Enhanced influenza A H1N1 T cell epitope recognition and cross-reactivity to protein-O-mannosyltrans

Narcolepsy type 1 (NT1) is a chronic neurological disorder having a strong association with HLA-DQB1*0602, thereby suggesting an immunological origin. Increased risk of NT1 has been reported among children or adolescents vaccinated with AS03 adjuvant-supplemented pandemic H1N1 influenza A vaccine, Pandemrix. Here we show that pediatric Pandemrix-associated NT1 patients have enhanced T-cell immunity against the viral epitopes, neuraminidase 175–189 (NA175–189) and nucleoprotein 214–228 (NP214–228), but also respond to a NA175–189-mimic, brain self-epitope, protein-O-mannosyltransferase 1 (POMT1675–689). A pathogenic role of influenza virus-specific T-cells and T-cell cross-reactivity in NT1 are supported by the up-regulation of IFN-γ, perforin 1 and granzyme B, and by the converging selection of T-cell receptor TRAV10/TRAJ17 and TRAV10/TRAJ24 clonotypes, in response to stimulation either with peptide NA175–189 or POMT1675–689. Moreover, anti-POMT1 serum autoantibodies are increased in Pandemrix-vaccinated children or adolescents. These results thus identify POMT1 as a potential autoantigen recognized by T- and B-cells in NT1.

Narcolepsy type 1 (NT1) is a rare, chronic brain disorder, characterized by excessive daytime sleepiness, cataplexy and disturbed nocturnal sleep1,2,3. An exceptionally high association of NT1 with the human leukocyte antigen (HLA) class II allele DQB1*0602, and polymorphisms in the genes encoding the T-cell receptor (TCR) α-chain (TRA), tumor necrosis factor-α (TNF-α) promoter, TNF receptor 2 and P2Y11 receptor, strongly suggest an immunological origin of the disease4,5,6,7,8. In particular, T-cell mediated mechanisms leading to impaired hypocretin (HCRT) signaling have been implicated in the development of NT14,9,10,11. Increased risk of NT1 has been reported from several European countries, among children and adolescents vaccinated during 2009/10 with the AS03 adjuvant-supplemented pandemic H1N1 influenza A vaccine (Pandemrix®)12,13,14,15. We previously reported that Pandemrix-associated NT1 patients showed enhanced antibody responses to influenza A virus proteins present in Pandemrix vaccine16. This suggested that vaccinees who later developed NT1 may also have mounted aberrant T-cell responses to Pandemrix. Further, we hypothesized that CD4+ T-cells primed by influenza A (H1N1) virus peptides cross-react with CNS autoantigens, and orchestrate an immune-mediated attack on the hypocretin neuronal network in NT1.

Here we demonstrate enhanced T helper 1 (Th1)-cell/cytotoxic T-cell responses in Pandemrix-associated NT1 patients against distinct viral NA and NP peptide epitopes. Patient-derived T-cells also respond against a self-epitope in brain-expressed protein-O-mannosyltransferase 1 (POMT1), that represents a peptide mimic of a T-cell epitope of NA strongly recognized in patients. Peripheral blood mononuclear cell (PBMC) gene expression profiles are closely matched, and TCR repertoire analyses detect upregulation of identical clonotypes in response to either NA or POMT1 peptide stimulation, thus supporting molecular mimicry and a pathogenic role of virus-directed, autoreactive T-cell clonotypes in NT1. Finally, vaccinees have increased antibody levels against human POMT1, suggesting that Pandemrix vaccination triggers POMT1 autoimmunity. These findings provide a link between influenza A (H1N1) virus-directed T-cell immunity and the development of autoimmunity in NT1.

