α-Synuclein-specific T cell reactivity is associated with preclinical and early Parkinson’s disease

A diagnosis of motor Parkinson’s disease (PD) is preceded by a prolonged premotor phase with accumulating neuronal damage. Here we examined the temporal relation between α-synuclein (α-syn) T cell reactivity and PD. A longitudinal case study revealed that elevated α-syn-specific T cell responses were detected prior to the diagnosis of motor PD, and declined after. The relationship between T cell reactivity and early PD in two independent cohorts showed that α-syn-specific T cell responses were highest shortly after diagnosis of motor PD and then decreased. Additional analysis revealed significant association of α-syn-specific T cell responses with age and lower levodopa equivalent dose. These results confirm the presence of α-syn-reactive T cells in PD and show that they are most abundant immediately after diagnosis of motor PD. These cells may be present years before the diagnosis of motor PD, suggesting avenues of investigation into PD pathogenesis and potential early diagnosis.


Statistics
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For hierarchical and complex designs, identification of the appropriate level for tests and full reporting of outcomes Estimates of effect sizes (e.g. Cohen's d, Pearson's r), indicating how they were calculated Our web collection on statistics for biologists contains articles on many of the points above.

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Data analysis
For manuscripts utilizing custom algorithms or software that are central to the research but not yet described in published literature, software must be made available to editors/reviewers. We strongly encourage code deposition in a community repository (e.g. GitHub). See the Nature Research guidelines for submitting code & software for further information.

Data
Policy information about availability of data All manuscripts must include a data availability statement. This statement should provide the following information, where applicable: -Accession codes, unique identifiers, or web links for publicly available datasets -A list of figures that have associated raw data -A description of any restrictions on data availability Alessandro Sette David Sulzer Mar 13, 2020 No open source or custom code was used to collect the data in this study.
Fluorospot spot counting was performed using computer-assisted image analysis on the AID iSpot version 7 (Aid Diagnostica GMBH, Strassberg, Germany). Flow cytometry data was acquired on a BD LSRII flow cytometer and analysis was done using FlowJo X Software version 10 (FlowJo LLC, Ashland, OR). HLA typing was performed by an ASHI-accredited laboratory at Murdoch University. HLA alleles were determined through a proprietary allele calling algorithm and analysis pipeline (IIID HLA analysis suite; www.iiid.com.au/laboratory-testing/) using the latest IMGT HLA database as reference (www.imgt.org). HLA association odds ratios and relative frequencies were calculated using the RATE program (www.iedb.org), Statistical analyses was performed using GraphPad Prism version 7 and GraphPad Quickcalcs.
All data generated or analyzed during this study are included in this article and its Supplementary Information. For HLA typing information from the freely available international immunogenetics information system (http://www.imgt.org), and an ASHI-accredited HLA allele caller software pipeline, IIID HLA Analysis suite (http:// nature research | reporting summary

