Page 324 - 2022_01-Haematologica-web

Letters to the Editor
Ikaros deficiency is associated with aggressive BCR-ABL1 B-cell precursor acute lymphoblastic leukemia independent of the lineage and develop- mental origin
The B-cell lineage is established from waves of differ- entiation that begin in the fetal liver (FL) and continue in the postnatal bone marrow (BM). In mice, FL lym- phopoiesis gives rise to B1 cells, which produce natural polyreactive immunoglobulin M (IgM) antibodies and are implicated in innate immunity. Conversely, BM lym- phopoiesis favors conventional B2-cell development over B1, and is essential for adaptive immunity.1,2 The features that distinguish B1- and B2-cell differentiation remain partially understood. Ikaros, encoded by IKZF1, is a key transcriptional regulator of B lymphopoiesis in mice and humans.3 It promotes B2-cell development at multiple stages, including large pre-B-cell (Hardy fraction C') differentiation where it antagonizes IL-7/STAT5 sig- naling.4 Ikaros is also a tumor suppressor in B2 progeni- tors, and deleterious IKZF1 alterations are prominent in BCR-ABL1+ B-cell precursor acute lymphoblastic leukemias (BCP-ALL) with constitutively active STAT5.5 While Ikaros is likewise important for fetal and adult B1- cell development, the exact stage where it is required has remained unclear, due to a dearth of in-depth knowledge about B1-cell differentiation. Whether Ikaros acts as a tumor suppressor in B1 cells, or in fetal B cells in general, is currently unknown. Here we show that B1-cell differ- entiation parallels that of B2 cells in terms of phenotype and cell cycle status. We show that Ikaros promotes B1- cell differentiation at a stage equivalent to fraction C' of B2 cells, by modulating the expression of genes involved in cell proliferation and migration, pre-B-cell receptor
(BCR) signaling, Ig-HC rearrangement and IL-7/STAT5 signaling. We also show that Ikaros inhibits the expan- sion of murine BCR-ABL1+ B1 progenitors from the FL and BM, and suggest that this function might be con- served in human FL-derived B cells.
In order to determine the role of Ikaros in adult B1-cell differentiation, we analyzed the BM of Ikf/fMb-1-Cre+ conditional knockout (cKO) and Ikf/fMb-1-Cre- wild-type (WT) mice (Figure 1A), where the Cre recombinase is controlled by the endogenous Cd79a promoter.4 While the percentage of WT B1 progenitors (Lin-IgM- CD93+CD19+B220-/lo) remained low with age, the same population in the cKO mice was ~20x higher until 4 weeks of age, before decreasing to WT levels by week 16 (Figure 1B). This increase was not due to a compensatory decrease in B2 progenitors, as the percentage of BM B2 progenitors (B220+CD19+CD43+) was stable over time (Online Supplementary Figure S1A). Ikaros loss in B1 pro- genitors was confirmed by intracellular staining (Online Supplementary Figure S1B). These results showed that Ikaros is required for differentiation in B1 progenitors from an early pro-B1 cell stage.
In order to pinpoint the stage where Ikaros promotes B1-cell differentiation, we dissected the above B1 pro- genitor cell population, by analyzing Ig-HC rearrange- ment, and evaluating CD24 and BP-1 expression, typi- cally used to define B2-cell development.6 WT or cKO B1 progenitors were heterogenous with unrearranged, germline HC genes, and rearranged DJ as well as proxi- mal and distal VDJ genes (Figure 2A). WT B1 progenitors were mostly CD24-BP-1- and CD24+BP-1-, resembling Hardy fractions A and B among B2 progenitors (Figure 2B). Interestingly, cKO cells were mainly CD24+BP-1+ and CD24++BP-1+, and resembled B2 fractions C and C'. Hence, B1 progenitors are more heterogenous than pre-
A
B
Figure 1. B1 progenitors accumulate in Ikaros conditional knockout bone marrow. (A) Representative flow cytometry analysis of bone marrow (BM) B1 progen- itors (Lin-IgM-CD93+CD19+B220-/lo) from 4-week-old mice. Numbers correspond to % of parental population. (B) % BM B1 progenitors in wild-type (WT) or condi- tional knockout (cKO) mice with age (D: days; W: weeks after birth). Significance was calculated using unpaired Student's t-test. ns: not significant; *P<0.05; ***P<0.001; ****P<0.0001. Ikf/fMb-1-Cre mice4 were housed in specific pathogen-free (SPF) conditions or kept in single-ventilated cages. IgM: immunoglob- ulin M.
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haematologica | 2022; 107(1)