Fig 1.
Transient increase of DN1 and decrease of DN2, DN3 showed in the Foxn1lacZ thymus.
(A). Representative profiles of CD25 and CD44 on gated total CD4-CD8- DN thymocytes at day 21 in +/Z, Z/Z and Z/N mice. (B-E). Percentages of DN1, DN2, DN3, and DN4 populations at various time points are summarized in the histogram. Each assay and time point represents at least 5 individuals. One-way ANOVA results between Z/Z, Z/N test groups and +/Z control group at various time point: *P <0.05, **P <0.01, ***P <0.001. Bars indicate means ± SEM.
Fig 2.
The kinetic change of DN1a,b, and DN1c,d subsets showed an increased B potential in the thymic progenitors in Foxn1lacZ mutants.
(A). The representative profiles of CD24 and cKit on gated Lin-DN1 population at day 14 in +/Z, Z/Z, and Z/N mice. DN1a-e subsets gates in +/Z. (B, C). Percentage of DN1a,b (DN1a plus DN1b) (B) and DN1c,d (DN1c plus DN1d) (C) at various analysis time points. (D, E). Total cell number of DN1a,b (D) and DN1c,d (E) at various analysis time points. (F). The ratio of DN1c,d versus DN1a,b cells. Each assay and time point represented at least three individuals. One-way ANOVA results between Z/Z, Z/N test groups and +/Z control group at various time point: *P <0.05, **P <0.01, ***P <0.001. Bars indicate means ± SEM.
Fig 3.
Thymic B cells formed a transient wave around day 28 in the thymus.
(A). Histograms show the representative staining of CD19, NK1.1 and CD3 on gated DN1 thymocytes in +/Z, Z/Z and Z/N mice. (B). The summary of the percentage of CD19+, NK1.1+ and CD3+ cells in the DN1 subset. (C-E). Kinetic change of the percentage of thymic B cells in DN1 subset cell (C), total thymic B-cell number (D), and the ratio of thymic B cells to total thymocyte number (E) in +/Z, Z/Z, and Z/N mice. Each assay time point represents at least five individuals. (F-G). The kinetic change of the percentage of total B cells (CD19+), mature B cells (CD19+CD24lo) and immature B cells (CD19+CD24hi) in spleen (F) and BM (G) cells in BL6 mice. Each assay time point represents at least three individuals. One-way ANOVA results between Z/Z or Z/N test group and +/Z control group at various time point: *P <0.05, **P <0.01, ***P <0.001, ****P<0.0001. Bars indicate means ± SEM.
Fig 4.
Young BM cells generated more thymic B cells than adult BM cells due to an age-dependent B potential revealed by the Foxn1lacZ thymus.
1x107 T and B-depleted BM cells from age indicated CD45.1 mice were transferred into sub-irradiated CD45.2 +/Z, Z/Z, and Z/N mice. Thymic B cells from the donor (CD45.1) and host (CD45.2) were analyzed 12 days later. (A). Gating for donor and host CD19+ cells in the DN1 subset. (B). Day 14 wild-type BM cells were transferred into 2-month old mice, genotypes of host recipients are indicated to the left of each pair of panels. Profiles of CD19 and CD24 staining within the DN1 subset are shown. Percentages of CD19+ cells in DN1 subsets in donor and host are summarized in the graphs to the right. Data are representative of three independent experiments, (+/Z: n = 7, Z/Z: n = 5, Z/N: n = 4). (C). 3 month-old wild-type BM cells were transferred into 20-day old Z/N mice. Data are representative of two independent experiments, (n = 4). (D). Competitive transfer experiment of mixed BM cells. 5 x 106 BM cells from 3 months CD45.1mice and 5 x 106 BM cells from day 20 CD45.2 mice were mixed and then transferred into day 20 lethal irradiated Z/N (n = 4) mice. The CD19 and CD24 staining profile were measured on the DN1 subset 12 days later. (E). BM cells from 14, 22-day and 2.5-month were transferred into sublethally-irradiated 20-day old Z/N mice, and profiled for CD19 and CD24 staining. Percentage of donor CD19+ cells in DN1 subsets and total thymic B cells are shown in the bar graphs. Data are representative of four independent experiments, (14d n = 5, 22d n = 3, 2.5M n = 4). Student’s t-test results: *P <0.05, **P <0.01, ***P <0.001. Bars indicate means ± SEM.
