Promiscuous binding of CLIP-peptide to distinct HLA-I alleles, representing four supertypes.

<p>The CLIP peptide RMATPLLMQALPM (peptide 3) was tested for binding affinity in a competition-based cellular peptide binding assay. The four tested HLA-I alleles (HLA-A0201, -A0301, -B0702 and -B4002) harbor a completely distinct binding pocket and bind different peptide ligands. CLIP peptide shows intermediate to high binding affinity to all of these. Separate positive control peptides efficiently bind to their respective HLA allele: GILGFVFTL (A0201 peptide), QVPLRPMTYK (A0301 peptide), SPSVDKARAEL (B0702 peptide) and GEFGGFGSV (B4002 peptide) with IC50 values of 3.7, 0.2, 0.7 and 0.2, respectively <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034649#pone.0034649-Kessler1" target="_blank">[21]</a>. The peptide concentration started at 100 µg/ml for HLA-A0201, -A0301 and -B4002 and 25 µg/ml for HLA-B0702, followed by a serial dilution of a factor two. Exact IC50 values of the CLIP peptides are depicted in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034649#pone-0034649-t002" target="_blank">Table 2</a>.</p

Surface display of the CLIP epitope on HLA-II-negative leukemic cells and influence of the invariant chain on HLA-I expression.

<p>(A) Surface expression of CLIP, HLA-DR, total HLA-II (‘HLA-DRPQ’) and invariant chain (‘CD74’) of myeloid cells from an acute promyelocytic leukemia (APL) patient, as determined by flow cytometry. Myeloid cells were defined as CD45^dim/SSC^low/int and expression thresholds were set according to unstained myeloid cells. (B) Quantitative analysis on frequencies of myeloid cells from APL patients that express CLIP (n = 9), HLA-DR (n = 9), total HLA-II (n = 6) and CD74 (n = 6). Frequencies indicate percentage tumor cells that reach threshold expression based on unstained leukemic cells. (C) The effect of invariant chain Ii down-modulation in KG-1 (CLIP^-) and THP-1 (CLIP⁺) leukemic cells on HLA-I expression at the cell surface. Intracellular staining (ICS) of Ii (PIN1.1) and surface staining of HLA-I (W6/32) were compared between Ii siRNA-transduced and non-transduced cells.</p

HLA-I binding affinity of eluted peptides derived from the invariant chain.

*<p>See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034649#pone-0034649-g002" target="_blank">Figure 2</a> for amino acid position in the invariant chain protein.</p>†<p>IC50 is the concentration used to obtain half maximal competition and represents the mean value of two independent experiments.</p>‡<p>Binding affinity is classified according to the following IC50 cut-off values: high affinity, ≤5 µM; intermediate (int) affinity, 5–15 µM; low affinity, 15–100 µM; no binding, >100 µM <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034649#pone.0034649-Kessler1" target="_blank">[21]</a>.</p

Invariant chain-derived peptides identified in isolated HLA-I molecules of B-LCLs.

<p>Peptide elutions of purified HLA-I molecules from EBV-transformed B-LCLs resulted in the identification of five peptides originating from the invariant chain. HLA-I purification and subsequent mass spectrometry analysis are described in <i>Materials and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034649#s2" target="_blank">Methods</a></i>. Of note, peptide 3 and 4 are located in the CLIP region, known for universal binding to HLA-II molecules.</p

HLA-I binding affinity of peptide length variants located in the CLIP region of the invariant chain.

*<p>Peptide found with HLA-I elutions.</p>†<p>Amino acid (AA) position in the invariant chain protein.</p>‡<p>Predicted HLA-I allele to which the peptide binds. Peptide binding prediction was done with netMHC (<a href="http://www.cbs.dtu.dk/services/NetMHC" target="_blank">http://www.cbs.dtu.dk/services/NetMHC</a>). Binding predictions can be made for peptide lengths between 8 and 11 for all alleles with a novel approximation algorithm using artificial neural networks trained on 9-mer peptides <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034649#pone.0034649-Lundegaard1" target="_blank">[26]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034649#pone.0034649-Lundegaard2" target="_blank">[27]</a>. Only peptides are shown with a predicted binding affinity of 500 nM or stronger.</p>§<p>IC50 is the concentration used to obtain half maximal competition and represents the mean value of two independent experiments.</p>¶<p>Binding affinity is classified according to the following IC50 cut-off values: high affinity, ≤5 µM; intermediate (int) affinity, 5–15 µM; low affinity, 15–100 µM; no binding, >100 µM <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034649#pone.0034649-Kessler1" target="_blank">[21]</a>.</p

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_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

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

<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