Single-cell transcriptomics in bone marrow delineates CD56GranzymeK subset as intermediate stage in NK cell differentiation.

Human natural killer (NK) cells in lymphoid tissues can be categorized into three subsets: CD56CD16, CD56CD16 and CD69CXCR6 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 CD56GzmK subset was identified that shared features with CD56 and CD56GzmK NK cells based on transcriptome, phenotype (NKG2ACD16KLRG1TIGIT) and functional analysis in bone marrow and blood, supportive for an intermediate subset. Pseudotime analysis positioned CD56, CD56GzmK and CD56GzmK 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.