Abstract
Narcolepsy is a chronic sleep disorder caused by the loss of neurons that produce hypocretin. The close association with HLA-DQB1*06:02, evidence for immune dysregulation and increased incidence upon influenza vaccination together suggest that this disorder has an autoimmune origin. However, there is little evidence of autoreactive lymphocytes in patients with narcolepsy. Here we used sensitive cellular screens and detected hypocretin-specific CD4+ T cells in all 19 patients that we tested; T cells specific for tribbles homologue 2—another self-antigen of hypocretin neurons—were found in 8 out of 13 patients. Autoreactive CD4+ T cells were polyclonal, targeted multiple epitopes, were restricted primarily by HLA-DR and did not cross-react with influenza antigens. Hypocretin-specific CD8+ T cells were also detected in the blood and cerebrospinal fluid of several patients with narcolepsy. Autoreactive clonotypes were serially detected in the blood of the same—and even of different—patients, but not in healthy control individuals. These findings solidify the autoimmune aetiology of narcolepsy and provide a basis for rapid diagnosis and treatment of this disease.
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Data availability
The data presented in this manuscript are included in the paper and its Supplementary Information. TCR sequences from samples listed in Supplementary Table 2 are available as a .txt file. The sequences have also been deposited in the ImmunoAccess database (http://clients.adaptivebiotech.com/pub/Latorre-2018-nature; https://www.doi.org/10.21417/B73H0P).
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Acknowledgements
We thank all patients and their families for their participation in the study. We thank A. Sette and C. Lindestam Arlehamn (La Jolla Institute for Allergy and Immunology) for providing the human cytomegalovirus and Epstein–Barr virus peptide pools, G. Nepom and W. Kwok (University of Washington) and R. Martin and M. Sospedra (University Hospital Zurich) for providing DR2a- and DR2b-transfected B cell lines, the Servizio Tipizzazione of the Policlinico San Matteo, University of Pavia, for HLA typing, H. Hidalgo for logistical support and L. Sallusto for discussions and support. This work was supported by the European Research Council grant (no. 323183, PREDICT, to F.S.) and the Swiss National Science Foundation grants (no. 149475 and no. CRSII3_154483 to F.S.). F.S. and the Institute for Research in Biomedicine are supported by the Helmut Horten Foundation.
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Nature thanks B. Kornum, E. Unanue and the other anonymous reviewer(s) for their contribution to the peer review of this work.
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Contributions
D.L. performed experiments with assistance from E.A., F.M., A.C. and S.J. U.K., J.M., F.Z., R.K., M.M. and C.L.B. recruited participants, performed clinical evaluation and collected biological samples. D.J. performed cell sorting. M.F. performed bioinformatics analysis. M.T. supervised HLA typing. F.S. and C.L.B. conceived and supervised the project. F.S., D.L., C.L.B., U.K., M.T., B.B. and A.L. wrote the manuscript. All authors provided input and reviewed the manuscript.
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Extended data figures and tables
Extended Data Fig. 1 Screening of memory CD4+ T cell library from patients with NT1 or NT2 and healthy controls.
a–c, Memory CD4+ T cell screening for Influvac. Memory CD4+ T cell libraries from patients with NT1 or NT2 and control donors shown in Fig. 2 were also screened for their capacity to proliferate in response to the influenza vaccine Influvac, used as a positive control. On day 4, proliferation was measured after a 16-h pulse with [3H]thymidine. The number of tested T cell lines per donor is indicated on top of the graphs and the proliferation of individual T cell lines (each line is represented by a single dot) is shown as Δc.p.m. value. Positive lines were defined as Δc.p.m. ≥ 2,000 (horizontal dotted line) and SI ≥ 3. d, The frequency of Influvac-specific memory CD4+ T cells in patients with narcolepsy and controls (NT1, n = 13; NT2, n = 3; control, n = 12, biologically independent samples) is shown. Dots represent frequency in each donor, boxes are quartile values, whiskers represent the highest and lowest values, and lines represent the median values. Results are presented as precursor frequency per million memory CD4+ T cells. Data were analysed using two-tailed Mann–Whitney U-test. NS, not significant (P values > 0.05).
Extended Data Fig. 2 Autoreactive memory CD8+ T cells in patients with NT1 or NT2 as detected using the T cell library method.
a–c, f–h, Memory CD8+ T cells were polyclonally expanded and screened for their capacity to proliferate in response to HCRT peptide pool (a–c) or CMV + EBV peptide pool (f–h), used as positive control, in the presence of irradiated autologous B cells. On day 4, proliferation was measured after a 16-h pulse with [3H]thymidine. The number of tested T cell lines per donor is indicated on top of the graphs and the proliferation of individual T cell lines (each represented by a single dot) is shown as Δc.p.m. value. Positive responses were defined as Δc.p.m. ≥ 2,000 (horizontal dotted line) and SI ≥ 3. d, The Δc.p.m. values of HCRT-positive T cell lines (NT1, n = 6; NT2, n = 20; control, n = 2, biologically independent samples) from patients (PT) and controls (C) are shown. e, i, The frequency of HCRT-specific (e) and CMV + EBV-specific (i) cells per million memory CD8+ T cells in patients with narcolepsy and controls is shown (e, NT1, n = 10; NT2, n = 3; control, n = 9, biologically independent samples; i, NT1, n = 10; NT2, n = 2; control, n = 9, biologically independent samples). Dots represent frequency in each donor, boxes are quartile values, whiskers represent the highest and lowest values, and lines represent the median values. Results are presented as precursor frequency per million memory CD8+ T cells. Data were analysed using two-tailed Mann–Whitney U-test. NS, not significant (P values > 0.05).