Fig 5.
Most thymic B cells in the Foxn1lacZ thymus have an immature phenotype.
(A). Representative profiles of CD19 and B220 staining (left panels) and CD24 and CD43 (right panels) on gated B cells in thymocytes from +/Z, Z/Z, and Z/N mice as indicated to the left. (B,C) Same analysis of splenocytes (B) and BM cells (C) from +/Z mice. (D). The ratio of CD19loB220lo immature B (im-B) to CD19hiB220hi mature B (m-B) cells in thymus, spleen and BM cells. (E-F). The percentage of CD43-/loCD24lo (E) and CD43lo/hiCD24hi (F) B subsets in total thymic B cells. (G). Histograms show overlapping profiles of Ly51, CD93, CD25, MHCII or IgM staining on gated thymic B cells from +/Z, Z/Z and Z/N mice. All assays were performed at day 30. Data are representative of five individual experiments, (+/Z: n = 7, Z/Z: n = 5, Z/N: n = 8). One-way ANOVA: *P <0.05, **P <0.01, ***P <0.001, ****P<0.0001. ns: not significant. Bars indicate means ± SEM.
Fig 6.
Thymic progenitor B cells accumulate in the cortex and originate in the Foxn1lacz mutant thymus.
(A). Immunofluorescence staining of sections from +/Z, Z/Z and Z/N thymus at day 30 for CD19 (red) and β5t (green) (top row), or CD19 (red) and K5 (green) (bottom row). a,b. Insets show digitally enlarged images of thymic B cells in the medulla in Z/Z (a) and subcapsular zone in Z/N mice (b). Scale bar = 0.1mm. (B). Analysis of CD45.1+ wild-type donor progenitor B cells (B220+CD19+ CD24+CD43+/loIgM-) in thymus, spleen, and BM at 21 days after transfer into sublethally irradiated adult CD45.2 +/Z and Z/N mice (top panels: PBS injection control; middle and bottom panels: CD45.1+ cells transferred into +/Z and Z/N mice). (C). Summary of CD45.1 cell numbers from the experiment in (B). Data are representative of two independent experiments, (PBS: n = 3, cells in +/Z: n = 2, in Z/N: n = 4). One-way ANOVA: **P <0.01, ***P <0.001. ns: not significant. Bars indicate means ± SEM.
Fig 7.
A decrease of MHCII, Dl4, and Ccl25 but an increase in Il7 in the Foxn1lacZ thymus.
(A). The gate for CD45-Epcam+MHCII+ TEC. (B-E). TECs sorted from day 8, 15, 22, 30 and 40 thymi were analyzed for gene expression of MHCII (B), Dl4 (C), Ccl25(D) and Il7 (E) respectively by qPCR. The gene expression in TECs at all time points were normalized to day 8 +/Z control values, which were set as 1. Each time point represents at least five individuals. (F). TECs were sorted into MHCIIlo and MHCIIhi populations from day 22 thymi. Il7 gene expression in each sorted population is shown to the right. (G). TECs were sorted into four populations defined as MHCIIlo, MHCIIhi, cTECs (UEA-1-), and mTECs (UEA-1+) from day 30 thymi. Il7 expression levels in these sorted populations is shown in the histogram. (+/Z: n = 2, Z/Z: n = 3 in F and G). Student’s t-test results between +/Z control and Z/Z cells: *P <0.05, **P <0.01, ***P <0.001. Bars indicate means ± SEM.