Influenza A H1N1 virus T-cell epitope screen in Pandemrix-vaccinated HLA-DQ6.2 transgenic mice, and HLA-DQB1*0602 positive individuals

To map influenza A (H1N1) T-cell epitopes in Pandemrix-associated NT1, 15-mer peptides covering hemagglutinin (HA), NA, and NP of influenza (A/reassortant/NYMC X-179A (California/07/2009 × NYMC X-157)(H1N1)) vaccine virus with 12 amino acid overlap were produced. The experimental design for T-cell epitope discovery is illustrated in Fig. 1. First, we screened T-cell responses to peptide pools (Supplementary Data 1) using spleen cells from Pandemrix-vaccinated HLA-DQ6.2 transgenic mice17. Upregulation of interferon-γ (IFN-γ) and/or interleukin-2 (IL-2) mRNA (RT-qPCR) or protein (fluorescent multiplex bead-based immunoassay, FMIA) were used as read-out (Fig. 2a–c). As the second step, T-cell reactivity to single peptides from pools stimulating IFN-γ or IL-2 upregulation was tested in spleen cells from Pandemrix-vaccinated HLA-DQ6.2 mice (Supplementary Fig. 1A–C) and in peripheral blood mononuclear cells (PMBC) from Pandemrix-vaccinated HLA-DQB1*0602 positive individuals (sleep clinic control patients without a diagnosis of NT1; Fig. 2d–f; Supplementary Table 1). Single T-cell peptides from HA, NA or NP that stimulated IFN-γ upregulation either in mice, or in HLA-DQB1*0602 positive individuals, were identified as antigens for subsequent T-cell assays comparing NT1 patients and controls (Supplementary Table 2).

Identification of influenza A H1N1 virus T-cell epitopes in Pandemrix-associated NT1 patients

To dissect the influenza A (H1N1)-directed T-cell immunity, PBMC from a discovery cohort of well-characterized Finnish pediatric cases of Pandemrix-associated NT1, and from healthy age-matched, Pandemrix-vaccinated control children (Table 1, Supplementary Tables 3A, B), were stimulated with a panel of pre-selected 15-mer peptides from HA, NA or NP (Supplementary Table 2). Interestingly, two viral T-cell epitopes were differentially recognized and induced high IFN-γ and IL-2 responses in Pandemrix-associated NT1 patients in comparison to healthy vaccinees. These epitopes were AWSASACHDGINWLT (NA178–192) and KTRIAYERMCNILKGKFQ (NP214–231). Epitopes AFAMERNAGSGIIIS (HA271–285) and MERNAGSGIIISDTP (HA274–288), previously associated with NT118, were also recognized, but the IFN-γ and IL-2 responses were relatively low, and no significant differences between patients and healthy vaccinees were observed (Fig. 3a–f, Supplementary Fig. 2A–F)

Next, the AWSASACHDGINWLT (NA178–192) and KTRIAYERMCNILKG (NP214–228) epitopes were mapped in a validation cohort of Finnish pediatric Pandemrix-associated NT1 patients, and from healthy age-matched, Pandemrix-vaccinated controls (Table 1, Supplementary Tables 3A, B). Stimulations with overlapping 15-mer peptides in T-cell assays showed that AWSASACHDGIN (NA178–189) and IAYERMCNILKG (NP217–228) are T-cell core epitopes (Fig. 4a, d, Supplementary Fig. 3A, D). Interestingly, the AWSASACHDGIN (NA178–189) epitope includes WSASACHD (NA179–186), an influenza A (H1N1) B-cell epitope, predicted by phage display-mimotope variation analysis to react with sera from Pandemrix-associated NT1 patients19.

