The in vitro derivation of phenotypically mature and diverse B cells from immature spleen and bone marrow precursors.
ABSTRACT: The capacity of immature B cells of the spleen and bone marrow to differentiate in vitro into cells representing mature end stage cells was investigated using B-cell activating factor belonging to the TNF family (BAFF) and Notch pathway activators. Immature splenic and bone marrow B cells were found, in the presence of both of these activators, to mature into cells with follicular mature (FM) and marginal zone (MZ) cell phenotypes. Such cells were functionally responsive to B-cell-specific activation. The derivation in vitro of cells with an MZ phenotype was more robust from CD23(-) populations than CD23(+) immature/transitional B cells, suggesting a direct immature/T1 B cell to MZ cell differentiation pathway. Transcript analysis of the in vitro-derived B-cell populations demonstrated expression profiles similar to maturing B cells in vivo. FACS-purified populations of B220(+)CD19(+)CD21(-)CD23(-) cells from bone marrow of 2-wk-old mice gave rise to populations of CD21(+)CD23(-) cells with MZ cell phenotypes as well as CD21(+)CD23(+) cells with FM cell phenotypes in percentages similar to those found in vivo. These data suggest that the commitment to an MZ and FM B cell phenotype is set prior to immature B-cell release from the marrow.
Project description:Marginal zone (MZ) B cells, identified as surface (s)IgM(high)sIgD(low)CD23(low/-)CD21(+)CD38(-) B cells, were purified from human spleens, and the features of their V(D)J gene rearrangements were investigated and compared with those of germinal center (GC), follicular mantle (FM) and switched memory (SM) B cells. Most MZ B cells were CD27(+) and exhibited somatic hypermutations (SHM), although to a lower extent than SM B cells. Moreover, among MZ B-cell rearrangements, recurrent sequences were observed, some of which displayed intraclonal diversification. The same diversifying sequences were detected in very low numbers in GC and FM B cells and only when a highly sensitive, gene-specific polymerase chain reaction was used. This result indicates that MZ B cells could expand and diversify in situ and also suggested the presence of a number of activation-induced cytidine deaminase (AID)-expressing B cells in the MZ. The notion of antigen-driven expansion/selection in situ is further supported by the VH CDR3 features of MZ B cells with highly conserved amino acids at specific positions and by the finding of shared ("stereotyped") sequences in two different spleens. Collectively, the data are consistent with the notion that MZ B cells are a special subset selected by in situ antigenic stimuli.
Project description:We have analyzed miRNA expression profile in FO and MZ B cells to identified differentially expressed miRNAs between this two subsets that could be potencially involved in the regulation of terminal B cell differentiation Spleens from CD19-Creki/+Dicerfl/+ and CD19-Creki/+Dicerfl/fl mice were collected and after erythrocyte lysis, splenocytes were stained with anti-B220, anti-CD21 and anti-CD23 antibodies. B220+CD21brightCD23+ (MZ) and B220+CD21+CD23bright (FO) B cell populations were sorted and total RNA of purified populations was extracted with TRIzol. miRNA microarray hybridations were performed on Mouse miRNA Microarray platform (Agilent Technologies).
Project description:The neurogenic locus notch homolog protein (Notch)-2 receptor is a determinant of B-cell allocation, and gain-of-NOTCH2-function mutations are associated with Hajdu-Cheney syndrome (HCS), a disease presenting with osteoporosis and acro-osteolysis. We generated a mouse model reproducing the HCS mutation (Notch2HCS), and heterozygous global mutant mice displayed gain-of-Notch2 function. In the mutant spleen, the characteristic perifollicular rim marking the marginal zone (MZ), which is the interface between the nonlymphoid red pulp and the lymphoid white pulp, merged with components of the white pulp. As a consequence, the MZ of Notch2HCS mice occupied most of the splenic structure. To explore the mechanisms involved, lymphocyte populations from the bone marrow and spleen were harvested from heterozygous Notch2HCS mice and sex-matched control littermates and analyzed by flow cytometry. Notch2HCS mice had an increase in CD21/35highCD23- splenic MZ B cells of approximately fivefold and a proportional decrease in splenic follicular B cells (CD21/35intCD23+) at 1, 2, and 12 months of age. Western blot analysis revealed that Notch2HCS mutant splenocytes had increased phospho-Akt and phospho-Jun N-terminal kinase, and gene expression analysis of splenic CD19+ B cells demonstrated induction of Hes1 and Hes5 in Notch2HCS mutants. Anti-Notch2 antibodies decreased MZ B cells in control and Notch2HCS mice. In conclusion, Notch2HCS mutant mice have increased mature B cells in the MZ of the spleen.
