Fig. 2
From: Overexpression of wild type RRAS2, without oncogenic mutations, drives chronic lymphocytic leukemia
CD5 + IgM+ B cells are oligoclonal and can be transferred to wild type mice. a Two-parameter flow cytometry of the expression of CD19 and CD5 in blood cells of 35 wk-old control and Rosa26-RRAS2fl/flxmb1-Cre (mb1-Cre+) mice. b Quantification of the number of CD5 + IgM+ B cells (CD19+) in the blood of 30–35 wk-old control (n = 10) and mb1-Cre (n = 47) mice. Unpaired t-test with Welch’s correction. c Dot plot representation of the quantification of CD5 + IgM+ B cells (CD19+) in the blood of mb1-Cre mice (n = 14) and its evolution over time. Data points were adjusted to an exponential growth. d Kaplan-Meier survival plot of Rosa26-RRAS2fl/flxmb1-Cre (mb1-Cre+) mice (n = 22) and WT control C57BL/6 J mice (n = 40) allowed to age in the same housing conditions. Median survival for mb1-Cre = 13.57 months; median survival for WT controls = 32.39 months. P < 0.0001, long-rank Mantel-Cox test and Gehan-Breslow-Wilcoxon test. e Experimental setup for the adoptive transfer experiment. Total B cells from mouse donors bearing the hematopoietic cell marker allele CD45.2 were purified and inoculated i.v. in the tail vein of sublethaly irradiated wild type recipient of the same strain (C57BL/6) but bearing the CD45.1 allele. Donor control mice refer to Rosa26-RRAS2fl/fl without Cre recombinase 11 wk-old and the donor problem mice corresponded to 16 wk-old Rosa26-RRAS2fl/fl x mb1-Cre. f Quantification by flow cytometry of the expression of CD45.2 and CD5 within the CD19+ population of inoculated CD45.1+ mice (n = 8) and bled at the indicated time points. In this figure, control mice refer to Rosa26-RRAS2fl/fl without Cre recombinase. Two-way ANOVA test. g PCR results of using specific forward oligonucleotides for different VH families and a constant reverse oligonucleotide against the J region of the immunoglobulin heavy chain gene. The specific VH families analyzed in each case are indicated on top of each section of the agarose gel. H2O is used as a negative control. In each VH family, BM corresponds to bone marrow cells of a wild-type 12 wk-old mouse, T corresponds to sorted naïve thymocytes (no recombination) from a wild type 6 wk-old mouse, and each column corresponds to the spleen of an individual mb1-Cre mouse of ages between 35 and 40 weeks. h Representative flow cytometry plot showing the presence of GFPlow and GFPhigh populations in the spleen of a 30 wk-old Rosa26-RRAS2fl/flxmb1-Cre mouse. i RT-qPCR for the differentially expressed RRAS2 gene in the CD19 + CD5+ GFPhigh and GFPlow populations sorted from spleens of n = 7 30–35 wk-old Rosa26-RRAS2fl/flxmb1-Cre mice compared to follicular B cells of n = 6 30 wk-old WT mouse controls. One-way ANOVA test. j Dot plot representation of GFPhigh CD5+ leukemic B cell evolution in blood from mb1-Cre mice over time, showing each mouse individually (n = 14). Data points were adjusted to a linear fit. k Paraffin-embedded lung tissue section stained with hematoxylin-eosin, showing representative lymphocytic infiltration in one out of three 35 wk-old Rosa26-RRAS2fl/flxmb1-Cre mice. l Percentage of IgM + CD5+ B cells within the lymphoid population infiltrating the lungs of n = 2 35 wk-old Rosa26-RRAS2fl/flxmb1-Cre mice compared to n = 2 32 wk-old Rosa26-RRAS2fl/fl control ones. m Bar plot of the distribution of lymphoid cells between the GFPlow and GFPhigh populations in the lungs and lymph nodes (LN) of 20 wk-old Rosa26-RRAS2fl/flxmb1-Cre mice (n = 2)