B cell–specific Notch1 hyperactivation accelerates CLL onset. (A-C) Analysis of Notch gain-of-function BM chimeras. Ten million CD45.2⁺ T cell–depleted BM cells isolated from IgH.TEμ;RNIC^lox/+ and IgH.TEμ;RNIC^{Δ/+ mb1-Cre} donors were transplanted into lethally irradiated CD45.1⁺ congenic recipients (n = 5 for IgH.TEμ;RNIC^lox/+ control and n = 10 for IgH.TEμ;RNIC^{Δ/+ mb1-Cre} experimental chimeras). (A) Relative reconstitution of BM chimeras 1 and 7 months (mos) posttransplantation (ATBM) is shown. Percentages of CD45.2⁺ cells in PBLs of IgH.TEμ;RNIC^lox/+ controls (blue, n = 5) and IgH.TEμ;RNIC^{Δ/+ mb1-Cre} chimeras (red, n = 10) are plotted. (B) Percentages of animals developing CLL over time post-ATBM within the IgH.TEμ;RNIC^lox/+ (in blue, n = 5) and IgH.TEμ;RNIC^{Δ/+ mb1-Cre} (in red, n = 10) cohorts are depicted. A mouse is considered sick when >5% of CLL cells (CD19⁺CD5⁺) are detected within CD45.2⁺ PBLs. P values were calculated using paired Student t test: *P = .0222. (C) Representative flow cytometric analysis of PBLs stained for CD19, B220 and CD5 derived from control IgH.TEμ;RNIC^lox/+ (left) and IgH.TEμ;RNIC^{lox/+ mb1-Cre} (right) chimeras are shown 6 mos posttransplantation. The CD5 and eGFP histograms, reflecting NIC expression (lower) are gated on CD19⁺B220⁺ B cells (dashed line) and CD19⁺B220⁻ CLL cells (solid line). The plot in the top right shows the percentages of CLL cells (CD19⁺CD5⁺) within PBLs of respective chimeras as indicated (IgH.TEμ;RNIC^lox/+ in blue, n = 5; IgH.TEμ;RNIC^{Δ/+ mb1-Cre} in red; n = 10). (D-F) Analysis of aging cohorts composed of 10 IgH.TEμ;RNIC^{xΔ/+ mb1-Cre} mice and 10 IgH.TEμ;RNIC^lox/+ littermate controls. Disease progression was followed until animals reached end-stage disease. (D) Percentages of IgH.TEμ;RNIC^{Δ/+ mb1-Cre} (red, n = 10) and IgH.TEμ;RNIC^lox/+ mice (blue, n = 10) developing CLL over time is shown. A mouse was considered sick when >10% of CLL-like cells (CD19⁺CD5⁺) were present in total PBLs. P values were calculated using paired Student t test: *P = .0152. (E) The Kaplan-Meier method was applied to monitor survival of IgH.TEμ;RNIC^lox/+ (in blue, n = 10) and IgH.TEμ;RNIC^{Δ/+ mb1-Cre} (in red, n = 10) animals. (F) Representative flow cytometric profiles of PBLs stained for CD19, B220, and CD5 derived from 3-mos-old IgH.TEμ;RNIC^lox/+ control (left) and IgH.TEμ;RNIC^{Δ/+ mb1-Cre} mice (right). The CD5 and eGFP, reflecting NIC expression, histograms (lower) are gated on CD19⁺B220⁺ B cells (dashed line) and CD19⁺B220⁻ CLL cells (solid line). On the far right plot, the percentages of CLL cells (CD19⁺CD5⁺) within PBLs derived from corresponding mice as indicated (IgH.TEμ;RNIC^lox/+ in blue, n = 10 and IgH.TEμ;RNIC^{Δ/+ mb1-Cre} in red, n = 10). The Kaplan-Meier method was applied to monitor survival of Rag2^−/−γc^−/− recipients transplanted with either (G) 10 000 or (H) 1000 sorted CD19⁺B220⁻ spleen-derived CLL cells isolated from either IgH.TEμ;RosaNIC^{Δ/+ mb1-Cre} mice (red line, n = 4 [G]; n = 7 [H]) or from IgH.TEμ;RosaNIC^lox/+ control animals (blue line n = 5 [G]; n = 7 [H]). CLL cells were sorted and pooled from 3 donors of each genotype with comparable disease stage.