TOSO-Deficient B Cells Show An Impaired NF-κB and Migration Signaling In Vivo

Our group firstly demonstrated that TOSO (FAIM3) is over-expressed in CLL compared to healthy B cell subsets as well as other B cell lymphomas. Furthermore, we detected a significant correlation of high TOSO expression to high lymphocyte count, unmutated IgVH status and Binet C, which are all markers for poor prognosis. TOSO has been described as pro-survival gene also in other settings. However, its mode of action is discussed controversially.

Therefore, we aimed to elucidate the role of TOSO in B-cell specific gene expression by creating a knockdown mouse model. CD40 ligation and B cell receptor (BCR) activation influences TOSO expression and the fact that transcriptional regulation of TOSO is still unknown, we were eager to determine transcriptional factors that are directly responsible for the alterable TOSO levels.

Faim3-floxed C57BL/6 FLP deleter mice were crossbred with CD19 specific Cre recombinase expressing mice. B-cells from the TOSO^CD19−/− (KO) mice were isolated and gene expression was analyzed via mRNA based Illumina microchip array. Convincing results were verified by flow cytometry and blood count was carried out in addition.

To determine the promoter region of TOSO, three overlapping DNA fragments (containing either NF-κB, Bcl-6 or both binding sites) upstream of the transcription start site of the first TOSO exon were cloned into a luciferase reporter vector lacking a promoter. Those constructs were transfected into HeLa cells. After 24 hours luciferase assays were performed. The involvement of NF-κB in the regulation of TOSO transcription was measured by TNFα stimulation of transfected cells prior to luminescence measurement. Targeted mutagenesis of the NF-κB binding site was performed to confirm the data. In addition, Bcl-6 expression vector was co-transfected for evaluation of repressing influence on TOSO expression.

In order to cover the functional part of TOSO, we generated a B-cell specific TOSO^CD19−/− mouse model. Downstream effects of TOSO were validated via microarray-based gene expression analysis. Results displayed a clear clustering of deregulated genes compared to control mice. Nearly 400 genes showed expression alterations; genes involved in the NF-κB pathway and migration processes were downregulated in TOSO^CD19−/−. These results were confirmed by flow cytometry analysis. The TOSO KO displayed also relevant effects on the hematopoietic system. Lymphocyte (p=0,0048), neutrophil (p=0,0007) and red blood cell counts (p=0,0051) were significantly decreased in the TOSO^CD19−/− mice. Most important, the B-cell count was significantly reduced in TOSO-deficient settings (n=9; p=0,032).

Since TOSO level seems to be so important for such fundamental pathways, investigation of gene expression regulation is mandatory. In situ analysis of the TOSO promoter region revealed NF-κB and Bcl-6 as promising results. Luciferase reporter assays including targeted mutagenesis confirmed the positive regulation of NF-κB and the repressing influence of Bcl-6 on TOSO expression significantly.

We reveal for the first time a TOSO-dependent expression profile. We identified TOSO-dependent deregulated genes, which were involved in NF-κB signaling and migration, suggesting that TOSO represents an important factor in these pathways. Additionally, TOSO KO caused a decrease of peripheral B-cells in vivo. Furthermore, we identified NF-κB and Bcl-6 to regulate the TOSO expression in an opposite manner.

No relevant conflicts of interest to declare.