BF Fox-3 HLA merge A 3

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1 Supplementary Figure 1 F Fox-3 merge 3 SN TRL L P L TRL SN P Supplementary Figure 1. Series of low magnification brightfield and immunofluorescence images (Fox-3 in green/, and in red). ircles indicate neuro that have NM (see brightfield images) and are immunoreactive for both Fox-3 (green) and (red). rrows indicate blood vessels. Squares indicate the presence of nuclei of NM - neuro, which are negative for. Photomicrographs illustrate both a region with NM + neuro (top row) and an area within the same section with NM - (bottom row) of the SN. oistent with TH staining results, immunolabel appears mainly in blood vessels and in NM + cells, while neuro devoid of NM are also devoid of, both in the SN () and in the L (). The intraneuronal label of Fox-3 in SN neuro is coistent with previous reports 1. Scale = 50 µm. F = brightfield; TRL = control.

2 Supplementary Figure 2 TH F merge SN L F 8+ merge Supplementary Figure 2. ) rightfield and immunofluorescence images of a VT neuron (NM - ) with (red) and TH (green). Scale = 10 µm. ) V-VIP immunostaining of a SN section devoid of NM + neuro showing in blood vessels (arrows) but not in glia or neuro. Scale = 50 µm ) dditional immunoelectron microscopy images demotrating antigenicity to within NM granules of SN and L (white arrows). Scale = 500 nm. ) ouble immunofluorescence in the SN of human of a postmortem P sample. NM was observed under brightfield microscopy. appears in red and 8 + T cells in green. Scale = 50 µm. Each experiment was repeated at least in triplicate. Within each individual experiment, each condition was also performed at least three times; F = brightfield.

3 Supplementary Figure 3 ontrol ontrol IFN-γ TH MH-I merge Expression of MH-I (%) IFN-γ PS LPS α-syn NM VM x Str 2 * * E Proliferation (rbitrary Units) F 1 No cells s SN neuro TRL PEPTIE Neuronal survival TRL OT-1 cells conditioned medium 10 Neuronal survival 6 4 WT astrocytes KO astrocytes Veh Tcs IFN/SIIN/Tcs Supplementary Figure 3. ) ultured motor neuro derived from hes treated with either the vehicle (PS) or human IFN-γ. No MH- I was observed in any condition. Green = synapsin, blue = choline acetyltraferase, a marker for motor neuro and red = MH-I. Scale = 50 µm. ) ouble TH/MH-I immunolabel in unfixed postnatally-derived cultured SN neuro imaged by confocal microscopy. ontrol neuro exhibited no plasma membrane MH-I immunolabel. neuron (TH: green) exposed to IFN-γ presented MH-I. Scale = 30 µm. ) omparison of the expression of MH-I after exposing primary VM, cortical and striatal neuro to medium from microglia pre-stimulated with LPS, NM or α-syn. ata are presented as the mean ± SEM ( = non significant; p < 0.01, One-way NOV with Tukey post-hoc test). ) OT-1 cell proliferation as measured by rdu incorporation. SIINFEKL peptide alone did not activate OT-1 cell proliferation in the absence of other cells, but SIINFEKL peptide activated OT-1 cell proliferation in the presence of either s, or with IFN-γ exposed VM neuronal cultures. ata are presented as the mean ± SEM ( = non significant; * p < 0.001, One-way NOV with Tukey post-hoc test). E) Neuronal survival after incubating VM neuro with or without OT-1 cell conditioned medium after seven days of culture. ata are presented as the mean ± SEM ( = non significant, two-tailed Student s T-test). F) Neuronal survival of wild type VM neuro plated on astrocytes from wild type of β2m KO mice after the addition of IFN-γ, SIINFEKL and OT-1 cells. ata are presented as the mean ± SEM ( = non significant, two-tailed Student s T-test). Each experiment was repeated at least in triplicate and within each experiment, each condition was also performed at least three times. For each condition, neuro on n = 24 fields at 20X were quantified. TRL = control; IFN = interferon gamma; SIIN = SIINFEKL peptide; Veh = vehicle.

4 Supplementary Figure 4 F TH merge F TH β2m merge 17 + S1 S1 S1+P S1 S1+P β2m 11 β2m Omission of primary antibody (green channel) E + WT WT WT KO KO KO WT WTWT KO KO KO + ( Ka) Supplementary Figure 4. ) rightfield and fluorescence images in the human SN showing double immunostaining for,, and and TH and β2m and TH after pre-adsorbing each primary antibody with its correspondent neutralizing peptide. NM was identified under brightfield illumination. The staining for was fully blocked within the neuro and partially blocked in the blood vessels, where the levels of are much higher than in the neuro. β2m labelling was totally blocked in neuro and in blood vessels, and only some inespecific precipitate could be observed in the form of dots. Scale = 100 µm. ) Representative western blots of and β2m expression in samples from the SN of a human control individual. single band was observed at Ka for and at 12 Ka for β2m. ) blot (upper panel): the first lane shows the positive control, second and third lanes show our sample blotted with the primary antibody and fourth lane shows our sample blotted with the primary antibody pre-adsorbed with the blocking peptide. In the β2m blot (lower panel), the first lane shows the positive control, second lane shows our sample blotted with the primary antibody and third lane show our sample blotted with the primary antibody pre-adsorbed with the blocking peptide. ) Immunofluorescence images of 8 + expression in human spleen sectio at different magnificatio and the correspondent negative control (omission of primary antibody). alibration bars = 50 μm (small zoom) and 10 μm (large zoom). E) Representative western blot of MH-I expression in samples from brain homogenates from wild type versus β2m KO mice. Positive control was loaded on both sides of the gel. Left panel shows the uncropped film. F = brightfield; P = blocking peptide; + = positive control; S1 = sample.

5 Supplementary References 1. annon, J.R., & Greenamyre, J.T. NeuN is not a reliable marker of dopamine neuro in rat substantia nigra. Neurosci. Lett. 464, (2009).