P-241: Progression from smouldering myeloma to myeloma is accompanied by an increase in CD56bright bone marrow resident NK cells with reduced NK cell cytotoxic capacity

Introduction: Natural killer (NK) cells are crucial in the innate immune response against tumour progression. Whilst the immune microenvironment in myeloma (MM) is reported to be increasingly dysfunctional through disease evolution, information on whether and how the NK cell compartment alters with progression is limited. We aimed to study NK cell phenotype and function in disease progression, based on primary samples from trial patients, using single-cell RNA-sequencing (scRNAseq) and functional studies.

Methods: Freshly isolated bone marrow (BM) mononuclear cells from precursors and newly diagnosed MM enrolled in clinical trials (COSMOS, Smouldering Myeloma (SMM) n=88 and Monoclonal Gammopathy of Undetermined Significance (MGUS) n=17), and MM (RADAR, n=48) were analysed by flow cytometry for tumour infiltration (CD138+) and NK cell subsets (CD3-CD56+). Functional capacity of patient NK cells toward tumour target was assessed by cytotoxicity assays against the NK sensitive cell line K562 (n=16). Finally, we performed scRNAseq on 18 patients and integrated our data with previously published studies of BM and peripheral blood to analyse a total of 68,665 NK cells from 191 subjects, including samples from healthy donors and individuals with MGUS, SMM and MM.

Results: We observed a stepwise increase in the proportion of BM NK cells with a CD56^bright phenotype through progression from MGUS through SMM to MM using flow cytometry. Interestingly, there was a positive correlation between abundance of the NK CD56^bright subset, and tumour infiltration in precursor conditions (r=0.39, p< 0.001) but not in MM.

Functional studies revealed a reduced cytotoxic response towards tumour target in BM NK cells of MM patients when compared to SMM, with reduced expression of the degranulation marker CD107a (p=0.002). Immunomodulatory response of MM NK cells was also significantly lower, with reduced expression of XCL1/2 (p=0.02) and CCL4 (p=0.01) compared to SMM NK cells.

Using scRNA-seq, we identified a subset of CD56^bright NK cells found only in BM samples. In addition to XCL1 and GZMH, found on the classical CD56^bright population, this subset also had high expression of CD69 and CXCR6, consistent with BM residency. These resident NK cells had a significantly lower score for a cytotoxic gene set, including cytolytic molecules PRF1, GZMB and GNLY, compared to the classical CD56^bright population (p=5.6x10^-11) suggesting reduced cytotoxic capacity. With disease progression, we found an increase in the proportion of this BM-specific CD56^bright population which was significant between SMM and MM (padj=0.008).

Conclusions: We show for the first time that progression to myeloma is accompanied by an enrichment of the CD56^bright NK cell compartment, specifically the BM-specific NK CD56^bright CXCR6+ CD69+ subset with a reduced cytotoxic profile. Further work will clarify if these changes in the NK compartment contribute to the loss of host immune control at disease progression.