Differential T-cell responses to influenza A H1N1 epitopes in NT1 patients and HLA-DQB1*0602 positive or negative healthy vaccinees

T cell responses to the epitopes NA175–189, NA178–192, and NP214–228 were stronger in NT1 patients than in either HLA-DQB1*0602 positive or negative controls (combination of NT1 patient discovery and validation cohorts; Fig. 4b, c, e, Supplementary Fig. 3B, C, E). Homozygosity for HLA-DQB1*0602 did not appear to augment responses to these T-cell peptides beyond the levels reached in HLA-DQB1*0602 heterozygous NT1 patients. In contrast, T-cell responses against HA271–285 and HA274–288 were not increased in NT1 patients in comparison to controls, while responses against NP217–231 were significantly enhanced in NT1 patients only when compared to HLA-DQB1*0602 negative, but not positive controls (Supplementary Fig. 4A–D, Fig. 4f, Supplementary Fig. 3F). The results indicated that NT1 patients mounted unusually strong T-cell responses against NA175–192 and NP214–228 viral epitopes in response to Pandemrix vaccination, and that this was not simply a result of restriction by HLA molecules present on the DRB1*1501-DRB5*0101-DQA1*0102-DQB1*0602haplotype. These findings were consistent with our previous report of enhanced antibody responses in NT1 patients against viral proteins from Pandemrix, also in comparison to healthy vaccinees carrying the HLA-DQB1*0602 allele16.

Search for human protein sequences with homology to identified influenza A H1N1 T-cell epitopes from NA and NP

To identify possible cross-reactive self-epitopes, we performed basic local alignment of 12-mer peptides against human proteome (blastp). Sequence similarity searches for IAYERMCNILKG (NP217–228) and ESVAWSASACHD (NA175–186) produced top hits for syntrophin gamma-1 (YEIMCKILK, SNTG1338–346; E-value 2.0, 75% query cover, 78% identity) and protein-O-mannosyltransferase 1 (VAWYSSACH, POMT1681-689; E-value 0.083, 75% query cover, 78% identity). Both proteins are known to be expressed in the human brain.

Discovery of a putative influenza A H1N1 virus cross-reactive T-cell self-epitope in Pandemrix-associated NT1 patients

To test immunological cross-reactivity, we performed T-cell assays with overlapping 15-mer peptides covering the two possible self-epitopes identified by blastp in SNTG1 and POMT1 (Supplementary Table 2). Interestingly, increased IFN-γ responses to peptide IFSALVVAWYSSACH (POMT1675–689) were demonstrated in NT1 patients (p < 0.01, Fig. 5a; Supplementary Fig. 5A). This suggested that POMT1 could represent an autoantigen in NT1. As seen for T-cell responses to peptide NA175–189, homozygosity for HLA-DQB1*0602 did not appear to augment responses to POMT1675–689 beyond the levels reached in HLA-DQB1*0602heterozygous NT1 patients. In contrast, peptide RAEKTFSVYEIMCKI (SNTG1330–344) was recognized only by one NT1 patient, and by vaccinated controls (n.s., Fig. 5b; Supplementary Fig. 5B).

IFN-γ responses to peptide NA175–189 and POMT1675–689, or NA178–192 and POMT1675–689,showed correlation, further suggesting cross-reactivity of specific, virus-directed T-cells with this self-epitope in NT1 (Spearman’s rank correlation coefficients rs = 0.511 and rs = 0.778, respectively (p = 0.078; **p = 0.007)).

Transcriptomic analyses of PBMC stimulated with influenza A H1N1 NA175–189 or human POMT1675–689 indicate activation of T helper 1-cell and cytotoxic T-cell responses with cross-reactivity in Pandemrix-associated NT1