Project description:We have analyzed miRNA expression profile in FO and MZ B cells to identified differentially expressed miRNAs between this two subsets that could be potencially involved in the regulation of terminal B cell differentiation Overall design: Spleens from CD19-Creki/+Dicerfl/+ and CD19-Creki/+Dicerfl/fl mice were collected and after erythrocyte lysis, splenocytes were stained with anti-B220, anti-CD21 and anti-CD23 antibodies. B220+CD21brightCD23+ (MZ) and B220+CD21+CD23bright (FO) B cell populations were sorted and total RNA of purified populations was extracted with TRIzol. miRNA microarray hybridations were performed on Mouse miRNA Microarray platform (Agilent Technologies).
Project description:The Mef2 family transcriptional regulator Mef2c (myocyte enhancer factor 2c) is highly expressed in maturing bone marrow and peripheral mature B-cells. To evaluate the role of this transcription factor in B-cell development, we generated a B-cell-specific conditional deletion of Mef2c using the Mb-1-Cre transgene that is expressed during the early stages of immunoglobulin rearrangement. Young mice possessing this defect demonstrated a significant impairment in B-cell numbers in bone marrow and spleen. This phenotype was evident in all B-cell subsets; however, as the animals mature, the deficit in the peripheral mature B-cell compartments was overcome. The absence of Mef2c in mature B-cells led to unique CD23+ and CD23- subsets that were evident in Mef2c knockout primary samples as well as Mef2c-deficient cultured, differentiated B-cells. Genome-wide expression analysis of immature and mature B-cells lacking Mef2c indicated altered expression for a number of key regulatory proteins for B-cell function including Ciita, CD23, Cr1/Cr2 and Tnfsf4. Chromatin immunoprecipitation analysis confirmed Mef2c binding to the promoters of these genes indicating a direct link between the presence (or absence) of Mef2c and altered transcriptional control in mature B-cells.
Project description:We have characterized a distinct, late transitional B cell subset, CD21(int) transitional 2 (T2) B cells. In contrast to early transitional B cells, CD21(int) T2 B cells exhibit augmented responses to a range of potential microenvironmental stimuli. Adoptive transfer studies demonstrate that this subset is an immediate precursor of both follicular mature and marginal zone (MZ) B cells. In vivo, a large percentage of CD21(int) T2 B cells has entered the cell cycle, and the cycling subpopulation exhibits further augmentation in mitogenic responses and B cell-activating factor of the TNF family (BAFF) receptor expression. Consistent with these features, CD21(int) T2 cells exhibit preferential responses to BAFF-facilitated homeostatic signals in vivo. In addition, we demonstrate that M167 B cell receptor (BCR) idiotypic-specific B cells are first selected within the cycling CD21(int) T2 population, ultimately leading to preferential enrichment of these cells within the MZ B cell compartment. These data, in association with the coordinate role for BAFF and microenvironmental cues in determining the mature BCR repertoire, imply that this subset functions as a unique selection point in peripheral B cell development.
Project description:BAFF is an important prosurvival cytokine for mature B cells. However, previous studies have shown that BAFFR is already expressed at the immature B cell stage, and that the prosurvival protein Bcl-2 does not completely complement the B cell defects resulting from the absence of BAFFR or BAFF. Thus, we hypothesized that BAFF also functions to aid the differentiation of nonautoreactive immature B cells into transitional B cells and to promote their positive selection. We found that BAFFR is expressed at higher levels on nonautoreactive than on autoreactive immature B cells and that its expression correlates with that of surface IgM and with tonic BCR signaling. Our data indicate that BAFFR signaling enhances the generation of transitional CD23(-) B cells in vitro by increasing cell survival. In vivo, however, BAFFR signaling is dispensable for the generation of CD23(-) transitional B cells in the bone marrow, but it is important for the development of transitional CD23(-) T1 B cells in the spleen. Additionally, we show that BAFF is essential for the differentiation of CD23(-) into CD23(+) transitional B cells both in vitro and in vivo through a mechanism distinct from that mediating cell survival, but requiring tonic BCR signaling. In summary, our data indicate that BAFFR and tonic BCR signals cooperate to enable nonautoreactive immature B cells to differentiate into transitional B cells and to be positively selected into the naive B cell repertoire.
