Supplementary MaterialsSupplementary Information 41467_2019_14118_MOESM1_ESM. Tables. Technical scRNA-seq information and data tables with details of the included patients are included as Supplementary Tables. The source data underlying Figs.?3f, g, 4bCf and Supplementary Figs.?1aCe, 4c, d, 10bCf are provided in the Source Data file. Abstract Cerebrospinal fluid (CSF) protects the central nervous system (CNS) and analyzing CSF aids the diagnosis of CNS diseases, but our understanding of CSF leukocytes remains superficial. Here, using single cell transcriptomics, we identify a specific location-associated composition and transcriptome of CSF leukocytes. Multiple sclerosis (MS) C an autoimmune disease of the CNS C increases transcriptional diversity in blood, but increases cell type diversity in CSF including a higher abundance of cytotoxic phenotype T helper cells. An analytical approach, named cell set enrichment analysis (CSEA) identifies a cluster-independent increase of follicular (TFH) cells potentially driving the known expansion of B lineage cells in the CSF in MS. AMG 073 (Cinacalcet) In mice, TFH cells accordingly promote B cell infiltration into the CNS and the severity of MS animal models. Immune mechanisms in MS are thus highly compartmentalized and indicate ongoing local T/B cell conversation. and and and and and gene family) corresponded to naive B cells (B1; and and and and value (?log10) based on beta-binomial regression (Methods). Horizontal line indicates significance threshold. Cluster key: pDC, plasmacytoid dendritic cells (DC); mDC1, myeloid DC type 1; Mono1, monocyte cluster 1 preferentially blood-derived; Mono2, monocyte cluster 2 preferentially CSF-derived; gran, granulocytes; Tdg, T cells; CD8na, nonactivated CD8+ T cells; CD8a, activated CD8+ T cells; Tregs, regulatory CD4+ T cells; CD4, CD4+ T cells; NK, natural killer cells; MegaK, megakaryocytes; B1/B2, B cell subsets; plasma, plasmablasts. Source data for (c) listing the differential expression values for all those cells merged are provided in Supplementary Dataset?1. Source data for (d, e) listing the differential expression values for CSF vs. blood are provided in Supplementary Dataset?2. CSF leukocytes exhibit a specific composition and transciptome CSF cells have not been characterized with unbiased approaches. We therefore next analyzed the compartment-specific cell type composition identified AMG 073 (Cinacalcet) by unbiased scRNA-seq in CSF compared to blood. As expected for CSF4,18, non-hematopoietic cells (e.g., neurons, glia, and ependymal cells), megakaryocytes, granulocytes, and RBCs (removed from final AMG 073 (Cinacalcet) clustering) were absent or strongly reduced compared to blood (Fig.?1d, e, Supplementary Fig.?3a, CR2 b). We also found CD56dim NK1 cells reduced among CSF cells, while the NK2 cluster was not different (Fig.?1d, e). Both the mDC1 and mDC2 clusters had a significantly higher proportion in CSF than in blood (Fig.?1d, e). Notably, mDC1 cells expressed markers indicating cross-presenting capacity (and (ref. 19) Fig.?1c). Among T cells, total CD4 cells and Tregs were more abundant in the CSF, while CD8 T cell clusters were not different (Fig.?1d, e). Flow cytometry confirmed this unique composition of CSF leukocytes (Supplementary Fig.?4aCc). Cell proportions in CSF and blood did not correlate by either scRNA-seq or flow cytometry supporting an independent regulation of their cell composition. In summary, we confirmed a highly compartment-specific composition of CSF cells and identified an enrichment of mDC1 and Tregs in the CSF. We also found a CSF-specific pattern of myeloid lineage cells. The Mono2 cluster was almost exclusively CSF-derived (Fig.?1d, Supplementary Fig.?2c) and canonical markers indicated an intermediate CD14+FCGR3A/CD16int phenotype (Fig.?1c) as described for CSF20. It also expressed a unique transcriptional signature, AMG 073 (Cinacalcet) including genes previously identified in classical (and and (ref. 22)), microglia (and (ref. 23)), and CNS border-associated macrophages (and (ref. 24,25)) previously identified in rodents. In a systematic comparison (Methods), the Mono2 gene signatures resembled homeostatic microglia described previously26 (Supplementary Fig.?14aCd). We thus identified a distinct phenotype of CSF monocytes. We next aimed to identify further compartment-specific gene expression signatures on a per cluster level (Supplementary Table?5). We focussed on genes identified independently as differentially expressed (DE) by two methods (MannCWhitney test, edgeR27) and supported by Bayesian model comparison in single-cell variational inference.