Figure 3.

Single cell RNA sequencing (scRNA-Seq) of peripheral and teratoma-associated B cells. CD19⁺CD20⁺ cells were sorted from Patient 2 and Patient 4 (initial) teratoma samples and their paired PBMCs. (A) UMAP dimensionality reduction plot of scRNA-Seq data from B cells showing cluster identity, using Seurat. Curated cell type identity is labelled. (B) B cells coloured by the BCR constant region isotype that is maximally expressed by each cell and (C) by the rate of variable region mutations from germline BCR sequences, as estimated by HighVQuest (log₁₀ number of mutations per kilobase). This showed a median of 1.65 versus 1.20 log₁₀ mutations per kilobase of variable (V) region in a comparison of switched versus unswitched memory B subsets (P = 7.49 × 10^–100). (D) Violin plot of variable region mutation rate by BCR constant region and a bar plot of Wilcoxon rank sum differences in the number of mutations per kilobase of V regions between IGHA1/2 and IGHG1 (95% CIs indicated by error bars). (E) Bar plot showing the log₂ fold difference of expanded BCR clonotypes between teratoma and PBMCs (95% CIs based on 1000 downsamplings of the larger set of BCR clonotypes in each case). (F) UMAP plot of B cells coloured by k-nearest neighbours tissue of origin (k = 200) with red representing OT and grey PBMCs. The inset shows log₂ fold enrichment of cells with IGHA1 constant region expression within cluster 6 from 10 000 permutations (P = 0.03). (G) UMAP plot of B cells coloured by the expression level of top genes enriched within cluster 6. These data have been normalized to the total gene expression, multiplied by 10⁴ and finally 1 has been added to all values, so that 10⁰ equates to an original value of 0. HSP = heat shock protein; IGH = immunoglobulin heavy constant gene; TNF = tumour necrosis factor.