An incoherent regulatory network architecture that orchestrates B cell diversification in response to antigen signaling

Abstract

The B-lymphocyte lineage is a leading system for analyzing gene regulatory networks (GRNs) that orchestrate distinct cell fate transitions. Upon antigen recognition, B cells can diversify their immunoglobulin (Ig) repertoire via somatic hypermutation (SHM) and/or class switch DNA recombination (CSR) before differentiating into antibody-secreting plasma cells. We construct a mathematical model for a GRN underlying this developmental dynamic. The intensity of signaling through the Ig receptor is shown to control the bimodal expression of a pivotal transcription factor, IRF-4, which dictates B cell fate outcomes. Computational modeling coupled with experimental analysis supports a model of 'kinetic control', in which B cell developmental trajectories pass through an obligate transient state of variable duration that promotes diversification of the antibody repertoire by SHM/CSR in direct response to antigens. More generally, this network motif could be used to translate a morphogen gradient into developmental inductive events of varying time, thereby enabling the specification of distinct cell fates.

Figure 1

Modeling B cell fate dynamics. (A) Cell fate dynamics that are considered to underlie the generation of effector B cells. Activated B cells can directly differentiate into IgM-secreting plasma cells or undergo CSR/SHM before terminal differentiation. (B) GRN that orchestrates B cell fate dynamics. Genes specific for the CSR/SHM state are shown in green, and those specific for the plasma-cell state are shown in red. Arrows denote activation, and barred lines denote repression. A dotted line indicates that the regulatory interaction may be indirect. In the model, the expression of genes other than Pax5 and Bach2 is assumed to be contingent upon B cell activation; Bcl-6 and Bach2 are treated as a single species. (C, D) Typical trajectories in the [Blimp-1]-[AID] plane of the core deterministic model (Equation (1)) with parameters corresponding to the basic bistability (C) and kinetic-control scenarios (D). Expression coordinates for the indicated components are spaced at equal time intervals. (E, F) Duration of CSR/SHM state as a function of initial IRF-4 production rate. The time spent in the CSR/SHM state decreases with initial IRF-4 production rate (e_I) in the kinetic-control scenario (F) but is insensitive to e_I in the basic bistability scenario (E). A cell is considered to have entered the CSR/SHM state when its Pax5 concentration is above 3/4 of the peak value of Pax5 (around 1.5) and to have exited when Blimp-1 concentration is greater than a threshold ([Blimp-1]=3). Shading indicates standard deviations arising from noise in the multiscale simulations.

Figure 2

Experimentally testable predictions for B cell fate dynamics. (A, B) Relative numbers of plasma cells (red) and Ig-switched cells (green) predicted by the (A) basic bistability and (B) kinetic-control scenarios as a function of initial IRF-4 production rate (e_I). (C, D) Comparison of peak expression levels of Bcl-6/Bach2 (green) and Blimp-1 (red) genes in populations of cells with different initial rates of IRF-4 production in the (C) basic bistability and (D) kinetic-control scenarios. The peak is determined after the averages are calculated for consistency with the RT–PCR measurements.

Figure 3

Strength of BCR signaling determines the initial induction levels and subsequent dynamics of IRF-4 expression. (A) The dynamics of IRF-4 expression in response to BCR stimulation. NP-specific B cells were purified from spleens of B1-8i mice and stimulated with NP(12)-Ficoll in the presence of interleukin-2, -4, -5 and soluble CD40L. The expression of IRF-4 was measured over time using flow cytometry of cells labeled with the division-tracking dye CFSE. The two shaded gray bars highlight the IRF-4^lo- and IRF-4^hi-expressing cell populations. (B) The dynamics of IRF-4 expression in response to differing BCR signaling intensities. Purified NP-reactive B cells were stimulated with serial dilutions of NP(12)-Ficoll (initial concentration 0.01 ng/ml) or NP(109)-Ficoll (0.0001 ng/ml) as described above. IRF-4 expression was analyzed on day 1 (top panels) and day 3 (bottom panels). Data are representative of five experiments.

Figure 4

IRF-4 transduces BCR signal strength to control B cell fate. Purified splenic B cells from NP-specific B1-8i heavy chain knock-in mice were stimulated with NP(12)-Ficoll in the presence of interleukin-2, -4, -5 and soluble CD40L. Serial 10-fold dilutions of NP-Ficoll were used with an initial concentration of 0.01 ng/ml. (A) Differentiated cells were analyzed at day 4 and all flow cytometry plots are shown in relation to cell division (CFSE). The frequencies of plasmablasts (Syndecan-1⁺) and IgG1 class-switched cells are shown as a function of increasing antigen concentration. (B) BCR signal strength dictates B cell fate dynamics by modulating changes in the expression levels of key components of the GRN. RNA was isolated from B cells activated as described above on days 1, 2 and 3. Indicated transcripts were measured by quantitative RT–PCR; transcripts were normalized to Oct1 and used to calculate relative expression. Shown is the average expression from two independent experiments, with results for each gene in turn normalized by its highest expression across all conditions. (C) Peak levels of Bach2 (green) and Blimp-1 (red) expression from (B) as a function of antigen concentration.

Figure 5

Super-induction of IRF-4 regulates B cell fate dynamics in a manner predicted by the modeling. (A) Purified splenic B cells of the indicated genotype (Irf4^+/−ColA1^Irf4/Irf4Rosa26^+/M2rtTA) were stimulated with CD40L and IL-4 in the presence of varying concentrations of DOX (6.4–250 ng/ml). The frequencies of IgG1 class-switched cells and of Syndecan-1⁺ plasmablasts were determined at day 4 of activation, relative to cell division (CFSE dilution). The expression of IRF-4 as a function of DOX was monitored by flow cytometry. The average frequencies from three experiments is shown along with s.e.m. (B, C) Induction of IRF-4 expression by DOX results in changes in the expression levels of key components of the GRN. RNA was isolated and analyzed as described in Figure 4.

Figure 6

Inducible expression of IRF-4 is sufficient to regulate B cell fate in a manner predicted by the modeling. (A) Cell fate of purified splenic B cells of the indicated genotype (Irf4^−/−ColA1^Irf4/Irf4Rosa26^+/M2rtTA) were enumerated as in Figure 5A. (B, C) Expression levels of key components of the GRN as a function of IRF-4 induction as in Figure 5B and C.