Regulation of Multiple Myeloma Survival and Progression by CD1d

Downregulation of conventional HLA molecules from the surface of tumour cells is an important mechanism for tumour immune evasion, survival and progression. Whether CD1d, a non-conventional, glycolipid-presenting HLA class I-like molecule can affect tumour cell survival is not known. To test this we studied expression of surface CD1d on plasma cells from different stages of multiple myeloma (MM) using flow-cytometry. Expressing results as the ratio of the Geo MFI CD1d/isotype IgG1 we found that while CD1d expression was comparable between MGUS (n=8) and newly diagnosed MM patients (n=14; Geo MFI MGUS: 8.61±4.3 vs new MM: 7.1±4.72, p>0.05), in relapsed/advanced disease CD1d was significantly lower (Geo MFI:1.92±0.9, p<0.003 vs MGUS and new MM) and completely lost in 4 out of 5 myeloma cell lines at protein and RNA level. Further, 4 out of 8 paired, same-patient trephine biopsies stained with anti- CD1d showed drastic loss of CD1d expression in advanced compared to early disease. These results confirmed loss of CD1d expression during disease progression and suggested that CD1d impacts negatively on myeloma cell survival. Consistent with this, we found that engagement of CD1d by 2 different anti-CD1d mAbs and as compared to isotypic IgG or media control, induces cell death (i.e., Annexin+) of the CD1d-expressing B lymphoblastoid cell line C1R-CD1d, of myeloma cell lines with retrovirally restored expression of CD1d and purified, CD1d-expressing primary myeloma cells in a dose- and time-dependent manner, coincident with loss of mitochondrial membrane potential (MMP) as assessed by DioC3 staining. Biochemical analysis of relevant cell death pathways showed that MMP loss is associated with overexpression of the pro-apoptotic protein Bax but as demonstrated by immunoblotting and pharmacological inhibition it is caspase- independent. By introducing appropriate CD1d retroviral constructs into CD1d- myeloma cell lines we showed that anti-CD1d-induced cell death requires the cytoplasmic tail but not a Tyr residue critical for lysosomal sorting of CD1d. Finally, we found that anti-CD1d co-operates with anti-myeloma agents in the killing of myeloma cells. Thus, these findings provide evidence linking a novel function of CD1d in the regulation of cell death with tumour survival and progression and might have pathogenetic and therapeutic implications for other CD1d-expressing hematopoietic malignancies as well as myeloma.