Autoimmune response to C9orf72 protein in amyotrophic lateral sclerosis

Abstract

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease characterized by a progressive loss of motor neurons. Neuroinflammation is apparent in affected tissues, including increased T cell infiltration and activation of microglia, particularly in the spinal cord^1,2. Autoimmune responses are thought to have a key role in ALS pathology, and it is hypothesized that T cells contribute to the rapid loss of neurons during disease progression^3,4. However, until now there has been no reported target for such an autoimmune response. Here we show that ALS is associated with recognition of the C9orf72 antigen, and we map the specific epitopes that are recognized. We show that these responses are mediated by CD4⁺ T cells that preferentially release IL-5 and IL-10, and that IL-10-mediated T cell responses are significantly greater in donors who have a longer predicted survival time. Our results reinforce the previous hypothesis that neuroinflammation has an important role in ALS disease progression, possibly because of a disrupted balance of inflammatory and counter-inflammatory T cell responses⁴. These findings highlight the potential of therapeutic strategies aimed at enhancing regulatory T cells⁵, and identify a key target for antigen-specific T cell responses that could enable precision therapeutics in ALS.

Fig. 1. Immunophenotyping shows alterations in the T helper cell subsets associated with ALS.

a–c, Broad representation of lymphocyte subsets (a) and CD4 (b) or CD8 (c) memory T cell subsets in individuals with ALS (n = 19) and in healthy control (HC) donors (n = 20). NKT, natural killer T cell. d, Composition of functional T helper cell subsets defined on the basis of expression of the CCR6, CXCR3 and CCR4 subset markers, in the same donors as in a–c. P values from two-tailed Mann–Whitney tests and geometric mean ± 95% confidence intervals are shown. Fold change (FC) values represent the ratio of geometric values in the ALS–HC cohorts. Source data

Fig. 2. Cytokine responses towards neuroantigens in individuals with ALS and in healthy control individuals.

a, Total IFNγ, IL-5 and IL-10 responses detected against each pool in HC donors (n = 28) and individuals with ALS (n = 28). b–d, Production of IFNγ (b), IL-5 (c) and IL-10 (d) individual cytokines, in the same donors as in a. SFCs, spot-forming cells. e, Relative balance of average IFNγ, IL-5 and IL-10 responses in HC individuals and in individuals with ALS. f, IFNγ-mediated (left), IL-5-mediated (middle) and IL-10-mediated (right) T cell responses towards serially diluted concentrations of the C9orf72 peptide pool in HC individuals (n = 5) and individuals with ALS (n = 5). g, Corresponding peptide concentration required to achieve 50% of the maximum response (EC₅₀) in HC donors (n = 5) and those with ALS (n = 5). For one HC donor, the lack of IL-5 responses prevented the development of a dose–response curve. P values from two-tailed Mann–Whitney tests and geometric mean ± 95% confidence intervals are shown. Fold change values represent the ratio of geometric values in the ALS–HC cohorts. HC donors are shown in blue; ALS donors in red. Source data

Fig. 3. Mapping specific C9orf72 epitopes from responding individuals with ALS.

a, Distribution of recognized epitopes among the 100 peptides spanning the C9orf72 protein sequence, identified in 28 participants with ALS. b, Number of epitopes recognized by each patient with ALS (n = 28) (median of 3, range 0–26). Median and range are shown. c, Number of predicted binding events to the 27 most common HLA II allelic variants, by peptides recognized in three or more patients (n = 21), one or two patients (n = 51) or no patients (n = 23). Binding was predicted using the NetMHCIIpan 4.1 EL prediction method, using a cut-off for a binding event as a predicted binding percentile score threshold of 20% or less. P value from two-tailed Mann–Whitney test and mean ± 95% confidence intervals are shown. Source data

Fig. 4. IL-5- and IL-10-mediated T cell responses are associated with C9orf72 mutation status and predicted survival.

a–d, Total (a), IFNγ-mediated (b), IL-5-mediated (c) and IL-10-mediated (d) T cell responses towards C9orf72 in a validation cohort of participants with ALS carrying the C9orf72 mutation (n = 7) or not carrying any mutation linked to an increased risk of ALS (n = 7). e–h, Total (e), IFNγ-mediated (f), IL-5-mediated (g) and IL-10-mediated (h) T cell responses towards C9orf72 in individuals with ALS with a short (n = 6) or long (n = 11) predicted survival time. P values from two-tailed Mann–Whitney tests and geometric mean ± 95% confidence intervals are shown. Source data

Extended Data Fig. 1. Gating strategy for flow cytometry.

Gating strategies for broad immunophenotyping of T cell subsets in PBMCs. Related to data in Fig. 1.

