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

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-CD8dullCD56+ ‘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-CD8dullCD56+ 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-CD8dullCD56+ ‘memory-like NK cell subset’ with increased response upon secondary challenge in diabetics remains to be elucidated.