CD48: Friend or Foe in NK-Cell Immunosurveillance of Multiple Myeloma?

Introduction: The balance between activating and inhibitory receptors on NK cells regulates their ability to recognize and eliminate target cells. This balance's impact on myeloma (MM) progression and immune surveillance remains unclear. The 2B4 (CD244) receptor modulates NK cell cytotoxicity and cytokine production, containing four Immunoreceptor Tyrosine-based Switch Motifs (ITSMs) that interact with various adaptor molecules to relay both inhibitory and activating signals. CD244’s ligand, CD48, is a surface glycoprotein overexpressed in MM cells (Ferguson et al., Nat Comm 2022). While high CD48 expression in solid tumors often links to poor prognosis, its role in hematological malignancies is less clear. CD48 is believed to activate NK cells, and its absence may impair NK cell function, though this is not well elucidated.

Methods: We assessed the role of CD48 in MM using RNA-seq data from the coMMpass (IA19) study, analyzed with DESeq2, survminer, and survival packages in R. Public scRNA-seq data (GSE223060) was also analyzed with Seurat v4.3.0, comparing 53 MM patients (MMPt) to 7 healthy donor (HD) bone marrow samples. Harmony package was used for data integration. To identify transcription factors (TFs) binding to the CD48 locus, MMPt ATAC-seq and ENCODE ChIP-seq data were analyzed, informing an eXtreme Gradient Boosting (XGBoost) model. NK cells' role in MM progression was examined using an in vivo mouse model with the Vk*MYC cell line, where NK cell depletion was achieved with anti-NK1.1 antibody, and MM progression was monitored via bioluminescence.

Results: RNA-seq analysis of the CoMMpass dataset showed elevated CD48 expression in high-risk MM subtypes, such as t(4;14) and gain 1q21 (p-value < 0.0001). Higher CD48 expression correlated with poorer overall survival (top 20% vs. bottom 20% expression, p-value = 0.0044). This suggests that increased CD48 expression might impair NK cell immunosurveillance of MM, contrary to cell line-based CRISPR screens indicating CD48 can activate NK cells (Dufva et al., Immunity (2023); Liu et al., Nat Commun (2024)). Analysis of scRNA-seq data from 150,256 cells revealed a trend toward higher CD244 expression in MMPt NK cells compared to HD, with elevated levels of inhibitory phosphatases binding to ITSMs and triggering inhibitory NK cell signaling through CD244, such as SHIP1 (INPP5D), SHP1 (PTPN6), and EAT-2 (SH2D1B). SAP (SH2D1A), an activating phosphatase, was also higher in MMPt NK cells. Distinct NK cell subclusters were observed in MM patients, showing a phenotype similar to the adaptive NK CD56-dim phenotype with high expression of cytotoxic markers (GNLY, PRF1, NKG7, FGFBP2) and altered receptor expression, including downregulation of KLRB1 and KLRF1, and upregulation of inhibitory receptors KLRC2 and KLRC3. Notably, KLRC2 was predominantly upregulated in NK cell subclusters from MM patients absent in healthy donors. To explore potential drivers of CD48 expression in MM, ATAC-seq and ENCODE ChIP-seq data identified 89 TFs potentially binding the CD48 locus in primary MM samples. An XGBoost model indicated TFs associated with MM progression, such as IRF4, predicted CD48 expression (R^2 = 0.40). Using the syngeneic Vk*MYC mouse model, in vitro co-culture of CD48 knockout or CD48 overexpression Vk*MYC cells suggested that increased CD48 modestly enhanced KIL.C2 murine NK cell killing of tumor cells. In the immunocompetent Vk*MYC mouse model, NK cell depletion led to accelerated and more aggressive MM progression, highlighting the critical role of NK cells in tumor control.

Conclusions: NK cells play a crucial role in controlling MM progression. Although previous studies have focused on CD48’s role in NK cell activation using cells from HD, our findings suggest that CD48's effects on NK cells are complex and might be a significant prognostic factor in the MM microenvironment. Further research is needed to investigate NK cell surface markers related to dysfunction, such as CD244 and KLRC2, and to understand how increased CD48 influences MM evasion of NK cell-mediated surveillance, particularly in relation to adaptive NK cells and inhibitory phosphatases like SHIP1 and EAT-2.