Graded expression of interferon regulatory factor-4 coordinates isotype switching with plasma cell differentiation

Molecular mechanisms underlying the coordination of isotype switching with plasma cell differentiation are poorly understood. We show that interferon regulatory factor-4 (IRF-4) regulates both processes by controlling the expression of the Aicda and Prdm1 genes, which encode AID and Blimp-1, respectively. Genome-wide analysis demonstrated that Irf4(-/-) B cells failed to induce the entire Blimp-1-dependent plasma cell program. Restoration of AID or Blimp-1 expression in Irf4(-/-) B cells promoted isotype switching or secretion, respectively. IRF-4 was expressed in a graded manner in differentiating B cells and targeted Prdm1. Higher concentration of IRF-4 induced Prdm1 and consequently the transition from a germinal center gene expression program to that of a plasma cell. We propose a gene-regulatory network in which graded expression of IRF-4 developmentally coordinates isotype switching with plasma cell differentiation.     

Reduced expression of IL-12 p35 by SJL/J macrophages responding to Theiler's virus infection is associated with constitutive activation of IRF-3

Macrophages responding to viral infections may contribute to autoimmune demyelinating diseases (ADD). Macrophages from ADD-susceptible SJL/J mice responding to Theiler's Virus (TMEV) infection, the TLR7 agonist loxoribine, or the TLR4 agonist-LPS expressed less IL-12 p35 but more IL-12/23 p40 and IFN-beta than macrophages from ADD-resistant B10.S mice. While expression of IRF-1 and -7 was similar between B10.S and SJL/J TMEV-infected macrophages, SJL/J but not B10.S macrophages exhibited constitutively active IRF-3. In contrast to overexpressed IRF-1, IRF-5, and IRF-7, which stimulated p35 promoter reporter activity, overexpressed IRF-3 repressed p35 promoter activity in response to TMEV infection, loxoribine, IFN-gamma/LPS, but not IFN-gamma alone. IRF-3 lessened but did not eliminate IRF-1-stimulated p35 promoter activity. Repression by IRF-3 required bp -172 to -122 of the p35 promoter. The data suggest that pre-activated IRF-3 is a major factor in the differences in IL-12 production between B10.S and SJL/J macrophages responding to TMEV.     

Antibody class switching differs among SJL, C57BL/6 and 129 mice

Inbred mouse strains used for gene manipulation studies vary in many respects, including immune system function. These differences can interfere with data interpretation unless the mice are well backcrossed. Here, we show that antibody class switching to IgG3 in cultured splenic B cells from Swiss James Lambert (SJL) and 129/Sv mice is 2- to 6-fold less efficient compared with C57BL/6 (B6). Under optimal stimulation conditions, IgA switching is also 2- to 19-fold lower in SJL and 129/Sv B cells. Splenic B cells from SJL mice express higher levels of CD19 and CD21 compared with B6, and their CD21(high)CD23(low) B cells have little CD9 expression, suggesting atypical marginal zone (MZ) B cells. However, sort purification of splenic B cell subsets did not equalize in vitro class switching to IgG3 or IgA between SJL and B6. 129/Sv spleens have a 3-fold greater number of MZ B cells compared with B6, with similar CD9 expression. Poor IgG3 switching by 129/Sv B cells is specific to CD23(high) follicular B cells, whereas similar changes in IgA switching are seen in both CD21(high) and CD23(high) B cell subsets from 129/Sv. Therefore, the functions and phenotypes of mature B cells differ among three common strains of mice.     

