BCR sequence characteristics required for autonomous signaling. (A) Calcium flux assay of TKO cells transduced with wild-type or modified BCR of DLBCL cell lines TMD8 and DLBCL case 3267. HC: Heavy chain. LC: Light chain. wt: wild-type BCR expressed by cell lines and DLBCL biopsies. GL: IGHV of BCR reverted back to germ-line sequence. VRQ (valine-arginine-glutamine) > GAQ (glycine-alanine-glutamine): FR2 VRQ motif essential for autonomous BCR signaling reverted to GAQ. CDR3: 4391: TMD8 HCDR3 replaced with HCDR3 from DLBCL case 4391. CDR3:OCI-Ly3: 3267 HCDR3 replaced with HCDR3 from OCI-Ly3. Black triangles indicate administration of 4-OHT. Open triangles indicate anti-Ig heavy chain crosslinking. (B) Signaling strength of wild-type (wt) or modified BCR of DLBCL cell line TMD8 and DLBCL cases 3267, 3844, and 3872 as calculated from calcium flux assays of TKO cells. Modifications included reversion of IGHV back to its corresponding germ-line sequence (GL), replacing of the original HCDR3 with HCDR3 from closely related, non-signaling DLBCL BCR as indicated, and change of the constant region from IgM to IgG1. Bars represent mean of two repetitive measurements per sample. Comparisons between BCR signaling strengths were performed using one-sided unpaired t test. (C) Phospho-flow cytometry of 4-OHT–treated TKO cells transduced with wild-type or modified BCR of DLBCL cell line TMD8 or DLBCL case 3267. Bars represent MFI of AF647/APC after normalization to the MFI induced by BCR crosslinking with anti-Ig. The dotted line indicates the relative MFI measured after anti-Ig crosslinking. Dark bars: wild-type BCR. Bright bars: BCR reconverted to germ-line sequence. Error bars indicate standard deviation of AF647/APC MFI of all measured cells. Comparisons by unpaired t test showed P < 0.0001 between wild-type and GL BCR for all proteins. (D) Growth support assay of CRISPR-modified U2932 cells with BCR from TMD8 and DLBCL 4391. Proliferation is measured as cellular doubling time. k.o.: BCR KO. wt: wild-type TMD8 IGHV. GL: TMD8 IGHV reverted back to germ-line sequence. -VRQ: FR2 VRQ motif essential for autonomous BCR signaling reverted to GAQ. Dotted line indicates mean doubling time of unmodified wild-type U2932 cells. Error bars represent variation of triplicate measurements. Comparisons between experimental groups were performed using two-sided unpaired t test. (E) Growth support assay of CRISPR-modified U2932 cells with BCR from Karpas 422 (K422) and DLBCL 3267. Proliferation is measured as cellular doubling time. k.o.: BCR KO. wt: wild-type 3267 IGHV. GL: 3267 IGHV reverted back to germ-line sequence. -VRQ: FR2 VRQ motif essential for autonomous BCR signaling reverted to GAQ. Dotted line indicates mean doubling time of unmodified wild-type U2932 cells. Error bars represent variations of triplicate measurements. Comparisons between experimental groups were performed using two-sided unpaired t test. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ns, not significant.
Figure 3.

BCR sequence characteristics required for autonomous signaling. (A) Calcium flux assay of TKO cells transduced with wild-type or modified BCR of DLBCL cell lines TMD8 and DLBCL case 3267. HC: Heavy chain. LC: Light chain. wt: wild-type BCR expressed by cell lines and DLBCL biopsies. GL: IGHV of BCR reverted back to germ-line sequence. VRQ (valine-arginine-glutamine) > GAQ (glycine-alanine-glutamine): FR2 VRQ motif essential for autonomous BCR signaling reverted to GAQ. CDR3: 4391: TMD8 HCDR3 replaced with HCDR3 from DLBCL case 4391. CDR3:OCI-Ly3: 3267 HCDR3 replaced with HCDR3 from OCI-Ly3. Black triangles indicate administration of 4-OHT. Open triangles indicate anti-Ig heavy chain crosslinking. (B) Signaling strength of wild-type (wt) or modified BCR of DLBCL cell line TMD8 and DLBCL cases 3267, 3844, and 3872 as calculated from calcium flux assays of TKO cells. Modifications included reversion of IGHV back to its corresponding germ-line sequence (GL), replacing of the original HCDR3 with HCDR3 from closely related, non-signaling DLBCL BCR as indicated, and change of the constant region from IgM to IgG1. Bars represent mean of two repetitive measurements per sample. Comparisons between BCR signaling strengths were performed using one-sided unpaired t test. (C) Phospho-flow cytometry of 4-OHT–treated TKO cells transduced with wild-type or modified BCR of DLBCL cell line TMD8 or DLBCL case 3267. Bars represent MFI of AF647/APC after normalization to the MFI induced by BCR crosslinking with anti-Ig. The dotted line indicates the relative MFI measured after anti-Ig crosslinking. Dark bars: wild-type BCR. Bright bars: BCR reconverted to germ-line sequence. Error bars indicate standard deviation of AF647/APC MFI of all measured cells. Comparisons by unpaired t test showed P < 0.0001 between wild-type and GL BCR for all proteins. (D) Growth support assay of CRISPR-modified U2932 cells with BCR from TMD8 and DLBCL 4391. Proliferation is measured as cellular doubling time. k.o.: BCR KO. wt: wild-type TMD8 IGHV. GL: TMD8 IGHV reverted back to germ-line sequence. -VRQ: FR2 VRQ motif essential for autonomous BCR signaling reverted to GAQ. Dotted line indicates mean doubling time of unmodified wild-type U2932 cells. Error bars represent variation of triplicate measurements. Comparisons between experimental groups were performed using two-sided unpaired t test. (E) Growth support assay of CRISPR-modified U2932 cells with BCR from Karpas 422 (K422) and DLBCL 3267. Proliferation is measured as cellular doubling time. k.o.: BCR KO. wt: wild-type 3267 IGHV. GL: 3267 IGHV reverted back to germ-line sequence. -VRQ: FR2 VRQ motif essential for autonomous BCR signaling reverted to GAQ. Dotted line indicates mean doubling time of unmodified wild-type U2932 cells. Error bars represent variations of triplicate measurements. Comparisons between experimental groups were performed using two-sided unpaired t test. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ns, not significant.

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