October 2018
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Life sciences Behavioural & social sciences Ecological, evolutionary & environmental sciences
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Life sciences study design
All studies must disclose on these points even when the disclosure is negative. No sample size calculation was performed since we did not have an expected frequency of response. The final sample size used was based on the samples available from the recruitment sites.
Data was excluded from Fluorospot analysis according to predefined criteria of either cell death in culture preventing further experiments, or a failed PHA response in the assay (<100 SFC per million cells) The correlation between alpha-synuclein reactivity and time since diagnosis was confirmed in an independent patient cohort. Each individual sample was tested in one experiment.
Participants were allocated into experimental groups based on PD diagnosis or HC. For cohort 1 the patients were from all recruitment sites with a spread of time since diagnosis between 0-28 years. The validation cohort were patients from UAB diagnosed 0-8 years ago. Samples studied in flow cytometry were selected based on cells available following the Fluorospot assays.
For the AD studies individuals were allocated into each group based on AD diagnosis or HC. Assignments to each respective cohort was based on the clinical diagnosis.
Individuals performing the experiments and collecting the raw data were blinded to cohort assignment PD vs HC or AD vs HC. Individuals analyzing the data were not blinded since the comparisons were dependent on knowing the respective cohort.
For Fluorospot antibodies from Mabtech (Sweden) was used as described in materials and methods section.  Mouse anti-human IFNg (clone 1-D1K): https://www.mabtech.com/products/anti-human-ifn-gamma-antibody-1-d1kpurified-3420-3#tabs-min-1 mouse anti-human IL-5 (clone TRFK5): https://www.mabtech.com/products/anti-human-il-5-antibody-trfk5-purified-3490-3 mouse anti-human IL-10 (clone 9D7): https://www.mabtech.com/products/anti-human-il-10-antibody-9d7-purified-3430-3 IFNg-BAM (7-B6-1-FS-BAM) followed by anti-BAM-490, IL-5-WASP (5A10-WASP) followed by anti-WASP-640, IL-10-biotin (12G8biotin) followed by Streptavidin-550: https://www.mabtech.com/products/human-ifn-gamma-il-10-il-5-fluorospotplusfsp-010708 1. PD participants were enrolled on the basis of the following inclusion criteria: moderate to advanced PD; 2 of: rest tremor, rigidity, and/or bradykinesia, PD diagnosis at age 47-75, dopaminergic medication benefit, enrollment age 50-90, and ability to provide informed consent. The exclusion criteria were: atypical parkinsonism or other neurological disorders, history of cancer within the past 3 years (not skin), autoimmune disease (except thyroid), and chronic immune-modulatory therapy. Age-matched HC were selected on the basis of age 50-90 and ability to provide informed consent. Exclusion criteria were the same as for PD donors, and in addition we excluded self-reported genetic factors (i.e., PD in first-degree blood relative). In the LJI cohort (n=11 PD), Parkinson's disease was self-reported. These individuals were excluded from the correlations with clinical parameters, since those results were not available. Therefore, the conclusions in this manuscript are not impacted by this cohort. AD subjects from CUMC were recruited according to NIA-AA criteria. They were recruited after at least 2 clinical visits. They had neuropsychological testing and in some cases positive biomarkers (SPECT scan, FDG PET scan, CSF or amyloid scan). The HC went through assessments for neurological and neuropsychological testing. They had 2 consecutive years with normal neuropsychological testing. The AD cohort recruited by Precision Med were diagnosed according to NINCDS-ADRDA criteria. They underwent MRI/CT scans to rule out other causes of cognitive decline. They exhibit deficits in two or more areas of cognition and have progressive worsening of memory and other cognitive functions. The HC are self-reported without evidence for decline in cognitive functions.
All participants provided written informed consent for participation in the study. In the case of the AD cohort all the participants or their authorized representatives provided written informed consent. Ethical approval was obtained from the Institutional review boards at La Jolla Institute for Immunology (LJI; protocol numbers VD-167, VD-124, VD-118, VD-155, and VD-187), Rush University Medical Center (RUMC; Office of research affairs number 16042107-IRB01), University of California San Diego (UCSD; protocol number 161224) and University of Alabama (UAB; protocol number IRB-300001297) and CUMC (protocol number IRB-AAAQ9714).
PBMCs were purified from whole blood using Ficoll-Paque density gradient centrifugation. PBMCs were then thawed and stimulated with alpha-synuclein peptide pool for 14 days. Upon harvest cells were stimulated again with alpha-synuclein epitope pool.

BD LSR II
Diva software for collection and FlowJo X for analysis.
No sorting was performed for this study.
Phenotyping of alpha-synuclein specific T cells: Non-lymphocytes/monocytes and doublet cells were eliminated by forward and side scatter, and dead cells by Live/Dead stain. Cells were gated based on their cytokine expression and then cytokine-expressing cells were gated based on their CD3+ and CD56+ expression. CD3+ T cells were gated based on their CD4 and CD8 expression (CD8+, CD8+CD4+, CD4+, CD8-CD4-), and other populations based on CD3-CD56-and other markers as indicated; CD14-CD19+ (B