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Letters to the Editor
Myeloma natural killer cells are exhausted and have impaired regulation of activation
Multiple myeloma (MM) is an immunotherapy respon- sive disease. Treatment strategies including immune- modulatory drugs lenalidomide and pomalidomide, bi-specific t-cell engagers (BiTE), and antibodies targeting myeloma surface proteins SLAMF7 (elotuzumab) or CD38 (daratumumab and isatuximab) and chimeric anti- gen receptor T cells have been effective.1-4 Currently, myeloma-targeting antibodies against CD38 and SLAMF7 mediate their effect in part, via natural killer (NK) cells as key effectors.1,2 However, NK cells from myeloma patients have decreased functional responses to myeloma in vitro.5 Despite this, myeloma targeting anti- bodies that are reliant on NK-cell mediated cytotoxicity have been successful in treating MM patients.2
In order to understand this further, we explored NK- cell differentiation and function in newly diagnosed MM patients (NDMM) and, for the first time, gene expression profiles of NK-cell subsets from refractory relapsed MM (RRMM) patients. These analyses revealed that underly- ing NK-cell intrinsic properties explain this myeloma patient NK-cell dysfunction. We also characterized whether NK-cell dysfunction was rescued following induction therapy with lenalidomide and dexamethasone and post-autologous stem cell transplantation (ASCT) to understand whether myeloma-targeting antibodies such as elotuzumab could be used at these time points.
We compared peripheral blood and bone marrow NK cells from a NDMM patient cohort consecutively treat- ed in the context of a prospective phase II clinical LIT- VACC trial (clinicaltrials gov. Identifier: ACTRN12613000344796)6 or RRMM patient cohort from the RevLite trial (clinicaltrials gov. Identifier: NCT00482261)7 to healthy donor (HD) NK cells (for details see Online Supplementary Figure S1).
We first confirmed that RRMM and NDMM patients have a higher percentage of terminally differentiated CD57+ NK cells compared to HD both in the peripheral blood and bone marrow (Online Supplementary Figure S1C and D). These RRMM NK cells are dysfunctional. In contrast, HD CD56dimCD16+KIR+CD57+ NK cells are highly cytotoxic and secrete increased levels of interfer- on-γ (IFN-γ) in response to contact with targets.8 In order to explore reasons for this difference, principal component analysis of RNA sequencing data showed RRMM patient NK-cell gene expression profile (GEP) was distinct from HD NK cells (Figure 1A; Online Supplementary Figure S2A). Differential GEP analysis revealed numerous genes either down- or up-regulated in patient or HD CD57+ NK cells (Online Supplementary Figure S2B). When CD57+ NK cells from myeloma patients and HD were compared, we revealed differen- tially expressed genes (DEG) (n=133 and 533 DEG respectively), where 97 DEG were common to both RRMM patients and HD (Online Supplementary Figure S2C). Of the 36 DEG unique to patient CD57+ NK cells, 13 were up-regulated and 23 were down-regulated (Online Supplementary Figure S3C). When NK cell-specif- ic genes were examined, we found decreased expression of genes associated with CD16 cleavage such as ADAM17 in RRMM patient NK cells, increased expres- sion of genes associated with cytotoxicity and activa- tion such as PRF1, GZMB, NCR1, NCR2, and increased expression of novel immune checkpoint genes, CISH and TIGIT (Figure 1B; Online Supplementary Figure S2E). Cytokine-inducible SH2-containing protein (CIS, encod-
ed by CISH) is a critical negative regulator of IL-15 sig- naling and inhibits cytotoxicity against tumor cells.9
Gene set enrichment analysis (GSEA) revealed genes related to NK-cell activation pathways were significantly up-regulated in RRMM patient NK cells compared to HD NK cells, suggesting that NK cells from patients are con- stitutively more activated (Figure 1C). This finding was also true when comparing NK-cell activation pathways between RRMM patient and HD CD57– NK cells or CD57+ NK cells (Figure 1C and D). However, genes relat- ed to pathways regulating NK-cell activation (IL23A, IL23R, GAS6, IL18, IL15, AXL, FLT3LG, TICAM1 and PLDN) were downregulated in CD57+ NK cells from RRMM patients, suggesting dysregulation of patient NK cell activation (Figure 1C and E). GSEA enrichment plots highlight significantly increased MM patient NK-cell acti- vation, yet co-existing suppression of positive regulation of these activation pathways (Figure 1F). ADAM17 tran- script levels also correlated negatively with NK-cell acti- vation in RRMM patients as compared to HD (Online Supplementary Figure S2D). Taken together, these data indicate MM patient CD57+ NK cells are constitutively more activated than their normal donor counterparts. However, they lack expression of key regulators of NK- cell activation and have increased levels of the NK-cell immune checkpoint molecules CIS and TIGIT, suggesting an ‘exhausted’ state.
We next explored whether NK-cell chronic activation and low levels of ADAM17 observed in the GEP data in Figure 1 would affect the capacity of NK cells to respond via CD16 or SLAMF7 mediated signaling. In order to do this, peripheral blood mononuclear cells (PBMC) from NDMM and RRMM patients and HD were co-cultured with OPM2 myeloma targets and the anti-human SLAMF7 antibody, elotuzumab. In this context, activated NK cells were expected to down-regulate CD16 due to cleavage by ADAM17,10 and this would be evident by a reduction in the percentage of CD56dimCD16+ NK cells. Only HD NK cells significantly decreased the percentage of CD56dimCD16+ NK cells in response to elotuzumab (Figure 2A and B, left panel), which was inhibited in the presence of an ADAM17 inhibitor (Figure 2C). Whilst there was a trend to decreased CD56dimD16+ NK cells in NDMM patients, this did not reach significance. We observed a similar result for terminally differentiated CD56dimCD57+ NK cells. HD NK cells were responsive to activation via OPM2 cells and elotuzumab and signifi- cantly reduced the percentage of CD56dimCD57+ NK cells (Figure 2B, right panel). In the same conditions, untreated NDMM NK cells showed a trend for decreased percent- age of CD56dimCD57+CD16+ NK cells (P=0.051), whereas RRMM NK cells were relatively unresponsive. No differ- ence was observed in the percentage of CD56dimCD16+ NK cells in RRMM patients in the presence of ADAM17 inhibitor (Figure 2C). Prior studies demonstrated no loss of NK cells in PBMC treated with elotuzumab at higher concentrations than used in our assays,11 suggesting frat- ricide was unlikely to occur. Our data supports this as the SLAMF7 levels on NK cells between MM patients and HD were similar (Online Supplementary Figure S3A). Subsequently, NK cell subsets were examined for degran- ulation (CD107a+) in the presence of OPM2 cells and elo- tuzumab. Of all NK cell subsets, only the CD56dimCD16– NK cells degranulated at significantly higher levels in HD compared to both groups of MM patients (Figure 2C; Online Supplementary Figure S3B). A similar trend was also observed for the HD versus myeloma patient CD57+ NK cells, but this did not reach significance. These results suggest that low levels of ADAM17 may lead to consti-
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haematologica | 2021; 106(9)