HLA-DQB1*05 subtypes and not DRB1*10:01 mediates risk in anti-IgLON5 disease

Abstract

Anti-IgLON5 disease is a rare and likely underdiagnosed subtype of autoimmune encephalitis. The disease displays a heterogeneous phenotype that includes sleep, movement and bulbar-associated dysfunction. The presence of IgLON5-antibodies in CSF/serum, together with a strong association with HLA-DRB1*10:01∼DQB1*05:01, supports an autoimmune basis. In this study, a multicentric human leukocyte antigen (HLA) study of 87 anti-IgLON5 patients revealed a stronger association with HLA-DQ than HLA-DR. Specifically, we identified a predisposing rank-wise association with HLA-DQA1*01:05∼DQB1*05:01, HLA-DQA1*01:01∼DQB1*05:01 and HLA-DQA1*01:04∼DQB1*05:03 in 85% of patients. HLA sequences and binding cores for these three DQ heterodimers were similar, unlike those of linked DRB1 alleles, supporting a causal link to HLA-DQ. This association was further reflected in an increasingly later age of onset across each genotype group, with a delay of up to 11 years, while HLA-DQ-dosage dependent effects were also suggested by reduced risk in the presence of non-predisposing DQ1 alleles. The functional relevance of the observed HLA-DQ molecules was studied with competition binding assays. These proof-of-concept experiments revealed preferential binding of IgLON5 in a post-translationally modified, but not native, state to all three risk-associated HLA-DQ receptors. Further, a deamidated peptide from the Ig2-domain of IgLON5 activated T cells in two patients, compared with one control carrying HLA-DQA1*01:05∼DQB1*05:01. Taken together, these data support a HLA-DQ-mediated T-cell response to IgLON5 as a potentially key step in the initiation of autoimmunity in this disease.

Keywords: HLA; IgLON5; T cell; autoimmune encephalitis; autoimmunity.

Figure 1

HLA-association with three conserved HLA-DQ5 haplotypes. (A) The first two principal components (PC1, PC2) are shown to highlight the ethnic diversity of subjects included in the study. Cases were matched at a 1:8 ratio to controls from a set of 2503 controls by principal component analysis. [B(i)] HLA association of anti-IgLON5 disease across HLA class I and II is shown unconditioned, (ii) under conditioned exclusion of HLA-DRB1*10:01, (iii) HLA-DRB1*10:01, HLA-DRB1*01:01 and HLA-DRB1*01:02 and (iv) HLA-DRB1*10:01, HLA-DRB1*01:01, HLA-DRB1*14:01, HLA-DRB1*14:54 and HLA-DRB1*14:04. (C) Sankey plot shows conservation of risk-associated HLA-DRB1-DQA1-DQB1 haplotypes among cases.

Figure 2

Delayed age of onset correlates with ranked HLA-risk and HLA-DQ5 dosage. (A) Age at disease onset, (B) sex demographics and (C) major clinical features reported are shown for cases carrying (i) HLA-DQA1*01:05-DQB1*05:01, (ii) HLA-DQA1*01:01-DQB1*05:01, (iii) HLA-DQA1*01:04-DQB1*05:03 or (iv) none of the aforementioned haplotypes. In A, coloured, filled circles indicate individual cases, whereas black filled markers denote homozygous carriers of the given haplotypes (Supplementary Table 2). In C, the numbers in the centres of the pie charts indicate the total numbers of cases reporting the given clinical feature. (D) Age at disease onset is shown for carriers (i) homozygous for HLA-DQ5, (ii) heterozygous for HLA-DQ5 without HLA-DQ6, (iii) heterozygous for HLA-DQ5:∼ with HLA-DQ6:∼ and (iv) not carrying HLA-DQ5:∼. For D(i), age range = 49–80 years (x͂ = 60.5 years); D(ii), age range = 34–91 years (x͂ = 62.0 years); D(iii), age range = 54–70 years (x͂ = 65.0 years); D(iv), age range = 59–81 years (x͂ = 71.5 years). n = total number of cases, μ_age = mean age, x͂ = median age, *P < 0.05, **P < 0.01.

