Molecular Interactions Between NK Cells and Acute Leukemic Cells: KIR2DL5 Drastically Limits NK Cell Responses

Abstract

Natural Killer (NK) cells naturally recognize and eliminate leukemic cells. However, the molecular interactions that govern these responses are diverse due to the large number of activating and inhibitory NK receptors that modulate NK functions and the diversity of corresponding ligands that are differentially expressed in acute lymphoblastic and myeloblastic leukemias. We identified resting NKG2A⁺ NK cells and NKG2A⁺KIR⁺ NK cell subsets as the most effective in eliminating lymphoid and myeloid leukemic cells respectively. The NKG2A⁺KIR^±CD57^- cell subsets show high expression of activating receptors and a functional transcriptomic profile, but differ in KIR2DL5 expression. The frequency of KIR2DL5⁺ NK cells increases with the number of expressed KIR. Furthermore, KIR2DL5 is preferentially co-expressed with KIR2DL1 and is negatively regulated by NKG2A. Of note, CD57 expression, regardless of the NK cell subset considered, is associated with reduced receptor expression, consistent with its reduced cytotoxic potential. Furthermore, molecular interactions between NK cells and leukemic cells influence NK cell responses, particularly the inhibitory KIR2DL5-PVR axis. The integration of these data is of importance for the optimization of NK cell-based immunotherapies, as the selection of NK cell donors represents a key parameter for the improvement of these therapies.

Keywords: Acute leukemia; Heterogeneity; NK cells.

Fig. 1

Functional and phenotypic characterization of NK cell subsets in response to lymphoid and myeloid leukemic cells. Schematic representation of the method used to characterize the function and phenotype of NK cell subsets (A). Scatter plots illustrating the impact of HLA-C environment (C1C1, C1C2, C2C2) (B) and KIR genotypes (AA, B +) (C) on the degranulation of 24 NK cell subsets, defined by NKG2A, KIR2DL1, KIRDL2/L3, KIR3DL1, CD57 and NKG2C expression, against lymphoid and myeloid cell lines, taking into account the representativeness of NK cell subsets. Bar graphs comparing the relative MFI of activating and inhibitory receptors on NK cell subsets defined on the basis of NKG2A, KIR2DL1 and KIR2DL2/3 (KIR) expression (n = 40) (D) and the frequency of KIR3DL2 (n = 40) and KIR2DL5 (n = 20) on NK cell subsets defined on the basis of NKG2A, KIR2DL1 and KIR2DL2/3 expression (E). Relative MFI represents the ratio of the MFI of each receptor on the MFI of isotype control. Statistical significance is indicated as follows: *p < 0.05, **p < 0.01, ***p < 0.001 and ****p < 0.0001. Comparisons were made by Mann–Whitney and Kruskall-Wallis tests. Bar graphs represent mean ± SD

Fig. 2

Transcriptomic signature of NK^neg, NK^2A+ and NK^2A+KIR+ cell subsets. Schematic representation of the method used for the transcriptomic study (A). Three-way Venn diagram showing the number of shared and uniquely expressed transcripts between NK^neg, NK^2A+ and NK^2A+KIR+ (B). Volcano plots showing the differential gene expression analysis of the comparisons of NK^2A+ vs NK^neg (C), NK^2A+KIR+ vs NK^neg (D), and NK^2A+ and NK^2A+KIR+ (E). Representation of upregulated (green) and downregulated (red) key transcripts for the comparisons of NK^2A+ vs NK^neg (F), NK^2A+KIR+ vs NK^neg (G), and NK^2A+ and NK^2A+KIR+ (H). The size of the circle corresponds to the value of log2FoldChange

Fig. 3

ALL and AML cells exhibit differential expression patterns of ligands for key NK receptors. Schematic representation of the method used to characterize the phenotype of leukemic cell patients (A). Swimmer’s plot of patient outcomes (n = 33) (B). Bar graphs comparing the MFI of various ligands expressed at the cell surface of leukemic cells from ALL or AML patients (C). K-mean clustering of ligands expressed by leukemic cells from ALL and AML patients (D). Bar graph comparing the ligand expression profiles of the three clusters identified by K-mean clustering (E). Statistical significance is indicated as follows: *p < 0.05, **p < 0.01, ***p < 0.001 and ****p < 0.0001. Comparisons were made by multiple Mann–Whitney (Bonferroni-Dunn method) and Kruskall-Wallis tests. Bar graphs represent mean ± SD

