High affinity germinal center B cells are actively selected into the plasma cell compartment

Abstract

A hallmark of T cell-dependent immune responses is the progressive increase in the ability of serum antibodies to bind antigen and provide immune protection. Affinity maturation of the antibody response is thought to be connected with the preferential survival of germinal centre (GC) B cells that have acquired increased affinity for antigen via somatic hypermutation of their immunoglobulin genes. However, the mechanisms that drive affinity maturation remain obscure because of the difficulty in tracking the affinity-based selection of GC B cells and their differentiation into plasma cells. We describe a powerful new model that allows these processes to be followed as they occur in vivo. In contrast to evidence from in vitro systems, responding GC B cells do not undergo plasma cell differentiation stochastically. Rather, only GC B cells that have acquired high affinity for the immunizing antigen form plasma cells. Affinity maturation is therefore driven by a tightly controlled mechanism that ensures only antibodies with the greatest possibility of neutralizing foreign antigen are produced. Because the body can sustain only limited numbers of plasma cells, this "quality control" over plasma cell differentiation is likely critical for establishing effective humoral immunity.

Figure 1.

GC and antibody responses to low and high affinity antigens. SW_HEL B cells were adoptively transferred into CD45.1 congenic mice and challenged with HEL^WT-SRBC, HEL^3X-SRBC, or mock antigen. (A) Splenocytes from recipient mice were analyzed by flow cytometry for expression of CD45.2, IgG1, and the ability to bind HEL^WT. The percentage of donor-derived HEL^WT-binding GC B cells that had switched to IgG1 was determined and plotted over the course of the response. Data points represent individual recipients, and lines connect the means. (B) Splenocytes from recipient mice (n = 3) were pooled and stained with CD45.2, syndecan-1, and HEL^WT. Single CD45.2⁺, HEL^WT-binding, GC (syndecan-1⁻) B cells were sorted, and the targeted Ig heavy chain variable gene was PCR amplified and sequenced. Points represent the frequency of somatic mutations in individual clones (integers). A statistically significant difference in mean mutation frequency per clone (bars) was detected at day 15 and appeared close to significant at day 10 but was not significant at day 5. (C) Sera were collected from recipient mice, and the concentration of anti-HEL^WT IgG1 antibodies was measured by ELISA. Data points represent individual recipients, and lines connect the means. (D) Affinity maturation of the IgG1 antibody response to HEL^3X-SRBC. IgG1 antibodies in pooled sera from recipient mice (n = 5) on day 15 of the responses to HEL^WT-SRBC and HEL^3X-SRBC were analyzed for their ability to bind HEL^WT and HEL^3X in parallel ELISAs.

Figure 2.

Tracking affinity-based selection in vivo. Splenocytes from the response shown in Fig. 1 were analyzed by flow cytometry for their expression of CD45.2, IgG1, and the ability to bind the mutant antigen HEL^3X, and the analogue data are presented on logarithmic axes. The frequency of donor-derived (CD45.2⁺) cells as a proportion of total splenocytes and the mean fluorescence intensity (mfi) of HEL^3X binding are shown. CD45.2⁺ cells were also analyzed for HEL^3X binding counterstained against IgG1 (to correct for surface Ig expression level) to show a population of high affinity HEL^3X-binding IgG1⁺ cells that emerges on day 10 and dominates the response to HEL^3X-SRBC by day 15 (oval gate). The frequency of these high affinity cells as a proportion of donor-derived cells is shown. Concatenated data are representative of five mice in each group.

Figure 3.

Selection of a high affinity Y53D somatic mutation in response to HEL^3X-SRBC. (A) SHM analysis of targeted Ig heavy chain loci of responding SW_HEL GC B cells was performed on day 15 of the responses to HEL^WT-SRBC and HEL^3X-SRBC, as described in Materials and methods. The codon encoding Y53 was found to be extensively mutated in the HEL^3X-SRBC response (86% of sequences) and replaced by an aspartate residue (Y53D) in the majority (82%) of cases. (B) ELISA showing the binding of soluble HEL^3X to plate-bound recombinant wild-type (HyHEL10^WT) and Y53D-mutated (HyHEL10^Y53D) HyHEL10 IgG1 antibodies. The Y53D mutation increased the affinity of HyHEL10 for HEL^3X by ∼85-fold. (C) ELISAs showing the relative binding of soluble recombinant HyHEL10^WT and HyHEL10^Y53D antibodies to plate-bound HEL^WT versus HEL^3X. All four binding assays were run in parallel.

Figure 4.

Affinity threshold for post-GC plasma cell differentiation. (A) SW_HEL B cells were adoptively transferred and challenged with HEL^3X-SRBC, and individual donor-derived GC B cells and plasma cells were sorted for SHM analysis, as described in Materials and methods. The proportion of donor-derived GC B cells and plasma cells with the Y53D_HyHEL10 mutation on days 5, 10, and 15 of the response are shown. From left to right, SHM frequencies were 28, 1, 98, 61, 137, and 157 mutations per 10⁴ bp. (B) SW_HEL B cells with (SW_HEL × Blimp ^gfp/+) and without the Blimp-GFP reporter gene were adoptively transferred and challenged with HEL^3X-SRBC as in Fig. 1. Splenocytes harvested on day 10 were analyzed by flow cytometry for expression of GFP, CD45.2, IgG1, and HEL^3X binding, and digital data are presented using biexponential axes. (left) Plots show HEL^3X binding versus IgG1 gated on CD45.2⁺ donor-derived cells. (right) GFP expression by high (top oval gate) and low (bottom oval gate) affinity cells are shown. A distinct population of Blimp-GFP–expressing plasma cells is only evident in high affinity cells in mice that received SW_HEL × Blimp ^gfp/+ B cells. Profiles represent concatenated data with equal contributions from five individual recipient mice. Similar results were obtained in two independent experiments.