Protocol for in vitro BCR-mediated plasma cell differentiation and purification of chromatin-associated proteins

Abstract Abstract 1109 Plasma cell differentiation is initiated by antigen stimulation of B cell receptor (BCR). Until BCR stimulation, BLIMP1, a master regulator of plasma cell differentiation, is suppressed by PAX5, a key transcriptional repressor to maintain B cell identity. After BCR stimulation, upregulation of BLIMP1 and subsequent suppression of PAX5 by BLIMP1 are observed and thought to be the trigger of plasma cell differentiation; however, the trigger that derepresses BLIMP1 expression is yet to be revealed. Here, we demonstrated PAX5 phosphorylation by ERK1/2, the main component of BCR signal, in vitro and in vivo. The sites of PAX5 phosphorylation were identified by PCR mutagenesis assay. In luciferase reporter assays, transcriptional repression on BLIMP1 promoter by PAX5 was canceled by PAX5 phosphorylation. Furthermore, transcriptional repression by phosphorylation-defective mutant of PAX5 was attenuated by CA-MEK1 co-expression to a significantly lesser extent than that by wild-type PAX5, indicating its resistance to ERK1/2 signal-dependent cancelation of the transcriptional repression (Figure A). Finally, BCR stimulation induced strong ERK1/2 activation, phosphorylation of endogenous PAX5 (Figure B), and upregulation of BLIMP1 mRNA expression in B cells. These phenomena were inhibited by U0126, MEK1 inhibitor. These data imply that PAX5 phosphorylation by BCR signal is the initial event in plasma cell differentiation (Figure C). Disclosures: Naoe: Kyowa-Hakko Kirin.: Research Funding; Dainipponn-Sumitomo Pharma.: Research Funding; Chugai Pharma.: Research Funding; Novartis Pharma.: Honoraria, Speakers Bureau; Zenyaku-Kogyo: Research Funding; Otsuka Pharma.: Research Funding.

Download Full-text

Heme regulates B-cell differentiation, antibody class switch, and heme oxygenase-1 expression in B cells as a ligand of Bach2

Blood ◽

10.1182/blood-2010-07-296483 ◽

2011 ◽

Vol 117 (20) ◽

pp. 5438-5448 ◽

Cited By ~ 44

Author(s):

Miki Watanabe-Matsui ◽

Akihiko Muto ◽

Toshitaka Matsui ◽

Ari Itoh-Nakadai ◽

Osamu Nakajima ◽

...

Keyword(s):

Cell Differentiation ◽

B Cells ◽

B Cell ◽

Heme Oxygenase ◽

Plasma Cell ◽

Heme Oxygenase 1 ◽

Plasma Cell Differentiation ◽

Class Switch ◽

Antibody Class

Abstract Heme binds to proteins to modulate their function, thereby functioning as a signaling molecule in a variety of biologic events. We found that heme bound to Bach2, a transcription factor essential for humoral immunity, including antibody class switch. Heme inhibited the DNA binding activity of Bach2 in vitro and reduced its half-life in B cells. When added to B-cell primary cultures, heme enhanced the transcription of Blimp-1, the master regulator of plasma cells, and skewed plasma cell differentiation toward the IgM isotype, decreasing the IgG levels in vitro. Intraperitoneal injection of heme in mice inhibited the production of antigen-specific IgM when heme was administered simultaneously with the antigen but not when it was administered after antigen exposure, suggesting that heme also modulates the early phase of B-cell responses to antigen. Heme oxygenase-1, which is known to be regulated by heme, was repressed by both Bach2 and Bach1 in B cells. Furthermore, the expression of genes for heme uptake changed in response to B-cell activation and heme administration. Our results reveal a new function for heme as a ligand of Bach2 and as a modulatory signal involved in plasma cell differentiation.

Download Full-text

High affinity germinal center B cells are actively selected into the plasma cell compartment

Journal of Experimental Medicine ◽

10.1084/jem.20061254 ◽

2006 ◽

Vol 203 (11) ◽

pp. 2419-2424 ◽

Cited By ~ 207

Author(s):

Tri Giang Phan ◽

Didrik Paus ◽

Tyani D. Chan ◽

Marian L. Turner ◽

Stephen L. Nutt ◽

...