Fig. 4

Expression profile of KIR2DL5 on resting and activated NK cells. Mean frequencies of KIR2DL5⁺ NK cells from 29 healthy blood donors expressing KIR2DL5A*001 allotype. Eighty individuals are CMV seronegative and 11 are CMV seropositive (A). Bar graphs comparing the mean frequency of CD57⁻ vs CD57⁺ NK cells that express KIR3DL2 and KIR2DL5 (n = 40) (B). Bar graphs comparing the mean frequency of NK cells expressing KIR2DL5 following the number of expressed KIRs (0 to 3) and NKG2A expression (n = 20) (C). Mean frequency of CD3⁻ CD56⁺ cells, described as NK cells, determined for 6 KIR2DL5A*001⁺ healthy blood donors by flow cytometry using specific mAbs described in methods at days 0, 7, 14 and 17 following stimulation with the irradiated (HLA-A*03:01, A*26:01, B*07:02, C*07:02) EBV-B cell line (EBV-01/10) or cytokines (CK) including IL-12 (10 ng/ml), IL-15 (10 ng/ml) and IL-18 (100 ng/ml) (D). Representative density plots showing the NK cell expansion with feeder or cytokine stimulation at day 0 and day 14 (E). Geometric mean fluorescent intensity (MFI) of marker (TIGIT, CD96 and DNAM-1) expressed on NK cells at day 0, 7, 14 and 17 following stimulation with the irradiated EBV-01–10 cell line or CK (F). Mean frequencies of KIR2DL5⁺ NK cells at day 0, 7, 14 and 17 following stimulation with the irradiated EBV-01–10 cell line or CK (G). Representative density plots showing the coexpression of KIR2DL5 with TIGIT and DNAM-1 on NK cells expanded after feeder or cytokine stimulation at day 0 and day 14 (H). Means are indicated with error at all kinetic points. Statistical significance is indicated as follows: *p < 0.05, **p < 0.01, ***p < 0.001 and ****p < 0.0001. Comparisons were made by Mann–Whitney and Kruskall-Wallis tests. Bar graphs represent mean ± SD

Fig. 5

KIR2DL5-PVR axis inhibits NK cell degranulation against PVR + leukemic cells. Schematic representation of the degranulation assay performed on 10 NK cell subsets co-expressing or not NKG2A, KIR2DL1, KIR2DL3 and KIR2DL5 against primary leukemic cells and leukemic cell lines expressing or lacking PVR (A). Histograms showing the PVR and Nectin-2 expression on primary leukemic AML6 cells (B). Bar graphs comparing the degranulation of KIR2DL5⁻ vs KIR2DL5⁺ NK cells against primary leukemic AML6 cells (C). Bar graphs comparing the degranulation of KIR2DL5⁻ vs KIR2DL5⁺ NK cells against primary leukemic AML6 cells preincubated with or without an anti-PVR mAb (D). Bar graphs comparing the degranulation of KIR2DL5⁻ vs KIR2DL5⁺ NK cells against PVR- leukemic cells (Raji, Daudi) and PVR + cell lines (NB4, KG1, Molt4, H9) (E). Histograms showing the PVR and Nectin-2 expression on PVR^KO KG1 cell line generated by CRISPR-Cas9 technique (F). Bar graph comparing the degranulation of total NK cells against the PVR^WT vs PVR^KO KG1 cell lines (G). Bar graph comparing the degranulation of KIR2DL5⁻ and KIR2DL5⁺ NK cell subsets against the PVR^WT vs PVR^KO KG1 cell lines (H). Statistical significance is indicated as follows: *p < 0.05. Comparisons were made by multiple Mann–Whitney and Kruskall-Wallis tests. Bar graphs represent mean ± SD