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

RESEARCH ARTICLE Identification of GAD65 AA 114-122 reactive ’memory-like’ NK cells in newly diagnosed Type 1 diabetic patients by HLA-class I pentamers Valentina Perri1☯, Elena Gianchecchi1☯¤, Loredana Cifaldi2, Marsha Pellegrino1, Ezio Giorda3, Marco Andreani4, Marco Cappa5, Alessandra Fierabracci1* a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 1 Type 1 Diabetes Centre, Infectivology and Clinical Trials Research Department, Bambino Gesù Children’s Hospital, IRCCS, Rome, Italy, 2 Pediatric Hematology and Oncology Department, Bambino Gesù Children’s Hospital, IRCCS, Rome, Italy, 3 Research Laboratories, Bambino Gesù Children’s Hospital, IRCCS, Rome, Italy, 4 Laboratory of Immunogenetics and Transplant Biology, IME Foundation, Polyclinic of Tor Vergata, Rome, Italy, 5 Endocrinology Department, Bambino Gesù Children’s Hospital, Rome, Italy ☯ These authors contributed equally to this work. ¤ Current address: Vismederi Srl, Siena, Italy * OPEN ACCESS Citation: Perri V, Gianchecchi E, Cifaldi L, Pellegrino M, Giorda E, Andreani M, et al. (2017) Identification of GAD65 AA 114-122 reactive ’memory-like’ NK cells in newly diagnosed Type 1 diabetic patients by HLA-class I pentamers. PLoS ONE 12(12): e0189615. https://doi.org/10.1371/ journal.pone.0189615 Editor: Massimo Pietropaolo, Baylor College of Medicine, UNITED STATES Received: July 24, 2017 Accepted: November 29, 2017 Published: December 13, 2017 Copyright: © 2017 Perri et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability Statement: All relevant data are within the paper and its Supporting Information files. Funding: This work was supported by the Italian Ministry of Health Ricerca Corrente RC 201602P003714 (AF). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing interests: The authors have declared that no competing interests exist. 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+ PLOS ONE | https://doi.org/10.1371/journal.pone.0189615 December 13, 2017 1 / 22 ’Memory-like’ NK cells in Type 1 diabetic patients ‘memory-like NK cell subset’ with increased response upon secondary challenge in diabetics remains to be elucidated. Introduction 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]. This multifactorial disorder develops in human leukocyte antigen (HLA) genetically predisposed individuals with the contribution of still unknown environmental factors and stochastic events [2]. In the disease pathogenesis several immunotypes play important roles i.e. autoreactive CD4+ and CD8+ T cells, autoantibodies producing B lymphocytes and innate immunity components [3]. 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]. Nevertheless, 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. This tool allowed the specific detection of autoreactive T cells by means of HLA A 02:01 GAD65 AA 114–122 pentamers [6,7]. The possibility to detect these specialized cell populations would also 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]. Multimer tools could also nowadays foster investigations aimed to discover specific immunotypes of unexpected role in the disease pathogenesis. As regards, despite autoimmune conditions are indeed principally due to B and T lymphocytes, recent studies provide evidence that also natural killer (NK) cells play a significant role in the promotion and/or maintenance of altered adaptive immune responses; these cells are indeed involved in the establishment of peripheral tolerance and immune regulation in preventing disease onset [9,10]. In the pathogenetic process, NK cells realize cross-talk with dendritic cells (DCs) and T cells through different receptor-ligand interacti (...truncated)