Fixation and permeabilization kit

For intracellular p24 staining, U1-shCTH and U1-shNT cells were stimulated with PMA (5 ng/ml), washed with PBS followed by fixation and permeabilization using a fixation and permeabilization kit (eBiosciences). Permeabilized cells were then incubated with 50 μl of 1:100 dilution of phycoerythrin (PE)-conjugated mouse anti-p24 monoclonal antibody (KC57-RD1; Beckman Coulter, Inc) for 30 min at room temperature. After incubation, the cells were washed two times and the fluorescence of stained samples was acquired using BD FACSVerse flow cytometer (BD Biosciences). The data were analyzed using FACSuite software (BD Biosciences).

For T cell phenotyping, pancreas, pLN, and peripheral LN were processed and stained as described (6 (link)). The mAbs used were as follows: anti-CD4-BV711, anti-CD4-FITC, anti-CD8a-BV786, anti-CD8a-FITC, anti-Thy1.1-PerCP, anti-CD62L-APC, anti-INFγ-PE, anti-CD45RB-PE, and anti-CD107a-PE (BD PharMingen); anti-CD25-APC-eFluor780, anti-Granzyme B-PE, anti-KLRG1-PE-Cy7, and anti-FoxP3-PerCP-Cy5.5 (eBioscience). Cells were analyzed on a FACSCanto II or a LSR Fortessa apparatus using Diva software (BDB). Isotype-matched antibodies were used as controls. Intracellular Granzyme B and Foxp3 staining was performed using the Fixation and Permeabilization Kit (eBioscience). Intracellular IFNγ and IL-2 staining was performed after 5 h restimulation with HA peptide using the Cytofix/Cytoperm Kit (BD PharMingen) (6 (link)).

PBMCs were defrosted (see Supplementary Methods) and cultured for 48 h in 200 μL of RPMI cell culture media with glutamine (Gibco 21875-034) in a 96-well round bottom plate. The test wells were stimulated with either CpG DNA (1:10 000) (Cambridge Bioscience #Hycult HC4039) and CD40 ligand (1:5000) (R and D systems, 6245-CL-050) or alpha-synuclein fibrils for 48 h. Five hours prior to removal from culture, phorbol 12-myristate 13-acetate (PMA) (50 ng/mL) (Sigma-Aldrich #P8139), ionomycin (500 ng/mL) (Sigma-Aldrich #I0634) and brefeldin A (BFA) (5 μg/mL) (BioLegend #420601) (PIB) were added to the CD40L/CpG wells and to separate intermediate (PIB) wells. BFA was added to the alpha-synuclein and unstimulated wells.
Cells were removed from culture at 48 h and centrifuged at 350g for 5 min. Surface staining was done as per the protocol above excluding the fixation step. Cells were fixed and permeabilized prior to intracellular staining using the eBiosciences fixation and permeabilization kit as per manufacturer’s instructions [Fix/Perm concentrate (00-5123-43) Fix/Perm Diluent (00-5223-56) and 10 × Perm buffer (00-8333-56)]. See Supplementary Methods for a list of antibodies. Flow cytometry was run within 2–4 h.

For surface staining, cells were harvested, washed, and stained with the PE- or FITC-conjugated anti-mouse CD4 antibodies in the presence of Fc blocker (BioLegend). For intracellular staining, cells were stimulated with phorbol myristic acetate (50 ng/ml, Sigma Aldrich), ionomycin (1 μg/ml, Sigma Aldrich), and brefeldin A (1 μg/ml, Sigma Aldrich) for 4–6 h in complete RPMI 1640 medium. Then, cells were fixed and permeabilized overnight using Fixation and Permeabilization kit (eBioscience, SanDiego, CA, USA), followed by incubating with FITC-conjugated anti-mouse IL-17 antibody, PE-conjugated anti-mouse Foxp3 antibody, and APC-conjugated anti-mouse IFN-γ antibody. The stained cells were detected by flow cytometry (FACS Calibur, BD Biosciences, San Jose, CA, USA), and the data were analyzed by FlowJo software (Tree Star, Ashland, OR, USA).