Page 235 - 2021_09-Haematologica-web

Letters to the Editor
A
C
B
DE
Figure 1. Gene expression analysis of natural killer (NK) cell subsets from refractory relapsed multiple myeloma patient and donor peripheral blood mononu- clear 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 log2 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 com- pared to curated NK-related gene sets from MSigDB (lower panel). Red arrows indicate analyses depicted in heatmaps and running enrichment score (ES) analy- sis. 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).
F
haematologica | 2021; 106(9)
2523