Copy number variations across the blood-brain barrier in multiple sclerosis

Abstract

Objective: Multiple sclerosis (MS) is a neuroinflammatory disease where immune cells cross the blood-brain barrier (BBB) into the central nervous system (CNS). What predisposes these immune cells to cross the BBB is still unknown. Here, we examine the possibility that genomic rearrangements could predisposespecific immune cells in the peripheral blood to cross the BBB and form sub-populations of cells involved in the inflammatory process in the CNS.

Methods: We compared copy number variations in paired peripheral blood mononuclear cells (PBMCs) and cerebrospinal fluid (CSF) cells from MS patients. Thereafter, using next generation sequencing, we studied the T-cell receptor beta (TRB) locus rearrangements and profiled the αβ T cell repertoire in peripheral CD4⁺ and CD8⁺ T cells and in the CSF.

Results: We identified deletions in the T-cell receptor alpha/delta (TRA/D), gamma (TRG), and TRB loci in CSF cells compared to PBMCs. Further characterization revealed diversity of the TRB locus which was used to describe the character and clonal expansion of T cells in the CNS. T-cell repertoire profiling from either side of the BBB concluded that the most frequent clones in the CSF samples are unique to an individual. Furthermore, we observed a difference in the proportion of expanded T-cell clones when comparing samples from MS patients in relapse and remission with opposite trends in CSF and peripheral blood.

Interpretation: This study provides a characterization of the T cells in the CSF and might indicate a role of expanded clones in MS pathogenicity.

Figure 1

Whole genome CNVs between PB and CSF cells from the seven MS patients (14‐036, 14‐087, 14‐250, 14‐265, 14‐131, 14‐155, and 09‐073) and two HC (15‐237 and 15‐138). The upper panel shows the CNV frequency plot for all nine individuals with blue upward bars or red downward bars indicating more copy numbers in the PB or more copy numbers in the CSF cells respectively, with the chromosome numbers indicated at the top of the image. The panel below shows the annotation tracks for genes, exons, CNVs & miRNA according to a reference database. The lower panel shows the CNV between PB and CSF for each individual. CNV, copy number variation; PB, peripheral blood; CSF, cerebrospinal fluid; HC, heathy controls.

Figure 2

Validation of CNVs in CSF cells using Taqman copy number assays targeting genes in the TRG, TRB, and TRA regions. CSF cells are compared to PBMCs and PBMCs in each individual was used as a calibrator in the CopyCaller™ Software (Applied Biosystems) to calculate the CN of the target genes, that is, for each target gene the CN in CSF cells is calculated in comparison to CN in the PBMCs. (A) Showing the individual variations in copy numbers of the target genes. (B) Comparing the copy numbers of the target genes of MS (n = 8) to non‐MS (n = 4). Wilcoxon rank‐sum test was used for the statistical testing and generating the p values presented in the figure. ns, not significant; CNVs, copy number variations; CSF, cerebrospinal fluid; CN, copy number; TRG, T‐cell receptor gamma; TRA, T‐cell receptor alpha; TRB, T‐cell receptor beta; PBMCs, peripheral blood mononuclear cells; MS, multiple sclerosis.

Figure 3

CNVs in the TCR regions in paired CD4⁺, CD8⁺ T, and CSF cells. The PBMCs in each individual was used as a calibrator in the CopyCaller™ Software (Applied Biosystems) to calculate the CN of the target genes, that is, for each target gene the CN in the CD4⁺ and CD8⁺ T cells is calculated in comparison to the CN in the PBMCs. CNVs, copy number variations; CSF, cerebrospinal fluid; TCR, T‐cell receptor; CN, copy number; PBMCs, peripheral blood mononuclear cells.

Figure 4

CNVs in the TCR regions in paired γδ T and CSF cells. PBMCs in each individual was used as a calibrator in the CopyCaller™ Software (Applied Biosystems) to calculate the CN of the target genes, that is, for each target gene the CN in the CSF and γ/δ T cells is calculated in comparison to the CN in the PBMCs. CNVs, copy number variations; CSF, cerebrospinal fluid; TCR, T‐cell receptor; CN, copy number; PBMCs, peripheral blood mononuclear cells.

Figure 5

T‐cell clone frequency distributions in CD4⁺, CD8⁺ T, and CSF cells. (A) Frequency distribution of all identified clones. Vertical dotted line marking the threshold of expansion of 0.1%. (B) Distribution of clones with a frequency ≥0.1%.

Figure 6

Proportion of expanded and non‐expanded clones in CD4⁺ T cells, CD8⁺ T cells, and CSF cells. Clones with a frequency of either ≥0.1 or <0.1% of all clones are considered expanded and non‐expanded respectively, calculated as the number of clones of the respective category compared to the total sum of clones per sample. The bars represent the median of the expanded and non‐expanded clones per cell compartment. Differences in the proportion of expanded clones between the three cell compartments were tested using Wilcoxon rank‐sum test. CSF, cerebrospinal fluid.

Figure 7

Difference in the proportion of expanded clones between MS patients under relapse or remission in CD4⁺, CD8⁺ T, and CSF cell compartments. Expanded clones are clones with a frequency of ≥0.1% and the proportion of expanded clones is calculated as the number of expanded clones compared to the total sum of clones per sample. The bars represent the median of the expanded clones per cell compartment. Differences of the proportion of expanded clones between relapse and remission were tested using Wilcoxon rank‐sum test. ns, not significant; CSF, cerebrospinal fluid.