Antigen Complexed with a TLR9 Agonist Bolsters c-Myc and mTORC1 Activity in Germinal Center B Lymphocytes

Abstract

The germinal center (GC) is the anatomical site where humoral immunity evolves. B cells undergo cycles of proliferation and selection to produce high-affinity Abs against Ag. Direct linkage of a TLR9 agonist (CpG) to a T-dependent Ag increases the number of GC B cells. We used a T-dependent Ag complexed with CpG and a genetic model for ablating the TLR9 signaling adaptor molecule MyD88 specifically in B cells (B-MyD88^- mice) together with transcriptomics to determine how this innate pathway positively regulates the GC. GC B cells from complex Ag-immunized B-MyD88^- mice were defective in inducing gene expression signatures downstream of c-Myc and mTORC1. In agreement with the latter gene signature, ribosomal protein S6 phosphorylation was increased in GC B cells from wild-type mice compared with B-MyD88^- mice. However, GC B cell expression of a c-Myc protein reporter was enhanced by CpG attached to Ag in both wild-type and B-MyD88^- mice, indicating a B cell-extrinsic effect on c-Myc protein expression combined with a B cell-intrinsic enhancement of gene expression downstream of c-Myc. Both mTORC1 activity and c-Myc are directly induced by T cell help, indicating that TLR9 signaling in GC B cells either enhances their access to T cell help or directly influences these pathways to further enhance the effect of T cell help. Taken together, these findings indicate that TLR9 signaling in the GC could provide a surrogate prosurvival stimulus, "TLR help," thus lowering the threshold for selection and increasing the magnitude of the GC response.

FIGURE 1.. mRNA-seq analysis of WT and B-MYD88⁻ NP⁺ GC B cells shows increased c-Myc and mTORC1 gene expression signatures.

(A) Enumeration of GC B cell percentages and total cell numbers from draining lymph nodes of WT and B-MYD88⁻ animals at D14 postimmunization. Representative of four independent experiments with at least three mice per group analyzed by a two-tailed Student t test, *p < 0.05, ***p < 0.0005. (B) Volcano plots comparing gene expression fold changes to p values for all genes expressed in at least one sample after DESeq2 analysis. Red dots in each panel indicate genes associated with the given metabolic/synthetic complex listed. (C) GSEA plots for hallmark gene sets for mTORC and c-Myc gene signatures enriched in WT transcriptional data. (D) GSEA plot from curated gene sets showing enrichment of rapamycin- and serum-sensitive genes in WT samples. (E) GSEA plot from curated gene sets showing enrichment of c-Myc target genes in WT samples.

FIGURE 2.. Dual BCR/TLR9 signaling positively regulates c-Myc expression in naive and GC B cells.

(A) Overlay of representative histograms showing c-Myc-GFP expression in response to anti–IgM-CpG stimulation for 24 h of WT c-Myc-GFP and B-MYD88⁻c-Myc-GFP naive B cells. (B) Enumeration of percent GFP-positive and MFI of cells in (A) (shown is a single experiment representative of three independent experiments). (C) Overlay of representative histograms showing GFP fluorescence gating on live, singlet, CD19⁺, Fas⁺, IgD^lo, and GL7⁺ GC cells of c-Myc-GFP^+/+ mice immunized with NP-CGG-CpG or NP-CGG-Non D14 postimmunization. (D) Enumeration of percent and number of GFP+ GC B cells from NP-CGG-CpG- and NP-CGG-Non-immunized c-Myc-GFP^+/+ mice (three combined experiments with at least three mice per group). (E) Enumeration of percent and number of GFP-positive cells from NP-CGG-CpG–immunized B-MYD88⁻ and WT mice D14 (representative experiment of three independent experiments). **p < 0.005, ****p < 0.0001. NS, p < 0.05 by one-way ANOVA and Holm–Sidak multiple comparison test.

FIGURE 3.. Dual BCR/TLR9 signaling positively regulates mTORC1 activity measured by ribosomal protein S6 phosphorylation in naiveand GC B cells.

(A) Overlay of representative histograms showing pS6(ser240/244) levels in response to anti–IgM-CpG for 24 h in WT and B-MYD88⁻ naive B cells. (B) Enumeration of cell size by forward light scatter (FSC-A), percent pS6(ser240/244)⁺, and MFI of those cells gated positively for pS6(ser240/244) cells for WT and B-MYD88⁻ naive B cells stimulated with anti–IgM-CpG and anti–IgM-Non. (C) Enumeration of cell size from D14 NP-CGG-CpG–immunized B-MYD88⁻ and WT non-GC and GC B cells (representative of four independent experiments). (D) Phospho-flow MFI of mTORC1 downstream target pS6(ser240/244) D14 NP-CGG-CpG–immunized B-MYD88⁻ and WT B cells normalized to mean of B-MYD88⁻ GC MFI (data are combined from three experiments with at least three mice per group). (E) Phospho-flow MFI of mTORC1 downstream target pS6(ser240/244) D14 NP-CGG-CpG-and NP-CGG-Non-immunized mice normalized to mean MFI of NP-CGG-Non MFI (combined from two independent experiments with at least four mice per condition). *p < 0.05, **p < 0.005, ****p < 0.0001. NS, p < 0.05 by one-way ANOVA and Holm–Sidak multiple comparison test.

FIGURE 4.. CpG stimulation of GC B cells ex vivo increases mTORC1 signaling to similar levels as anti-CD40 stimulation.

(A) Representative histograms comparing phospho-flow pS6(ser240/244) staining of 4 h ex vivo–stimulated naive and GC B cells isolated from i.p. NP-CGG alum–immunized mouse spleens at day 12 to D14 (three mice per experiment performed in three separate experiments). (B) Enumeration of experiments from (A), showing level of pS6(ser240/244) expression in naive and GC B cells from (A). (C) Overlays of c-Myc-GFP expression comparing no stimulation to the respective stimulation in naive and GC B cells after 4 h. (D) Enumeration of the relative c-Myc-GFP MFI increase normalized to no stimulation of the samples in (C) (representative of three independent experiments with at least three mice per experiment). *p < 0.05, ***p < 0.001, ****p < 0.0001. NS, p < 0.05 by one-way ANOVA and Tukey multiple comparison test.