Tolerance Induction in Diabetogenic BDC-2.5 T cells

CD4 T cells play a critical role in promoting the development of autoimmunity in Type 1 Diabetes (T1D). The diabetogenic CD4 T cell clone BDC-2.5, originally isolated from a non-obese diabetic (NOD) mouse, has been widely used to study the contribution of autoreactive CD4 T cells and relevant antigens to autoimmune diabetes. Recent work from our lab has shown that the antigen for BDC-2.5 T cells is a hybrid insulin peptide (2.5HIP) consisting of an insulin C-peptide fragment fused to a peptide from chromogranin A (ChgA), and that endogenous 2.5HIP-reactive T cells are major contributors to autoimmune pathology in NOD mice. The objective of this study was to determine if poly(lactide-co-glycolide) (PLG) nanoparticles (NPs) loaded with the 2.5HIP antigen (2.5HIP-PLG) can tolerize BDC-2.5 T cells. Infusion of 2.5HIP-PLG NPs was found to prevent diabetes in an adoptive transfer model by impairing the ability of BDC-2.5 T cells to produce pro-inflammatory cytokines through induction of anergy, leading to an increase in the ratio of Foxp3+ regulatory T cells to IFN-γ+ effector T cells. This work is the first to use a hybrid insulin peptide, or any neoepitope, to re-educate diabetogenic T cells and may have significant implications for the development of an antigen-specific therapy for T1D patients. Sequencing of FACS-sorted BDC-2.5 TCR-transgenic, CD4-positive, Foxp3-negative (GFP-negative) effector T cells (Teffs) was performed in order to determine the mechanisms of tolerance induction. Activated BDC-2.5 T cells were adoptively transferred into NOD.scid mice and Teffs were sorted from spleen/lymph nodes of 2.5HIP-PLG treated or untreated mice (pooled from 4 mice per group) 6 days after T cell transfer. RNA-Seq data are from 1 experiment without replicates. An up-regulation of anergy related genes in tolerized Teffs was found.CD4⁺ T细胞（CD4 T cells）在促进1型糖尿病（Type 1 Diabetes, T1D）的自身免疫发病进程中发挥关键作用。最初从非肥胖糖尿病（non-obese diabetic, NOD）小鼠体内分离得到的致糖尿病性CD4⁺ T细胞克隆BDC-2.5，已被广泛用于探究自身反应性CD4⁺ T细胞及相关抗原在自身免疫性糖尿病中的作用。本实验室近期研究表明，BDC-2.5 T细胞的靶抗原为杂交胰岛素肽（2.5HIP），该肽由胰岛素C肽片段与嗜铬粒蛋白A（chromogranin A, ChgA）来源的肽段融合而成；同时，内源性2.5HIP反应性T细胞是NOD小鼠自身免疫病理损伤的主要介导因子。本研究旨在验证负载2.5HIP抗原的聚乳酸-羟基乙酸共聚物（poly(lactide-co-glycolide), PLG）纳米颗粒（nanoparticles, NPs，即2.5HIP-PLG NPs）是否能够诱导BDC-2.5 T细胞产生免疫耐受。研究发现，经2.5HIP-PLG NPs输注可通过诱导BDC-2.5 T细胞失能（anergy），削弱其分泌促炎细胞因子的能力，从而在过继转移模型中阻止糖尿病的发生，同时提升Foxp3阳性调节性T细胞（Foxp3+ regulatory T cells）与干扰素-γ阳性效应T细胞（IFN-γ+ effector T cells）的比例。本研究首次利用杂交胰岛素肽或任意新表位（neoepitope）重编程致糖尿病性T细胞，该成果可为1型糖尿病患者的抗原特异性治疗开发提供重要参考。为阐明免疫耐受的诱导机制，本研究对经荧光激活细胞分选（fluorescence-activated cell sorting, FACS）得到的BDC-2.5 T细胞受体（TCR）转基因、CD4阳性、Foxp3阴性（GFP阴性）的效应T细胞（effector T cells, Teffs）进行了测序。具体操作如下：将活化的BDC-2.5 T细胞过继转移至NOD.scid小鼠体内，于细胞转移后6天，从2.5HIP-PLG处理组与未处理组小鼠（每组均混合4只小鼠的组织样本）的脾脏及淋巴结中分选效应T细胞。本研究的RNA测序（RNA-Seq）数据来自1次无生物学重复的实验，结果显示，经诱导产生免疫耐受的效应T细胞中，与T细胞失能相关的基因表达显著上调。