IL-21 acts directly on B cells to regulate Bcl-6 expression and germinal center responses

Abstract

During T cell-dependent responses, B cells can either differentiate extrafollicularly into short-lived plasma cells or enter follicles to form germinal centers (GCs). Interactions with T follicular helper (Tfh) cells are required for GC formation and for selection of somatically mutated GC B cells. Interleukin (IL)-21 has been reported to play a role in Tfh cell formation and in B cell growth, survival, and isotype switching. To date, it is unclear whether the effect of IL-21 on GC formation is predominantly a consequence of this cytokine acting directly on the Tfh cells or if IL-21 directly influences GC B cells. We show that IL-21 acts in a B cell-intrinsic fashion to control GC B cell formation. Mixed bone marrow chimeras identified a significant B cell-autonomous effect of IL-21 receptor (R) signaling throughout all stages of the GC response. IL-21 deficiency profoundly impaired affinity maturation and reduced the proportion of IgG1(+) GC B cells but did not affect formation of early memory B cells. IL-21R was required on GC B cells for maximal expression of Bcl-6. In contrast to the requirement for IL-21 in the follicular response to sheep red blood cells, a purely extrafollicular antibody response to Salmonella dominated by IgG2a was intact in the absence of IL-21.

Figure 1.

IL-21–deficient mice form detectable GCs after immunization, but the kinetics of the GC is altered. (A and B) ELISA analysis of total serum IgG1 (A) and IgE (B) in unimmunized mice of the indicated genotypes. (C) Flow cytometric contour plots and graphical analysis of GL-7⁺Fas⁺ GC cells gated on live B220⁺ lymphocytes from Il21^+/+ and Il21^−/− mice at the indicated time points after SRBC immunization. Each symbol represents one mouse. (D) Photomicrographs of spleen sections stained for IgD (brown) and PNA (blue) from Il21^+/+ and Il21^−/− mice 8 d after immunization. Data are representative of two independent experiments (n ≥ 4 per group). Bars, 200 µm. (E) Flow cytometric histograms and graphical analysis of PNA binding on GL-7⁺Fas⁺B220⁺ GC B cells from Il21^+/+ and Il21^−/− mice 7 d after SRBC immunization. Statistically significant differences are indicated (*, P ≤ 0.05; **, P ≤ 0.01). Data are representative of two independent experiments, each symbol represents one mouse, and tops of bars are drawn through the median values. MFI, mean fluorescence intensity; ns, not significant.

Figure 2.

IL-21–deficient mice form Tfh cells after immunization, but their maintenance is impaired. (A) Flow cytometric contour plots and graphical analysis of CXCR5⁺PD-1⁺ Tfh cells gated on CD4⁺ B220⁻ live lymphocytes from Il21^+/+ and Il21^−/− mice at the indicated time points after SRBC immunization (percentages are shown). (B) Photomicrographs of spleen sections taken from Il21^+/+ (top) and Il21^−/− (bottom) mice 8 d after immunization with SRBCs. In all panels, IgD is stained in brown; blue color stains indicate PNA binding (left), CD3 (middle), and PD-1 (right). Bars, 200 µm. Statistically significant differences are indicated (*, P ≤ 0.05). Data are representative of two independent experiments, each symbol represents one mouse, and tops of bars are drawn through the median values. ns, not significant.

Figure 3.

