ABSTRACT
Differentiation of B cells into antibody-secreting cells (ASCs), plasmablasts and plasma cells is regulated by a network of transcription factors. Within this network, factors including PAX5 and BCL6 prevent ASC differentiation and maintain the B cell phenotype. In contrast, BLIMP-1 and high IRF4 expression promote plasma cell differentiation. BLIMP-1 is thought to induce immunoglobulin secretion, whereas IRF4 is needed for the survival of ASCs. The role of IRF4 in the regulation of antibody secretion has remained controversial. To study the role of IRF4 in the regulation of antibody secretion, we have created a double knockout (DKO) DT40 B cell line deficient in both IRF4 and BCL6. Although BCL6-deficient DT40 B cell line had upregulated BLIMP-1 expression and secreted antibodies, the DKO cell line did not. Even enforced BLIMP-1 expression in DKO cells or IRF4-deficient cells could not induce IgM secretion while in WT DT40 cells, it could. However, enforced IRF4 expression in DKO cells induced strong IgM secretion. Our findings support a model where IRF4 expression in addition to BLIMP-1 expression is required to induce robust antibody secretion.
Subject(s)
Antibodies/immunology , Antibody Formation/genetics , Avian Proteins/genetics , B-Lymphocytes/immunology , Interferon Regulatory Factors/genetics , Positive Regulatory Domain I-Binding Factor 1/genetics , Animals , B-Lymphocytes/cytology , Cell Differentiation/immunology , Cell Line , Cell Proliferation , Chickens , Gene Knockout Techniques , Immunoglobulin M/biosynthesis , Immunoglobulin M/immunology , PAX5 Transcription Factor/genetics , Positive Regulatory Domain I-Binding Factor 1/immunology , Proto-Oncogene Proteins c-bcl-6/geneticsABSTRACT
The graded expression of transcription factor interferon regulatory factor 4 (IRF4) regulates B cell development and is critical for plasma cell differentiation. However, the mechanisms, by which IRF4 elicits its crucial tasks, are largely unknown. To characterize the molecular targets of IRF4 in B cells, we established an IRF4-deficient DT40 B cell line. We found that in the absence of IRF4, the expression of several molecules involved in BCR signalling was altered. For example, the expression of B cell adaptor for PI3K (BCAP) was upregulated, whereas the SHIP (SH2-containing Inositol 5?-Phosphatase) expression was downregulated. These molecular unbalances were accompanied by increased BCR-induced calcium signalling, attenuated B cell linker protein (BLNK) and ERK activity and enhanced activity of PI3K/protein kinase B (Akt) pathway. Further, the IRF4-deficient cells showed dramatically diminished cytoskeletal responses to anti-IgM cross-linking. Our results show that IRF4 has an important role in the regulation of BCR signalling and help to shed light on the molecular mechanisms of B cell development and germinal centre response.
Subject(s)
Avian Proteins/metabolism , B-Lymphocytes/physiology , Interferon Regulatory Factors/metabolism , Receptors, Antigen, B-Cell/metabolism , Actin Cytoskeleton/metabolism , Adaptor Proteins, Signal Transducing/genetics , Adaptor Proteins, Signal Transducing/metabolism , Animals , Avian Proteins/genetics , Calcium Signaling/genetics , Cell Line , Chickens , Extracellular Signal-Regulated MAP Kinases/metabolism , Gene Expression Regulation/genetics , Gene Knockout Techniques , Interferon Regulatory Factors/genetics , Intracellular Signaling Peptides and Proteins/metabolism , Oncogene Protein v-akt/metabolism , Phosphatidylinositol 3-Kinases/metabolism , Phosphoric Monoester Hydrolases/genetics , Phosphoric Monoester Hydrolases/metabolism , Phosphorylation/genetics , Protein-Tyrosine Kinases/metabolism , Syk KinaseABSTRACT
Effective humoral immunity depends on B cells, plasma cells and follicular helper T cells (TFH) and secreted high-affinity antibodies. The differentiation of mature B cell into plasma cells is ultimately hardwired in a regulatory network of transcription factors. This circuitry is responding to extracellular stimuli, which leads to production of higher-affinity antibodies after germinal centre (GC) reaction. The understanding of the transcriptional regulation of GCs and the initiation of plasma cell differentiation is becoming increasingly clear. It is evident that transcriptional repressor Blimp-1 can drive the plasma cell differentiation, but the initiation of plasma cell differentiation in GCs is likely coupled to the loss of B cell characteristics maintained by transcription factors Pax5 and Bcl6.
