Fig 2: miR-17 plays a central role in regulating the pro-B to pre-B transition.(a) Outline of experimental strategy. HSCs were enriched from the Mb1tKO donors, transduced with miRNA-expressing lentiviruses and mixed with bone marrow cells from the congenic WT mice, to generate mixed bone marrow chimeras. Reconstituted mice were analysed 8 weeks later. Ratios of lentivirus-transduced cells (GFP+) versus the WT donor-derived cells (CD45.1+) in pro-B cells (B220+cKit+) and pre-B cells (B220+CD25+) were quantified. (b) Representative flow cytometry plots of pro-B cells and pre-B cells in the bone marrow of recipient mice of indicated groups, showing contributions from WT-derived cells (CD45.1+) and lentivirus-transduced cells (GFP+). A reconstitution group with control virus-transduced CD45.2+ WT HSCs mixed with the CD45.1+ WT BM cells is included for comparison. (c) Changes in ratios of GFP+ cells versus CD45.1+ cells during the pro-B to pre-B transition. The dash line marks the pre-B/pro-B ratio in Mb1tKO mice. Data are representative (b) or pooled (c) from 6 independent experiments (mean±s.e.m. in c) with n=3 (WT-Vector), 8 (tKO-Vector and 4 × 17), 11 (FL), 7 (del17 and 4 × 19), 4 (del18, del19 and del92) or 6 (4 × 18 and 4 × 92) in b,c.

Fig 3: Zfp335-deficient thymocytes undergo a developmental block during DN3 to DN4 transition.(A–C) Thymi were harvested from 6- to 8-week-old WT and KO mice. The different stages of DN thymocytes in WT and KO mice were measured by flow cytometry (n = 4). (A) Representative FACS plots of DN1 (CD25-CD44+), DN2 (CD25+CD44+), DN3 (CD25+CD44-), and DN4 (CD25-CD44-) thymocytes. (B) The percentages of DN3 and DN4 thymocytes. (C) The numbers of DN3 and DN4 thymocytes. (D–E) WT and KO DN3 thymocytes were cultured with OP9-DL1 feeder cells in vitro in the presence of IL-7 (1 ng/ml) and Flt3L (5 ng/ml) for 2 days. The expression of CD44 versus CD25 was measured by flow cytometry (n = 3). (D) Representative FACS plots of DN3 and DN4 thymocytes. (E) The ratio of DN3 to DN4 thymocytes 2 days post culture in vitro. (F–G) A mixed population of WT and KO DN3 thymocytes at a 1:4 ratio was cocultured with OP9-DL1 feeder cells in vitro in the presence of IL-7 (1 ng/ml) and Flt3L (5 ng/ml) for 2 days. The expression of CD44 versus CD25 was measured by flow cytometry (n = 3). (F) Representative FACS plots of DN3 and DN4 thymocytes. (G) The ratio of DN3 to DN4 thymocytes 2 days post culture in vitro. (H, I) Full chimeric mice were generated by transplanting WT (CD45.1+) or KO (CD45.2+) bone marrow progenitor cells into lethally irradiated (8.5 Gy) WT recipients (CD45.1+CD45.2+). Six weeks after transplantation, thymi from recipient mice were harvested. The expression of CD44 versus CD25 was measured by flow cytometry (n = 4). (H) Representative FACS plots of DN3 and DN4 cells. (I) The ratio of DN3 to DN4 thymocytes. (J, K) Full chimeric mice were generated by transplanting a mixed population of WT (CD45.1+) and KO (CD45.2+) bone marrow progenitor cells at a 1:4 ratio into lethally irradiated (8.5 Gy) WT recipient mice (CD45.1+CD45.2+). Six weeks after transplantation, thymi from recipient mice were harvested. The expression of CD44 versus CD25 was measured by flow cytometry (n = 4). (J) Representative FACS plots of DN3 and DN4 thymocytes. (K) The ratio of DN3 to DN4 thymocytes. Results represent three independent experiments. *p < 0.05, **p < 0.01, and ***p < 0.001. Figure 3—source data 1.Figure 3B, C. The percentages and numbers of DN3 and DN4 thymocytes from WT and KO mice.Figure 3E–G. The ratio of DN3 to DN4 thymocytes 2 days post culture in vitro. Figure 3I–K. The ratio of DN3 to DN4 thymocytes in chimeric mice transferred with WT and KO bone marrow cells.

Fig 4: 89Zr-CD25 IgG uptake in mouse cells in vitro and in mouse lymphoma in vivo. (A) CD25 expression (left) and anti-mouse 89Zr-CD25 IgG binding (right) in mouse thymus Treg lymphocytes and EL4 mouse lymphoma cells. Cell uptake was compared by using equivalent amounts of cells. Blocking was done by adding 0.5 μM of unlabeled anti-CD25 IgG. (B) Representative coronal (left) and transaxial (right) PET images at 5 days of EL4 tumor-bearing immunocompetent C57/Bl6 mice administered with anti-mouse 89Zr-CD25 IgG alone (top) or co-injected and 0.8 mg of unlabeled anti-CD25 IgG for blocking (middle). Biodistribution data are shown (bottom) as the mean ± S.E of values obtained from two independent experiments (n = 6 per group) N.S., not significant.

Fig 5: CD25 expression and 89Zr-CD25 IgG binding to human lymphoma cells. (A) Western blots of CD25 protein and anti-human 89Zr-CD25 IgG uptakes in H9, Jurkat, and SUDHL1 lymphoma cells (left). Complete blocking of SUDHL1 cell binding by 0.5 μM of unlabeled anti-CD25 IgG (right). (B) Lindmo binding assays for determining the immunoreactive fraction. Five concentrations of SUDHL1 cells were used, from 0.5 to 16 million cells/ml. The final concentration of 89Zr-CD25 IgG was 50 ng/ml, and nonspecific binding was assessed with 5 μg of unlabeled anti-CD25 IgG. A conventional plot of specific and nonspecific binding over total applied radioactivity, as a function of increasing cell concentration is shown (left). A double inverse plot was then drawn using the same data as total applied radioactivity over specific binding, as a function of the inverse cell concentration (right). The immunoreactive fraction was determined through linear extrapolation to the ordinate. Data represent the mean ± S.E of values from two independent experiments [total n = 5 per group; uptake in (A)], or the mean ± S.D of data from a single representative experiment [n = 3 per group; (B)].