Fig 2: Rad21 deficiency increases self-renewal and leads to impaired and myeloid-skewed differentiation of HSPCs. (A–D) Freshly isolated LSK cells were infected with an shRNA against Rad21 shRad21 or a control virus (shLuci) in culture and transplanted into lethally irradiated mice together with noninfected LSK cells from the same culture. (A and B) The number of HSCs (GFP+CD150+CD34-LSK cells) derived from infected LSK cells in the primary recipients (A) and the secondary recipients (B) that were transplanted with total BM cells of the primary recipients at 4 mo after the first transplantation. The dots represent individual recipient mice (n = 4–5 mice per group per experiment, n = 3 independent experiments, one of the three repeat experiments is shown, the other experiments showed similar results). Statistical values were calculated by Welch’s t test after log transformation. (C) The chimerism of GFP+ cells from the primary recipients was analyzed by FACS at 4 mo after transplantation in the indicated populations. HSC: CD150+CD34-LSK; MPP: CD34+LSK; CMP: common myeloid progenitor (Lin-c-Kit+Sca-1-CD34+Fc?R-); GMP: granulocyte macrophage progenitor (Lin-c-Kit+Sca-1-CD34+Fc?R+); MEP: megakaryocyte erythrocyte progenitor (Lin-c-Kit+Sca-1-CD34-Fc?R-), CLP: common lymphoid progenitor (Lin-c-KitlowSca-1lowIL-7Ra+Flt3+), Lin-: lineage- cells; BM: total BM cells. n = 4–5 mice per group per experiment, three independent experiments, one of the three experiments is shown, the other experiments showed similar results. (D) PB cells from the primary recipients were analyzed by FACS. The histogram shows the percentage of myeloid and lymphoid cells in GFP+CD45+ cells in the PB at the indicated time points after transplantation (n = 4–5 mice per group for each experiment, n = 3 independent experiments, one of the three experiments is shown; the other experiments showed similar results). Statistical significance for myeloid cells was assessed by Welch’s t test after logit transformation of the data. (E and F) LSK cells from young (2 mo old) wild-type mice were transduced with shLuci or shRad21 virus followed by transplantation into 2–3-mo-old recipients. 8 wk after transplantation, recipient mice were sacrificed and total BM was transplanted into secondary recipients. 4 wk after secondary transplantation, BM cells were analyzed to investigate the chimerism of shLuci- or shRad21-LSK cells in BM. Chimerism of shLuci- or shRad21-infected LSK cells in PB was monitored every 4 wk after each round of transplantation. (E) The data show the relative change of GFP+ cells in PB from the recipients at indicated time points after transplantation. The initial transduction efficiency was 71% for shLuci-LSK and 54% for shRad21-LSK (n = 5 recipient mice per group, per experiment in the primary transplantation, n = 10 recipient mice per group, per experiment in the secondary transplantation, n = 2 independent experiments, one of the two experiments is shown; the other experiment showed a similar result). (F) The histograms depict the chimerism of GFP+ cells in the indicated populations of the secondary recipients at 4 wk after transplantation. The dots represent individual mice (n = 4 mice per group for each experiment, n = 2 independent experiments, one of the two experiments is shown, the other experiment showed a similar result). Data in all panels of this figure depict mean ± SD; *, P < 0.05; ***, P < 0.001; ****, P < 0.0001.

Fig 3: Mll-AF4+VEC-Cre+ aging mice have a higher proportion of CD3+ T-lymphoid cells in the spleen. (A) Experimental layout. Proportions of (B) CD11b+Gr1+, (C) B220+CD19+IgM+, (D) B220+CD19+IgM–, and (E) CD3+ cells in the peripheral blood of the control and Mll-AF4+VEC-Cre+ aged cohort at two time points. Proportions of (F) CD11b+Gr1+, (G) B220+CD19+IgM+, (H) B220+CD19+IgM–, and (I) CD3+ in the bone marrow and spleen of the control and Mll-AF4+VEC-Cre+ cohort at the end of study (18 months old). Peripheral blood counts of (J) white blood cells, (K) red blood cells, and (L) platelets of the control and Mll-AF4+VEC-Cre+ cohort at the end of study (18 months). Statistical differences were assessed using a nonparametric Mann–Whitney U test with a bilateral p value (*p < 0.05, **p < 0.01, ***p < 0.001).

