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Effects of STAP-2 on B-cell recovery
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Figure 4. STAP-2 impairs B-cell differentiation and pre-B-cell proliferation. (A) LSK cells derived from STAP-2 transgenic (Tg) marrow were cultured under stromal- cell free, B-cell differentiation-supporting conditions. Flow cytometry was used to evaluate differentiation. (Left) Representative gating for Mac1+ or Gr1+ myeloid and B220+ CD19+ B cells. The number of myeloid and B cells recovered were used to calculate yields per input cell (fold expansion). (B and C) Lin– Sca1+ cKitlow Flk2high IL7Rα+ CLP derived from Tg were cultured without (B) or with (C) OP9 stromal cells under B-cell conditions, and the production of B220+ CD19+ B cells was analyzed. (D) In colony-forming unit (CFU pre-B) assays, the ability to generate pre-B colonies was evaluated. (E) The stromal cell-free culture system was used to evaluate the proliferation of pre-B cells and differentiation to IgM+ immature B cells. Similar results were obtained in three independent experiments. The results are shown as mean±standard deviation. Statistical significances relative to wild-type (WT) control were determined by unpaired two-tailed Mann-Whitney tests: *P<0.05; **P<0.01. KO: knockout.
We analyzed HSPC in BM one month after transplanta- tion via flow cytometry and found that there were no dif- ferences in the number of each subset of multipotent HSPC and early lymphoid progenitors between WT and KO donors (Figure 1D). In contrast, STAP-2 deficiency significantly increased pre-B-cell subset. These results indicate that STAP-2 in B progenitors is involved with B- cell recovery under regenerative conditions.
It is known that self-renewal capacity and lineage com- mitment in HSPC change according to the type of hematopoietic stress to which they are exposed;1,2 there- fore, the effects of STAP-2 on aged hematopoiesis were also analyzed. In aged KO mice, no abnormalities were observed with respect to hematopoietic parameters in BM or peripheral blood (Online Supplementary Figure S1). In old KO LSK-transplanted mice, comparable long-term engraftment was observed. We found B-cell reconstitu- tion from aged HSPC was also accelerated by STAP-2 deletion (Figure 1E).
Collectively, our findings indicate that STAP-2 sup- presses B-cell recovery during stress hematopoiesis at the pre-B-cell stage in BM, while STAP-2 does not affect the functions of multipotent HSPC.
STAP-2 is critical for B-cell differentiation during stress hematopoiesis
To further investigate the role of STAP-2 in early B lym- phopoiesis, we generated transgenic mice that overex- press STAP-2 under the control of an Em enhancer and Lck proximal promoter. This promoter could drive
expression of the inserted cDNA in lymphoid lineage cells in BM, T-cell precursors in thymus, and peripheral mature lym- phoid cells. The consistent upregulation from the com- mon lymphoid progenitor (CLP) stage to mature lym- phoid cells in lymph nodes was confirmed with RT-PCR (Figure 2A). Tg mice showed no apparent defects, and had normal life-spans.
We conducted a thorough analysis of marrow hematopoiesis in STAP-2 Tg mice that were 8-16 weeks old. No abnormalities were found with respect to mar- row cellularity or proportions and numbers of HSPC and CLP (Figure 2B and C). In contrast, numbers of B220+ B progenitors were significantly reduced (Figure 2B). By immunophenotyping B-cell development, the reduction of all BM B-lineage-committed progenitor stages (pre-pro- B, pro-B and pre-B) was confirmed (Figure 2C). Analyses of the proliferation and apoptosis status showed no sta- tistical differences among WT, KO, and Tg mice under steady states (Figure 2D). Dendritic cells that can be gen- erated from CLP, were also affected by STAP-2 overex- pression (Figure 2E). These findings suggest that STAP-2 has a critical function in maintaining the integrity of the B-progenitor compartment.
Although overexpression of STAP-2 in B precursors seemed to increase the sensitivity to subtle stress under homeostasis, peripheral B-cell cellularity could be main- tained through compensatory mechanisms, and the sizes of spleen were rather enlarged (Figure 3A and B). We therefore investigated whether B-cell recovery is compro-
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