To further characterize T-cell responses against the epitopes from influenza A (H1N1) virus NA and human POMT1, and to identify putative pathogenic pathways in NT1, we studied the gene expression levels of viral NA175–189- or human POMT1675–689-stimulated PBMC from Pandemrix-associated NT1 patients and Pandemrix-vaccinated healthy controls. In RNA sequencing, stimulation induced a clear response in NT1 patients versus healthy controls. Altogether 74 genes were differentially expressed, either in response to NA175–189 (49 genes), or POMT1675–689 (28 genes; overlap of 3 genes, **p < 0.01; Fig. 6, Supplementary Fig. 6, Supplementary Data 2). The results further demonstrated that the gene expression profiles of PBMC from NT1 patients responding to these two peptides were closely matched (Fig. 6), and that no genes were differentially expressed in PBMC from NT1 patients stimulated with either one of the peptides (p-level < 0.05). These findings confirmed the correlation analyses for IFN-g responses, and supported T-cell cross-reactivity between NA and POMT1. The list of 74 differentially expressed genes contained several genes closely associated with cytotoxic T-cell function, e.g., IFN-γ, chemokines/chemokine ligands known to be associated with IFN-γ, perforin 1, and granzyme B. A focused view onto T-cell effector molecules also showed that T-cells from individual NT1 patients responded similarly to stimulation either with viral NA175–189 or human POMT1675–689, consistent with cross-reactivity of specific, influenza A (H1N1) virus-directed T-cells with the autoantigen POMT1 in NT1 (Fig. 7). This was exemplified most clearly by two strong responders, NT1 patients P003 and P015 (Figs. 6, 7), both characterized clinically by rapid onset of disease with the development of excessive daytime sleepiness and cataplexy within 4 weeks from vaccination with Pandemrix.

In addition, RNA sequencing of FACS-sorted, NA175–189-stimulated CD4+ and CD8+ T-cell populations from a single pediatric, Pandemrix-associated NT1 patient (P003) revealed upregulation of IFN-γ, perforin 1, granzyme B, chemokine (C–C motif) ligand 4, and chemokine (C motif) ligand in CD8+ T-cells, while the gene expression profile of CD4+ T-cells from the same patient was characterized mainly by IFN-γ (Fig. 6; FACS gating and sort strategy shown in Supplementary Fig. 7). Overall, gene expression in the NA175–189-stimulated CD8+ T-cell sample matched the profile obtained from NA175–189-stimulated PBMC, suggesting that peptide NA175–189 might be presented by both HLA class I and II. This finding was also consistent with the notion that cytotoxic T-cells are a key effector cell population mediating immunity against influenza A virus20.

T-cell receptor sequencing demonstrates converging selection of influenza A H1N1 NA175–189- and human POMT1675–689-reactive clonotypes in Pandemrix-associated NT1

To characterize T-cell responses against the putative cross-reactive epitopes at the clonal level, we sequenced TCR α-chains (TRA) and β-chains (TRB) from RNA of viral NA175–189- or human POMT1675–689-stimulated PBMC from Pandemrix-associated NT1 patients and Pandemrix-vaccinated healthy controls. TRA variable (TRAV), TRA joining (TRAJ), TRB variable (TRBV), TRB diversity (TRBD), and TRB joining (TRBJ) gene segment usage did not differ significantly between peptide-stimulated and medium control samples, neither in patients nor controls (Supplementary Data 3). We identified TRA clonotypes that were upregulated in ≥3 NT1 patient samples, stimulated either with NA175–189 or POMT1675–689peptides (Fig. 8). Among these public TRA clonotypes, six clonotypes significantly increased in abundance both when stimulated with NA175–189, or POMT1675–689 (proportion of false positives <0.05), further supporting T-cell cross-reactivity. Most strikingly, clonotypes CVVSAIKAAGNKLTF-TRAV10/TRAJ17 and CVVSAMTTDSWGKFQF-TRAV10/TRAJ24 were upregulated in 3 of 8 (3 of 8) NA175–189-stimulated, and 5 of 10 (4 of 10) POMT1675–689-stimulated samples. In NT1 patients P003 and P015, characterized by T helper 1 (Th1)-cell/cytotoxic T-cell gene expression signatures in response to stimulation with NA175–189 or POMT1675–689, both clonotypes were upregulated in response to either of the two peptides. Three additional clonotypes were found, significantly more abundant after stimulation with at least one of the two peptides, and with high similarity to either of the two clonotypes above: CVVITIKAAGNKLTF-TRAV10/TRAJ17, significantly upregulated in POMT1675–689-stimulated samples, including P003; CVVSGMTTDSWGKLQF-TRAV10/TRAJ24, upregulated in NA175–189-stimulated samples, including P015; and CVVSGMTTDSWGKFQF-TRAV10/TRAJ24, upregulated in both NA175–189- and POMT1675–689-stimulated samples, including both P003 and P015. These results demonstrated converging selection of NA175–189- and/or POMT1675–689-reactive T-cell clones using TRAV10/TRAJ17 or TRAV10/TRAJ24 gene segments in Pandemrix-associated NT1 patients.

