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. 2023 Dec 9;16:17562864231211077. doi: 10.1177/17562864231211077

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© The Author(s), 2023

This article is distributed under the terms of the Creative Commons Attribution-NonCommercial 4.0 License (https://creativecommons.org/licenses/by-nc/4.0/) which permits non-commercial use, reproduction and distribution of the work without further permission provided the original work is attributed as specified on the SAGE and Open Access pages (https://us.sagepub.com/en-us/nam/open-access-at-sage).

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Figure 2. — Bulk transcriptomic analysis of flow cytometry-sorted B cells, CD4⁺ and CD8⁺ T cells from frozen PBMC of 12 RRMS patients at BL, after 6 and 12 months after cladribine therapy initiation (6M, 12M). (a) Volcano plots displaying significantly down- (LFC < −1, blue) and upregulated (LFC > 1, yellow) DEG (adjusted p-value ⩽0.05) comparing 12 months after cladribine therapy initiation to BL of B cells (highlighted are immune-relevant genes), CD4⁺ and CD8⁺ T cells. (b) Molecular degree of perturbation scores for B cells, CD4⁺ and CD8⁺ cells were calculated for all three time points, with the respective BL group as reference based on the cohort filtered for matched data (n = 10). (c) Pathway enrichment analysis in B cells. DEG for the time points 6M and 12M (each in comparison to BL) were used as input; the resulting pathways were classified as ‘significant enrichment upon reconstitution’ (i.e. only altered after 12M, p_adj < 0.05) or ‘significant enrichment upon depletion’ (i.e. altered after 6M and 12M, p_adj < 0.05) depending on their enrichment patterns. (d) Enrichment analysis of GO, BP based on differential expression analysis of bulk RNA sequencing data. Enrichment analysis based on significantly downregulated (c) and upregulated (d) DEG of B cells comparing 12M to BL. Shown are the top 20 GO enrichment terms for BP, including respective gene counts. GeneRatio stands for relevant genes for the tested term divided by total input genes. Immune-relevant pathways are marked in bold italic, B cell-relevant pathways in blue bold italic. Adjusted p-values were calculated through a hypergeometric distribution. Dot sizes correspond to the number of genes from the input lists that are enriched for each GO term. # = somatic recombination of immune receptors built from immunoglobulin superfamily domains.

BL, baseline; BP, biological process; DEG, differentially expressed gene; GO, gene ontology; LFC, log2 fold change; padj, adjusted p-value; PBMC, peripheral blood mononuclear cells; RRMS, relapsing-remitting multiple sclerosis.