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

Background and aims: Autoreactive T cells are a hallmark of type 1
diabetes (T1D) pathogenesis and represent key mediators of islet autoimmunity.
Insulitic lesions from both T1D donors and NOD mice are
enriched with CD8+ Tcells which lead to beta-cell destruction. Previous
studies demonstrated that the human leukocyte antigen (HLA)-A2-restricted
zinc transporter 8 (ZnT8)186-194 beta-cell epitope is preferentially
targeted 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 such
ZnT8186-194-reactive CD8+ Tcell in T1D. Therefore, the aim of this study
was to identify ZnT8186-194-reactive cells in the pancreas of T1D donors
using HLA-A2 multimer (MMr) immunostaining.
Materials and methods: The in-situ MMr immunohistochemical detection
method used herein has been previously reported and validated. OCT
frozen pancreatic sections from n=4 T1D, n=4 islet-specific autoantibodies
positive (aAb+) and n=4 islet-specific autoantibodies negative
(CTR) non-diabetic donors were obtained from the nPOD network. PE
(Phycoerythrin)-coupled ZnT8186-194MMrs recognizing autoreactive
CD8+ 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 detect
MMr binding.
Results: We investigated whether ZnT8186-194-reactive cells were detected
in pancreata from nPOD donors. Sections from all 4 T1D cases analyzed
and 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 number
of positive cells in T1D cases and aAb+ cases compared with CTR;
moreover, an increased number of MMr+ cells were found in islets of
T1D cases compared with aAb+ donors. Parallel staining of sections with
control MMrs loaded with an irrelevant MelanA peptide did not detect
any 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 unprecedented
role for these cells in destructive insulitis during autoimmune diabetes