Because P4-type ATPases have previously been implicated in phosphatidylserine (PS) asymmetry in yeast31, we hypothesized that increased surface PS exposure and consequent PS-mediated phagocytosis could account for diminished numbers of B cells in mutant mice33. in the bone marrow shortly after birth7. As a result, MZ and B-1 B cells predominate in the periphery of mutant mice, while follicular B cell numbers are severely reduced. In the absence of IL-7 or its common gamma chain (c)-associated receptor component IL-7R , B cell development is usually arrested in the adult bone marrow shortly after cells acquiresurface B220, yet before they express CD19 or CD24 (refs. 8,9). Expression of the transcription factor early B cell factor (EBF) via STAT5 signaling appears to be important at this stage, since overexpression of EBF or constitutively active STAT5 can overcome the developmental arrest in mutant progenitors9,10. Other data suggests that rather than inducing transcription of (ref. 11). Whichever the mechanism, IL-7 and EBF are both key determinants of B cell differentiation12,13, and little else is known of the molecules and microenvironments that induce and maintain their expression in the adult. We (and our colleagues14) describe here an essential role for the previously uncharacterized P4-type ATPase ATP11C in early B cell FLN2 differentiation. ATP11C was redundant during B cell development in the fetal liver, yet essential in the context of adult bone marrow, where it was required for optimal Calcifediol monohydrate responses to IL-7 and sustained expression of pedigree. Percentages (b, c, e) and numbers (d) of B cell subsets in bone marrow (b), spleen (c) and peritoneal cavity (e). Hardy fractions in bone marrow (A-F) were gated as follows: A (B220+CD43+BP-1?CD24?); B (B220+CD43+BP-1?CD24+); C (B220+CD43+BP-1+CD24+); D (B220+CD43?IgM?IgD?); E (B220+CD43?IgM?IgD+); F (B220+CD43?IgM+IgD+). The C’ fraction (B220+CD43+BP?1+CD24hi) was not resolved. Fractions A and B-D may also be gated as CD19? and CD19+ populations among B220+IgM?cells, respectively (b). IgM+ splenocytes were divided into the following subsets: T1 (CD93+CD23?); T2 (CD93+CD23+IgMint); T3 (CD93+CD23+IgMhi); MZ (CD93?CD23?IgMhiCD21hi); Fo (CD93?CD23+). Peritoneal lymphocytes were divided into B-2 (CD19+B220hi), B-1 (CD19+B220lo?int), B-1a (CD5+CD43+) and B-1b (CD5?CD43?) subsets. (f) IgM allotype expression on CD19+ blood lymphocytes from wild-type and mice on a (C57BL/6 BALB/c)F2 (IgMa/b) background. Data are representative of three (a to e), or one (f) impartial experiments using three mice per genotype. Each symbol represents an individual mouse. Among B cell progenitors in the bone marrow, mutants had reduced numbers of cells beginning at the pre-pro-B to pro-B transition (Hardy fraction A [B220+CD43+BP-1?CD24?] to B [B220+CD43+BP-1?CD24+]16) (Fig. 1b), with a severe deficiency of immature B cells (Hardy fraction E [B220+CD43?IgM?IgD+]). In the spleen, mice had one-tenth the normal number of CD19+ cells, largely due to a lack of follicular and transitional subsets (Fig. 1c,d), although numbers of MZ B cells and Thy1.2+ cells and were normal. Numbers of B-1 cells, the predominant population of B cells in the peritoneal cavity, Calcifediol monohydrate were reduced by Calcifediol monohydrate a factor of three in mutant mice (Fig. 1d,e), while numbers of peritoneal B-2 cells were reduced by a factor of ten. B cells in the blood of mutant mice had undergone normal allelic exclusion at the locus (Fig. 1f). Despite a reduction in follicular B cell numbers, the B cells that remained appeared largely functional, and retained the capacity to produce all major immunoglobulin isotypes (Fig. 2a), as well as the ability to generate specific antibodies to T-independent and T-dependent immunogens (here, 4-hydroxy-3-nitrophenylacetyl (NP)-conjugated Ficoll and NP-chicken gamma globulin, NP-CGG, Fig. 2b,c, respectively). However, 50% less NP-specific IgM and high-affinity IgG1 was produced in response to NP-Ficoll and alum-precipitated NP-CGG immunization, respectively. Open in a separate window Physique 2 Immunoglobulin secretion in mice. (a) Total immunoglobulins as measured in the serum of 12C24 Calcifediol monohydrate week-old naive mice. NP-specific antibodies were measured 7, 14 or 28 days after immunization of 12 week-old mice with NP-Ficoll (b) or alum-precipitated NP-CGG (c). Different capture antigens were used to discriminate between total (NP23-BSA) and high-affinity (NP4-BSA) IgG1 in response to NP-CGG. Each symbol represents an individual mouse from one experiment. values indicated under plots were calculated by unpaired groups. ns, not significant ( 0.05). Cell-intrinsic failure of adult B lymphopoiesis To determine the cellular origin of the phenotype, irradiated mutant mice were reconstituted with an equal mix of mutant and wild-type bone marrow. Although the Thy1.2+ compartment was reconstituted equally, less than Calcifediol monohydrate 1% of CD19+ cells were of mutant origin (Fig. 3a), indicating that the defect was intrinsic to B cell progenitors but did not appreciably affect T cell development. Even.