T cell responses towards PINK1 and ⍺-synuclein are elevated in prodromal Parkinson's disease - Fluorospot data

Antigen-specific T cell reactivity in PBMCs were studied using the Fluorospot assay PBMCs were co-cultured with alpha-synuclein, (⍺-syn) PINK1, or Epstein-Barr virus peptide pools and subsequently expanded in vitro over a two-week period. Cultures were supplemented with IL-2 (10 U/mL) every 4 days. After two weeks, expanded cells were harvested and were re-stimulated in triplicates with the same peptide pools, dimethylsulfoxide (DMSO; as a negative control) and phytohemagglutinin (PHA; as a positive control) and then processed for a dual-color (interferon-gamma and IL-5) Fluorospot assay (Mabtech). Spot forming cells (SFC) were captured using an IRIS Fluorospot reader (Mabtech) and results were analyzed using Excel (Microsoft). To be considered positive, antigen-specific T cells had >100 SFC per million PBMC, p-value <0.05 and >2 fold increase when compared to the DMSO control. Note: “ND” refers to “no data”, as some samples either did not have the adequate amount of cells for testing or did not pass our SFC quality control criteria. The "Sum of ⍺-syn and PINK1 IFNγ SFC/10^6 cultured cells" and "Sum of ⍺-syn and PINK1 IL-5 SFC/10^6 cultured cells" was only calculated for donors screen with both PINK1 and ⍺-syn. In the Unified Parkinson’s Disease Rating Scale (UPDRS) part III and the Montreal Cognitive Assessment (MoCA) tests columns, donors not tested were assigned "Not tested".本研究采用荧光斑点（Fluorospot）检测技术，对外周血单个核细胞（peripheral blood mononuclear cells, PBMCs）中的抗原特异性T细胞反应性展开分析。 将外周血单个核细胞与α-突触核蛋白（alpha-synuclein, α-syn）、PINK1或EB病毒（Epstein-Barr virus）肽库共培养，随后于体外进行为期两周的扩增培养。每4天向培养体系中补加终浓度为10 U/mL的白细胞介素-2（IL-2）。两周后收集扩增后的细胞，以相同肽库、二甲基亚砜（dimethylsulfoxide, DMSO，阴性对照）及植物血凝素（phytohemagglutinin, PHA，阳性对照）进行三次重复刺激，随后采用双色（干扰素-γ与白细胞介素-5）荧光斑点检测法（Mabtech）完成样本处理。使用IRIS荧光斑点读数仪（Mabtech）捕获斑点形成细胞（spot forming cells, SFC），并采用Excel（Microsoft）对检测结果进行分析。抗原特异性T细胞阳性判定标准为：每百万外周血单个核细胞中斑点形成细胞数大于100、p值小于0.05，且相较于DMSO阴性对照，斑点形成细胞数增幅达2倍以上。 注：“ND”代表“无数据”，部分样本因未获得足够用于检测的细胞，或未通过本研究的斑点形成细胞质量控制标准，故标注为ND。 “α-突触核蛋白与PINK1干扰素-γ斑点形成细胞数/每10^6培养细胞之和”及“α-突触核蛋白与PINK1白细胞介素-5斑点形成细胞数/每10^6培养细胞之和”仅针对同时接受PINK1与α-突触核蛋白筛选的供体进行计算。 在统一帕金森病评定量表第三部分（Unified Parkinson’s Disease Rating Scale part III, UPDRS III）与蒙特利尔认知评估（Montreal Cognitive Assessment, MoCA）测试列中，未接受检测的供体标注为“未检测”。