An atlas of infiltrated B-lymphocytes in breast cancer revealed by paired single-cell RNA-sequencing and antigen receptor profiling

Abstract

While it has been well-recognized that T-cell mediated adaptive cellular immunity plays important roles in cancer immune response and tumor control, the roles of B lymphocytes in tumor development and therapy have only been proposed until recently, and are still mostly controversial. To gain mechanistic insights into the origin and dynamics of tumor infiltrated immune cells, especially B lymphocytes, we combine single-cell RNA-sequencing and antigen receptor lineage analysis to characterize a large number of triple-negative breast cancer (TNBC) infiltrated immune cells and present a comprehensive atlas of infiltrated B-lymphocytes in TNBC, the most aggressive breast cancer subtype. We demonstrate that TNBC infiltrated B cells showed more mature and memory B cell characteristics, as well as high clonality and extensive IgH class switching recombination and somatic hypermutations. The B cell signatures based on single-cell RNA-seq results are significantly associated with improved survival for TNBC patients and provide better prognostication than classic single B cell markers (CD19 or CD20). Further dissection of the mechanisms regulating the functions and dynamic distribution of tumor infiltrated B cell populations will provide new clues for tumor immunotherapy.

Whole Transcriptome Analysis Reveals Heterogeneity in B Cell Memory Populations in Patients With Juvenile Idiopathic Arthritis-Associated Uveitis

“…Head-to-head comparison of JIA cases with and without uveitis revealed 387 DEGs at nominal significance ( Supplementary Table S7 ) and no genes at a FDR of 5%. However, recent single cell analyses has revealed that the peripheral blood B cell compartment contains ∼10 functionally distinct B cell subsets ( 22 , 27 ). Because the peripheral B cell fraction is dominated by naive B cells ( Figure 1A ), we hypothesized that B cell subset-specific gene expression from relatively rarer cell types in blood, may be drowned out in bulk transcriptome analysis.…”

“…Despite this limitation, this study may guide future studies into relevant B cell subsets. We exploited marker genes used to mark B cell subsets in peripheral blood mononuclear cells in recently reported single-cell studies ( 22 , 27 ). We would like to emphasize that this set of genes is by no means exhaustive, but showed to be instrumental in our study to reclassify cases, because we showed that the newly identified subgroup of JIA-uveitis cases displayed genuine enrichment for memory B cell gene circuits as supported CIBERSORT analysis and distinct gene profiles as shown by differential expression analysis.…”

“…We extracted expression data for 13 marker genes that distinguish peripheral blood B cell subsets as determined by single-cell sequencing analysis ( 22 ) and also include genes encoding IgA, IgM, and IgG (17 genes in total, see Supplementary Table S3 ), because previous studies have linked immunoglobulin expression to juvenile idiopathic arthritis-associated uveitis ( 23 ). Differences in the expression of these genes was considered to indicate changes in (memory) B cell subsets in blood, which would be evident by using this set of genes to conduct principal component analysis with the factoextra package in R. Using the first two principal components, we reclassified patients into new groups.…”

Assigning Immunoglobulin Class from Single-Cell Transcriptomes in IgA1-Secreting Versus Membrane Subpopulations

“…Given the overlap in calls among the different heavy chain isotypes, we needed to find the best route to assign the appropriate antibody isotype. A previous publication analyzing transcriptome and VDJ clonality in B cells from breast cancer tissue found similar issues with assigning isotype class and used a cut-off value of tenfold higher than the next highest expressing heavy chain isotype in that cell [ 21 ]. However, because each cell line likely has a unique proportion of immunoglobulin producers in the total population, using a predetermined expression cutoff would not work well.…”

Unraveling Heterogeneity in Epithelial Cell Fates of the Mammary Gland and Breast Cancer

“…It remains to be determined whether more sophisticated analysis tools such as single-cell potency analysis [121], or higher sequencing depth per cell and larger cell numbers are required to unambiguously define the MaSC as a distinct cell type (Figure 5). Recent years have also seen the emergence of studies that applied scRNAseq to unravel complexity of breast cancer [122,123], including the immune environment of breast tumors [124,125], and response to Herceptin therapy that targets EGFR2 (Her2) [126]. More is certainly to come as scRNAseq and its analysis becomes more mainstream and affordable.…”