A complete list of both public and private clonotypes using TRAV10/TRAJ17 and TRAV10/TRAJ24, identified in NT1 patients or healthy controls, is provided in Fig. 9a, b. The results demonstrate that in addition to the identified public clonotypes using TRAV10/TRAJ17 (CVVSAIKAAGNKLTF, CVVITIKAAGNKLTF) and TRAV10/TRAJ24 (CVVSAMTTDSWGKFQF, CVVSGMTTDSWGKLQF, CVVSGMTTDSWGKFQF), several highly similar clonotypes were present in peptide-stimulated samples from various NT1 patients, including CVVSMIKAAGNKLTF-TRAV10/TRAJ17, CVVSSMTTDSWGKFQF-TRAV10/TRAJ24, CVVTVMTTDSWGKFQF-TRAV10/TRAJ24, and CVVSSLTTDSWGKFQF-TRAV10/TRAJ24. The identified public clonotypes were not exclusive to NT1 patients, but occasionally seen in healthy controls, too. In particular, the HLA-DQB1*0602-negative healthy control HC27 shared several of the clonotypes upregulated in NT1 patient samples stimulated with NA175-189 or POMT1675-689. However, in contrast to NT1 patients, HC27 did not show an increase in expression of inflammatory cytokines/ chemokines in response to peptide stimulation (compare Fig. 4a, b; 5a; 7). Therefore, these TRAV10–TRAJ17 and TRAV10–TRAJ24 clonotypes might have contributed to an inflammatory reaction in NT1 patients, but their functional phenotype could be effectively regulated when present in healthy controls.

Applying the same search criteria as used for TRA, we also identified a smaller number of TRB clonotypes that were upregulated in ≥3 NT1 patient samples, stimulated either with NA175–189 or POMT1675–689 peptides (Supplementary Fig. 8). Among these public TRB clonotypes, again one clonotype significantly increased in abundance in both NA175–189- or POMT1675–689-stimulated patient samples, further supporting T-cell cross-reactivity. This clonotype was CASSEAGQGAYEQYF-TRBV6-1/TRBJ2–7 (upregulated in P003, P015, and P023).

Discovery of POMT1 autoantibodies in Pandemrix-vaccinated children

Finally, we studied levels of plasma antibodies to POMT1 by radioimmunoassay (RIA), a sensitive and specific liquid-phase method for the detection of autoantibodies recognizing conformational epitopes21. Autoantibodies to POMT1 were increased in Pandemrix-vaccinated children (both in NT1 patients and healthy controls) in comparison to unvaccinated control children, suggesting that Pandemrix induced autoimmunity to POMT1 (****p < 0.0001, Fig. 10; Table 1).

Genetic studies strongly implicate HLA-DQ6.2-restricted CD4+ T-cells in the pathogenesis of NT14,5. A central role for CD4+ T-cells is seen in many autoimmune disorders, e.g., in celiac disease and type 1 diabetes. Both disorders show exceptionally strong HLA class II associations22, to be surpassed still in NT1. HLA class II-associated autoimmune diseases are considered antigen driven (e.g., by wheat gliadin in celiac disease), although tissue pathology is not mediated by CD4+ T-cells only. Other immune cells are involved in tissue destruction, and antigen spreading leads to broad autoimmunity. In Pandemrix-associated NT1, we hypothesized that CD4+ T-cells primed by influenza A (H1N1) virus epitopes are disease drivers, cross-react with CNS autoantigens, and orchestrate an immune-mediated attack against the hypocretin neuronal network. Therefore, we focused our study on influenza A (H1N1) T-cell epitopes that are recognized in the context of HLA-DQ6.2, although HLA-DQ6.2 peptide binding was not directly tested. Due to limitations in the availability of blood samples from pediatric, Pandemrix-associated NT1 patients, we started to search for dominant T-cell epitopes using Pandemrix-primed mice that expressed HLA-DQ6.2 as the only MHC class II allele, and Pandemrix-vaccinated, HLA-DQB1*0602-positive individuals. Choosing this strategy, the selected T-cell peptides were enriched for HLA-DQ6.2-binding, but likely included both MHC class II- and/or class I-binding peptides.

