環境管理計画への市民参加とその規定因としてのエンパワーメント

<p>DMPC/2/siRNA treatment of Jurkat T cells, 48 hours from the beginning of transfection.</p

Identification of GAD65 AA 114-122 reactive 'memory-like' NK cells in newly diagnosed Type 1 diabetic patients by HLA-class I pentamers

<div><p>Type 1 diabetes is an autoimmune disease, in which pancreatic β cells are destroyed by autoreactive T cells in genetically predisposed individuals. Serum beta cell autoantibody specificities have represented the mainstay for classifying diabetes as autoimmune-mediated and for stratifying risk in first-degree relatives. In recent years, approaches were attempted to solve the difficult issue of detecting rare antigen-specific autoreactive T cells and their significance to etiopathogenesis such as the use of the MHC multimer technology. This tool allowed the specific detection of increased percentages of GAD65 autoreactive T cells by means of HLA A*02:01 GAD65 AA 114–122 pentamers in newly diagnosed diabetics. Here we provide evidence that GAD65 AA 114–122 pentamers can depict a GAD65 AA114-122 peptide expandable population of functionally and phenotypically skewed, preliminary characterized CD3^-CD8^dullCD56⁺ ‘memory-like’ NK cells in PBMC of newly diagnosed diabetics. Our data suggest that the NK cell subset could bind the HLA class I GAD65 AA 114–122 pentamer through ILT2 inhibitory receptor. CD107a expression revealed increased degranulation of CD3^-CD8^dullCD56⁺ NK cells in GAD65 AA 114–122 and FLU peptide expanded peripheral blood mononuclear cells of diabetics following GAD65 AA 114–122 peptide HLA A*02:01 presentation in respect to the unpulsed condition. CD107a expression was enriched in ILT2 positive NK cells. As opposite to basal conditions where similar percentages of CD3^-CD56⁺ILT2⁺ cells were detected in diabetics and controls, CD3^-CD56⁺CD107a⁺ and CD3^-CD56⁺ILT2⁺CD107a⁺ cells were significantly increased in T1D PBMC either GAD65 AA 114–122 or FLU peptides stimulated after co-culture with GAD65 AA 114–122 pulsed APCs. As control, healthy donor NK cells showed similar degranulation against both GAD65 AA 114–122 pulsed and unpulsed APCs. The pathogenetic significance of the CD3^-CD8^dullCD56⁺ ‘memory-like NK cell subset’ with increased response upon secondary challenge in diabetics remains to be elucidated.</p></div

Similar percentages of GAD65 AA 114–122 HLA A*0201 pentamer reactive cells are circulating in newly diagnosed vs long-term T1D patients.

<p>Relative percentages of CD3⁺CD8^bright (A), CD3⁺CD8^dull (B), total CD3⁺CD8⁺ (C) and CD3^-CD8^dull cells (D) in PBMC of newly diagnosed (ND, circle dots) vs long-term T1D patients (LT, square dots); horizontal bars, average values are reported.</p

Sex, age and diabetes-related autoantibodies profile (GAD65, IA2, IAA) in newly diagnosed T1D patients at disease onset used to define percentages of GAD65 pentamer reactive NK cells and their phenotypical characterization.

<p>Sex, age and diabetes-related autoantibodies profile (GAD65, IA2, IAA) in newly diagnosed T1D patients at disease onset used to define percentages of GAD65 pentamer reactive NK cells and their phenotypical characterization.</p

Unchanged healthy donor NK cell degranulation in response to GAD65 AA 114–122 peptide-pulsed APCs.

<p>Degranulation of CD3^-CD56⁺ILT2⁺ NK cells of PBMC from HD patients, expanded with GAD65 AA 114–122 peptide, measured as CD107a cell-surface expression following stimulation with APCs, either left unpulsed or GAD65 AA 114–122 peptide-pulsed. A representative experiment out of two performed is shown. K562 cells were used as positive control. The percentage of CD3^-CD56⁺CD107a⁺ NK cells (upper panel) and of CD3^-CD56⁺CD107a⁺ILT2⁺ (lower panel) is indicated for each condition.</p

Increased percentage of CD3^-CD8^dullGAD65 AA 114–122 pentamer reactive cells after GAD65 AA 114–122 peptide stimulation in T1D patients compared to healthy controls.

<p>PBMC from healthy blood donors and T1D patients, were GAD65 AA 114–122 peptide stimulated, stained with CD3, CD8 and GAD65 AA 114–122 HLA A*02:01 pentamers, then analyzed by FACS to determine the percentages of GAD65 pentamer reactive cells in CD3⁺CD8^bright, CD3⁺CD8^dull, total CD3⁺CD8⁺ and CD3^-CD8^dull subsets (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0189615#pone.0189615.s001" target="_blank">S1 Fig</a>). In the graph, horizontal bars represent the mean percentage of CD3^-CD8^dull GAD65 AA 114–122 reactive cells and each symbol represents an individual: circle dots represents the normal and square dots the diabetic. Percentages of CD3^-CD8^dull GAD65 reactive cells refer to analyzed events within flow-cytometry gates as shown in representative dot plots in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0189615#pone.0189615.s001" target="_blank">S1 Fig</a>. GAD65⁺ = reactive with PE-labelled GAD65 AA 114–122 pentamer. N = number of T1D patients and HD controls analyzed.</p

Correlation of GAD65 pentamer reactive cells with metabolic markers.

<p>(A) Direct correlation with C-peptide values; (B) Inverse correlation with HbA1c values; (C) Inverse correlation with insulin requirements.</p

Correlation of GAD65 pentamer reactive cells with Abs levels.

<p>(A) No correlation with GAD65 Abs; (B) No correlation with IA2 Abs; (C) No correlation with IAA.</p

Similar percentage of total and ILT2 expressing NK cells between healthy donors and T1D patients.

<p>The percentage of CD3^-CD56⁺ cells (A) and of CD3^-CD56⁺ILT2⁺ cells (B) were assessed by flow cytometric analyses of IL-2 alone treated PBMC. NK cell phenotype of 14 healthy donors (HD, circle plots) was compared with that of 14 T1D patients (square dots); horizontal bars, average values are shown. No significant differences are reported (KS test, unpaired t test).</p

Increased susceptibility of GAD65 AA 114–122 peptide-pulsed APCs to T1D NK cell-mediated recognition associates with NK cell-ILT2 expression.

<p>Degranulation of CD3^-CD56⁺ILT2⁺ NK cells of PBMC from T1D patients, expanded with GAD65 AA 114–122 or FLU peptides, measured as CD107a cell-surface expression following stimulation with APCs, either left unpulsed or GAD65 AA 114–122 peptide-pulsed. (A) A representative experiment out of two performed is shown. K562 cells were used as positive control. The percentage of CD3^-CD56⁺CD107a⁺ NK cells (upper panel) and CD3^-CD56⁺CD107a⁺ILT2⁺ (lower panel) is indicated for each condition. (B) Summary of CD3^-CD56⁺CD107a⁺ and (C) CD3^-CD56⁺ILT2⁺CD107a⁺ NK cell percentage of four T1D PBMC, expanded with GAD65 AA 114–122 or FLU peptides, following stimulation with APCs, either left unpulsed (circle dots) or GAD65 AA 114–122 (GAD65)-pulsed (square dots); horizontal bars, average values are reported.</p