Fig 4: Vav-Cre Mta2fl/fl Mice Recapitulate the BM B cell Developmental Defects of Mta2?/? Mice(A) BM cells from mice with indicated Mta2 status stained for immature B (B220loIgM+), mature B (B220hiIgM+), pro-B (B220+IgM-CD43+), and pre-B (B220+IgM-CD43-) cell populations. Numbers indicate percentages of specific cell subsets in parental populations.(B) Cell numbers of different BM B cell subsets (from two femurs) in 2- to 3-month-old Vav-Cre Mta2fl/fl and littermate or age-matched control mice (n = 16 for Vav-Cre Mta2fl/fl and control mice; t test; **p < 0.01, ***p < 0.001).(C) qPCR results indicating efficiencies of Vav-Cre-mediated deletion of Mta2 allele in indicated cell populations (n = 4 for each cell subsets; t test; ***p < 0.001).(D) Representative FACS data showing percentages of Hardy Fr. A to C within the pro-B population in Vav-Cre Mta2fl/fl and control mice. The average numbers of each fractions are plotted (n = 9 for each genotype; t test; **p < 0.01).(E) qPCR results indicating relative abundances of different immunoglobulin heavy chain gene rearrangements in pro-B cells isolated from mice with indicated genotypes (n = 4 for each genotype).(F) Representative FACS data showing percentages of large (L) and small (S) pre-B (B220+IgM-CD43-) cells in Vav-Cre Mta2fl/fl and control mice. The average numbers of each type of pre-B cells from mice with indicated genotypes are plotted (n = 13 for Vav-Cre Mta2fl/fl and littermate control mice; t test; ***p < 0.001).(G) qPCR results indicating relative abundances of different VJ rearrangements in small pre-B cells isolated from mice with indicated genotypes (n = 4 for each genotype; t test; *p < 0.05, **p < 0.01, ***p < 0.001).

Fig 5: Chronic Infection Is a Potent Long-Term Driver of B Cell Responses(A) We infected syngeneic recipient mice with rARM* or rCl13* on day 0, and 20 days later, we adoptively transferred CTV-labeled KL25HL-AIDrep B cells. TAM was administered on the day of transfer (day 20) and on day 23.(B and C) Five and 15 days after adoptive transfer (day 25 and day 35), we analyzed KL25HL-AIDrep B cells by flow cytometry for AID reporting (EYFP+) and GC differentiation (GL7 expression). Representative FACS plots in (B) are gated on adoptively transferred proliferated B cells (CD45.1+ CD45.2- CTVlo B220+ CD138- cells, gating strategy in Figure S4). The average proportional representation of EYFP+GL7– (red), EYFP+GL7+ (orange), EYFP–GL7+ (magenta), and EYFP–GL7– (blue) subsets is displayed in pie charts. Absolute numbers of these same cell populations are shown in (C). Bars represent means ± SEM, n = 3–5.(D) The concentration of KL25 IgG in the serum of KL25HL-AIDrep B cell recipients was determined by GP-1 ELISA. Background GP-1-specific IgG levels in control mice without KL25HL-AIDrep B cell transfer were at least 4-fold lower than in the respective groups of recipients. Symbols show individual mice.(E) Histological sections from spleens of rCl13* and rARM* infected mice on day 25. Scale bar: 50 µm. n = 3, N = 2.Two-way ANOVA with Bonferroni’s post-test for multiple comparisons (C), unpaired two-tailed Student’s t test (D). *p < 0.05, **p < 0.01. See also Figure S4.