Single cell transcriptomics of cerebrospinal fluid cells from patients with recent-onset narcolepsy.

Narcolepsy is a rare cause of hypersomnolence and may be associated or not with cataplexy, i.e. sudden muscle weakness. These forms are designated narcolepsy-type 1 (NT1) and -type 2 (NT2), respectively. Notable characteristics of narcolepsy are that most patients carry the HLA-DQB1*06:02 allele and NT1-patients have strongly decreased levels of hypocretin-1 (synonym orexin-A) in the cerebrospinal fluid (CSF). The pathogenesis of narcolepsy is still not completely understood but the strong HLA-bias and increased frequencies of CD4+ T cells reactive to hypocretin in the peripheral blood suggest autoimmune processes in the hypothalamus. Here we analyzed the transcriptomes of CSF-cells from twelve NT1 and two NT2 patients by single cell RNAseq (scRNAseq). As controls, we used CSF cells from patients with multiple sclerosis, radiologically isolated syndrome, and idiopathic intracranial hypertension. From 27,255 CSF cells, we identified 20 clusters of different cell types and found significant differences in three CD4+ T cell and one monocyte clusters between narcolepsy and multiple sclerosis patients. Over 1000 genes were differentially regulated between patients with NT1 and other diseases. Surprisingly, the most strongly upregulated genes in narcolepsy patients as compared to controls were coding for the genome-encoded MTRNR2L12 and MTRNR2L8 peptides, which are homologous to the mitochondria-encoded HUMANIN peptide that is known playing a role in other neurological diseases including Alzheimer's disease.

Antigen-specific immune reactions by expanded CD8+ T cell clones from HLA-B*27-positive patients with spondyloarthritis.

Spondyloarthritis (SpA) is a chronic inflammatory disease that is tightly linked to HLA-B*27 but the pathophysiological basis of this link is still unknown. It is discussed whether either the instability of HLA-B*27 molecules triggers predominantly innate immune reactions or yet unknown antigenic peptides presented by HLA-B*27 induce adaptive autoimmune reactions by CD8+ T cells. To analyze the pathogenesis of SpA, we here investigated the T cell receptor (TCR) usage and whole transcriptomes of CD8+ single cells from synovial fluid of HLA-B*27-positive SpA patients and HLA-B*27-negative controls. In HLA-B*27-positive patients, we confirmed preferential expression of several TCR ß-chain families, found even more restricted usage of particular TCR α-chains, assigned matching TCR αß-chain pairs with homologous CDR3-sequences, and detected identical TCR-chains in different patients. Gene expression analyses by single cell mRNAseq revealed that genes specific for the tissue resident memory phenotype, exhaustion, and apoptosis were particularly highly expressed in expanded clonotypes from HLA-B*27-positive SpA patients. Together, several independent lines of evidence argue in favor of an (auto)antigenic peptide related pathogenesis.

Antigen-Specific TCR Signatures of Cytomegalovirus Infection.

CMV is a prevalent human pathogen. The virus cannot be eliminated from the body, but is kept in check by CMV-specific T cells. Patients with an insufficient T cell response, such as transplant recipients, are at high risk of developing CMV disease. However, the CMV-specific T cell repertoire is complex, and it is not yet clear which T cells protect best against virus reactivation and disease. In this study, we present a highly resolved characterization of CMV-specific human CD8+ T cells based on enrichment by specific peptide stimulation and mRNA sequencing of their TCR ß-chains (TCRß). Our analysis included recently identified T cell epitopes restricted through HLA-C, whose presentation is resistant to viral immunomodulation, and well-studied HLA-B-restricted epitopes. In eight healthy virus carriers, we identified a total of 1052 CMV-specific TCRß sequences. HLA-C-restricted, CMV-specific TCRß clonotypes dominated the ex vivo T cell response and contributed the highest-frequency clonotype of the entire repertoire in two of eight donors. We analyzed sharing and similarity of CMV-specific TCRß sequences and identified 63 public or related sequences belonging to 17 public TCRß families. In our cohort, and in an independent cohort of 352 donors, the cumulative frequency of these public TCRß family members was a highly discriminatory indicator of carrying both CMV infection and the relevant HLA type. Based on these findings, we propose CMV-specific TCRß signatures as a biomarker for an antiviral T cell response to identify patients in need of treatment and to guide future development of immunotherapy.