Extended Data Fig. 3 Cytokine production and gene expression by autoreactive CD4+ T cell clones from patients with narcolepsy.
a, b, HCRT-specific (a, n = 26 biologically independent samples) and TRIB2-specific (b, n = 18 biologically independent samples) CD4+ T cell clones derived from patients with narcolepsy were stimulated with HCRT peptide pool (a) or TRIB2 peptide pool (b) presented by irradiated autologous B cells. Cytokines released in the 48-h culture supernatants were quantified by bead-based multiplex assay. Data represent the mean + s.e.m. c, d, mRNA expression levels for 579 genes in HCRT-specific (c, n = 6 biologically independent samples; n = 3 from P1 and n = 3 from P8) and TRIB2-specific (d, n = 6 biologically independent samples; n = 4 from P22 and n = 2 from P14) CD4+ T cell clones were measured using NanoString technology. The top 50 expressed genes are shown. Data represent the mean ± s.e.m.
Extended Data Fig. 4 Evaluation of T cell cross-reactivity with influenza virus antigens and MHC restriction of autoreactive T cell clones.
a, b, HCRT-specific (a, n = 61 biologically independent samples) and TRIB2-specific (b, n = 14 biologically independent samples) CD4+ T cell clones from patients with narcolepsy were stimulated in the presence of irradiated autologous B cells pulsed with HCRT peptide pool, TRIB2 peptide pool or influenza vaccine (Influvac) and—for some clones (n = 29, HCRT-specific and n = 9, TRIB2-specific)—with haemagglutinnin (HA) peptide pool. Each clone represents an individual clonotype. The c.p.m. values indicate the proliferation of autoreactive T cell clones measured after a 16-h pulse with [3H]thymidine are shown. c, The proliferative response of HCRT-specific, HLA-DR-restricted CD4+ T cell clones (n = 9 biologically independent samples) after stimulation with irradiated autologous B cells or a DRB1*15:01-transfected B cell line in absence or presence of HCRT peptide pool, is shown. Proliferation was measured on day 3 after a 16-h pulse with [3H]thymidine.
Extended Data Fig. 5 Clonotypic analysis of blood memory CD4+ T cells.
a, b, TCR Vβ sequencing was performed on memory CD4+ T cells ex vivo after sorting from peripheral blood of the indicated patients with narcolepsy (a) and healthy controls (b). The frequency distribution of all TCR Vβ clonotypes (n indicates total number of clonotypes) is shown. In these samples, no sequences of autoreactive T cell clones were found. The number of sequenced clonotypes was comparable in patients and controls; a range of 14,812–58,900 (39,494 ± 10,514 (mean ± s.d.)) and a range of 18,731–44,004 (33,354 ± 7,831 (mean ± s.d.)), respectively. TCR sequencing data are available as Supplementary Information.
Extended Data Fig. 6 Clonotype sharing of blood and CSF CD4+ and CD8+ T cells from patients with narcolepsy.
a, TCR Vβ sequencing was performed on memory CD4+ T cells ex vivo after sorting from blood, and on CD4+ T cells sorted from in vitro-expanded CSF T cells. The comparison of clonotype frequency distribution of CD4+ T cells from CSF (y axis) and blood (x axis) from the same patient is shown. Coloured circles represent autoreactive clonotypes (red, HCRT-specific; green, TRIB2-specific). b, TCR Vβ sequencing was performed on memory CD8+ T cells ex vivo after sorting from blood, and on CD8+ T cells sorted from in vitro-expanded CSF T cells. The comparison of clonotype frequency distribution of CD8+ T cells from CSF (y axis) and blood (x axis) from the same patient is shown. The total number of clonotypes in each sample is indicated on the axis. Values on top of the plots represent the number and the percentage of shared clonotypes between the two samples, calculated on the total number of clonotypes found in the CSF. TCR sequencing data are available as Supplementary Information.
Supplementary information
Supplementary Tables
This file contains Supplementary Tables 1-3.
Supplementary Data
This file contains TCR sequencing data.
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Latorre, D., Kallweit, U., Armentani, E. et al. T cells in patients with narcolepsy target self-antigens of hypocretin neurons. Nature 562, 63–68 (2018). https://doi.org/10.1038/s41586-018-0540-1
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DOI: https://doi.org/10.1038/s41586-018-0540-1
Keywords
- Hypocretin (HCRT)
- HCRT Neurons
- Tribbles Homologue 2 (TRIB2)
- Clonotypes
- Narcolepsy Type
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