Fig 2: BMI1 Prevents the Upregulation of p53 Signaling and Maintains Large Pre-B Cell Cycling(A) RNA sequencing (RNA-seq) MA (log ratio and mean average) plot of Bmi1+/+ versus Bmi1-/- pro-B cells. Known targets of p53 are depicted in red.(B) qRT-PCR expression analysis of indicated genes from sorted pro-B cells of indicated mice. Each point represents one animal; the line represents the mean. n = 4 for Bmi1+/+ animals; n = 5 for Bmi1-/- animals.(C) Frequency of apoptotic (as indicated by annexin V positivity) pro-B cells and pre-B cells of indicated mice. Each point represents one animal; the line represents the mean. n = 5 for each genotype.(D) Frequency of large pre-B cells present in the pre-B cell compartment of indicated mice. Each point represents one animal; the line represents the mean. n = 6 for each genotype.(E) FACS plot of developing B cells in the bone marrow of indicated mice. Shown is the frequency of each cell population in the bone marrow of indicated animals. Representative of 3 independent experiments.(F) Frequency of B220+ cells in the spleen and peripheral blood of indicated mice. Each point represents one animal; the line represents the mean. n = 3 for each genotype.***p < 0.001, **p < 0.01, *p < 0.05. PB, peripheral blood.See also Figure S4 and Table S1.

Fig 3: ASXL1-MT expands the pLT-HSC compartment along with dysregulated hematopoiesis during aging.a Enumeration of white blood cells (WBC), hemoglobin (Hb), and platelets (Plt) in peripheral blood of aged Vav-Cre ASXL1-MT KI mice (n = 13 (Control) and 12 (ASXL1-MT)). b The frequency of myeloid cells (CD11b+), B cells (B220+), and T cells (CD3+) in peripheral white blood cells of aged Vav-Cre ASXL1-MT KI mice (n = 13 (Control) and 12 (ASXL1-MT)). c The frequency of LSK cells, MPPs, ST-HSCs, and LT-HSCs in bone marrow cells of young and aged Vav-Cre ASXL1-MT KI mice (n = 6 (Young) and 5 (Aged)). Representative FACS plot (left panel) and summarized data (right panel) are shown. d The experimental design for competitive transplantations. 200 LT-HSCs isolated from aged Vav-Cre ASXL1-MT KI mice and age-matched control mice were transplanted into lethally irradiated recipient mice with 4 × 105 whole bone marrow cells. e Levels of donor chimerism in peripheral blood were analyzed at the indicated weeks after transplantation (n = 7 (Control) and 8 (ASXL1-MT)). f 6 months after transplantation, the frequency of donor-derived cells in peripheral blood (PB), whole bone marrow cells, Lin- cells, Lin-c-kit+Sca1-(LK) cells, MPPs, ST-HSCs, and LT-HSCs were analyzed (n = 6 (Control) and 8 (ASXL1-MT)). Data are mean ± s.e.m. Data are mean ± s.d. unless otherwise noted. Data are assessed by two-tailed Student’s t-test (a, b, e, f) or one-way ANOVA with Tukey–Kramer’s post-hoc test (c). *P = 0.05, **P = 0.01, ***P = 0.001.

Fig 4: Generation of a KI mouse model bearing a pre-rearranged PGT121 ? Strategy for the insertion of PGT121 pre-rearranged VJ into the mouse Ig ? locus. Targeting DNA donor with 5' (3.9 kb) and 3' (3.9 kb) homology arms to the C57BL/6 WT mouse Ig ? locus, murine promoter, leader, and the human PGT121 light chain VJ sequences are located between two homology arms. Two sgRNAs, 18 and 11, were targeted at J4-J5 region of Ig ? locus. CRISPR/Cas9-mediated HDR leads to the insertion of the promoter and PGT121 sequences into the C57BL/6 mouse genome. V segments, enhancers, and the kappa constant regions are shown in gray and labeled appropriately. Yellow rectangles represent J segments; dark blue oval represents the V?4-53 promoter (P); light blue line represents the inserted segment and red rectangles show the rearranged PGT121 VJ. “T” represents TaqMan probe. WT probes were used for the detection of WT allele, Leader probes were used for the detection of the 5'end of the insertion, the specific probes were used for the detection of pre-arranged VJ insertion for PGT121 (probe sequences, see Appendix Table S1).A fragment of genomic DNA (2.2 kb) was amplified by PCR and in vitro sgRNA-guided Cas9-mediated cleavage assay was performed with each of the sgRNAs. sgRNA-targeting sites are indicated by arrows, genomic DNA size is indicated by asterisk.B220+ single B cells from peripheral blood of three PGT121 LC KI naïve mice were sorted. B220+ B-cell populations and their frequencies are shown in FACS plots (left panel). Ig light chains from single-cell sorted B cells were PCR amplified and sequenced. The resulting IGLV libraries were compared to the PGT121 LC reference sequence. The pie charts indicate the frequency of IGLV sequences identical to human PGT121 (red) and mouse IGLV (gray).

Fig 5: GC responses on Day 8 in adoptively transferred mice with rare precursor frequency Germinal center (GC) response induced by eOD-GT8 60mer immunization in WT mice adoptively transferred with CD45.2 B cells from CLK21, CLK09, CLK19 mice on Day 8. 8-week-old CD45.1 mice were transferred with 5 × 105 (1 in 104) of isolated CD45.2 B cells from CLK21, CLK09 and CLK19 KI one day before the immunization with eOD-GT8 60mer, on day 8, the splenocytes were isolated and detected by FACS. First column shows the frequency of total GC (CD38loCD95+) among B cells gated from SSL, the second column shows the frequency of CD45.2 B cells among total GC, third column shows the frequency of eOD-GT8 binding B cells among GC CD45.2 B cells; fourth column shows the frequency of eOD-GT8 binding B cells that do not bind the VRC01 epitope-knockout, eOD-GT8-KO; diagrams at right show eOD-GT8 KD affinities for CLK21 (green, affinity 440 nM), CLK09 (blue, affinity 350 nM) and CLK19 (orange, affinity 1.8 µM).Quantification of GCs, CD45.2 cells and CD45.2 binder frequency in adoptively transferred mice. X-axis represents CLK21, CLK09 and CLK19 adoptively transferred mice group that immunized with eOD-GT8 60mer. Y-axis represents the frequency of total GC (left) among B cells, the frequency of CD45.2 cells within GCs and the frequency of eOD-GT8-CD45.2 binders. Each circle represents one mouse. n = 5 mice/group for CLK21 and CLK09, n = 3 mice/group for CLK19. Bars indicate geometric mean and geometric SD from mice in each group.Immunohistochemistry of Day 8 spleen sections for host mouse receiving 5 × 105 isolated CD45.2 B cells from CLK09 (left) and CLK19 (right) KI mouse. Green, B220; Blue, CD3; White, CD45.2; Red, GL7. White arrows indicate the CD45.2 B cells.