IRF-1 and NF-kappaB p50/cRel bind to distinct regions of the proximal murine IL-12 p35 promoter during costimulation with IFN-gamma and LPS

LPS and IFN-gamma, which activate NF-kappaB cRel/p50 and IFN regulatory factor-1 (IRF-1), respectively, costimulate expression of the IL-12 p35 subunit in macrophages. The murine p35 promoter proximal to exon 2 is active during costimulation with IFN-gamma and LPS because it contains kappaB and IRF elements (E) with significant homology to the human p35 promoter. IFN-gamma or LPS stimulate nuclear localization of IRF-1 or cRel/p50, respectively, in the RAW 264.7 macrophage cell line. EMSAs reveal that IFN-gamma/LPS stimulates within 2 h, in RAW 264.7 cells or peritoneal macrophages, nuclear localization of proteins that target nt -137/-93 of the p35 promoter. DNA affinity assays utilizing nuclear extracts from RAW 264.7 cells show that NF-kappaB cRel and p50 bind to the kappaB-E within nt -122 to -93 of the p35 exon 2 promoter while IRF-1 binds to the IRF-E within nt -157 to -113 but not the one within nt -122 to -93. In addition, p50/cRel attachment to the kappaB-E was not dependent upon IRF-1 association with the IRF-E, and vice versa. Chromosome immunoprecipitation assays confirm inducible recruitment of IRF-1 and cRel to the endogenous p35 exon 2 promoter in both RAW 264.7 and primary macrophages costimulated with IFN-gamma and LPS. IFN-gamma, IFNgamma/LPS, or overexpression of IRF-1 plus cRel activated the wild-type p35 promoter reporter but not the p35 promoter reporter mutated at nt -110/-101 or in the presence of IRF-1 siRNA. Thus, cRel with IRF-1 induce p35 expression through a small region of the p35 exon 2 promoter during IFN-gamma and LPS costimulation of macrophages.     

B cell‐intrinsic MyD88 signaling controls IFN‐γ‐mediated early IgG2c class switching in mice in response to a particulate adjuvant

Adjuvants improve the potency of vaccines, but the modes of action (MOAs) of most adjuvants are largely unknown. TLR‐dependent and ‐independent innate immune signaling through the adaptor molecule MyD88 has been shown to be pivotal to the effects of most adjuvants; however, MyD88's involvement in the TLR‐independent MOAs of adjuvants is poorly understood. Here, using the T‐dependent antigen NIPOVA and a unique particulate adjuvant called synthetic hemozoin (sHZ), we show that MyD88 is required for early GC formation and enhanced antibody class‐switch recombination (CSR) in mice. Using cell‐type‐specific MyD88 KO mice, we found that IgG2c class switching, but not IgG1 class switching, was controlled by B cell‐intrinsic MyD88 signaling. Notably, IFN‐γ produced by various cells including T cells, NK cells, and dendritic cells was the primary cytokine for IgG2c CSR and B‐cell intrinsic MyD88 is required for IFN‐γ production. Moreover, IFN‐γ receptor (IFNγR) deficiency abolished sHZ‐induced IgG2c production, while recombinant IFN‐γ administration successfully rescued IgG2c CSR impairment in mice lacking B‐cell intrinsic MyD88. Together, our results show that B cell‐intrinsic MyD88 signaling is involved in the MOA of certain particulate adjuvants and this may enhance our specific understanding of how adjuvants and vaccines work.     

T‐bet is a new synergistic meeting point for the BCR and TLR9 signaling cascades

The importance of the BCR and TLR9 in autoimmunity and in the production of auto‐antibodies is well established but the underlying molecular mechanism still needs to be determined. Here, we aim to characterize the BCR‐TLR9 cross‐talk by its effect on T‐bet, as T‐bet is activated and regulated by both receptors and has an important role in class‐switching to pathological IgG2a in mice. Using primary mouse B cells, we demonstrate that T‐bet expression is synergistically elevated by the cross‐talk between the BCR and TLR9. To test the effect of this synergy on IgG2a‐switching, the levels of switched B cells were checked by functional tests. We found that BCR costimulation had no additional effect on TLR9‐induced IgG2a expression, however the expression of Rad51 was synergistically increased. To check the biological significance of the synergy, we compared T‐bet expression in B cells from healthy and collagen‐induced arthritis mice but no differences were found. Taken together, we demonstrate here that signaling cascades driven by the BCR and TLR9 have a newly identified meeting point at T‐bet. The two cascades act synergistically on T‐bet; however additional signals may be needed to induce prolonged functional responses such as class‐switch recombination.