Expression of unique gene signature distinguishes TCRαβ⁺/BCR⁺ dual expressers from CD3⁺CD14⁺ doublets

Abstract

Increasing evidence shows pathophysiological significance of rare immune cells, necessitating the need for reliable and proper methods for their detection and analysis. We have recently identified a new lymphocyte that coexpresses lineage markers of T-and B-cells including T cell receptor and B cell receptor (called dual expressers, DEs). Because of the peculiar phenotype of DEs, we used multiple techniques to authenticate their identity (fluorescence-activated cell sorting [FACS], scRNAseq, EBV cell lines, and imaging flow cytometry). In an recent article published in this journal, Burel and colleagues successfully detected DEs using FACS and imaging microscopy. Yet they claimed, based on the profile of what they called naturally occurring CD3 + CD14 + T cell/monocyte complexes that the scRNAseq signature of DEs resembles that of cell-cell complexes. Serious flaws in their analysis, however, invalidate their conclusions. Unlike the CD3 + CD14 + complexes, DEs have a distinct identity due to expression of a unique set of signature genes. Without a clear explanation, Burel and colleagues excluded these genes from their analysis, thereby effectively

Distinguishing cell–cell complexes from dual lineage cells using single‐cell transcriptomics is not trivial

“…But when specifically testing for genes that were differentially expressed in CD3+CD14+ doublets compared to T cells or monocytes, we can identify a distinct expression pattern (Figure 1A) and cell type score (Figure 1B) in CD3+CD14+ doublets compared to singlet monocytes and T cells. These results were highly similar to the expression pattern of the gene signature of DE cells when compared to singlet B cells and T cells, as displayed in Figure 1B from Jie et al [4]. Genes in the signature of CD3+CD14+ doublets included several genes associated with cell adhesion (ITGA5, SKAP2), cell proliferation (CCDC6, ETS1, NCOR2, PIN1, STAT5B), and the inflammatory p38 MAPK signaling pathway (MAPKAKP2, RELL1, SRF, TCF3) (full gene list in Table S1).…”

“…Thus, it is likely that the permutation test results from Jie et al are only driven by a few outliers and are not a good representation of the overall DE population. This is further supported by the heatmap representation in Figure 1C from their correspondence [4], where the expression of B-and T-cellspecific genes seems overall of much lower magnitude in DE cells…”

“…In their first publication [3] as well as their most recent report [4], Hamad and colleagues identified the gene signature of DE cells on a small number of cells (n = 77, split into three groups: DE cells, singlet B cells and singlet T cells, GSE129112) and did not perform a validation step in an independent dataset. This is problematic, as single‐cell transcriptomics is known to show a lot of variability across cells, samples and cohorts, and it is often too easy to identify “false positive” gene signatures, especially in small datasets with less than 100 cells.…”

“…When performing principal component analysis (PCA) on the scRNAseq data of sorted DE cells, B cells, and T cells originally reported in [3] (GSE129112), Jie et al found that DE cells presented a much higher dispersion than B cells and T cells, and not positioned halfway, as B cell‐T cell complexes would [4]. The high dispersion of DE cells suggests the presence of outliers (as was already noted in the previous paragraph) and prompted us to further look into the heterogeneity of this cell population.…”

“…In their correspondence, Jie et al claim that unlike CD3+CD14+ doublets, DE cells have a unique gene signature compared to singlet T cells and B cells, and thus represent a novel cell type [4]. We did not test for a specific gene signature in CD3+CD14+ doublets in our original manuscript, as the purpose of our study was to demonstrate that when looking at genes unique to monocytes or T cells, CD3+-CD14+ doublets have positive expression for both set of genes, and thus appear to have a mixed lineage nature (figure 1 in [1]).…”