Figure 3

Risk-associated HLA-DQ5 molecules preferentially bind IgLON5 in a deamidated form. (A) The entire length and sequence of the IgLON5 peptide is shown, highlighting sites of post-translational modification (PTM) as predicted by MusiteDeep, NetPhos3.1, NetNGlyc1.0 and mass spectrometry data obtained from Itoh et al. N_glyc = N-linked glycosylation, S_phos = serine phosphorylation, T_phos = threonine phosphorylation and Y_phos = tyrosine phosphorylation. (B) Schematic shows how peptide-HLA binding is determined using competition binding assay. (C) Results from competition binding assay are shown for (i) HLA-DQA1*01:05-DQB1*05:01, (ii) HLA-DQA1*01:01-DQB1*05:01, (iii) HLA-DQA1*01:04-DQB1*05:03 and (iv) HLA-DRA1*01:01-DRB1*10:01. Light bars show IgLON5 15mer peptides in native, and dark bars in PTM form (see legend at bottom). The y-axis (log scale) shows mean fluorescence intensity (mfi) of IgLON5 peptide together with biotinylated-competitor in competition, divided by the mfi of biotinylated-competitor alone. For i–iii, bio-PLXC1, and for iv, bio-HTSF1 was used as biotinylated-competitor. Along the x-axis, enumerated 15-mer IgLON5 peptides, sequentially encompassing the entire length of the IgLON5 protein, are shown (see Supplementary Table 3 for a full list of enumerated peptides). The dotted lines show the thresholds for weak (0.5) and strong (0.25) binders. (D) Binding of HLA-DQA1*01:05-DQB1*05:01 (green bars, left in each group), HLA-DQA1*01:01-DQB1*05:01 (purple bars, middle in each group) and HLA-DQA1*01:04-DQB1*05:03 (yellow bars, right in each group) to the five peptides (i) ⁵³SCFIDEHVTRVAWLN⁶⁷, (ii) ¹²⁵VYLIVHVPARIVNIS¹³⁹, (iii) ¹⁴¹PVTVNEGGNVNLLCL¹⁵⁵, (iv) ¹⁴⁵NEGGNVNLLCLAVGR¹⁵⁹ and (v) ¹⁴⁹NVNLLCLAVGRPEPT¹⁶³ is shown, with asparagine residues (N) in bold in an unmodified (‘native’, left), glycosylated (‘GLcNac’, middle) or deamidated (‘deamidated’, right) form. Dotted lines as for C; y-axis is also as described for C but linear. (E) Dose-dependent change in binding of peptides described in D(i–v) when deamidated at bold N residues (i.e. mimicking aspartic acid, D). The y-axis shows dose-dependent change in binding as a ratio relative to baseline concentration used in D (40 μM of IgLON5 peptide), given the variation of IgLON5 peptide concentrations shown along the x-axis. Error bars in C and D, and shaded regions in E, represent the standard error of the mean (sem). All assays were repeated at least in duplicate.

Figure 4

CD4⁺ T-cell reactivity and phenotypic profiling. (A) Schematic shows how peptide-HLA-spheromers are assembled and used to isolate antigen-specific CD4⁺ T cells. (B) Proportion of CD4+ T cells reactive to ¹²⁵VYLIVHVPARIVDIS¹³⁹ in one control and two patients. (C) Neighbourhood clustering of isolated single cells by delineation into individuals [control (green), Patient 1 (purple), Patient 2 (pink)]. (D) T-cell markers. (i) Dot plot displays the expression levels of T-cell subset markers related to distinct transcriptional states in the control versus patient (Patients 1 and 2) group and (ii) related to distinct activation states. (E) T-cell receptor sequencing from antigen-specific cells isolated from control (green), Patient 1 (purple) and Patient 2 (pink) display the frequency of gene segments (i) TRAV, (ii) TRAJ, (iii) TRBV and (iv) TRBJ. (F) Pairings of (i) TRAV-TRAJ and (ii) TRBV TRBJ are shown. For B, C, E and F, see legend for colour coding and descriptions. T_naive = naive; T_em = effector; T_emra = highly effector; T_reg = regulatory.