Extended Data Fig. 2. Donors with ALS have an IL-5-biased T cell response.

Proportion of IFNγ (a) and IL-5 (b) releasing T cells among all cytokine-releasing cells in response to C9orf72 in healthy controls (HC; n = 28) and donors with ALS (n = 28). (c) Ratio of the number of cells releasing IFNγ and IL-5. Total (d), IFNγ- (e), IL-5- (f), and IL-10-mediated (g) T cell responses towards C9orf72 in ALS, HC, Parkinson’s disease (PD; n = 15), and Alzheimer’s disease (AD; n = 15) cohorts (ALS (n = 28) and HC (n = 28) are the same as in Fig. 2 and panels a–c). Proportion of IFNγ- (h) and IL-5-releasing (i) T cells among all cytokine-releasing cells, and ratio of the number of cells releasing IFNγ and IL-5 (j), in response to C9orf72 in HC, ALS, AD, and PD cohorts. P-values from two-tailed Mann–Whitney tests from comparisons of two groups, and from Kruskal-Wallis tests followed by Dunn’s multiple comparisons test for comparison of four groups. Only significant p-values are shown, the complete list of p-values can be found in the Source Data file. Geometric mean ± 95% confidence interval is shown. Fold change (FC) values represent the ratio of geometric values in ALS/HC. Source data

Extended Data Fig. 3. Phenotyping of cytokine-secreting cells.

Gating strategies for the identification of cytokine-expressing T cells 24 h post C9orf72 peptide pool restimulation of 2-week cell culture, in healthy controls (HC; n = 30) and donors with ALS (n = 29). Related to data in Fig. 4.

Extended Data Fig. 4. Characterization of C9orf72-reactive T cells.

Frequency of CD4 and CD8 T cells in HC (a) and ALS (b), as well as cytokine-expressing CD4 and CD8 T cells in HC individuals (n = 15) (c) and patients with ALS (n = 15) (d) 6 h post C9orf72 peptide pool restimulation of 2-week culture. Cytokine expression in CD4 (e) and CD8 (f) T cells in patients with ALS and HC individuals after C9orf72 peptide pool restimulation. (g) Overall sum of % CD4 and CD8 responses by the different cytokines. Ratios of the frequency of CD4 T cells expressing IFNγ and IL-4 (h), IFNγ and IL-10 (i), and IFNγ and cells expressing IL-4 or IL-10 (j). P-values are from two-tailed Mann–Whitney test, and geometric mean ± 95% confidence interval and fold change (FC) values are shown. Source data

Extended Data Fig. 5. Association between C9orf72 peptide immunogenicity and HLA binding.

(a) Correlation between response frequency and number of predicted binding events for each peptide (n = 95) to the 27 most common HLA II allelic variants. Correlation is indicated by Spearman r and p-value. (b) Comparison of median binding rank to the 27 most common HLA II allelic variants for peptides recognized (n = 72) or not recognized (n = 23) in 28 patients with ALS. The most recognized peptide, aa56–DGEITFLANHTLNGE, is highlighted in red. P-value from two-tailed Mann–Whitney tests and geometric mean ± 95% confidence interval are shown. (c) Predicted binding events for each peptide from C9orf72, TDP-43, and SOD1, to the 27 most common HLA II allelic variants. P-values from two-tailed Kruskal-Wallis tests followed by Dunn’s multiple comparison test, and median ± 95% confidence interval are shown. Source data

Extended Data Fig. 6. Correlation between C9orf72 responsiveness and clinical and biological variables.

(a) Difference in magnitude of response between ALS samples from male (n = 24) and female (n = 16) individuals. P-value from two-tailed Mann–Whitney tests and geometric mean ± 95% confidence interval are shown. Correlation between C9orf72 response and age (n = 40) (b), time since onset (n = 31) (c), and time since diagnosis (n = 40) (d) against the magnitude of C9orf72-specific reactivity. Correlation between ALSFRS-R score and Total (e), IFNγ- (f), IL-5- (g), and IL-10- (h) mediated C9orf72 responses in donors with ALS (n = 33). Correlation is indicated by Spearman r and p-value. Total (i), IFNγ- (j), IL-5- (k), and IL-10-mediated (l) T cell responses towards C9orf72 for participants with ALS carrying the C9orf72 mutation (n = 3), compared to the remaining participants with ALS, either carrying other mutations (ataxin-2, CHCHD10, FIG4, NEK1, SOD1, and TBK1) (n = 12), or carrying none of those mutations (n = 10), or those for which the genetic data were not available (unknown; n = 15). P-values from two-tailed Mann–Whitney tests and geometric mean ± 95% confidence interval are shown for histograms. Source data