We found that T-cell responses against two NA- and NP-derived epitopes were enhanced in children and adolescents with Pandemrix-associated NT1 in comparison to Pandemrix-vaccinated controls. Further, our study provided evidence for influenza A (H1N1) virus-directed T- and B-cell cross-reactivity with human POMT1. Homology of a viral NA peptide sequence and a human POMT1 sequence was suggested by basic local alignment. We could show that IFN-γ secretion in response to NA175–189/NA178–192- and POMT1675–689-stimulation was correlated, and that T cell cytokine/chemokine gene expression patterns were closely matched. This was exemplified by two strong responders, patients P003 and P015, who both developed clinical narcolepsy with cataplexy within one month from Pandemrix vaccination. TCR repertoire analyses in NT1 patients revealed increased abundance of seven public clonotypes in response to stimulation both with NA175–189 and POMT1675–689 in NT1 patients. In addition, vaccination with Pandemrix induced anti-POMT1 autoantibodies in children. These results were consistent with cross-reactivity of specific, virus-directed T-cells and antibodies with the autoantigen POMT1 in Pandemrix-associated NT1.

The reasons remain unknown for the strongly enhanced T-cell reactivity to different influenza A (H1N1) protein fragments demonstrated in this study in patients who developed NT1 after Pandemrix vaccination. Our earlier study showed that viral proteins contained in the Pandemrix antigen suspension formed complexes, which may have contributed to the enhanced reactivity to both NA- and NP-derived epitopes16. Alternatively, and not mutually exclusive, enhanced T- and B-cell reactivity in NT1 patients may be attributed to immunogenetic predispositions6,7,8,23, resulting in hyper-responsiveness to pro-inflammatory signals and AS03 adjuvant-supplemented influenza A (H1N1) priming.

Specific HLA class I alleles, most consistently HLA-A*11:01, account for additional genetic risk contributions in NT1, independent from HLA-DQB1*06:0224,25. Cytotoxic CD8+ T cells, likely acting in concert with CD4+ Th1-cells, are frequently implicated in the pathogenesis of autoimmune diseases, including the neurological disorders multiple sclerosis26 and NT125. In this study of Pandemrix-associated NT1, cytokine and chemokine gene expression by PBMC in response to stimulation with influenza A (H1N1) NA175–189 or human POMT1675–689matched closely, and revealed a cytotoxic T cell signature, characterized by IFN-γ, IFN-γ−associated genes, perforin 1, and granzyme B. This phenotype was consistent with CD8+ T-cells, but also unconventional, cytotoxic CD4+ T-cells, previously observed in influenza A infection20. In FACS-sorted T cell fractions, available for study from one Pandemrix-associated NT1 patient only, perforin 1 and granzyme B expression was upregulated in NA175–189-stimulated CD8+ T-cells, but not in CD4+ T cells. Thus, NA175–189 peptide is most likely binding to both HLA class I and II molecules.

