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. 2014 Jul 1;111(26):9585-90.
doi: 10.1073/pnas.1402485111. Epub 2014 Jun 16.

Regulation of germinal center responses and B-cell memory by the chromatin modifier MOZ

Kim L Good-Jacobson  1 , Yunshun Chen  2 , Anne K Voss  3 , Gordon K Smyth  4 , Tim Thomas  3 , David Tarlinton  1
Affiliations

Regulation of germinal center responses and B-cell memory by the chromatin modifier MOZ

Kim L Good-Jacobson et al. Proc Natl Acad Sci U S A. .

Abstract

Memory B cells and long-lived bone marrow-resident plasma cells maintain humoral immunity. Little is known about the intrinsic mechanisms that are essential for forming memory B cells or endowing them with the ability to rapidly differentiate upon reexposure while maintaining the population over time. Histone modifications have been shown to regulate lymphocyte development, but their role in regulating differentiation and maintenance of B-cell subsets during an immune response is unclear. Using stage-specific deletion of monocytic leukemia zinc finger protein (MOZ), a histone acetyltransferase, we demonstrate that mutation of this chromatin modifier alters fate decisions in both primary and secondary responses. In the absence of MOZ, germinal center B cells were significantly impaired in their ability to generate dark zone centroblasts, with a concomitant decrease in both cell-cycle progression and BCL-6 expression. In contrast, there was increased differentiation to IgM and low-affinity IgG1(+) memory B cells. The lack of MOZ affected the functional outcome of humoral immune responses, with an increase in secondary germinal centers and a corresponding decrease in secondary high-affinity antibody-secreting cell formation. Therefore, these data provide strong evidence that manipulating epigenetic modifiers can regulate fate decisions during humoral responses, and thus could be targeted for therapeutic intervention.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
Histone acetyltransferase MOZ is important for GC formation. Mb-1Cre/+ (WT control) and Mozfl/flMb-1Cre/+ mice were immunized with NP–KLH/alum. (A and B) B cells (CD19+IgDlo) were stained for antigen-specific GCs (NPintFashi) at multiple time points postimmunization. (A) Representative flow cytometric plot of gating profiles for GC B cells. (B–D) Frequency (B) and number (C) of GC B cells and (D) frequency of GC B cells that are IgG1+. Mb-1Cre/+, black bars; Mozfl/flMb-1Cre/+, white bars. Data are combined from three experiments (day 7) and two per time point (day 13 and day 27); n = 5–10 per genotype. Error bars indicate ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. (E and F) Immunohistological analysis of splenic sections stained with (E) B220 (blue) and IgG1 (red) or (F) B220 (red) and PNA (blue) day 7 postimmunization; representative of three spleens per genotype. Original magnification: 5×.
Fig. 2.
Fig. 2.
MOZ regulates proliferation and zonal formation within the GC. (A–D) GC B cells were assessed for DZ and LZ proportions using CD86 and CXCR4 to differentiate these zones by flow cytometry. (A) NP+FashiIgDlo B cells were assessed for zonal formation at day 7 postimmunization in Mb-1Cre/+ and Mozfl/flMb-1Cre/+. (B–D) Ratio of DZ to LZ GC B cells (B) and number of DZ (C) and LZ (D) GC B cells. Mb-1Cre/+, black bars; Mozfl/flMb-1Cre/+, white bars. Data are combined from three (day 7) and two (day 13) experiments; n = 5–10 per genotype. Error bars indicate ± SEM. *P < 0.05, **P < 0.01, ****P < 0.0001. (E and F) Cell-cycle analysis of day 7 sort-purified GC B cells from Mb-1Cre/+ (black bars) and Mozfl/flMb-1Cre/+ (white bars). Data are combined from two experiments; n = 6 per genotype. Error bars indicate ± SEM. *P < 0.05, **P < 0.01.
