Modifications to endow non-signaling BCR with autonomous signaling activity. (A) Calcium flux assay of TKO cells transduced with modified BCR of DLBCL cell line Karpas 422 (K422) and DLBCL case 4391. BCR sequences were modified by HCDR3 exchange with a closely related, autonomously signaling BCR as indicated. HC: Heavy chain. LC: Light chain. Black triangles indicate administration of 4-OHT. Open triangles indicate anti-Ig heavy chain crosslinking. (B) Signaling strength of non-signaling BCR after HCDR3 exchange with an autonomously signaling BCR as calculated from calcium flux assays of TKO cells. Symbols represent arithmetic means of two consecutive measurements. Solid lines indicate identity of non-signaling BCR. Dashed lines indicate identity of HCDR3. WT: wild-type non-signaling BCR. +CDR3: Non-signaling BCR with exchanged HCDR3. The dotted line indicates the upper limit of BCR signaling strength of non-signaling BCR. (C) Growth support assay of CRISPR-modified U2932 cells with wild-type or modified BCR of DLBCL cell line Karpas 422 (K422). Proliferation is measured as cellular doubling time. k.o.: BCR KO. wt: wild-type K422 HCDR3. 3267: HCDR3 replaced from closely related, autonomously signaling BCR from DLBCL case 3267. Comparisons between experimental groups were performed using two-sided unpaired t test. The dotted line indicates mean doubling time of unmodified wild-type U2932 cells. (D) Growth support assay of CRISPR-modified U2932 cells with wild-type or modified BCR of DLBCL 4391. Proliferation is measured as cellular doubling time. k.o.: BCR knock-out. wt: wild-type 4391 HCDR3. TMD8: HCDR3 replaced from closely related, autonomously signaling BCR from cell line TMD8. Comparisons between experimental groups were performed using a two-sided unpaired t test. The dotted line indicates mean doubling time of unmodified wild-type U2932 cells. (E) Signaling strength of non-signaling BCR from primary DLBCL cases 3882 (IgG1), 4391 (IgG2), 3567 (IgG2), and 3850 (IgG4) to IgM as calculated from calcium flux assays of TKO cells. The dotted line indicates the upper limit of BCR signaling strength of non-signaling BCR.
Figure 4.

Modifications to endow non-signaling BCR with autonomous signaling activity. (A) Calcium flux assay of TKO cells transduced with modified BCR of DLBCL cell line Karpas 422 (K422) and DLBCL case 4391. BCR sequences were modified by HCDR3 exchange with a closely related, autonomously signaling BCR as indicated. HC: Heavy chain. LC: Light chain. Black triangles indicate administration of 4-OHT. Open triangles indicate anti-Ig heavy chain crosslinking. (B) Signaling strength of non-signaling BCR after HCDR3 exchange with an autonomously signaling BCR as calculated from calcium flux assays of TKO cells. Symbols represent arithmetic means of two consecutive measurements. Solid lines indicate identity of non-signaling BCR. Dashed lines indicate identity of HCDR3. WT: wild-type non-signaling BCR. +CDR3: Non-signaling BCR with exchanged HCDR3. The dotted line indicates the upper limit of BCR signaling strength of non-signaling BCR. (C) Growth support assay of CRISPR-modified U2932 cells with wild-type or modified BCR of DLBCL cell line Karpas 422 (K422). Proliferation is measured as cellular doubling time. k.o.: BCR KO. wt: wild-type K422 HCDR3. 3267: HCDR3 replaced from closely related, autonomously signaling BCR from DLBCL case 3267. Comparisons between experimental groups were performed using two-sided unpaired t test. The dotted line indicates mean doubling time of unmodified wild-type U2932 cells. (D) Growth support assay of CRISPR-modified U2932 cells with wild-type or modified BCR of DLBCL 4391. Proliferation is measured as cellular doubling time. k.o.: BCR knock-out. wt: wild-type 4391 HCDR3. TMD8: HCDR3 replaced from closely related, autonomously signaling BCR from cell line TMD8. Comparisons between experimental groups were performed using a two-sided unpaired t test. The dotted line indicates mean doubling time of unmodified wild-type U2932 cells. (E) Signaling strength of non-signaling BCR from primary DLBCL cases 3882 (IgG1), 4391 (IgG2), 3567 (IgG2), and 3850 (IgG4) to IgM as calculated from calcium flux assays of TKO cells. The dotted line indicates the upper limit of BCR signaling strength of non-signaling BCR.

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