Six TRA clonotypes, and one TRB clonotype, significantly increased in abundance in samples from Pandemrix-associated NT1 patients both when stimulated with NA175–189, or with POMT1675–689. Strikingly, public TRA clonotypes CVVSAIKAAGNKLTF-TRAV10/TRAJ17 and CVVSAMTTDSWGKFQF-TRAV10/TRAJ24 were upregulated in 37.5% of NA175–189-stimulated, and 40–50% of POMT1675–689-stimulated samples. In addition, we identified several public TRA clonotypes with high similarity to these two dominant clonotypes in samples from NT1 patients. These results suggest converging selection in different NT1 patients of NA175–189- and/or POMT1675–689-reactive T-cell clones using TRAV10/TRAJ17 or TRAV10/TRAJ24 gene segments. This indicates that these clonotypes are likely involved in the pathogenesis of NT1.

However, the identified public clonotypes were not exclusive to NT1 patients. Occasionally, they were seen in Pandemrix-vaccinated healthy controls, too. Interestingly, a HLA-DQB1*0602-negative healthy control shared both dominant TRAV10–TRAJ17 and TRAV10–TRAJ24 clonotypes, indicating that T-cells expressing these clonotypes were probably not HLA-DQ6.2-restricted. Therefore, TRAV10–TRAJ17 and TRAV10–TRAJ24 clonotypes might have contributed to an inflammatory reaction in NT1 patients, likely driven by CD8+ T-cells recognizing NA175–189 and putative cross-reactive POMT1675–689 (auto-) antigens, while the same clonotypes appeared effectively regulated when present in healthy controls. Use in future studies of single cell technology may allow the direct functional characterization of T-cell clonotypes in NT1 patients and controls.

Of special interest is the identification of CVVSAMTTDSWGKFQF-TRAV10/TRAJ24 as a dominant public clonotype, and of additional, highly similar TRAV10–TRAJ24 clonotypes, upregulated in NA175–189- or POMT1675–689-stimulated samples from NT1 patients. Han et al. described a single nucleotide polymorphism (SNP) associated with NT1, rs1483979. This SNP was located within the TRAJ24 segment and changed an amino acid within the CDR3 peptide-binding site of the TCR (F8L)27. Of the top public TRAV10–TRAJ24 clonotypes detected in samples from NT1 patients in our study, two encoded for phenylalanine (F), and one for leucine (L). All TRAJ24 (and 3 of 4 TRAJ17) clonotypes that were upregulated in response to NA175–189- or POMT1675–689-stimulation shared TRAV10 gene segment usage. These clonotypes differed from TRAV2–TRAJ2418 and TRAV6–TRAJ2428 clonotypes, previously isolated from NT1 patients using HLA-DQ6.2-HCRT tetramers. To our knowledge, clonotypes using TRAV10, TRAJ17 or TRBV6–1/TRBJ2–7 have not been described in NT1 patients before. The dominant public TRAV10–TRAJ17, TRAV10–TRAJ24, and TRBV6–1/TRBJ2–7 clonotypes described here represent potential biomarkers for NT1.

Among the identified T-cell epitopes, IAYERMCNILKG (NP217–228; Immune Epitope Database ID 145824), derived from the NYMC X-157 vaccine strain, is shared between different influenza A virus strains. In contrast, ESVAWSASACHD (NA175-186; ID 97390) and MERNAGSGIIISDTP (HA274-288; ID 188723) are carried mainly by influenza A (H1N1) virus strains. A recent study by Luo et al. demonstrated an increased frequency of T-cells specific for HA273–287 in NT1 patients in comparison to HLA-DQB1*0602 positive controls18. However, a report by Schinkelshoek et al. found no significant increase in T cell reactivity against HA273–287 in NT1 patients in comparison to HLA-DQB1*0602 positive controls29. Peptides HA271–285 and HA274–288 were recognized by patients with NT1 and healthy vaccinees in our study cohort, confirming HA273–287 as an influenza A (H1N1) T-cell epitope. Yet again, we did not see a clear-cut difference between NT1 patients and healthy vaccinees, neither in comparison to HLA-DQB1*0602 positive or negative controls, questioning the role of HA273–287-reactivity in the pathogenesis of NT1.

