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ZBP1 regulates myeloma cell proliferation via IRF3/IRF4
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Figure 1. Restricted and constitutive ZBP1 expression in normal and myeloma plasma cells. (A) ZBP1 mRNA expression as assessed in four multiple myeloma cell lines (MMCL) and in the erythromyeloid cell line K562 by quantitative polymerase chain reaction (n=3; data shown as mean ± standard error of mean). (B) ZBP1 expression in indicated MMCL as assessed by immunoblotting using GAPDH as the loading control. Two main isoforms ~48 and ~40 kDa are detected in MMCL. (C, D) ZBP1 mRNA and protein expression in six myeloma patient-derived bone marrow plasma cells purified for CD138+ marker using CD138 immunomagnetic microbeads, and U266 MMCL shown as control. (E) Immunohistochemistry on paraffin-embedded tonsil tissue section. The panel on the left depicts low and high magnification of a germinal center in which expression of PAX5 and ZBP1 is mutually exclusive in most parts indicating ZBP1 expression in plasma cells that are negative for PAX5. The panel on the right depicts subepithelial tonsillar plasma cells showing co-expression of IRF4 (MUM.1) and ZBP1. (F) Immunohistochemistry for ZBP1 expression in paraffin-embedded bone marrow tissue sections from two MM patients.
dataset) (Online Supplementary Figure S1A, B). While there is no difference in ZBP1 expression between normal PC and the whole cohort of myeloma PC, ZBP1 expression is sig- nificantly higher in the hyperdiploid subgroup of myeloma PC (Arkansas GSE4581 microarray dataset) (Online Supplementary Figure S1C). ZBP1 is universally expressed in primary myeloma PC (MMRF CoMMpass dataset; n=767 patients) (Online Supplementary Figure S1D) at similar levels as the PC-defining transcription factor PRDM1 (BLIMP1). In the human B-cell lineage, ZBP1 is expressed at a very low level in germinal center B cells (GCB) and a moderate level in naïve and memory B cells, but PC show by far the highest expression (Blueprint DCC Portal data) (Online Supplementary Figure S1E). We confirmed expression of ZBP1 mRNA and protein in MMCL (Figure 1A, B) and also in primary myeloma PC (Figure 1C, D) but not in other hematopoietic or epithelial cancer cells (Online Supplementary Figure S1F, G), normal blood lineage cells (Online Supplementary Figure S1H, I) or healthy non- hematopoietic tissues (Online Supplementary Figure S1J).
Immunohistochemistry of human tonsils and lymph nodes showed expression of ZBP1 primarily in a group of PAX5–IRF4+ GCB cells i.e., those committed to PC differ- entiation33,34 and in interfollicular and subepithelial IRF4+ PC, with low-level expression in mantle zone B cells (Figure 1E and Online Supplementary Figure S1K,L). In bone marrow, expression of ZBP1 is mostly restricted to myelo- ma and normal PC and is not present in other blood line- age cells (Figure 1F and Online Supplementary Figure S1K,M). These results show constitutive and restricted expression of ZBP1 in myeloma PC as well as in late GCB cells and normal PC.
ZBP1 is required for optimal T-cell-dependent humoral immune responses in mice
Consistent with a conserved expression pattern, a previ- ous transcriptome analysis of murine B-cell development identified Zbp1 as one of the signature genes that define transition from follicular B cells to plasmablasts and mature PC4 (Online Supplementary Figure S2). To determine
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