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Data supplements

Article Figures & Data

Figures

  • Figure 1.

    Impaired CD8+ and γδ T-cell development in Stat5a/bnull/null survivor mice. (A) Picture of a Stat5a/bnull/null survivor mouse (left) compared with a Stat5a/b+/+ littermate at the age of 4 weeks. Representative body weight values and numbers of Stat5a/bnull/null mice and littermates surviving up to day 8, weaning (day 21), or day 42 are depicted. (B) Numbers of CD4/CD8/γδTCR+ T lymphocytes in thymi of Stat5a/bnull/null, Stat5a/bΔN/ΔN, and respective littermate controls. (C) Flow cytometric analysis of CD4+, CD8+, and CD4+/CD8+ cells in thymi and lymph nodes of 5 Stat5a/bnull/null survivors and 2 individual Stat5a/bnull/+ and 2 Stat5a/b+/+ littermate controls. Data are summarized in bar graphs. Due to the small size of thymi and lymph nodes, cells were pooled and did therefore not allow generation of error bars (B-C).

  • Figure 2.

    Impaired CD8+ T-cell development in Stat5a/bf/f lck-cre mice. (A) Splenic cells of 3 individual Stat5a/bf/f lck-cre (nos. 1-3) and 2 Stat5a/bf/f (nos. 4-5) mice were magnetically sorted for Thy1.2+ cells, and Stat5a/b expression was assessed by Western blot analysis. (B) Representative flow cytometric profile of CD4+, CD8+, and CD4+/CD8y cells in thymus, blood, spleen, and lymph nodes of Stat5a/bf/f lck-cre mice and Stat5a/bf/f controls. Asterisks denote significant difference as determined by a paired t test. (C) Analysis of transcriptional expression of pim-1, CIS, and cyclin D2 genes by semiquantitative RT-PCR. Splenic T cells were stimulated for 4 hours with α-CD3 (1 μg/mL) and human IL-2 (hIL-2; 1000 U/mL) to induce Stat5a/b target gene transcription. (D) Schematic model for the role of Stat5a/b in T-cell developmental choices. Stages affected in Stat5a/bnull/null survivor mice and/or Stat5a/bf/f lck-cre mice are indicated. The time point of Cre-recombinase activation under the control of the distal lck promoter is also depicted.

  • Figure 3.

    B-cell development is arrested at the pre–pro-B-cell stage in Stat5a/bnull/null survivor mice. (A) Percentages of pre–pro-B, early pro-B, and late pro-B cells in bone marrow and (B) mature B cells in spleen and lymph node of 5 Stat5a/bnull/null survivors compared with 2 Stat5a/bnull/+ and 2 Stat5a/b+/+ controls. Due to the small body size, bone marrows were pooled and therefore did not allow generation of error bars. (C) Schematic model for maturation of B-cell developmental fractions A-F. As indicated, individual maturational stages were distinguished by differential surface expression of B220, CD43, CD19, BP-1, IgM, and IgD. The different blocks in Stat5a/bΔN/ΔN and Stat5a/bnull/null mice are indicated by vertical lines. CLP indicates common lymphoid progenitor.

  • Figure 4.

    B-cell development is arrested at the pre–pro-B-cell stage in Stat5a/bnull/null fetal liver–derived cultures. Fetal livers of 2 embryos of each genotype were pooled (ED 14) and cocultivated on an OP-9 fibroblast feeder layer in the presence of IL-7, Flt-3L, and SCF (10 ng/mL each). Outgrowth of pro-B-cell stages (fractions A-C), immature (fraction E), and mature (fraction F) B cells over a 12-day period is depicted.

  • Figure 5.

    Abelson-induced transformation is dependent on Stat5a/b in vitro. (A) Ab-MuLV–induced colony formation of Stat5a/b+/+, Stat5a/bnull/+, and Stat5a/bnull/null fetal liver cells in methylcellulose. Single-colony pictures of each phenotype are depicted in the bottom panels. Stat5a/bnull/null cells showed no ability to form growth factor–independent colonies. (B) Summary of data obtained from Ab-MuLV–induced colony formation assays represent means ± SEM of 4 embryos per genotype (each performed in triplicates). (C) Surface expression of B-lineage markers was verified by flow cytometric analysis (right; data of one representative CD19+ CD43+ colony is shown). (D) [3H]thymidine incorporation of fetal liver–derived Stat5a/b+/+ and Stat5a/bnull/+ Ab-MuLV–transformed cell lines. Stat5a/b-deficient fetal livers did not give rise to stable transformed cell lines. Data represent means ± SEM of 2 cell lines per genotype. cpm indicates counts per minute. (E) Ab-MuLV–induced colony formation of Stat5a/b+/+ (n = 2), Stat5a/bnull/+ (n = 2), and Stat5a/bnull/null (n = 5; pooled) survivor bone marrow cells in methylcellulose. Stat5a/bnull/null survivor cells showed no ability to form growth factor–free colonies. Experiment was performed in triplicates. Asterisks denote significant differences as determined by a one-way ANOVA followed by a Tukey test (A,C) or a paired t test (B).

  • Figure 6.

    Abelson-induced transformation is dependent on Stat5 in vivo. (A) Kaplan-Meier plot of Rag2–/– mice that received a transplant of either Stat5a/b+/+, Stat5a/bnull/+, or a 4:1 mixture of Stat5a/bnull/null and Stat5a/b+/+ freshly Ab-MuLV–transformed bone marrow cells (5 mice/group; 1 × 106 cells each mouse). Genotyping PCR analysis of mice used for 4:1 mixture is depicted. wt indicates wild type. (B) Immunoblotting for Stat5a/b of leukemic cells derived from bone marrow of Rag2–/– mice that received a transplant of either Stat5a/b+/+ or a 4:1 mixture of Stat5a/bnull/null/Stat5a/b+/+ bone marrow. (C) PCR analysis of ex vivo–derived cell lines. Representative data of bone marrow (BM)–, peripheral blood (PB)–, spleen-, and lymph node (LN)–derived leukemic cell lines of one Rag2–/– mouse that received a transplant of a 4:1 mixture of Stat5a/bnull/null/Stat5a/b+/+ bone marrow cells, which was later killed. All cultures derived from Rag2–/– mice that received transplants of Stat5a/bnull/null/Stat5a/b+/+ cells were Stat5a/b+/+ as determined by PCR analysis.

Tables

  • Table 1.

    Mean ratio of CD4+ to CD8+ T cells in thymi, peripheral blood, spleen, and lymph nodes of Stat5a/bf/f and Stat5a/bf/f lck-cre mice

    Ratio of CD4+/CD8+ cells
    S5f/fS5f/f lck-cre
    Thymus 4.1:1 11:1
    Lymph node 1.9:1 5:1
    Spleen 2.2:1 8:1
    Peripheral blood 1.5:1 6:1
  • Table 2.

    Ability of fetal liver and bone marrow cells to form bcr/abl p185- or Ab-MuLV–induced colonies and stable cell lines

    Colony formationTransformed cell lines
    bcr/abl transformation
        S5ΔN/+, BM Yes Yes
        S5ΔN/ΔN, BM Yes Yes
        S5+/+, FL Yes Yes
        S5null/+, FL Yes Yes
        S5null/null, FL No No
    Ab-MuLV transformation
        S5ΔN/+, BM Yes Yes
        S5ΔN/ΔN, BM Yes Yes
        S5+/+, FL Yes Yes
        S5null/+, FL Yes Yes
        S5null/null, FL No No
        S5+/+, BM Yes Yes
        S5null/+, BM Yes Yes
        S5null/null, BM No No