TY - ABST

T1 - In-situ immunohistochemical detection of ZnT8(186-194) reactive CD8(+) T cells in the pancreas of nPOD type 1 diabetic donors

AU - Sebastiani, G.

AU - Nigi, L.

AU - Culina, S.

AU - Afonso, G.

AU - Ventriglia, G.

AU - Buus, S.

AU - Dotta, F.

AU - Mallone, R.

PY - 2017

Y1 - 2017

N2 - Background and aims: Autoreactive T cells are a hallmark of type 1diabetes (T1D) pathogenesis and represent key mediators of islet autoimmunity.Insulitic lesions from both T1D donors and NOD mice areenriched with CD8+ Tcells which lead to beta-cell destruction. Previousstudies demonstrated that the human leukocyte antigen (HLA)-A2-restrictedzinc transporter 8 (ZnT8)186-194 beta-cell epitope is preferentiallytargeted by interferon-γ-producing CD8+ T cells in T1D patients.Although such autoimmune reactivity in T1D has been previously reported,information is lacking about the pancreatic localization of suchZnT8186-194-reactive CD8+ Tcell in T1D. Therefore, the aim of this studywas to identify ZnT8186-194-reactive cells in the pancreas of T1D donorsusing HLA-A2 multimer (MMr) immunostaining.Materials and methods: The in-situ MMr immunohistochemical detectionmethod used herein has been previously reported and validated. OCTfrozen pancreatic sections from n=4 T1D, n=4 islet-specific autoantibodiespositive (aAb+) and n=4 islet-specific autoantibodies negative(CTR) non-diabetic donors were obtained from the nPOD network. PE(Phycoerythrin)-coupled ZnT8186-194MMrs recognizing autoreactiveCD8+ T cells were loaded at 1μg/section and incubated at 4°C overnight.Rabbit anti-PE and Goat anti-Rabbit-HRP were used in order to detectMMr binding.Results: We investigated whether ZnT8186-194-reactive cells were detectedin pancreata from nPOD donors. Sections from all 4 T1D cases analyzedand from 2 of 4 aAb+ cases displayed ZnT8186-194 MMr+ cells,while all sections from CTR cases were negative. ZnT8186-194 MMr+cells were found scattered either within islets or the exocrine tissue.MMr+ cell count per each section analyzed revealed an increased numberof positive cells in T1D cases and aAb+ cases compared with CTR;moreover, an increased number of MMr+ cells were found in islets ofT1D cases compared with aAb+ donors. Parallel staining of sections withcontrol MMrs loaded with an irrelevant MelanA peptide did not detectany positive cells, confirming ZnT8 specificity.Conclusion: We detected for the first time the presence of ZnT8186-194-reactive cells in the pancreas of T1D donors, thus suggesting an unprecedentedrole for these cells in destructive insulitis during autoimmune diabetes

AB - Background and aims: Autoreactive T cells are a hallmark of type 1diabetes (T1D) pathogenesis and represent key mediators of islet autoimmunity.Insulitic lesions from both T1D donors and NOD mice areenriched with CD8+ Tcells which lead to beta-cell destruction. Previousstudies demonstrated that the human leukocyte antigen (HLA)-A2-restrictedzinc transporter 8 (ZnT8)186-194 beta-cell epitope is preferentiallytargeted by interferon-γ-producing CD8+ T cells in T1D patients.Although such autoimmune reactivity in T1D has been previously reported,information is lacking about the pancreatic localization of suchZnT8186-194-reactive CD8+ Tcell in T1D. Therefore, the aim of this studywas to identify ZnT8186-194-reactive cells in the pancreas of T1D donorsusing HLA-A2 multimer (MMr) immunostaining.Materials and methods: The in-situ MMr immunohistochemical detectionmethod used herein has been previously reported and validated. OCTfrozen pancreatic sections from n=4 T1D, n=4 islet-specific autoantibodiespositive (aAb+) and n=4 islet-specific autoantibodies negative(CTR) non-diabetic donors were obtained from the nPOD network. PE(Phycoerythrin)-coupled ZnT8186-194MMrs recognizing autoreactiveCD8+ T cells were loaded at 1μg/section and incubated at 4°C overnight.Rabbit anti-PE and Goat anti-Rabbit-HRP were used in order to detectMMr binding.Results: We investigated whether ZnT8186-194-reactive cells were detectedin pancreata from nPOD donors. Sections from all 4 T1D cases analyzedand from 2 of 4 aAb+ cases displayed ZnT8186-194 MMr+ cells,while all sections from CTR cases were negative. ZnT8186-194 MMr+cells were found scattered either within islets or the exocrine tissue.MMr+ cell count per each section analyzed revealed an increased numberof positive cells in T1D cases and aAb+ cases compared with CTR;moreover, an increased number of MMr+ cells were found in islets ofT1D cases compared with aAb+ donors. Parallel staining of sections withcontrol MMrs loaded with an irrelevant MelanA peptide did not detectany positive cells, confirming ZnT8 specificity.Conclusion: We detected for the first time the presence of ZnT8186-194-reactive cells in the pancreas of T1D donors, thus suggesting an unprecedentedrole for these cells in destructive insulitis during autoimmune diabetes

U2 - 10.1007/s00125-017-4350-z

DO - 10.1007/s00125-017-4350-z

M3 - Conference abstract in journal

C2 - 28795195

VL - 60

SP - S200-S201

JO - Diabetologia

JF - Diabetologia

SN - 0012-186X

IS - S1

ER -