Single-cell transcriptomics in bone marrow delineates CD56dimGranzymeK+ subset as intermediate stage in NK cell differentiation

Abstract

Human natural killer (NK) cells in lymphoid tissues can be categorized into three subsets: CD56^brightCD16⁺, CD56^dimCD16⁺ and CD69⁺CXCR6⁺ lymphoid tissue-resident (lt)NK cells. How the three subsets are functionally and developmentally related is currently unknown. Therefore, we performed single-cell RNA sequencing combined with oligonucleotide-conjugated antibodies against CD56, CXCR6, CD117 and CD34 on fresh bone marrow NK cells. A minor CD56^dimGzmK⁺ subset was identified that shared features with CD56^bright and CD56^dimGzmK^- NK cells based on transcriptome, phenotype (NKG2A^highCD16^lowKLRG1^highTIGIT^high) and functional analysis in bone marrow and blood, supportive for an intermediate subset. Pseudotime analysis positioned CD56^bright, CD56^dimGzmK⁺ and CD56^dimGzmK^- cells in one differentiation trajectory, while ltNK cells were developmentally separated. Integrative analysis with bone marrow cells from the Human Cell Atlas did not demonstrate a developmental connection between CD34⁺ progenitor and NK cells, suggesting absence of early NK cell stages in bone marrow. In conclusion, single-cell transcriptomics provide new insights on development and differentiation of human NK cells.

Keywords: bone marrow; natural killer cells (NK cells); pseudotime analysis; single-cell RNA sequencing; spectral cytometry.