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. 2021 Sep 1;106(9):2522-2526.
doi: 10.3324/haematol.2020.277525.

Myeloma natural killer cells are exhausted and have impaired regulation of activation

Criselle D'Souza  1 , Simon P Keam  2 , Han Xian Aw Yeang  3 , Michael Neeson  3 , Kelden Richardson  3 , Andy K Hsu  3 , Rachael Canfield  3 , Natalie Bezman  4 , Michael Robbins  5 , Hang Quach  6 , David S Ritchie  7 , Simon J Harrison  8 , Joseph A Trapani  9 , H Miles Prince  10 , Paul A Beavis  9 , Phillip K Darcy  9 , Paul J Neeson  11
Affiliations

Myeloma natural killer cells are exhausted and have impaired regulation of activation

Criselle D'Souza et al. Haematologica. .
No abstract available

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Figures

Figure 1.
Figure 1.
Gene expression analysis of natural killer (NK) cell subsets from refractory relapsed multiple myeloma patient and donor peripheral blood mononuclear cells reveals increased activation but loss of regulatory pathways in myeloma patient CD57+ NK cells. Refractory relapsed multiple myeloma (RRMM) patient and healthy donor (HD) NK cells were FACS-sorted to CD57+ and CD57– subsets, RNA extracted and RNA sequencing performed using the SMART-seq v4 low input RNA kit (Takara Bio USA) and sequenced on the NextSeq 550 sequencing system (Illumina, USA). The 36 samples, each containing on average 14,496,483 reads, were aligned using seqliner v0.7.1 to hg19 reference genome and quantified using Htseq v0.6.1 software. Normalization and differential expression analysis was performed with Limma-Voom in R v3.3.3 on a total of 20,850 genes. (A) Overarching differences in HD and myeloma NK cell subset GEP are depicted in two-dimensional principal component analysis (PCA) of patient or HD in four groups (n=6 per group). (B) Normalized log counts-per-million (cpm) transcript levels of B3GAT1 (CD57), ADAM17, PRF1 (perforin), GZMB (granzyme B), FCGR3A (CD16), SLAMF7, KIR3DL2 and KIR2DL1. Protein products are indicated in parentheses. Statistical analysis performed using Student’s t-test *P<0.05, ** P<0.01, ***P<0.001 and ****P<0.0001. (C) Schema showing directionality of GSEA comparisons performed between the four NK-cell groups (upper panel) and bubble chart of GSEA analysis NES and FDR scores when compared to curated NK-related gene sets from MSigDB (lower panel). Red arrows indicate analyses depicted in heatmaps and running enrichment score (ES) analysis. GSEA heatmaps for all replicates for (D) patient CD57+ vs. HD CD57+ cells in NK-cell activation pathways in GO, and (E) patient CD57+ vs. patient CD57– cells in GO: positive regulation of NK-cell activation pathway. (F) Running enrichment score (ES) analysis of panels (D) and (E).
Figure 2.
Figure 2.
CD56+ natural killer cells from multiple myeloma patients are hypo-responsive to elotuzumab-labeled myeloma cells. Peripheral blood mononuclear cells (PBMC) from healthy donors (n=9), and newly diagnosed mulptiple myeloma (NDMM) patients (n=10) or refractory relapsed MM (RRMM) patients (n=10) at baseline (pre-treatment) were cultured with OPM2 target cells in the presence of 10 μg/mL elotuzumab (elo) or human IgG1 (iso) isotype control. Shown in (A) histogram overlay of changes in CD16 expression on CD56dimCD16+ subset of natural killer (NK) cells. (B) Percentage distribution of NK-cell subsets (left panel) or percentage of CD16+ on CD56dimCD57+ NK cells (right panel) in healthy donor (HD), newly diagnosed MM and RRMM patient PBMC after treatment under the same conditions as above. (C) Percentage distribution of CD56dimCD16+ subset of NK cells in HD and RRMM patient PBMC after treatment under the same conditions as above in the presence or absence of ADAM17 inhibitor (n=5 per group) (D) Collated data for HD, NDMM and refractory relapsed (RR) MM patients (n=9-10 per group) showing CD107a degranulation by different NK-cell subsets. Data are pooled from four independent experiments. *P<0.05, Oneway ANOVA with Bonferroni post-hoc test.
Figure 3.
Figure 3.
Natural killer cells from newly diagnosed multiple myeloma patients show significantly lower myeloma antibody-dependent cellular cytotoxicity response post-induction therapy and post-autologous stem cell transplant. A standard 4-hour chromium release assay was used to assess natural killer (NK) cell function, adapted from Hsu et al. Peripheral blood mononuclear cells (PBMC) from healthy donors (HD) (n=8) and newly diagnosed multiple myeloma (NDMM) patients at the time points end of induction (EOI) and post-autologous stem cell transplant (post-ASCT), n=10 per group, were co-cultured with K562 target cells to determine NK cell natural cytotoxicity levels (A), or with OPM2 myeloma target cells and 10 μg/mL elotuzumab (Elo) (B) or human IgG1 isotype (iso) control (C) to determine antibody-dependent cellular cytotoxicity (ADCC) capacity. Cytotoxicity was assessed by chromium (51Cr) release assays and the data displayed as percentage of target cell lysis (A to C, left panels). Each line represents a non-linear regression curve for HD (blue line), or NDMM at EOI (green line) and post-ASCT (orange line) time points at the indicated effector:target (E:T) cells ratios (normalized for the percentage of NK cells). Inserted bar graphs on the right for (A), (B) and (C) show the NK E:T ratio required to achieve 40% target lysis (A, K562), 20% target lysis (B, OPM2 with elotuzumab) and 10% target lysis (C, OPM2 with isotype control) target lysis, extrapolated from the non-linear regression curves on the left. Each symbol represents an individual patient or HD. Data are pooled from five independent experiments. **P<0.01, * P<0.05, Student’s t-test.
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