(A) Flowchart of the analysis of human CSF by scRNA-Seq. Because CD4⁺ cells make up the majority of CSF cells, they were collected by magnetic beads and single CD4⁺ cells were isolated by flow cytometry. The remaining non-CD4⁺ cells were separately isolated by single-cell index sorting using different markers. Thus, the ratio of CD4⁺ and non-CD4⁺ cell numbers does not reflect the ratio of absolute cell numbers in the CSF samples. Nevertheless, ratios and cell numbers within the non-CD4⁺ populations are comparable. Whole transcriptomes of each single cell were determined by next-generation sequencing (NGS) with a read length of 2 × 150 bp that allows identification of the hypervariable regions of TCRs and BCRs together with their corresponding V families. Thus, not only transcriptome profiles of each single cell are determined, but also matching α:β TCR and H:L BCR chains. This allows tracking of distinct clones. (B) t-SNE projection of transcriptome data from 2752 single CSF cells and 332 PBMCs from 16 patients, profiled in 9 main clusters. For better visualization, background areas were shaded manually to indicate major cell populations, although some cells will appear in “foreign” areas. Each dot corresponds to one single cell, colored according to the respective cell cluster. DCs, pDCs, and monocytes were not specifically labeled during flow cytometry analyses; therefore up to 6 transcripts were used as discriminators and are listed next to each cluster. (C) Heatmap showing normalized mean expression levels of discriminative gene sets for T cell cluster I CD4⁺ (lane 1) and CD8⁺ cells (lane 2), and cluster II CD4⁺ (lane 3) and CD8⁺ cells (lane 4). (D) t-SNE projection of all index-sorted CD8⁺ T cells. (E) t-SNE projection of all index-sorted CD4⁺ T cells.