Mutation of the CD79A ITAM selectively affects tonic BCR signaling in GCB-DLBCL lines. (A) Schematic of the knock-in approach to fuse GFP to the 3′ end of CD79A, along with either mutation of both ITAM tyrosines (YY>FF) or no other change in CD79A (wild type [WT]). The sequence of the CD79A cytoplasmic tail shows the ITAM and its tyrosine residues. (B) BCR surface level and GFP expression in OCI-Ly7 cells 3 days after electroporation to knock in CD79A-GFP constructs. Both constructs yield 3 populations of cells, as marked in the top panel for the YY>FF construct, and GFP and BCR levels are correlated in knock-in cells. The bottom panel shows histograms of surface BCR level for the 3 populations in the top panel, as well as for knock-in cells with the WT construct, indicating that knock-in cells with the different constructs have equivalent BCR surface levels. (C) Calcium flux in response to BCR cross-linking with anti-IgM in CD79A-GFP knock-in OCI-Ly19 cells. (D) Absolute growth curves for BCR KO and CD79A-GFP knock-in cells, relative to the starting point 3 days after electroporation. The YY>FF ITAM mutation has effects similar to BCR KO in GCB-DLBCL lines. (E) Absolute growth curves, from a separate experiment, showing effect of single or dual CD79A ITAM tyrosine mutations in GCB-DLBCL lines. Panels D and E show the mean ± SD from 3 biological replicates.