Figure 3.
B cell-specific RBP-J deficiency reduces prevalence of CLL initiation. (A-C) Analysis of BM chimeras: Ten million CD45.2+ T cell–depleted BM cells isolated from IgH.TEμ;RBP-Jlox/lox and IgH.TEμ;RBP-JΔ/Δ mb1-Cre donors were transplanted into lethally irradiated CD45.1+ congenic recipients (n = 9 for IgH.TEμ;RBP-Jlox/lox control and n = 8 for IgH.TEμ;RBPΔ/Δ mb1-Cre experimental chimeras). (A) Relative reconstitution of BM chimeras 1 and 7 months (mos) posttransplantation (ATBM). Percentage of CD45.2+ cells in PBLs of control IgH.TEμ;RBP-Jlox/lox (blue, n = 9) and the RBP-J-deficient IgH.TEμ;RBP-JΔ/Δ mb1-Cre chimeras (red, n = 8) is shown. (B) Percentage of animals developing CLL over time post-ATBM within control IgH.TEμ;RBP-Jlox/lox (blue, n = 9) and IgH.TEμ;RBP-JΔ/Δ mb1-Cre (red, n = 8) chimeras is depicted. A mouse is considered sick when >5% of CLL-like cells (CD19+CD5+) are detected within CD45.2+ PBLs. P values were calculated using paired Student t test: *P = .0212. (C) Representative flow cytometric analysis of PBLs stained for CD19, B220 and CD5 derived from control IgH.TEμ;RBP-Jlox/lox (left) and the RBP-J-deficient IgH.TEμ;RBP-JΔ/Δ mb1-Cre (right) chimeras 10 months posttransplantation. The CD5 histograms (lower) are gated on CD19+B220+ B cells (dashed line) and CD19+B220− CLL cells (solid line). The far right panel plots the percentages of CLL cells (CD19+CD5+) within PBLs of respective BM chimeras 10 months posttransplantation. P values were calculated using unpaired Student t test: *P = .0406. Analysis of aging cohorts composed of 9 IgH.TEμ;RBP-Jlox/lox littermate controls and 10 IgH.TEμ;RBP-JΔ/Δ mb1-Cre mice. Disease progression was followed for 400 days. (D) Percentages of IgH.TEμ;RBP-Jlox/lox (blue, n = 9) and IgH.TEμ;RBP-JΔ/Δ mb1-Cre mice (red, 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 analyzed by flow cytometry. P values were calculated using paired Student t test: ****P < .0001. (E) The Kaplan-Meier method and log-rank (Mantel-Cox) test were applied to monitor survival of IgH.TEμ;RBP-Jlox/lox (blue line, n = 9) and IgH.TEμ;RBP-JΔ/Δ mb1-Cre (red, n = 10) mice. Mantel-Cox test: ****P < .0001. (F) Representative flow cytometric profiles of PBLs stained for CD19, B220, and CD5 derived from 6-month-old control IgH.TEμ;RBP-Jlox/lox (left) and RBP-J deficient IgH.TEμ;RBP-JΔ/Δ mb1-Cre animals (right). The CD5 histograms (lower) are gated on CD19+B220+ B cells (dashed line) and CD19+B220− CLL cells (solid line). The far right panel plots the percentages of CLL cells (CD19+CD5+) within PBLs of the respective animal cohorts as indicated. P values were calculated using unpaired Student t test: *P = .0032. (G) RBP-J deletion PCR performed on DNA isolated from sorted CLL cells from moribund IgH.TEμ;RBP-Jlox/lox control and 2 moribund IgH.TEμ;RBP-JΔ/Δmb1-Cre mice as well as from sorted CD19+B220+ B cells from 2 healthy IgH.TEμ;RBP-JΔ/Δ mb1-Cre mice. lox, loxP RBP-J allele; Δ, recombined RBP-J allele.