IL-21R expression is required on both GC B cells and Tfh cells for their maintenance but is dispensable for their formation. (A and B) Gating strategy (A) and bar graphs (B) of mixed bone marrow chimeras containing a 1:1 ratio of control Il21r^+/+CD45.1/Il21r^+/+CD45.2 or Il21r^+/+CD45.1/Il21r^−/−CD45.2 bone marrow. Spleens from unimmunized mice (A; and B, top) or from mice immunized with SRBCs 6 or 14 d previously (B, middle and bottom) were analyzed by flow cytometry for the percentage of B220⁺ B cells that are CD45.2⁺ (B220⁺), the percentage of GL-7⁺Fas⁺ GC cells among B220⁺ cells that are CD45.2⁺ (GC cells), the percentage of CD4⁺ Th cells that are CD45.2⁺ (CD4⁺), and the percentage of CXCR5^highPD-1^high Tfh cells among CD4⁺ cells that are CD45.2⁺ (Tfh cells). Each bar represents a single recipient mouse; individual mice have been numbered and placed in the same order in each of the plots. Statistically significant decreases are indicated (*, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001). Data are representative of two independent experiments. (C and D) Gating strategy (C) and dot plots (D) of the proportion of B220⁺, GC (B220⁺Fas⁺GL-7⁺), and IgG1⁺ GC (B220⁺Fas⁺GL-7⁺IgG1⁺) cells derived from the CD45.2 compartment of mixed chimeras reconstituted with a 1:1 ratio of either CD45.1 Il21r^+/+/CD45.2 Il21r^+/+ (left) or CD45.1 Il21r^+/+/CD45.2 Il21r^−/− (right) bone marrow before (C; and D, top) or 6 d after SRBC immunization (D, bottom). In D, each number represents a single recipient mouse from two separate groups of the chimeric mice indicated above the left and right panels.

Figure 4.

IL-21 contributes to affinity maturation but is dispensable for EM B cell formation. (A) Representative flow cytometric contour plots of donor CD45.1 SW_HEL cells from the spleens of Il21^+/+ or Il21^−/− recipient mice 10 d after adoptive transfer and immunization with HEL^3X SRBCs. Contour plots show IgG1⁺ cells binding with high (top oval gates) and low (bottom oval gates) affinity to HEL^3X (percentages are shown). (B) Bar graphs show HEL^{3X hi} IgG1⁺ CD45.1 cells (top oval gates in A) as a proportion of all HEL^3X-binding IgG1⁺ cells (square gates in A). This figure is representative of two independent experiments (n ≥ 3 mice per group. (C) Ratio of total Tfh (CXCR5^highPD-1^high CD4⁺) cells to total HEL-binding donor (CD45.1) GC B cells (Fas⁺GL-7⁺). Two independent experiments are represented in this plot. (D) Representative flow cytometric contour plots 10 d after adoptive transfer of SW_HEL B cells into Il21^+/+ or Il21^−/− mice and immunization with HEL^3X SRBCs gated on donor CD45.1 and HEL-binding cells (percentages are shown). The gates show GC cells (HEL^int binding) and EM B cells (HEL^hi binding). (E) Phenotype of GC B cells and EM cells gated as in D. (F and G) Total number of donor HEL^int-binding GC B cells per spleen (F) and the proportion of donor HEL^hi-binding EM B cells (G) in mice of the indicated genotypes gated as in D. Each symbol represents one mouse and tops of bars are drawn through the median values. Statistically significant differences are indicated (**, P ≤ 0.01).

Figure 5.

Lack of IL-21R signaling reduces the expression of Bcl-6 in GC B cells. (A) Flow cytometric contour plots indicating the gating strategy for non-GC and GC B220⁺ cells (left) and BCL-6 expression on GL-7⁺ (middle) and Fas⁺ (right) B cells. (B and C) Histogram overlays (B) and bar graphs (C) showing the fluorescence intensity of BCL-6 staining in GC and non-GC B cells as gated in A derived from the CD45.1 Il21r^+/+ or CD45.2 Il21^−/− compartment of CD45.1 Il21r^+/+/CD45.2 Il21r^−/− mixed bone marrow chimeras 6 d after SRBC immunization. The horizontal dashed line highlights the median levels of Bcl-6 found in non-GC B cells. Each set of two bars represents the data from the CD45.1 and CD45.2 compartments of a single mouse. MFI, mean fluorescence intensity.

Figure 6.

Lack of IL-21 does not alter Salmonella pathogen clearance or production of anti-Salmonella humoral immunity. (A) ELISA analysis of the titers of Salmonella-specific IgM, IgG1, IgG2a^b, IgG2b, and IgG3 in sera from Il21^+/+(white bars) and Il21^−/− (black bars) mice 12 d after Salmonella infection. (B and C) Dot plots of spleen weights (B) and the number of bacteria per liver (C) in Il21^+/+ and Il21^−/− mice 12 d after infection with Salmonella. Data are representative of two independent experiments. Each symbol represents one mouse and tops of bars are drawn through the median values. nd, not detected.