Myd88^L252P induces MCD lymphoma-associated dependencies in spontaneous splenic GCBs. (A-B) Cas9-expressing TMD8 (MCD-DLBCL) cells were transduced with vectors expressing sgRNA directed against endogenous heavy chain and rescued with vectors expressing autologous or heterologous IgV_H from BCRs from TMD8 itself or GCB-DLBCL cell lines (OCI-Ly-19 or BJAB) or spontaneous splenic or immunized GCBs from Cr2-cre Myd88^L252P fused to the mouse IgM constant region; percent rescue relative to autologous heavy chain was assessed 6 days later. Data in panel B are pooled from 5 independent experiments; each bar represents a distinct heavy chain, 15 IgV_H were cloned from unimmunized and 6 IgV_H were cloned from SRBC-immunized Cr2-cre Myd88^L252P/+ GCBs (see Methods). (C-D) Frequency of CD45.2⁺ cells among follicular B cells (FoB), IgM⁺, or IgM^– GCBs in Cr2-cre Myd88^L252P/+ mixed BM chimeras were treated with ibrutinib for 3 days. Data in panel D are pooled from 2 independent experiments with a total of 10 mice per group. (E-F) Irradiated hosts were reconstituted with Rosa26^Cas9 or Cr2-cre Myd88^L252P/+Rosa26^Cas9 BM that was transduced with vectors expressing control sgRNA or sgRNA targeting Tlr9 and the fluorescent reporter Ametrine. The ratio of sgRNA⁺ (Ametrine⁺) GCBs (GCB) relative to sgRNA⁺ (Ametrine⁺) FoB in unimmunized spleen was assessed 12 weeks later. Data in panel F are pooled from 2 independent experiments with a total of 10 mice per group. (G-H) Frequency of CD45.2⁺ cells among FoB, IgM^– GCBs, or IgM⁺ GCBs in Cr2-cre Myd88^L252P/+ mixed BM chimeras were treated with antibiotics (ampicillin 1 g/L, vancomycin 0.5 g/L, neomycin 1 g/L, and metronidazole 1 g/L) in drinking water for 12 weeks. Data in panel H are from one experiment with 9 and 10 mice per group, respectively. *P < .05, **P < .01, ***P < .001, unpaired two-tailed t test.