Macrophage-dependent senescence induction in MyD88-L265P– and CARD11-L244P–mutant lymphomas. (A) Fraction of F4/80⁺/CD11b⁺/GFP⁻/B220⁻ macrophages within the overall population of viable cells in freshly isolated single-cell suspensions from n ≥ 3 individual enlarged lymph node samples per indicated genotype (with error bars denoting the standard deviation) analyzed by flow cytometry. Notably, macrophages were GFP⁻ in all cases, that is, did not express the NF-κB–deregulating mutant. (B) Transcript-level expression of the macrophage chemoattractants Ccl2 (green) and Csf-1 (blue) in B220-sorted mutant-driven lymphoma cells relative to the empty vector cohort assessed by RNA-seq in the indicated numbers of samples and with error bars denoting the standard error in log₂ scale. Gray bars indicate nonsignificant results with a P ≥ .05. (C) ELISA to detect secreted CCL2 and CSF-1 in the supernatant of manifest Eµ-myc lymphomas (n = 5) stably transduced with either the GFP-encoding vector or the indicated mutants. After 10 days in culture, lymphoma cells were GFP-sorted and supernatants from 2 × 10⁵ GFP⁺ cells were collected (24 hours incubation time) and analyzed. Notably, mutant-expressing lymphoma cells do not show any signs of senescence induction in vitro (supplemental Figure 2F) (D) Fraction of viable macrophages (left; as in panel A) in MyD88-L265P–driven lymphomas with and without (n = 4 each) Clodronate administration in vivo. Corresponding fractions of senescent lymphoma cells by SA–β-gal staining of cytospin preparations from GFP⁺ lymphoma cell isolates (right). Scale bars, 100 µm. (E) TGF-β1 expression by immunohistochemistry (ematoxylin and eosin [H&E] stain) in tissue sections of NDM-driven Eµ-myc lymphomas. Representative photomicrographs of n = 3 independent analyses with percentage of positively stained area quantified for each genotype. Scale bars, 100 µm. (F) Detection of the fraction of senescent cells in MyD88-L265P-mutant lymphomas generated with or without stable coexpression of the dominant-negative TGF receptor (dnTGFR) by SA–β-gal staining of cytospin preparations, and Ki67 immunostaining of lymphoma sections in situ (H&E stain). Representative photomicrographs of 3 independent analyses; percentages indicate means ± standard deviation. Scale bars, 100 µm. Asterisks indicate significance (P < .05).