Abstract
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.
Highlights
Type 1 diabetes (T1D) is an autoimmune disease which results from destruction of the insulin-producing β cells present in the pancreatic islets of Langerhans [1]
As part of an extended GAD65 AA 114–122 human leukocyte antigen (HLA) class I pentamers analysis of PBMC from newly diagnosed diabetic patients recruited from Lazio region at the Endocrinology and Diabetes Unit at the Children’s Hospital Bambino Gesu, Rome [37,38] a subgroup of 23 HLA-AÃ02:01 positive pediatric patients (10 males and 13 females, age of onset range 2.11 to 17.10, mean 10.58 years) was dedicated in this study to statistically evaluate the percentages of GAD65 pentamer reactive natural killer (NK) cells in diabetics versus 23 controls (Table 1) while 15 (5 males and 10 females, age of onset range 4.8 years to 15 years, mean 10.5 years) were dedicated to pentamer reactive NK cell phenotypical characterization (Table 1)
Specificity of GAD65 AA 114–122 reactive T cells was confirmed in experiments of GAD65 AA 114–122 pentamer staining in PBMC of patients stimulated with the same GAD65 AA 114–122 peptide (GAD65)
Summary
Type 1 diabetes (T1D) is an autoimmune disease which results from destruction of the insulin-producing β cells present in the pancreatic islets of Langerhans [1]. For long-time, combination screenings of autoantibodies (Abs) directed against insulin (IAA), proinsulin, glutamic acid decarboxylase (GAD) isoforms GAD65, GAD67, the insulinoma-associated antigen (IA-2)/tyrosine phosphatase-like molecule IA-2 β [4] have represented the mainstay for classifying diabetes as autoimmune-mediated and for stratifying risk in first-degree relatives [5] These immune markers are not directly pathogenetic as opposite to autoreactive T cells [3], consistent with the notion that before and by the time of clinical disease onset these cells have received antigen-specific stimulation [6]. Following several attempts in evaluating autoreactive T cell responses that lacked diabetesspecificity, the use of the major histocompatibility complex (MHC) multimer technology [3] was introduced to solve the difficult issue of detecting in the peripheral blood rare antigen-specific autoreactive T cells and pinpoint their significance to disease onset and progression. The possibility to detect these specialized cell populations would offer the theoretical advantage of improved prediction strategies of disease as well as the opportunity to target them in immunomodulation therapies and foresee disease regression based on their physical disappearance or functional silencing [8]
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