Tet2 deficiency leads to the accumulation of clonal B220^lowB-cell population in a cell-autonomous fashion. (A) Monthly monitoring of blood cell count in Tet2^CD19-Cre− and Tet2^CD19-Cre+ mice. ***P < .001. (B) Monthly monitoring of CD19⁺ B220^low abnormal cell population in the blood of Tet2^CD19-Cre− and Tet2^CD19-Cre+ mice (left panel). Significance was tested using an unpaired Student t test. Time-dependent representation of the proportion of the B220^low population within the circulating B cells (CD19⁺) in the same mice as in panel A (right panel). Significance was tested using an unpaired Student t test. (C) Spleen weight of sacrificed mice at various time points was measured for both types of mice. Significance was tested using an unpaired Student t test. (D) Immunostaining of total splenocytes (polychromatic dot plot) from a representative sick Tet2^CD19-Cre+ mouse (same genotypes as above). Splenocytes were first stained with B220 and CD19 to define the B220⁺ and B220^low population of CD19⁺ cells (left polychromatic dot plot). Other dot plots show B220 expression together with the indicated antibody on the x-axis of the overlaid B220⁺CD19⁺ population (green) and B220^lowCD19⁺ population (violet). (E) Analyses of BCR rearrangement clonality in abnormal Tet2^CD19-Cre+ B cells. V-J junctions were PCR-amplified from cDNA of sorted B cells using VH family primers (VH1, VH2, VH3, VH5, VH6, and VH7, as indicated) and conjugated consensus JH primer²²  to assess the IgH CDR3 diversity. Top panels show Results shown are obtained from sorted Tet2^CD19Cre− B splenocytes (B220⁺CD19⁺) (top) and from age-matched Tet2^CD19-Cre+ sorted abnormal B cells (B220^lowCD19⁺) (bottom). (F) Transcription of selected genes important for B-cell differentiation differs between normal WT and Tet2-deficient tumor B cells. Histograms show quantitative reverse transcription (qRT)–PCR analysis results ΔΔCT-normalized by Abl1 expression levels. Results are an average of 3 independent duplicate experiments. The differences were validated in an extension cohort for 8 of the 11 genes. Significance was tested using the Mann-Whitney U test. *P < .05; **P < .01; ***P < .001. ns, not significant.