CD1d expression in primary myeloma cells ex vivo and in situ. (A) Surface expression of CD1d in bone marrow myeloma cells from patients in different stages of the disease was assessed by multicolor flow cytometry. Histograms are gated on plasma cells identified as CD138⁺CD38⁺CD45^low/− (dotted line represents isotype; continuous line, CD1d). (B) CD1d expression as assessed by the Geo MFI CD1d/isotypic IgG1 ratio (to account for the day-to-day variability of the isotype control) in MGUS (n = 8), newly diagnosed (n = 18), and relapsed patients (n = 17). Geo MFI CD1d/ IgG1 ratio was similar between MGUS and newly diagnosed patients (P > .05), but considerably lower in patients with relapsed/advanced disease compared with MGUS (P < .01) and newly diagnosed patients (P < .01). (C) Western blot analysis of CD1d in plasma cells selected with CD138 immunomagnetic beads from a MGUS, a newly diagnosed and a patient with relapsed MM. The CD1d-transduced B-cell line C1R-CD1d was used as a positive control. (D) Assessment of CD1d expression in myeloma cells by immunohistochemistry. For patients P5, P6, P8, and P11, paired bone marrow trephine biopsies from early (ie, at diagnosis or after first chemotherapy) and late (relapse after allogeneic hematopoietic stem cell transplantation) disease were stained with anti-CD1d; in P6, 3 consecutive trephine biopsies were studied; in P7, the late sample was skin plasmacytoma.