Kirstin M. Roundy

Defining In Vivo Transcription Factor Complexes of the Murine CD21 and CD23 Genes

The expression of the CD21 and CD23 genes is coincident with differentiation from transition 1 B cells (T1) to transition 2 B cells (T2). To define constituents controlling CD21 and CD23 expression, we conducted chromatin immunoprecipitation analyses for candidate transcription factors. We found constitutive binding of Oct-1, NFAT species, YY1, NF-κB-p52, Pax5, E2A, and RBP-Jκ to CD21 sequences and NF-κB-p52, Pax5, NFAT species, E2A, and RBP-Jκ to CD23 promoter sequences. Splenic T and B cell subsets displayed constitutive binding of YY1, NF-κB-p52, Pax5, and Oct-1 proteins to CD21 sequences in B cells but no specific binding of NFATc3 or Pax5 in T cells. Similarly, CD23 sequences demonstrated constitutive binding of NF-κB-p52 in splenic T and B cells but only Pax5 in B cells. Of the various NFAT species, only a subset were found forming constitutive DNA/protein complexes with the CD21, CD23, and IL-2 gene sequences. Maturing B cells in the marrow possess stable Pax5 complexes on CD19, CD21, and CD23 gene promoters in the nuclei of such cells, even though only CD19 is expressed. The similarity of genetic controlling elements between the CD21 and CD23 genes does not suggest a mechanism for alternative regulation of these genes; however, separation of splenic B cell subsets into T1, T2, marginal zone (MZ), and mature follicular B cells, followed by quantitative RT-PCR, demonstrated the lack of appreciable CD23 transcripts in CD21+ MZ cells. We propose an alternative derivation of MZ cells as maturing directly from T1 cells, leaving CD23 transcriptionally inactive in that lineage of cells.

Regulation of Murine Splenic B Cell CR3 Expression by Complement Component 3

Complement component C3 has established roles in both innate and adaptive immune responses. C3 cleavage products function in B cell activation through the complement receptors CD21/35. Phenotypes of Ab production between CD21/35−/− and C3−/− mice are not always congruent, implicating additional roles for C3 in B cell responses. To further characterize complement and complement receptors, we have identified a role for C3 in the regulation of CR3 on splenic B cells. Splenic B2 cells are not defined as expressing CR3, yet the analysis of splenic B cells from C3−/− animals demonstrate cell surface expression of CR3. B cells from both wild-type (WT) and C3−/− animals express CR3/CD11b/Itgam (integrin α M) gene transcripts although the level of such transcripts is 2- to 3-fold higher in B cells from the C3−/− animal vs WT cells. C3−/− and WT animals have similar B cell subpopulations with identical CR3 expression on B220− cells from the spleen, marrow, and lymph nodes. The C3-deficient environment is responsible for altered CR3 expression as WT splenic B cells transferred into C3−/− animals expressed cell surface CR3 within 48 h while transfer of C3−/− splenic B cells into WT animals depressed surface expression of CR3. Furthermore, transfer of C3-producing splenic macrophages into C3−/− mice depressed CR3 expression by resident B cells. These data suggest a role for C3 in influencing the level of expression of CR3 by modulating the transcript levels encoding the CD11b α integrin protein.

Bone marrow-induced Mef2c deficiency delays B-cell development and alters the expression of key B-cell regulatory proteins

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.

The in vitro derivation of phenotypically mature and diverse B cells from immature spleen and bone marrow precursors

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.

Deletion of putative intronic control sequences does not alter cell or stage specific expression of Cr2.

The expression of the mouse Cr2 gene has been shown to be restricted to mature B cells, follicular dendritic cells and, in some reports, to a minor population of activated T cells. In this report, we demonstrate that the expression of antigen(s) recognized by the anti-CR2 antibody on the surface of T cells is co-incident with T cell apoptotic death. Two distinct regions of the Cr2 gene have been implicated as critical for specific expression, the promoter region at the transcription start site and a control region within the first intron of the gene, approximately 1500 bp from the transcription start site. We have created a mouse that is lacking this intronic control sequence which, in the wild type (WT) mouse, contains multiple known binding sites for RBP-jkappa, Oct, NFAT and YY1 proteins. The analysis of this mouse named Cr2iDelta (Cr2 intron deletion) demonstrated normal tissue specific expression of the Cr2 gene including a lack of expression in mouse T cells. B cell expression of the Cr2 gene products, CR1 and CR2, is normal compared to WT, and the FDC of these mice continue to express Cr2 gene products. Therefore the intronic control region of the Cr2 gene, defined in transfection-based reporter gene assays as instrumental in controlling the cell specific expression profile of Cr2, does not influence the expression of the Cr2 gene in vivo nor alter the relative production of the CR1 and CR2 proteins via alternative slicing of Cr2 gene products.