Germinal center B cells govern their own fate via antibody feedback

Abstract

Affinity maturation of B cells in germinal centers (GCs) is a process of evolution, involving random mutation of immunoglobulin genes followed by natural selection by T cells. Only B cells that have acquired antigen are able to interact with T cells. Antigen acquisition is dependent on the interaction of B cells with immune complexes inside GCs. It is not clear how efficient selection of B cells is maintained while their affinity matures. Here we show that the B cells' own secreted products, antibodies, regulate GC selection by limiting antigen access. By manipulating the GC response with monoclonal antibodies of defined affinities, we show that antibodies in GCs are in affinity-dependent equilibrium with antibodies produced outside and that restriction of antigen access influences B cell selection, seen as variations in apoptosis, plasma cell output, T cell interaction, and antibody affinity. Feedback through antibodies produced by GC-derived plasma cells can explain how GCs maintain an adequate directional selection pressure over a large range of affinities throughout the course of an immune response, accelerating the emergence of B cells of highest affinities. Furthermore, this mechanism may explain how spatially separated GCs communicate and how the GC reaction terminates.

Figure 1.

Effects of antibody on affinity maturation. (a) Antibody feedback hypothesis: B cells, after proliferating and hypermutating their Ig genes, interact with antigens deposited on FDCs. As these antigens are masked by early low-affinity antibodies (blue), only B cells with higher-affinity BCRs can effectively compete for access to antigen. Successful BCR engagement consequently allows interaction with T cells. Higher-affinity antibodies (yellow), produced by GC-derived plasma cells, reenter GCs and restrict antigen access over time. (b and c) In silico simulation of GC development predicts a more efficient increase in antibody affinity (b) and a clear termination of the GC reaction (c) with antibody feedback. (d) Amount of high-affinity NP-specific IgG and ratio of high-affinity/total antibody in blood 10 d after immunization of μ_s^−/− mice immunized with low-affinity IC of NP-CGG. Horizontal bars indicate median, and each symbol corresponds to one mouse. Data are from one experiment. (e) GC development in IgH^μγ1 mice. (left) Representative spleen images days 10 and 21 after immunization showing PNA for GCs and IgM for follicular areas. Bar, 100 µm. (right) GC volumes 21 d after immunization. Box plots indicate median, 50%, and 100% range. Data are from two independent experiments with a total of seven or eight mice per group. **, P < 0.01; ***, P < 0.001.

Figure 2.

Affinity-dependent equilibrium of antibody inside and outside GCs. (a) Representative images of splenic B cell follicles from a time course after i.v. injection of low-affinity IgM^a-IC. IgM^a, endogenous IgM^b, and IgD to show follicles (F) were used. MZ, marginal zone; TZ, T zone. (b) Quantification of the staining intensity for IgM^a and IgM^b from a similar series of immunoenzymatically stained tissues shows appearance and replacement of the injected antibody. (left and middle) Each symbol represents one FDC network area in different GCs. (right) Mean ratio of IgM^a/IgM^b staining intensities with each symbol representing one animal. (c) Surface plasmon resonance from the four different NP-specific IgM^a monoclonal antibodies produced for this study. Based on the association and dissociation kinetics, antibody affinity was ranked as Low (clone Fab82; blue •) < IntLow (clone 2.315; yellow ◆) < IntHigh (clone 1.198; orange ▪) < High (clone 1.197; green ▴). The same labels are used throughout this manuscript. (d, left) IntLow-affinity IgM^a 5 d after IgM^a-IC immunization. Inset shows same staining at higher magnification. (right) Total NP-specific antibody showing B cells and IC in GCs, plasma cells in red pulp, and plasmablasts in GC vicinity (arrowhead). (e, left) IntHigh-affinity IgM^a 5 d after immunization. (right) Total NP-specific antibody. The arrowhead indicates GC-associated plasmablasts. G, GC; PC, plasma cell. Bars, 50 µm. (f) Semiquantitative analysis of IgM^a density on FDC networks. Each symbol corresponds to the median IgM^a density on FDC networks in one animal. Data are representative of three independent experiments. Horizontal bars indicate median. **, P < 0.01.

Figure 3.

Effects of antibody injection on established GCs. (a) IgM^a (blue) injected 3 d after immunization with NP-CGG was analyzed 48 h later. IgD is brown. F, follicle; TZ, T zone. (b) Quantitation of IgM^a levels on FDC networks for IgM^a injected 3 or 6 d after NP-CGG immunization, analyzed 2 d later (dotted line: background staining level). (c) Apoptotic B cells as indicated by active form of caspase 3. Bars, 25 µm. (d) Numbers of NP-binding GC B cells and NP-specific GC B cells staining for Annexin V 24 h after injection of NP-specific high-affinity antibody. (left) Representative FACS plot showing gating scheme. (right) Quantitative data from two independent experiments with a total of nine mice per group. (e) Density of apoptotic cells at different intervals after injection of antibody affinity in early stage (day 5) or later stage (day 8) GCs. (f) NP-specific GC sizes. (g) NP-specific GC-associated plasmablast output (Fig. 2, d and e, arrowheads). (h) NP₂/NP₁₅ binding ratio of NP-specific IgG in blood, expressed relative to median level of the day 5 control. Each symbol corresponds to one animal. Data are representative of three independent experiments. Horizontal bars show medians color coded as in i. Boxes are 50% range, and whiskers are 100% range. (i) In silico effect of antibodies of different affinities. Arrowheads indicate in silico antibody injection 3 (continuous lines) or 6 d (broken lines) after immunization. Vertical gray lines correspond to the time of analysis of the in vivo experiments. Data show means and standard deviation of 20 in silico experiments. (j) Change in IgG1 heavy chain germline RNA, Bcl-Xl, and Bcl2, but no significant change in AID expression in FACS-sorted GC B cells 6 or 12 h after antibody injection. Symbols for No Ab, IntLow, and High-affinity antibody groups correspond to individual animals. Data are representative of two independent experiments. Horizontal bars indicate median. nBC, naive B cells. *, P < 0.05; **, P < 0.01; ***, P < 0.001.