Shifting CD20 splicing from V3-to-V1 increases resistance to mosunetuzumab but not CART-20. (A) Diagram depicting the lentiviral vector transductions performed on OCI-Ly8 cells expressing a luciferase reporter. The MS4A1 gene was knocked out (CD20KO) and reconstituted with V1-, V2-, or V3-rCD20. The resistant CD20 (or rCD20) constructs have silent mutations in their CDS to avoid recognition by the CD20-targeting gRNA used in the knockout. (B) Isoform-specific RT-qPCR measurements of CD20 mRNA isoform relative to the RPL27 reference gene. (C) Measurement of CD20 protein levels by flow cytometry. On the left, it is quantitated as a log2 fold change in the MFI of total APC-CD20 relative to the unstained Ctrls. On the right, it is quantitated as the cell surface CD20 levels expressed as MESF. Each spot is an independent experiment. Representative histograms are included in each subpanel. (D) Viability of OCI-Ly8 cells after 24 hours of coculturing with CART-20 or untransduced donor T cells at 1:4 effector-to-target ratio. Cell viability (in percentage) is shown relative to that of Ctrl OCI-Ly8 cells that were not cocultured with either CART-20 or donor T cells. (E) OCI-Ly8 cell viability after 24 hours of coculturing with donor T cells and the indicated concentrations of mosunetuzumab. Cell viability (in percentage) is shown relative to that of the NT Ctrl. For panel D, data from 2 independent experiments using T cells from a single donor are shown. For panel E, data from 4 independent experiments with 2 unrelated donors are shown. In panel D and on the right of panel E, each spot represents the results from the replicate wells, with different colors denoting independent experiments. Horizontal bars represent the mean of all replicates. ∗∗P < .01; ∗∗∗P < .001 per the Kruskal-Wallis test (pwc: the Dunn test). (F) CD20 and PAX5 status per immunohistochemistry (IHC) (top) and transcript read abundance per RNA-seq (bar graphs) in paired pre- and postmosunetuzumab FL. TPM values quantify RNA-seq reads mapping to any exon in CD20 and PAX5. The relative abundance of V1 (red), V2 (green), V3 (blue), and V4 (yellow) is the ratio of sequencing reads mapping to the unique exon-exon junctions found in each 5′-UTR variant of CD20, as color-coded in the Reads spanning panel. (G) Micrographs corresponding to P29 pre and postmosunetuzumab FL samples. Formalin-fixed paraffin-embedded sections were IHC stained for CD20 (brown colorimetric detection with 3, 3'-diaminobenzidine [DAB]) using hematoxylin nuclear counterstain (blue). (H) Changes in ΔPSI values or in the ratio between reads including or excluding CD20 exons in the P29-post sample relative to the P29-pre sample. RNA-seq data were analyzed using the MAJIQ algorithm for all possible splicing changes in CD20, but only ΔPSI values above or below 0.05 are shown. (I) Sashimi plots depicting the density of exon-including and exon-skipping reads in the RNA-seq data corresponding to P29-pre and -post samples. FACS, fluorescence-activated cell sorting; H&E, hematoxylin and eosin.