Single-cell transcriptomics in bone marrow delineates CD56(dim)GranzymeK(+) subset as intermediate stage in NK cell differentiation

Human natural killer (NK) cells in lymphoid tissues can be categorized into three subsets: CD56(bright)CD16(+), CD56(dim)CD16(+) 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(dim)GzmK(+) subset was identified that shared features with CD56(bright) and CD56(dim)GzmK(-) NK cells based on transcriptome, phenotype (NKG2A(high)CD16(low)KLRG1(high)TIGIT(high)) and functional analysis in bone marrow and blood, supportive for an intermediate subset. Pseudotime analysis positioned CD56(bright), CD56(dim)GzmK(+) and CD56(dim)GzmK(-) 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.