Using HLA-DQ6.2 tetramers, the study by Luo et al. demonstrated the presence of CD4+ T-cells specific for C-terminally amidated, human HCRT54–66 and HCRT86–97 peptides, but not for native HCRT peptides, in NT1 patients and controls18. Furthermore, TCR sequencing analyses suggested cross-reactivity with HA. The recent study by Jiang et al., also using HLA-DQ6.2 tetramers, detected CD4+ T cells specific for native HCRT56–69 and HCRT87–100peptides in NT1 patients and controls28. In addition, Latorre et al. reported increased frequencies of predominantly HLA-DR-restricted CD4+ T cell clones recognizing much more diverse peptide epitopes of HCRT in NT1 patients. These clones did not show reactivity with HA. Notably, T-cell reactivity to HCRT was also detected in type 2 narcolepsy, which is not associated with HCRT-deficiency30. Thus, the current literature on T cell reactivity to HCRT in NT1 remains conflicting. In many autoimmune diseases, autoimmunity is directed against multiple autoantigens expressed in the target tissue. The results of our study do not exclude a role of autoimmunity to HCRT, but identify with POMT1 another autoantigen in NT1.

POMT1 is a glycosyltransferase anchored in the endoplasmic reticulum, catalyzing in complex with POMT2 the first step of O-mannosyl glycan synthesis. In humans, O-mannose-linked glycosylation is observed mainly in brain, peripheral nerve, and muscle glycoproteins. The best-known substrate of POMT1/POMT2 is α-dystroglycan, mediating the binding of extracellular matrix components such as laminin to the dystrophin complex. Congenital defects in POMT1 are a cause of muscular dystrophy-dystroglycanopathy types A1, B1, and C131. Some forms of muscular dystrophies, most clearly myotonic dystrophy type 1, are associated with rapid eye movement (REM) sleep dysregulation32. It is currently unknown whether POMT1 or its substrates are involved in neuronal sleep regulation, but this possibility could be explored in experimental studies.

In this study, T-cell reactivity with POMT1 was detected in Pandemrix-associated NT1 patients. Samples from unvaccinated NT1 patients were not available for comparisons. Currently, it remains unknown whether T cell reactivity against POMT1 can be observed in NT1 patients not exposed to influenza A (H1N1) vaccination or infection. Other questions to be addressed by future studies include the identities of the HLA alleles involved in the restriction of the dominant public TRAV10–TRAJ17, TRAV10–TRAJ24, and TRBV6–1/TRBJ2–7 clonotypes described, their function, antigenic peptide specificity and cross-reactivity, and their value for NT1 diagnosis.

In summary, this study provides evidence for an influenza A (H1N1) virus directed, POMT1 cross-reactive T-cell response in NT1, in the context of HLA-DQ6.2. We identified two dominant T-cell epitopes of influenza A (H1N1) NA and (NYMC X-157 vaccine strain) NP virus proteins in Pandemrix-associated NT1 patients. Patients mounted a vigorous Th1-cell/cytotoxic T-cell response against these epitopes and showed T-cell reactivity against a self-epitope in POMT1, an enzyme expressed in brain. The POMT1 epitope is a peptide mimic of a dominant T-cell epitope of influenza A (H1N1) NA in Pandemrix-associated NT1. IFN-γ, perforin 1, granzyme B, and chemokine gene expression in response to stimulation with T-cell peptides NA175–189 or POMT1675–689 was closely matched in individual NT1 patients. TCR repertoire analyses demonstrated converging selection of TRAV10/TRAJ17 and TRAV10/TRAJ24 clonotypes in NT1, upregulated in response to either NA175–189- or POMT1675–689-stimulation, thus supporting T-cell cross-reactivity and a pathogenic role of these clonotypes in NT1. Moreover, vaccinees had elevated antibody levels against human POMT1, suggesting that Pandemrix vaccination triggered an autoimmune response to POMT1. The results of this unbiased search identify POMT1 as an autoantigen recognized by T and B cells in NT1.