Cerebrospinal Fluid and Peripheral Blood Lymphomonocyte Single-Cell Transcriptomics in a Subject with Multiple Sclerosis Acutely Infected with HIV

Abstract

Signatures of neurodegeneration in clinical samples from a subject with multiple sclerosis (MS) acutely infected with HIV were investigated with single-cell transcriptomics using 10X Chromium technology. Sequencing was carried out on NovaSeq-TM, and the analysis was performed with Cell Ranger software (v 7.1.0) associated with a specifically established bioinformatic pipeline. A total of 1446 single-cell transcriptomes in cerebrospinal fluid (CSF) and 4647 in peripheral blood mononuclear cells (PBMCs) were obtained. In the CSF, many T-cell lymphocytes with an enriched amount of plasma cells and plasmacytoid dendritic (pDC) cells, as compared to the PBMCs, were detected. An unsupervised cluster analysis, putting together our patient transcriptomes with those of a publicly available MS scRNA-seq dataset, showed up-regulated microglial neurodegenerative gene expression in four clusters, two of which included our subject's transcriptomes. A few HIV-1 transcripts were found only in the CD4 central memory T-cells of the CSF compartment, mapping to the gag-pol, vpu, and env regions. Our data, which describe the signs of neurodegenerative gene expression in a very peculiar clinical situation, did not distinguish the cause between multiple sclerosis and HIV infection, but they can give a glimpse of the high degree of resolution that may be obtained by the single-cell transcriptomic approach.

Keywords: immune dysregulation in infectious diseases; interactions between infective agents and immune responses.

Figure 1

Uniform manifold approximation and projection (UMAP) plots representing cell clusters identified in patient’s CSF and PBMC single-cell transcriptomes. Color-coded cell clusters derived from single-cell transcriptomic analysis of CSF (A) and PBMC (B) samples are shown. To assess the cellular identity of each cluster, canonical cell markers were used (see Section 4).

Figure 2

Uniform manifold approximation and projection (UMAP) plots representing 0–15 clusters identified in unsupervised cluster analysis performed on the total CSF and PBMC transcriptomes. The 16 identified clusters are shown using different colors (see legend on the right) and the arrows indicate those displaying up-regulated neurodegenerative microglial gene expression.

Figure 3

Alignment track of HIV transcripts in single cells from the CSF across the annotated HIV subtype G (KU168277). On the top, the HIV reference sequence and annotated genes (in dark blue) are displayed. (A–C): the read coverage and stacked individual reads, represented as arrows, are shown in different panels enlarged below. Different colors within the reads highlight present nucleotide variations compared to the reference sequence: red is used for T, green is used for A, orange is used for G, and blue for C. Picture obtained from the IGV Web App.