Fig. 3: CLEC12A^hiCD9^lo basophils differentiate into CLEC12A^loCD9^hi basophils ex vivo and in vivo.

a–c FcεRIα^hiCD49b^lo and FcεRIα^loCD49b^hi subpopulations of basophils sort-purified from the bone marrow were separately cultured ex vivo for 1 or 2 days. In (a), the time course of their surface CLEC12A and CD9 expression is shown. In (b), the change in the frequency of CLEC12A^hi cells during the culture is shown (mean ± SEM, n = 3 each). In (c), the change in the number of live cells during the culture is shown. The red and blue bars correspond to the CLEC12A^loCD9^hi and CLEC12A^hiCD9^lo fractions, respectively (mean ± SEM, n = 3 each). Data in (a–c) are representative of at least three independent experiments. d FcεRIα^hiCD49b^lo and FcεRIα^loCD49b^hi subpopulations of basophils were separately isolated from the bone marrow of CD45.2⁺ C57BL/6 mice (5 × 10⁴ cells/mouse), mixed with CD45.1⁺ whole bone marrow cells (0.3 × 10⁶ cells/mouse), and intravenously administered to sublethally-irradiated CD45.1⁺ congenic C57BL/6 mice. One day after the cell transfer, bone marrow cells were prepared from recipient mice and subjected to flow cytometric analysis for the expression of CLEC12A and CD9 on CD45.2⁺ donor-derived basophils (bottom panels). For comparison, the CLEC12A and CD9 expression on transferred cells (Day0) is displayed in the upper panels. The frequency of CLEC12A^hi cells before and 1 day after the transfer in each experimental group is shown in the right panel where data are pooled from two experiments (mean ± SEM, n = 2 for FcεRIα^hi on D0, n = 3 for FcεRIα^lo on D0, n = 3 for FcεRIα^hi on D1, n = 5 for FcεRIα^lo on D1). Two-way ANOVA with Tukey’s multiple comparisons test was used for multiple comparisons (b, d).