DOI: 10.5061/dryad.h70rxwdrc
title: Data from: Single-cell peripheral immunoprofiling of Lewy body and Parkinson’s disease in a multi-site cohort

description: 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 three stimulants (LPS, IL-6, and IFNa) or remained at basal
 state, stained by 13 surface markers and 7 intracellular signal markers,
 and analyzed by flow cytometry, which generated 1,184 immune features
 after gating.  Results The model classified LBD from HC with an
 AUROC of 0.8790±0.06 and AUPRC of 0.806±0.06. Without retraining, the same
 model was able to distinguish LBD from ADD, NDC, and IDC. Model
 predictions were driven by pPLCγ2, p38, and pSTAT5 signals from specific
 cell populations under specific activation. The immune responses
 characteristic for LBD were not associated with other common medical
 conditions related to the risk of LBD or dementia, such as sleep
 disorders, hypertension, or diabetes. Conclusions and Relevance
 Quantification of PBMC immune response from multisite research
 participants yielded a unique pattern for LBD compared to HC, multiple
 related neurodegenerative diseases, and autoimmune diseases thereby
 highlighting potential biomarkers and mechanisms of disease.
description: 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 (BUV563), CD69 (BUV661), HLA-DR (BV480) (antibodies
 from BD Biosciences), CD3 (BV605) and CD45RA (BV570) (antibodies from
 BioLegend). Staining was done at room temperature for 30 min. After 2
 washes, cells were permeabilized with ice-cold methanol and were stored
 overnight at -80°C. Post permeabilization, cells were washed again, and
 intracellular staining was done with anti-pSTAT1 (AF488), pSTAT5 (PE-Cy7),
 pP38 (PE), pPLCγ2 (APC), pS6 (BV421), CD107b/Lamp2 (BV786), (antibodies
 from BD Biosciences) and Rab5 (PE-CF594) (from Santa Cruz Biotechnology)
 at room temperature for 30 min. After two further washes, the acquisition
 was performed on a BD Symphony A5 flow cytometer with a High Throughput
 Sampler (HTS) and analyzed using FlowJo software. 
description: # 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.csv**, which is
 linked to the gated flow data by 'Sample ID' column. The
 definition of the meta columns that are not NACC vocabulary can be found
 at **data_dictionary.csv**.  ## Description of the fcs data and file
 structure The data includes .fcs files of samples, the gated median
 values, and the associated metadata for each sample. The .fcs file name
 notation follows "export_**[Sample No.]*_*[Stimulating
 Condition]_**[Run No.]_Live_cells.fcs" ## Human subjects data All
 participants provided written informed consent to participate in the
 study, which followed protocols approved by the Stanford Institutional
 Review Board.