IGRP-specific T-cells lyse HLA-A2-positive islets <i>in-vitro.</i>

<p>Data represent the percent specific cytotoxicity against the indicated targets by IGRP_265–273-specific CD8 T cells. HLA-A2-restricted, tumor-antigen-specific CD8 T-cells were used as a control. ⁵¹Cr release from islets cultured in medium alone (<i>i.e</i>. spontaneous release) for each target was measured in 9 independent wells to calculate specific cytotoxicity as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0049213#s4" target="_blank">Materials and Methods</a>. Data are representative of 3 independent experiments.</p>*<p>indicates a significant difference (p<0.01) in cytotoxicity against NOD-<i>scid</i> islets tested at the same E:T ratio; ^$ indicates a significant difference (p<0.05) in cytotoxicity against NOD-<i>scid HHD</i> islets between IGRP-specific CD8 T-cells and control T-cells.</p

IGRP-specific T-cells are able to infiltrate and destroy beta-cells following intra-pancreatic injection into NOD-<i>scidIL2rγ^null HHD</i> mice.

<p>NOD-<i>scid IL2rγ^null HHD</i> recipient mice were injected i.v. with 20×10⁶ PBMC from an HLA-A*0201⁺ healthy donor. Two days later, these mice were injected intra-pancreatically with either 5×10⁶ IGRP-specific T-cells (left), 5×10⁶ control T-cells (middle) or were sham injected (right). Four weeks later, the pancreata were isolated and histologically examined. A, Sections of recipients of IGRP-specific or control T-cells, or of those receiving a sham injection were stained with H&E (upper panel) to visualize the histological integrity of the islets or stained for insulin (middle panel) to identify the beta cells or human CD45 (lower panel) to visualize the human T-cells. Similar data were obtained when staining sections for human CD8. B, Pancreatic sections were stained for insulin, cCaspase-3 to detect apoptotic cells and DAPI to identify nuclei. Individual fluorescence as well as an overlay is presented. Shown are representative examples of 5 mice per group.</p

IGRP_265–273-specific T-cells cloned from the peripheral blood of type 1 diabetic individuals.

<p><b>A,</b> PBMCs from a HLA-A*0201⁺ recent onset diabetic patients (left panel) and HLA A*0201⁺ healthy donors (right panel) were incubated with A2/IGRP tetramers, followed by incubation with anti-CD8. CD8/tetramer double positive T cells were only detected in PBMC obtained from type 1 diabetic individuals and were not detected in the blood obtained from healthy controls. <b>B,</b> CD8/tetramer double positive T-cells were sorted at one cell per well and clones were picked. IGRP-specific T-clones stained with IGRP specific tetramers were observed in wells derived from type 1 diabetic individuals (left panel). These clones did not bind control HLA-A2 tetramers (right panel). <b>C,</b> To assess their cytokine production profile, T-cells were incubated with IGRP peptide-pulsed or control peptide-pulsed HLA-A2 EBV-LCL on anti-IFNγ; anti-GrB and anti-IL10-coated ELISpot plates. Shown is the average number of spots of triplicate wells. Data are representative of 3 independent experiments. * indicates significant difference from controls, <i>P</i><0.01. <b>D,</b> IGRP-specific T-cells were incubated with control peptide-pulsed (dashed line) or IGRP peptide-pulsed HLA-A2 EBV-LCL in the presence of anti-CD107a (grey histogram) antibodies. As a control, T-cells were incubated with IGRP peptide-pulsed target cells in the presence of isotype control antibodies (black line) for 5 hours. T-cells were stained for CD8 and expression of CD107a was analyzed on CD8⁺ T-cells using flow cytometry. Results are representative of 2 independent experiments.</p

<i>In-vivo</i> lysis of peptide-pulsed HLA-A2 cell targets by IGRP-specific CD8 T-cells.

<p>Data represent the percentage of specific cytotoxicity against IGRP-peptide pulsed HLA-A2 EBV-LCL by IGRP-specific CD8 T-cells in two independent experiments. 4×10⁶ IGRP-specific T cells were injected intrasplenically. One day later, mice received an i.v. injection containing of a 1∶1 mixture of specific peptide-pulsed CFSE^hi target cells and control peptide-pulsed CFSE^lo target cells. At 20 hr, the ratio of CFSE^hi and CFSE^lo cells in the spleens was analyzed by flow cytometry.</p>*<p>indicates a significant difference (p<0.05) in cytotoxicity against control targets. In each experiment, 4 mice per group were used.</p