Transgenic expression of Spi‐C impairs B‐cell development and function by affecting genes associated with BCR signaling

Abstract

Summary Spi-C is an Ets family transcription factor closely related to PU.1 and Spi-B. Expression of Spi-C is developmentally regulated in the B cell lineage, but its function remains unknown. To determine the function of Spi-C in B cell development, we generated mice expressing a B cell-specific Spi-C transgene under the control of the IgH intronic enhancer. Spi-C transgenic mice had 50% fewer B cells than wild type littermates. Flow cytometric analyses showed that splenic transitional B cells and bone marrow pre-B or immature B cells from transgenic mice were dramatically reduced compared to those of wild type. Both nonspecific and Ag-specific serum IgM levels were significantly increased in transgenic mice, while serum IgG levels were significantly decreased compared to wild type. Spi-C transgenic B cells proliferated poorly after stimulation by anti-IgM or anti-CD40 in vitro, although they responded normally to LPS stimulation. Using real-time RT-PCR, we found that several BCR signaling related mediators were downregulated at pre-B cell and mature B cell stages in transgenic mice, while an inhibitor of BCR signaling was upregulated. Taken together, these data indicate that ectopic expression of Spi-C can impair B cell development and function by affecting genes associated with BCR signaling.

Regulation of Follicular B Cell Differentiation by the Related E26 Transformation-Specific Transcription Factors PU.1, Spi-B, and Spi-C

“…C57BL/6 mice were purchased as required from Charles River Laboratories (Pointe-Claire, Quebec, Canada). Sfpi1 +/2 , Spib 2/2 , PUB, and Em-Spi-C transgenic mice were previously generated and characterized, and genotyping was performed by PCR as described (18,20,24,26). To generate Em-Spi-C + PUB mice, Em-Spi-C transgenic stud males were bred with PUB females to generate Em-Spi-C + Sfpi1 +/2 Spib +/2 mice, which were back-crossed to PUB mice to generate Em-Spi-C + PUB mice.…”

“…For flow cytometry, mouse splenocytes were washed after hypotonic lysis with ammonium chloride solution and stained with allophycocyanin-conjugated anti-B220 (RA3-6B2), PE-conjugated anti-CD19 (1D3), PE-Cy5-conjugated anti-IgM (II/41), PE-conjugated anti-CD93 (AA4.1), FITC-conjugated anti-IgD (11)(12)(13)(14)(15)(16)(17)(18)(19)(20)(21)(22)(23)(24)(25)(26), FITC-conjugated anti-CD23 (B3B4), FITC-conjugated anti-CD21 (eBio8D9), FITC-conjugated anti-CD1d (1B1), or biotin-conjugated anti-IL-21R (eBio4A9). All Abs were purchased from eBioscience (San Diego, CA).…”

“…An Spi-C transgene rescues frequencies of CD23 + B cells in spleens of PUB mice To determine if the PUB phenotype could be complemented by ectopic expression of a closely related Ets transcription factor, we crossed PUB mice to Em-Spi-C transgenic mice (24). Spi-C is highly related to PU.1 and Spi-B and interacts with similar cognate DNA sequences (29,38).…”

“…Peripheral B cell differentiation has not been examined in detail in PUB mice, raising the issue of whether the immunodeficiency of these mice might be explained in part by selective loss of a particular B cell subset. Spi-C is expressed in mature B cells and in a subset of splenic macrophages and is highly expressed in splenic transitional B cells (21)(22)(23)(24). Mutation of the gene encoding Spi-C (Spic), or transgenic overexpression of Spi-C, causes various B cell differentiation abnormalities (24,25).…”

“…Spi-C is expressed in mature B cells and in a subset of splenic macrophages and is highly expressed in splenic transitional B cells (21)(22)(23)(24). Mutation of the gene encoding Spi-C (Spic), or transgenic overexpression of Spi-C, causes various B cell differentiation abnormalities (24,25). The objective of this study was to determine whether the Ets transcription factors PU.1 and Spi-B play roles in FO versus MZ B cell development.…”