions. In particular, mutations in CD40LG, CD40, AICDA, or UNG cause hyper-IgM (HIGM) syndrome, a heterogeneous group of primary immunodeficiencies. Follicular helper (Tfh) and follicular regulatory (Tfr) T cells play a key role in the formation and regulation of GCs, but their role in HIGM pathogenesis is still limited. Here, we found that compared to CD40 ligand (CD40L)- and activation-induced cytidine deaminase (AICDA)-deficient patients, circulating Tfh and Tfr cells were severely compromised in terms of frequency and activation phenotype in a child with CD40 deficiency. These findings offer useful insight for human Tfh biology, with potential implications for understanding the molecular basis of HIGM syndrome caused by mutations in CD40.</p