Single-cell peripheral immunoprofiling of Lewy body and Parkinson’s disease in a multi-site cohort

Background
Multiple lines of evidence support peripheral organs in the initiation or progression of Lewy body disease (LBD), a spectrum of neurodegenerative diagnoses that include Parkinson’s Disease (PD) without or with dementia (PDD) and dementia with Lewy bodies (DLB). However, the potential contribution of the peripheral immune response to LBD remains unclear. This study aims to characterize peripheral immune responses unique to patients with LBD at single-cell resolution to highlight potential biomarkers and increase mechanistic understanding of LBD pathogenesis in humans.
Methods
In a case-control study, peripheral mononuclear cell (PBMC) samples from research participants were randomly sampled from multiple sites across the United States. The diagnosis groups comprise healthy controls (HC, n=159164), LBD (n=110132), Alzheimer’s disease dementia (ADD, n=9798), other neurodegenerative disease controls (NDC, n=1921), and immune disease controls (IDC, n=14). PBMCs were activated with..., PBMCs were isolated by density-gradient centrifugation and cryopreserved. Post-thaw, cells were washed in a complete RPMI medium with benzonase. Cell viability as measured by Vi-Cell (Beckman Coulter) for all samples was above 90%. After resting for 2h at 37oC, PBMCs were either left unstimulated or stimulated with a panel of cytokines: IFNα (10,000 units/ml), IL-2 (50 ng/ml), IL-6 (50 ng/ml) and LPS (200 ng/ml) for 15 min, at 37oC. Stimulation was stopped by fixing cells with paraformaldehyde for 10 minutes at room temperature. After washing cells with PBS, samples were stained with LIVE/DEAD™ Fixable Blue Dead Cell Stain Kit, for UV excitation (from Invitrogen) for 15 min at room temperature. After live dead staining, cells were washed with wash buffer (Phosphate buffered saline, 2% Fetal bovine serum, 0.1% sodium azide), followed by surface staining with anti- CD4(BUV805), CD7 (AF780), CD8 (AF700), CD11b (BUV395), CD14 (BUV737), CD16 (BV750), CD19 (PerCP-Cy5.5), CD27 (BV711), CD56 (B..., , # Single-Cell Peripheral Immunoprofiling of Lewy Body Disease in a Multi-site Cohort

The code to analyze the median data can be found at: [https://github.com/tpjoe/flowADDF](https://github.com/tpjoe/flowADDF)

## Gated median value data

The gated data with median values and frequency can be found at **gated_median.csv** and **gated_medianCorrected.csv** for Site-corrected version using pyCombat. The column can be interpreted using the following nomenclature:

**Singlets/Live cells/B cells | Median (Comp-450_50 Violet I-A)--US**

Where,

* The text before \"|\" is the cell type with \"/\" indicating the hierarchy level of gating.
* Comp-450_50 Violet I-A is the intracellular signals where 
* Comp-450_50 = pS6
* Comp-780_60 = pSTAT5
* Comp-515_20 = pSTAT1
* Comp-586_15 = p38
* Comp-610_20 = Rab5
* Comp-660_20 = pPLCg2
* \--[...] = stimulation where there are US(unstim), LPS, IFNa, and IL-6.

## Meta data

The associated diagnoses and all metadata can be found in the **metadata.c..., All participants provided written informed consent to participate in the study, which followed protocols approved by the Stanford Institutional Review Board.

# 研究背景
多项证据表明，外周器官参与路易体病（Lewy body disease, LBD）的发生或进展过程。路易体病是一类神经退行性疾病谱系，涵盖不伴痴呆的帕金森病（Parkinson’s Disease, PD）、伴痴呆的帕金森病（PDD）以及路易体痴呆（DLB）。然而，外周免疫应答在路易体病发病过程中的潜在贡献仍未明确。本研究旨在以单细胞分辨率解析路易体病患者特有的外周免疫应答特征，以期发掘潜在生物标志物，并加深对人类路易体病发病机制的理解。

# 研究方法
本研究为病例对照研究，研究受试者的外周血单个核细胞（peripheral mononuclear cell, PBMC）样本从美国境内多个研究中心随机抽样获取。本研究的分组包括健康对照组（HC, n=159164）、路易体病组（LBD, n=110132）、阿尔茨海默病痴呆组（ADD, n=9798）、其他神经退行性疾病对照组（NDC, n=1921）以及免疫性疾病对照组（IDC, n=14）。
PBMC经[原文截断]刺激；PBMC通过密度梯度离心法分离并冻存。解冻后，细胞使用含苯并酶（benzonase）的完全RPMI培养基洗涤。所有样本经Vi-Cell（贝克曼库尔特，Beckman Coulter）检测的细胞活力均高于90%。
将细胞于37℃静置2小时后，将PBMC分为未刺激组，以及使用细胞因子组合：IFNα（10000 U/ml）、IL-2（50 ng/ml）、IL-6（50 ng/ml）与LPS（200 ng/ml），于37℃刺激15分钟的刺激组。刺激终止于室温下使用多聚甲醛固定细胞10分钟。经PBS洗涤细胞后，使用LIVE/DEAD™ 紫外激发型可固定蓝色死细胞染色试剂盒（Invitrogen）于室温染色15分钟。活死染色完成后，使用洗涤缓冲液（磷酸盐缓冲液、2%胎牛血清、0.1%叠氮化钠）洗涤细胞，随后使用以下抗体进行表面染色：抗CD4（BUV805）、CD7（AF780）、CD8（AF700）、CD11b（BUV395）、CD14（BUV737）、CD16（BV750）、CD19（PerCP-Cy5.5）、CD27（BV711）、CD56（B...）[原文截断]。

# 多中心队列路易体病的单细胞外周免疫谱分析
中位数数据分析代码可从以下网址获取：https://github.com/tpjoe/flowADDF

## 门控中位数数据
包含中位数数值与细胞频率的门控数据可从**gated_median.csv**与**gated_medianCorrected.csv**获取，其中后者为使用pyCombat进行位点校正后的版本。列名可通过以下命名规则解析：
> **单倍体/活细胞/B细胞 | 中位数(Comp-450_50 Violet I-A)--US**

其中：
* 「|」左侧为细胞类型，「/」用于标识门控的层级关系；
* Comp-450_50 Violet I-A 为细胞内信号标志物，具体对应关系如下：
* Comp-450_50 = pS6
* Comp-780_60 = pSTAT5
* Comp-515_20 = pSTAT1
* Comp-586_15 = p38
* Comp-610_20 = Rab5
* Comp-660_20 = pPLCg2
* 「--[...]」代表刺激条件，包含US（未刺激）、LPS、IFNα以及IL-6。

## 元数据
相关诊断信息及全部元数据可从metadata.c...[原文截断]获取。所有研究受试者均签署书面知情同意书，本研究方案经斯坦福大学机构审查委员会批准。