Natural Killer Large Granular Lymphocyte Leukemia (NK-LGLL) is a distinct LGLL subtype, accounting for 10-15% of LGLL cases. We present the first multimodal single-cell analysis of NK-LGLL in peripheral blood mononuclear cells from six patients and four healthy donors. We found that leukemic NK cells exhibited a CD3εposCD4negCD8αposCD45RAposCD16highTCRαβneg phenotype and upregulated cytotoxicity-related genes including GZMB, GZMH, GZMM, GZMA, and PRF1. Similar to T-LGLL, non-leukemic NK cells in patients showed greater cytotoxicity than those in healthy donors. Leukemic cells downregulated cytokine-receptor interaction genes while upregulating inhibitory receptors (PD1, TIM3) and multiple exhaustion markers. Somatic STAT3, TET2, or CCL22 mutations occur in ~70% of NK-LGLL cases. CCL22-mutant patients had a higher proportion of CD4⁺ T cells and CD14⁺ monocytes, with increased expression of CD56, CD57, and CD94, and the lowest leukemic cell fraction. TET2-mutated leukemic cells showed elevated CD57, reduced CD56 and CD94, and downregulated genes involved in cell adhesion. In contrast, STAT3-mutated leukemic cells exhibited the lowest CD57, CD56, and CD94 expression but the highest cytotoxicity and exhaustion marker expression. Hierarchical clustering of cell type-specific regulons identified CD8⁺ TEMRA cells as the closest non-leukemic counterpart, with leukemic cells displaying heightened expression of immune regulatory and cytotoxic TBX21 target genes. Cell-cell communication analysis revealed monocytes and dendritic cells as primary interaction partners of leukemic cells, while CCL22-mutant cells also engaged non-leukemic NK cells via CD44, HLA-E, and TGFB1. These findings define key features of NK-LGLL and provide a foundation for comparative analyses with T-LGLL.