B cell-specific RBP-J deficiency reduces prevalence of CLL initiation. (A-C) Analysis of BM chimeras: Ten million CD45.2+ T cell–depleted BM cells isolated from IgH.TEμ;RBP-Jlox/lox and IgH.TEμ;RBP-JΔ/Δ mb1-Cre donors were transplanted into lethally irradiated CD45.1+ congenic recipients (n = 9 for IgH.TEμ;RBP-Jlox/lox control and n = 8 for IgH.TEμ;RBPΔ/Δ mb1-Cre experimental chimeras). (A) Relative reconstitution of BM chimeras 1 and 7 months (mos) posttransplantation (ATBM). Percentage of CD45.2+ cells in PBLs of control IgH.TEμ;RBP-Jlox/lox (blue, n = 9) and the RBP-J-deficient IgH.TEμ;RBP-JΔ/Δ mb1-Cre chimeras (red, n = 8) is shown. (B) Percentage of animals developing CLL over time post-ATBM within control IgH.TEμ;RBP-Jlox/lox (blue, n = 9) and IgH.TEμ;RBP-JΔ/Δ mb1-Cre (red, n = 8) chimeras is depicted. A mouse is considered sick when >5% of CLL-like cells (CD19+CD5+) are detected within CD45.2+ PBLs. P values were calculated using paired Student t test: *P = .0212. (C) Representative flow cytometric analysis of PBLs stained for CD19, B220 and CD5 derived from control IgH.TEμ;RBP-Jlox/lox (left) and the RBP-J-deficient IgH.TEμ;RBP-JΔ/Δ mb1-Cre (right) chimeras 10 months posttransplantation. The CD5 histograms (lower) are gated on CD19+B220+ B cells (dashed line) and CD19+B220− CLL cells (solid line). The far right panel plots the percentages of CLL cells (CD19+CD5+) within PBLs of respective BM chimeras 10 months posttransplantation. P values were calculated using unpaired Student t test: *P = .0406. Analysis of aging cohorts composed of 9 IgH.TEμ;RBP-Jlox/lox littermate controls and 10 IgH.TEμ;RBP-JΔ/Δ mb1-Cre mice. Disease progression was followed for 400 days. (D) Percentages of IgH.TEμ;RBP-Jlox/lox (blue, n = 9) and IgH.TEμ;RBP-JΔ/Δ mb1-Cre mice (red, 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 analyzed by flow cytometry. P values were calculated using paired Student t test: ****P < .0001. (E) The Kaplan-Meier method and log-rank (Mantel-Cox) test were applied to monitor survival of IgH.TEμ;RBP-Jlox/lox (blue line, n = 9) and IgH.TEμ;RBP-JΔ/Δ mb1-Cre (red, n = 10) mice. Mantel-Cox test: ****P < .0001. (F) Representative flow cytometric profiles of PBLs stained for CD19, B220, and CD5 derived from 6-month-old control IgH.TEμ;RBP-Jlox/lox (left) and RBP-J deficient IgH.TEμ;RBP-JΔ/Δ mb1-Cre animals (right). The CD5 histograms (lower) are gated on CD19+B220+ B cells (dashed line) and CD19+B220− CLL cells (solid line). The far right panel plots the percentages of CLL cells (CD19+CD5+) within PBLs of the respective animal cohorts as indicated. P values were calculated using unpaired Student t test: *P = .0032. (G) RBP-J deletion PCR performed on DNA isolated from sorted CLL cells from moribund IgH.TEμ;RBP-Jlox/lox control and 2 moribund IgH.TEμ;RBP-JΔ/Δmb1-Cre mice as well as from sorted CD19+B220+ B cells from 2 healthy IgH.TEμ;RBP-JΔ/Δ mb1-Cre mice. lox, loxP RBP-J allele; Δ, recombined RBP-J allele.

Close Modal
This Feature Is Available To Subscribers Only

or Create an Account

Close Modal
Close Modal