Keyword(s):

Cell Differentiation ◽

B Cells ◽

Plasma Cell ◽

Plasma Cells ◽

Affinity Maturation ◽

The Body ◽

Plasma Cell Differentiation ◽

High Affinity

A hallmark of T cell–dependent immune responses is the progressive increase in the ability of serum antibodies to bind antigen and provide immune protection. Affinity maturation of the antibody response is thought to be connected with the preferential survival of germinal centre (GC) B cells that have acquired increased affinity for antigen via somatic hypermutation of their immunoglobulin genes. However, the mechanisms that drive affinity maturation remain obscure because of the difficulty in tracking the affinity-based selection of GC B cells and their differentiation into plasma cells. We describe a powerful new model that allows these processes to be followed as they occur in vivo. In contrast to evidence from in vitro systems, responding GC B cells do not undergo plasma cell differentiation stochastically. Rather, only GC B cells that have acquired high affinity for the immunizing antigen form plasma cells. Affinity maturation is therefore driven by a tightly controlled mechanism that ensures only antibodies with the greatest possibility of neutralizing foreign antigen are produced. Because the body can sustain only limited numbers of plasma cells, this “quality control” over plasma cell differentiation is likely critical for establishing effective humoral immunity.

Download Full-text

PAX5 Tyrosine Phosphorylation By Syk Co-Operatively Works with Its Serine Phosphorylation By ERK1/2 and Cancels PAX5-Dependent Repression of BLIMP1: A Mechanism of Antigen-Triggering Plasma Cell Differentiation through B Cell Receptor Signal

Blood ◽

10.1182/blood.v124.21.4349.4349 ◽

2014 ◽

Vol 124 (21) ◽

pp. 4349-4349

Author(s):

Yuichiro Inagaki ◽

Fumihiko Hayakawa ◽

Yuki Kojima ◽

Takanobu Morishita ◽

Naoto Imoto ◽

...

Keyword(s):

Cell Differentiation ◽

B Cell ◽

Plasma Cell ◽

Research Funding ◽

Cell Receptor ◽

B Cell Receptor ◽

Serine Phosphorylation ◽

Chugai Pharmaceutical ◽

Plasma Cell Differentiation

Abstract Pax5 is an essential transcription factor to maintain B cell identity. Pax5 is expressed in stages from pro-B to mature B cells and promotes the B cell differentiation program by transcriptional activation of many B cell receptor (BCR)-related genes such as CD19, CD79a, and BLNK. On the contrary, it inhibits plasma cell differentiation by suppressing the expression of BLIMP1 and XBP-1, transcription factors essential for plasma cell differentiation. After BCR stimulation by antigen, upregulation of BLIMP1 and XBP-1 and subsequent suppression of PAX5 by BLIMP1 were observed and thought to be the trigger of plasma cell differentiation. We previously demonstrated that serine phosphorylation of PAX5 by ERK1/2, a main component of BCR signal, attenuated the BLIMP1 suppression by PAX5 and that the PAX5 phosphorylation might be the initial event for plasma cell differentiation (Yasuda T et al, J Immunol. 2012; 188: 6127-34). Here, we investigated additional PAX5 phosphorylation by BCR signal and found that another BCR signal component, Syk, caused PAX5 phosphorylation in vitro (Figure A). We identified the tyrosines that were phosphorylated by Syk in vitro by making phosphorylation-defective mutants, and confirmed that Syk phosphorylated PAX5 at the same sites in vivo (Figure B). In the luciferase reporter assays, PAX5 tyrosine phosphorylation by Syk attenuated the BLIMP1 suppression by PAX5, similarly to its serine phosphorylation by ERK1/2, and both phosphorylations co-operatively worked for it (Figure C). Furthermore, we demonstrated that B cell receptor stimulation with anti-IgM antibody induced Syk and ERK1/2 activation, tyrosine and serine phosphorylation of endogenous Pax5, and upregulation of Blimp1 mRNA. These results suggested that PAX5 phosphorylations by Syk and ERK1/2 co-operatively work for the cancelation of transcriptional repression of Blimp1 by PAX5 after BCR activation by antigen. This might be a trigger of plasma cell differentiation. Our findings give a new insight into the regulation of the terminal differentiation of B cells. Figure 1 Figure 1. Disclosures Naoe: Zenyaku Kogyo: Research Funding; Dainippon Sumitomo Pharma: Research Funding; Kyowa Hakko Kirin Co. LTD: Research Funding; Chugai Pharmaceutical Co. LTD: Research Funding; Novartis Pharma,: Research Funding; Bristol-Myers Squibb: Research Funding; Otsuka Pharmaceutical Co. LTD: Research Funding; FUJIFILM Corporation: Research Funding. Kiyoi:Zenyaku Kogyo: Research Funding; Dainippon Sumitomo Pharma: Research Funding; Kyowa Hakko Kirin Co. LTD.: Research Funding; Chugai Pharmaceutical Co. LTD: Research Funding; Bristol-Myers Squibb: Research Funding; FUJIFILM Corporation: Research Funding.

Download Full-text