Fig. 3.
Fig. 3.
Reduced BCL-6 expression in the absence of MOZ. (A and B) Representative plots of BCL-6 expression versus Fas (arrow; A) in CD19+IgDlo cells, and BCL-6 expression in NP+FashiCD19+IgDlo cells (B), day 7 postimmunization. (C) Geometric mean fluorescence intensity (GMFI) of BCL-6 in NP+FashiCD19+IgDlo cells in Mb-1Cre/+ (black bars) and Mozfl/flMb-1Cre/+ (white bars). Data are combined from three (day 7) and two (day 13) experiments; n = 5–10 per genotype. Error bars indicate ± SEM. **P < 0.01.
Fig. 4.
Fig. 4.
MOZ is required in activated B cells during a humoral response. (A–D) Mozfl/flAicdaCre/+ (white bars) and AicdaCre/+ (WT control; black bars) were immunized with NP–KLH/alum. (A) GC B-cell frequency, (B) GC B-cell number, (C) DZ GC B-cell number, and (D) GMFI of BCL-6 in GC B cells were assessed at day 7 postimmunization. Data are combined from two experiments; n = 7 per genotype. Error bars indicate ± SEM. *P < 0.05, **P < 0.01. (E) Heatmap of RNA-sequencing data displayed in Table S1; each column is an independent sample obtained from pooled sort-purified day 7 GC B cells from either AicdaCre/+ or Mozfl/flAicdaCre/+ mice.
Fig. 5.
Fig. 5.
MOZ regulates memory formation. (A) Flow cytometric assessment of differentiation in the absence of Moz. NP+IgG1+CD19+Dumplo B cells were assessed for CD38 expression. (B and C) Frequency (B) and number (C) of NP+IgG1+ B cells in Mb-1Cre/+ (black bars) and Mozfl/flMb-1Cre/+ (white bars). (D–F) The frequency of CD38+ memory B cells within NP+IgG1+ (D), NP+IgG1+CD38+ frequency (E), and number (F) were assessed at multiple time points postimmunization. (G–J) ELISPOT analysis of NP+IgG1+ ASCs in the spleen (G and I) and BM (H and J) at multiple time points postimmunization. (I and J) Affinity of ASCs was assessed by calculating the ratio of NP2-binding to NP14–16-binding IgG1 ASCs. Data are combined from three experiments (day 7) and two per time point (day 13 and day 27); n = 5–10 per genotype. Error bars indicate ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.
Fig. 6.
Fig. 6.
B cell-intrinsic defect in the absence of MOZ. (A) Schematic representation of the BM chimera setup as detailed in Materials and Methods. BM chimeras in which irradiated Ly5.1 recipients were reconstituted with 50% Ly5.1 BM and 50% Mozfl/flAicdaCre/+ BM. (B–F) Flow cytometric assessment of the frequency of GC B cells (B), ratio of DZ to LZ within GC B cells (C), NP+IgG1+ B cells (D), CD38+ frequency of NP+IgG1+ B cells (E), and frequency of NP+IgG1+CD38+ B cells (F). Data are combined from two independent experiments; n = 7 mice per genotype. Error bars indicate ± SEM. **P < 0.01, ***P < 0.001.
Fig. 7.
Fig. 7.
Alteration in secondary responses in the absence of MOZ. To assess secondary responses, mice were immunized with NP–KLH precipitated in alum, rested for at least 6 wk, and then boosted with NP–KLH in PBS. Humoral responses were assessed 3 d postboost. (A and B) ELISPOT analysis of secondary NP+IgG1+ ASCs in the spleen in Mb-1Cre/+ (black bars) and Mozfl/flMb-1Cre/+ (white bars). High-affinity NP+IgG1+ ASCs (A) as well as all NP+IgG1+ ASCs (B) were assessed. (C) Flow cytometric assessment of GC B cells generated during a secondary response. (D and E) IgM+IgDlo memory B cells generated during a primary response 6 wk (D) or 7 d (E) postprimary immunization. Data are combined from two experiments; n = 4–7 per genotype. Error bars indicate ± SEM. *P < 0.05, **P < 0.01.
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