Expression of GPRC5D and BCMA on MM cells. (A) MM patient BM MNCs showing various profiles of GPRC5D and BCMA expression. Cells were gated using the CD138 plasma-cell marker (MI15; BioLegend) and antibodies to GPRC5D and BCMA to measure their surface expression (GPRC5D: 571961, R&D Systems; BCMA: 19F2, BioLegend). The left panel is representative of most patient samples surveyed, while the middle and right panels depict skewed expression for BCMA or GPRC5D, which was seen in a minority of patients (see panel B). (B) Expression of GPRC5D and BCMA surface protein in 51 BM MNC samples from MM patients on CD138⁺ cells as measured by flow cytometry. Each column represents an individual patient sample. Data are expressed as percent positivity of all CD138⁺ cells. (C) Generation of GPRC5D (top row) and BCMA (bottom row) KO H929 cells by CRISPR. Loss of signal can be seen in the KO cells compared with the isotype control (gray). (D) JNJ-64407564 (black circle) efficiently depleted H929 wild-type or BCMA KO cells, but not GPRC5D KO cells. Percent cytotoxicity and T-cell activation were measured as in Figure 2. A positive control BCMAxCD3 antibody (red triangle) killed wild-type H929 and GPRC5D KO cells but had no effect on BCMA KO cells. The negative control antibody NullxCD3 (blue square) had no effect on cytotoxicity or T-cell activation in all cell lines.