T cells from patients with Parkinson's disease recognize α-synuclein peptides

Abstract

Genetic studies have shown the association of Parkinson's disease with alleles of the major histocompatibility complex. Here we show that a defined set of peptides that are derived from α-synuclein, a protein aggregated in Parkinson's disease, act as antigenic epitopes displayed by these alleles and drive helper and cytotoxic T cell responses in patients with Parkinson's disease. These responses may explain the association of Parkinson's disease with specific major histocompatibility complex alleles.

Extended Data Figure 1. T cell reactivity against α-syn peptides (wild type and post-translationally modified)

Magnitude of responses, expressed as the total magnitude (SFC/10⁶ PBMC) of response per peptide/participant combination. Responses against any α-syn 15mer peptide spanning S129 and Y39, “any peptide”, PD (n=209), Control (n=132), and responses against individual α-syn 15mer peptides spanning S129 and Y39. Each dot represents a peptide/participant combination. Closed circles, PD (n=19); open circles, Control (n=12). Two-tailed Mann Whitney, *, p<0.05, **, p<0.01, ***, p<0.001. (A) IFNγ, (B) IL-5, (C) total (IFNγ and IL-5 combined) response.

Extended Data Figure 2. Characterization of α-syn specific responses in PD

a, Gating strategy. T cells were gated based on CD3 expression. Boolean gating was used to define cytokine-producing cells expressing CD4 and/or CD8. b, Percent total cytokine detected from CD3⁺ T cells in response to α-syn peptides. Each point represents one participant (n=9); median ± interquartile range is indicated. Dotted line indicates 0.05% cut-off for specific cytokine production by CD3⁺ T cells. c, Percentage of total cytokines produced for IFNγ, IL-4, IL-10, and IL-17. Each point represents one participant that exceeded the cut-off (n=6), median ± interquartile range is indicated. d, Percentage of total cytokines produced by CD4⁺, CD8⁺, CD4⁻CD8⁻, or CD4⁺CD8⁺ T cells. Each point represents one participant (n=6), median ± interquartile range is indicated.

Extended Data Figure 3. Specific T cell reactivity against native or fibrilized α-syn

Magnitude of responses, expressed as the average spots per 10⁶ PBMC, of response per protein/PD participant or peptide/PD participant combination (n=12 PD participants, each represented by a different symbol). The lines connect discrete values from each individual participant and are present to provide a means to compare responses within and between individuals. The difference between response to unstimulated compared to peptides, the native α-syn and PFF groups is significant by the Wilcoxon one-tailed test (values are shown in the figure). No significant difference (Wilcoxon two-tailed test) in response to PFF and native protein was apparent in this relatively small sample.

Extended Data Figure 4. HLA-DR surface expression across DRB1*15:01⁺ or DRB1*15:01⁻ PD and HC participants

(a) Gating strategy for FACS analysis. After eliminating non-lymphocytes and doublet cells by forward- and side-scatter, cells were gated based on HLA-DR expression. HLA-DR and CD3 expression of participant cells (black; HLA-DR antibody, red; isotype control) of PD patients that carry (b; n=5) and do not carry (c; n=5) DRB1*15:01 allele and HC that carry (d; n=3) and do not carry (e; n=5) DRB1*15:01 allele. 721.221 (f) and RM3 (g) cells are used as controls that do not and do express HLA class II, respectively. (h) Mean fluorescent intensities (MFI) ± standard deviations of HLA-DR expression for each participant cohort. (i) Percentage of living cells that express HLA-DR, mean ± SD.

Extended Data Figure 5. HLA class I surface expression across DRB1*15:01⁺ or DRB1*15:01⁻ PD and HC participants

(a) Gating strategy for FACS analysis. After eliminating non-lymphocytes and doublet cells by forward- and side-scatter, cells were gated based on HLA-ABC expression. HLA-ABC and CD3 expression of participant cells (black: HLA-ABC antibody, red: isotype control) of PD patients that carry (b; n=5) and do not carry (c; n=5) DRB1*15:01 allele and HC that carry (d; n=3) and do not carry (e; n=5) DRB1*15:01 allele. 721.221 (f) and RM3 (g) cells are used as controls that do not and do express HLA class I, respectively. (h) Mean fluorescent intensities (MFI) ± standard deviations of HLA-ABC expression for each participant cohort.

Figure 1. α-Syn autoimmune responses are directed against two regions

a Sequence of α-syn. Antigenic regions are highlighted with dashed red lines with amino acids Y39 and S129 in bold. (b–d) Magnitude of responses expressed as (SFC/10⁶ PBMC) per peptide/participant combination. Left panels; response to all overlapping native α-syn 15mer peptides in PD (n=733) and Control (n=372). Right panels indicate responses against specific 15mers. Grey shading indicates antigenic region containing Y39. (e–g). Magnitude of responses. Left panels; responses to all native and phosphorylated S129 α-syn 15mer peptides in PD (n=150) and Control (n=72). Right panels; responses against specific S129 peptides. Closed circles, PD (n=19, indicated by *, all other n=25); open circles, Control (n=12 participants). Two-tailed Mann Whitney, ns, not significant. (b,e) IFNγ, (c,f) IL-5, (d,g) total (IFNγ & IL-5) response. As many participants showed no response, many points are at the limit of resolution (100 SFC).

Figure 2. Reactivity to native and modified α-syn peptides in PD patients

(a–c) Magnitude of responses against native and modified α-syn 15mer S129 and Y39 region peptides as (SFC/10⁶ PBMC). Each point represents a peptide/participant combination. Closed circles, PD (n=403 peptide/participant combinations “any peptide”, KTKEGVLYVGSKTKE n=63 participants (^), modified peptides marked with * are tested in 19 participants, unmodified peptides are tested in n=41); open circles, control (n=228 any peptide, ^ n=36, *n=12 and unmodified peptides n=24)a, IFNγ, b, IL-5, c, total (IFNγ & IL-5 combined) responses. d, Combined IL-5 and IFNγ responses against individual native and modified α-syn peptides by PD. Black points, IFNγ responses; red points, IL-5 responses. Two-tailed Mann Whitney, *, p<0.05, **, p<0.01, ***, p<0.001. As many participants showed no response, many points are at the limit of resolution (100 SFC).

Figure 3. HLA association of Y39 epitope and identification of A*11:01 restricted 9–10aa length Y39 epitopes

a, Overlapping but largely independent associations between DRB1*15:01, DQB1*03:04 and A*11:01 for PD (13 participants) responding to the Y39 epitope. (b,c) Magnitude of responses by b, PD (n=19) and c, control participants (n=12), as (SFC/10⁶ PBMC) of response per peptide/participant combination to α-syn 9–10mer peptides spanning the protein. In some cases, response to overlapping peptides are combined, with additional residues of the longer peptide in parentheses. b, top panel, IFNγ; middle, IL-5; bottom, total (IFNγ & IL-5 combined) response. c, Total (IFNγ & IL-5 combined). As many participants showed no T cell response, many points are at the limit of resolution (100 SFC).