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V. Madan et al.
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Figure 5. Impaired B-cell lymphopoiesis in Asxl2-deficient mice. (A and B) Proportion (A) and absolute number (B) of cells at different stages of B-cell development in the bone marrow of >1-year old WT and Asxl2 KO mice (n=4-6). proB: CD43+B220+, preB: CD43–B220+IgM–, immature B (Imm. B): CD43–B220+IgM+, mature B (Mat. B): CD43–B220hiIgM+, Fraction A (Fr. A): CD24–BP1– proB cells, Fraction B (Fr. B): CD24+BP1– proB cells, Fraction C (Fr. C): CD24+BP1+ proB cells. Data are rep- resented as mean±Standard Error of Mean. *P<0.05, **P<0.01, ***P<0.001, ns: not significant.
ulation assay. ASXL2 deficiency resulted in poor reconsti- tution of lymphoid lineage (both B and T cells) compared with the WT cells (Figure 3G and H). The reconstitution of granulocytes was also impaired in mice transplanted with Asxl2-deficient LSK cells (Figure 3I), while the proportion of donor-derived monocytes was similar to the recipients transplanted with WT cells (Figure 3J). These repopulation assays demonstrated that ASXL2 is essential for differen- tiation into multiple hematopoietic lineages including the lymphoid lineage, prompting us to investigate the devel- opment of lymphoid lineage in the Asxl2 KO mice.
Impaired thymopoiesis in mice lacking ASXL2
A marked reduction of thymus size and cellularity was a consistent feature in >1-year old Asxl2 KO mice com- pared with the WT mice (Figure 4A). Flow cytometric analyses revealed a striking reduction in CD4+CD8+ dou- ble positive (DP) cells and a concomitant increase in the proportion and number of CD4–CD8– double negative (DN) cells in the thymi of Asxl2 KO mice (Figure 4B and C and Online Supplementary Figure S13A). Within the DN compartment, the proportion of DN1 (CD44+CD25–) sub- population was unchanged, whereas a significant reduc- tion was detected in the proportion of DN2 (CD44+CD25+) and DN3 (CD44–CD25+) subpopulations, indicating a partial block in differentiation of earliest thy- mocytes (Figure 4D and E). A large number of DN cells in the thymi of KO mice consisted primarily of CD25– cells,
present in the DN1 and DN4 gates (Figure 4D and Online Supplementary Figure S13B). Further characterization showed that the vast majority of cells in the DN gate were CD19+ (which contributed to 40% of total thymus-resid- ing hematopoietic cells), and the frequency and number of CD11b+ myeloid cells were also noticeably increased in the thymus of KO mice compared with the WT mice (Figure 4F and G and Online Supplementary Figure S13C-F).
Young Asxl2 KO mice exhibited decreased thymus cel- lularity, but displayed less pronounced effect on major thymic compartments compared with the >1-year old mice (Online Supplementary Figure S13G-I). These findings illustrate that ASXL2 is essential for maintaining normal thymopoiesis in mice and its deficiency leads to progres- sive impairment in thymocyte maturation, which is man- ifested by a partial block from the DN to the DP stage, and an accumulation of myeloid and B cells in the thymus of the old mice.
Defects in B-cell development in Asxl2 knockout mice We also examined the consequences of ASXL2 deficien- cy on B-cell development in the BM by examining the expression of several surface antigens, which define the advancing stages of B-cell maturation. While the young Asxl2 KO mice showed largely conserved subsets during B-cell lymphopoiesis, a notable decrease in the proportion of mature B cells (CD43–IgM+B220hi) compared with the WT mice was observed (Online Supplementary Figure S14).
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haematologica | 2018; 103(12)