PP4 is essential for germinal center formation and class switch recombination in mice.

Ming-Yu Chen,Yu-Wen Su,Wen-Jye Lin,Tse-Hua Tan,Guanni-Yi Yu,Ya-Ping Chen,Ming-Sian Wu,Javier M Di Noia

doi:10.1371/journal.pone.0107505

Abstract

PP4 is a serine/threonine phosphatase required for immunoglobulin (Ig) VDJ recombination and pro-B/pre-B cell development in mice. To elucidate the role of PP4 in mature B cells, we ablated the catalytic subunit of murine PP4 in vivo utilizing the CD23 promoter and cre-loxP recombination and generated CD23crePP4F/F mice. The development of follicular and marginal zone B cells was unaffected in these mutants, but the proliferation of mature PP4-deficient B cells stimulated by in vitro treatment with either anti-IgM antibody (Ab) or LPS was partially impaired. Interestingly, the induction of CD80 and CD86 expression on these stimulated B cells was normal. Basal levels of serum Igs of all isotypes were strongly reduced in CD23crePP4F/F mice, and their B cells showed a reduced efficiency of class switch recombination (CSR) in vitro upon stimulation by LPS or LPS plus IL-4. When CD23crePP4F/F mice were challenged with either the T cell-dependent antigen TNP-KLH or the T cell-independent antigen TNP-Ficoll, or by H1N1 virus infection, the mutant animals failed to form germinal centers (GCs) in the spleen and the draining mediastinal lymph nodes, and did not efficiently mount antigen-specific humoral responses. In the resting state, PP4-deficient B cells exhibited pre-existing DNA fragmentation. Upon stimulation by DNA-damaging drug etoposide in vitro, mutant B cells showed increased cleavage of caspase 3. In addition, the mutant B cells displayed impaired CD40-mediated MAPK activation, abnormal IgM-mediated NF-κB activation, and reduced S phase entry upon IgM/CD40-stimulation. Taken together, our results establish a novel role for PP4 in CSR, and reveal crucial functions for PP4 in the maintenance of genomic stability, GC formation, and B cell-mediated immune responses.

Highlights

Protein phosphatase 4 (PP4) is a complex and well-conserved holoenzyme containing a catalytic subunit that can bind to diverse regulatory subunits depending on the biological circumstances
For all the parameters used to evaluate DNA damage, including tail intensity, tail length, and Olive Tail Moment, scores were higher in mutant B cells than in WT B cells. These results demonstrate that PP4deficient B cells exhibit increased DNA fragmentation even in the resting state, and suggest that loss of PP4 results in severe DNA damage
We found that 14% and 8% of transgenic WT B cells from BCRHEL/CD23crePP4+/+ mice were in S phase upon hen egg lysozyme (HEL)- and IgM/CD40 Ab (CD40)-stimulation respectively

Summary

Introduction

Protein phosphatase 4 (PP4) is a complex and well-conserved holoenzyme containing a catalytic subunit that can bind to diverse regulatory subunits depending on the biological circumstances. We previously ablated PP4 in developing B cells using the mb-1/cre/loxP system and generated mb-1/cre/PP4F/F mice [21]. We found that this loss of PP4 impaired immunoglobulin (Ig) VDJ recombination and led to a profound block in B cell development at the pro-B/ pre-B cell stage [21]. In pro-B cells of these mutants, DNA breaks in the Ig loci were drastically increased, suggesting a role for PP4 in non-homologous end joining (NHEJ) DNA repair. Because mb-1/cre/PP4F/F mice do not produce mature B cells, they cannot be used to examine the role of PP4 in events triggered by antigen encounter

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Conclusion