Project description:Transcriptional control of marginal zone (MZ) and follicular (FO) B cell development remains incompletely understood. The transcription factor, IFN regulatory factor (IRF)8, is known to play important roles in the differentiation of early B cells. In this article, we demonstrate that IRF8 is also required for normal development of MZ and FO B cells. Mice with a conventional knockout of Irf8 (IRF8(-/-)) or a point mutation in the IRF association domain of IRF8 had increased numbers of MZ B cells. To determine the B cell-intrinsic effects of IRF8 deficiency, we generated mice with a conditional allele of Irf8 crossed with CD19-Cre mice (designated IRF8-conditional knockout [CKO]). These mice had enlarged MZ and increased numbers of MZ and FO B cells compared with controls. The FO B cells of CKO mice exhibited reduced expression of CD23 and moderately increased expression of CD21. Gene-expression profiling showed that increased B cell production in IRF8-CKO mice was associated with changes in expression of genes involved in regulation of transcription, signaling, and inflammation. Functional studies showed that IRF8-CKO mice generated normal Ab responses to T-independent and T-dependent Ags. Thus, IRF8 controls the expansion and maturation of MZ and FO B cells but has little effect on B cell function.
Project description:The pathogenic connection of type I IFN and its role in regulating the migration response of Ag delivery by B cells into lymphoid follicles in an autoimmune condition has not been well-identified. Here, we show that there was a significantly larger population of marginal zone precursor (MZ-P) B cells, defined as being IgM(hi)CD1d(hi)CD21(hi)CD23(hi) in the spleens of autoimmune BXD2 mice compared with B6 mice. MZ-P B cells were highly proliferative compared with marginal zone (MZ) and follicular (FO) B cells. The intrafollicular accumulation of MZ-P B cells in proximity to germinal centers (GCs) in BXD2 mice facilitated rapid Ag delivery to the GC area, whereas Ag-carrying MZ B cells, residing predominantly in the periphery, had a lower ability to carry Ag into the GCs. IFN-alpha, generated by plasmacytoid dendritic cells, induced the expression of CD69 and suppressed the sphingosine-1-phosphate-induced chemotactic response, promoting FO-oriented Ag transport by MZ-P B cells. Knockout of type I IFN receptor in BXD2 (BXD2-Ifnalphar(-/-)) mice substantially diffused the intrafollicular MZ-P B cell conglomeration and shifted their location to the FO-MZ border near the marginal sinus, making Ag delivery to the FO interior less efficient. The development of spontaneous GCs was decreased in BXD2-Ifnalphar(-/-) mice. Together, our results suggest that the MZ-P B cells are major Ag-delivery B cells and that the FO entry of these B cells is highly regulated by type I IFN-producing plasmacytoid dendritic cells in the marginal sinus in the spleens of autoimmune BXD2 mice.
Project description:IgD and IgM are produced by alternative splicing of long primary RNA transcripts from the Ig heavy chain (Igh) locus and serve as the receptors for antigen on naïve mature B lymphocytes. IgM is made selectively in immature B cells, whereas IgD is coexpressed with IgM when the cells mature into follicular or marginal zone B cells, but the transacting factors responsible for this regulated change in splicing have remained elusive. Here, we use a genetic screen in mice to identify ZFP318, a nuclear protein with two U1-type zinc fingers found in RNA-binding proteins and no known role in the immune system, as a critical factor for IgD expression. A point mutation in an evolutionarily conserved lysine-rich domain encoded by the alternatively spliced Zfp318 exon 10 abolished IgD expression on marginal zone B cells, decreased IgD on follicular B cells, and increased IgM, but only slightly decreased the percentage of B cells and did not decrease expression of other maturation markers CD21, CD23, or CD62L. A targeted Zfp318 null allele extinguished IgD expression on mature B cells and increased IgM. Zfp318 mRNA is developmentally regulated in parallel with IgD, with little in pro-B cells, moderate amounts in immature B cells, and high levels selectively in mature follicular B cells. These findings identify ZFP318 as a crucial factor regulating the expression of the two major antibody isotypes on the surface of most mature B cells.