Distinct Phenotypes of Islet Antigen-Specific CD4+ T Cells among the Three Subtypes of Type 1 Diabetes
Authors: CHUJO DAISUKE , KAWABE AKITSU , TAKAHASHI NOBUYUKI , MATSUSHITA MAYA , TSUTSUMI CHIHARU , HASEDA FUMITAKA , IMAGAWA AKIHISA , HANAFUSA TOSHIAKI , YAGI KUNIMASA , SHIMODA MASAYUKI , UEKI KOHJIRO , KAJIO HIROSHI 
Published: June 2018

Type 1 diabetes (T1D) is classified into three subtypes in Japan based on the manner of disease progression: acute onset (AT1D), slowly progressive (SP1D), and fulminant (FT1D). While cellular immunity is considered to play a major role in pancreatic β cell destruction in T1D, the phenotypes of autoreactive T cells in each subtype remain largely unknown. In this study, we performed an integrated assay that permits the determination of ex-vivo cellular immune responses and T cell repertoires specific for islet antigens. Briefly, peripheral blood mononuclear cells obtained from 20 AT1D, 17 SP1D, 18 FT1D patients, and 17 nondiabetics (ND) were stimulated for 48 h with GAD65, preproinsulin (PPI), IGRP, and ZnT8 peptide clusters, after which cytokine/chemokine secretion was measured (ex-vivo assay). Subsequently, peptide-reactive T cells were further expanded with IL-2 to identify CD4+ T cell subsets based on intracellular cytokine staining (ICS assay). In the ex-vivo assay, GAD65-specific IL-6 and IP-10 responses, and PPI-specific IP-10 responses were significantly upregulated in AT1D as compared to ND. Furthermore, GAD65-specific IL-1β and G-CSF, and PPI-specific G-CSF responses were significantly upregulated in FT1D when compared to ND. The ICS assay further revealed that GAD65- and PPI-specific CD4+ T cells were skewed towards Th1 cells in AT1D (P = 0.007 and P = 0.004, respectively), whereas GAD65- and IGRP-specific Th2 cells were more prevalent in SP1D (P = 0.01 and P = 0.03, respectively) than in ND. Interestingly, GAD65-specific Th1 cells were more abundant in SP1D with HLA-DR9 than in those without DR9 (P = 0.03). Of note, FT1D displayed significantly less Tr1 cells specific for all four antigens and GAD65-specific Th2 cells than ND.
In conclusion, the phenotypes of islet antigen-specific CD4+ T cells differ among the three T1D subtypes. These distinct phenotypes of pathogenic T cells may be associated with the manner of progression of β cell destruction.

Disclosure
D. Chujo: Research Support; Self; Mitsubishi Tanabe Pharma Corporation, Sanofi K.K., Kyowa Hakko Kirin Co., Ltd.. A. Kawabe: None. N. Takahashi: None. M. Matsushita: None. C. Tsutsumi: Research Support; Self; MSD K.K.. F. Haseda: None. A. Imagawa: Research Support; Self; Kowa Pharmaceutical, Sanwa Kagaku Kenkyusho Co., Ltd., Ono Pharmaceutical Co., Ltd., Sanofi K.K., Sumitomo Dainippon Pharma Co., Ltd., Taisho Pharmaceutical Co., Ltd., Kyowa Hakko Kirin Co., Ltd., Novo Nordisk A/S, Eli Lilly and Company, MSD K.K., Mitsubishi Tanabe Pharma Corporation, Shionogi & Co., Ltd., Nippon Boehringer Ingelheim Co. Ltd.. Speaker's Bureau; Self; Sanofi K.K., Novo Nordisk A/S, Mitsubishi Tanabe Pharma Corporation, Daiichi Sankyo Company, Limited, Nipro, Eli Lilly and Company, AstraZeneca, Nippon Boehringer Ingelheim Co. Ltd., Taisho Pharmaceutical Co., Ltd., Ono Pharmaceutical Co., Ltd., Sanwa Kagaku Kenkyusho Co., Ltd., Terumo Medical Corporation. T. Hanafusa: Speaker's Bureau; Self; Novo Nordisk Inc.. K. Yagi: None. M. Shimoda: None. K. Ueki: Speaker's Bureau; Self; Daiichi Sankyo Company, Limited, Eli Lilly and Company, Boehringer Ingelheim Pharmaceuticals, Inc., MSD K.K., Novo Nordisk Inc., Mitsubishi Tanabe Pharma Corporation, Kyowa Hakko Kirin Co., Ltd., Takeda. Research Support; Self; Takeda, Astellas, Novo Nordisk Inc.. H. Kajio: None.