ZnT8-Specific CD4⁺ T Cells Display Distinct Cytokine Expression Profiles between Type 1 Diabetes Patients and Healthy Adults

Determination of antigen-specific T cell repertoires in human blood has been a challenge. Here, we show a novel integrated approach that permits determination of multiple parameters of antigen-specific T cell repertoires. The approach consists of two assays: the Direct assay and the Cytokine-driven assay. Briefly, human PBMCs are first stimulated with overlapping peptides encoding a given antigen for 48 hours to measure cytokine secretion (Direct assay). Peptide-reactive T cells are further expanded by IL-2 for 5 days; and after overnight starvation, expanded cells are stimulated with the same peptides from the initial culture to analyze cytokine secretion (Cytokine-driven assay). We first applied this integrated approach to determine the type of islet-antigen-specific T cells in healthy adults. Out of ten donors, the Direct assay identified GAD65-specific CD4+ T cells in three adults and zinc transporter 8 (ZnT8)-specific CD4+ T cells in five adults. The intracytoplasmic cytokine staining assay showed that these islet-antigen-specific CD4+ T cells belonged to the CD45RO+ memory compartment. The Cytokine-driven assay further revealed that islet-antigen-specific CD4+ T cells in healthy adults were capable of secreting various types of cytokines including type 1 and type 2 cytokines as well as IL-10. We next applied our integrated assay to determine whether the type of ZnT8-specific CD4+ T cells is different between Type 1 diabetes patients and age/gender/HLA-matched healthy adults. We found that ZnT8-specific CD4+ T cells were skewed towards Th1 cells in T1D patients, while Th2 and IL-10-producing cells were prevalent in healthy adults. In conclusion, the Direct assay and the Cytokine-driven assay complement each other, and the combination of the two assays provides information of antigen-specific T cell repertoires on the breadth, type, and avidity. This strategy is applicable to determine the differences in the quality of antigen-specific T cells between health and disease.人体内血液中抗原特异性T细胞受体库的解析一直是一项学界难题。本研究提出一种全新的整合式研究方法，可实现对抗原特异性T细胞受体库多项参数的精准解析。该方法包含两种检测手段：直接检测法（Direct assay）与细胞因子驱动检测法（Cytokine-driven assay）。简言之，直接检测法的操作流程为：首先将人外周血单个核细胞（peripheral blood mononuclear cell, PBMC）与编码目标抗原的重叠肽段共同孵育刺激48小时，以此检测细胞因子的分泌情况。而细胞因子驱动检测法则需先通过白细胞介素-2（IL-2）将肽段反应性T细胞扩增培养5天；经过夜饥饿处理后，再使用初始培养体系中相同的肽段对扩增后的T细胞进行刺激，进而分析其细胞因子分泌情况。我们首先将该整合式方法应用于健康成年人胰岛抗原特异性T细胞的分型研究：在10名健康志愿者中，直接检测法在3名受试者体内检出了谷氨酸脱羧酶65（glutamic acid decarboxylase 65, GAD65）特异性CD4+ T细胞，在5名受试者体内检出了锌转运体8（zinc transporter 8, ZnT8）特异性CD4+ T细胞。胞内细胞因子染色检测结果显示，此类胰岛抗原特异性CD4+ T细胞均属于CD45RO+记忆性T细胞群。细胞因子驱动检测法进一步揭示，健康成年人体内的胰岛抗原特异性CD4+ T细胞可分泌多种类型的细胞因子，包括Ⅰ型、Ⅱ型细胞因子以及白细胞介素-10（IL-10）。随后，我们利用该整合式检测方法，探究了1型糖尿病（Type 1 diabetes, T1D）患者与年龄、性别、人类白细胞抗原（human leukocyte antigen, HLA）匹配的健康成年人之间，ZnT8特异性CD4+ T细胞的分型差异。研究结果显示，1型糖尿病患者体内的ZnT8特异性CD4+ T细胞呈现向Th1细胞极化的趋势，而健康成年人中则以Th2细胞及分泌IL-10的T细胞为主。综上，直接检测法与细胞因子驱动检测法可互为补充，二者联合使用可从广度、分型以及亲和力多个维度，全面获取抗原特异性T细胞受体库的相关信息。该研究策略可用于解析健康与疾病状态下，抗原特异性T细胞的质量差异。