Abstract

RESULTS AND DISCUSSION

To test the hypothesis that antibody feedback impacts the appearance of high-affinity B cell variants, a novel mathematical model of the GC reaction was developed that represents effects of soluble antibody with antibody concentration and affinity that is dependent on GC output. The model included masking of antigen by antibodies (using realistic on–off kinetics) and inhibition of uptake of antigen retained on follicular dendritic cells (FDCs), which impacts follicular T cell help (Meyer-Hermann et al., 2006). Both antibody feedback mechanisms, i.e., masking and retention, were made dependent on the affinity of antibodies produced by GC-derived plasma cells. With these parameters, the simulations revealed that antibody feedback accelerates affinity maturation (Fig. 1 b) and induces a timely end to the GC reaction (Fig. 1 c). To test these predictions, mice deficient in the secreted form of IgM (μ_s^−/− mice; Ehrenstein et al., 1998) were immunized with immune complex (IC) to induce B cell activation and IC localization into B cell follicles. These mice developed GCs and, as predicted in silico, 4-hydroxy-nitrophenyl (NP)–specific IgG was of significantly lower affinity during the early stages of the GC response (Fig. 1 d). As μ_s^−/− mice should still have antibody feedback through IgG, long-term development of GCs was followed in animals completely devoid of soluble Ig (IgH^μγ1 mice; Waisman et al., 2007). As predicted in silico, GC responses were longer lived in the complete absence of soluble antibody (Fig. 1 e).

The outcome of these experiments thus motivated us to test in vivo whether there is an affinity-dependent equilibrium of antibody inside and outside GCs. Primed C57BL/6 mice (allotype IgM^b) were immunized with ICs (IgM^a-IC) composed of NP coupled to chicken gamma globulin (CGG) and a nonmutated IgM^a antibody with low affinity to NP (clone Fab82, see Materials and methods). Exogenous IgM^a-IC localized in the marginal zone and B cell follicles within hours of administration and by 24 h deposited on the FDC networks (Fig. 2 a). Concomitantly, endogenous antibody was displaced from the FDC network (IgM^b in Fig. 2 [a and b]). 2 d after immunization, the time when the first NP-specific antibody–producing cells appear (Toellner et al., 1996), the injected antibody started to disappear and endogenous antibody reappeared on the FDC network (Fig. 2, a and b). 5 d after immunization, at the peak of the GC response, exogenous IgM^a antibody on FDCs was completely replaced by endogenous antibody (Fig. 2 a, bottom right).

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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.

To determine whether antibody replacement is dependent on the interaction between antibody and antigen, we used additional IgM^a monoclonal antibodies with affinities higher than clone Fab82. Two sets of IC with antibodies of lower intermediate (IntLow) and higher intermediate (IntHigh) affinities (Fig. 2 c) were created with NP-CGG and injected into primed IgM^b mice. In contrast to the low-affinity clone Fab82 (Fig. 2 a), both intermediate affinity antibodies were detectable in all mice in GCs 5 d after immunization (Fig. 2, d and e). Furthermore, significantly more antibodies were present in GCs of mice that had received the higher-affinity variant (IntHigh; Fig. 2, e [left] and f). Yet the total amount of NP-specific antibody on FDCs was similar whatever antibody had been injected (Fig. 2, d and e, right). These results demonstrate, for the first time, a dynamic process of antibody turnover within GC-localized IC and indicate that the replacement of antibody is affinity dependent.

To test whether antibody produced by plasma cells outside GCs can enter and affect B cell selection in established GCs, the IgM^a antibodies of either Low, IntLow, or highest (High) affinities were injected either 3 d (just after GCs had formed) or 6 d (when GC B cells had 3 d to mature to higher affinity) after immunization with soluble NP-CGG. The tissues were assessed 2 d later, i.e., day 5 or 8 (Fig. 3, a and b). 2 d after injection, Low-affinity antibody could not be detected on the FDC network of GCs, whereas both the IntLow- and High-affinity antibodies could be observed in the GCs (Fig. 3 a). The amount of antibody found correlated with the affinity of the antibody (Fig. 3 b). Importantly, maturation of the immune response affected antibody entry, as IntLow antibody was not able to enter mature GCs in substantial amounts after higher-affinity endogenous antibodies had been produced (Fig. 3 b, d8). Analyzing GC B cell differentiation revealed that when antibodies entered GCs, B cell selection was affected. Assessment of the active form of caspase-3 (Fig. 3 c) and Annexin V staining (Fig. 3 d) demonstrated increased apoptosis in NP-specific GC B cells. The amount of apoptosis correlated with the affinity of the injected antibody (Fig. 3 e). Injection of the IntLow-affinity antibody at early stages had less effect than High-affinity antibody, and it had no effect in mature GCs, supporting the hypothesis that antibody entering GCs competes with GC B cells in an affinity-dependent way. Increased apoptosis substantially reduced GC volumes (Fig. 3, a and f). Also, output from GCs was reduced with fewer plasmablasts present in the GC periphery (Fig. 3 g). Surprisingly, administration of the antibody variants even affected the affinity of endogenous antigen-specific IgG in blood. High-affinity antibody drove the response quicker to higher-affinity IgG (Fig. 3 h), but only when it was given at an early stage. Collectively, these observations demonstrate an increase in B cell selection stringency dependent on the affinity of antibody present inside GC.

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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.

We next asked whether these in vivo effects of antibody can be explained by competition for antigen using our new mathematical model. In silico experiments showed that constraining antigen access and uptake by the GC B cells are both necessary and sufficient to replicate the complex in vivo effects of antibody on B cell survival, GC volume, plasma cell output, and affinity (Fig. 3 i), supporting the conclusion that antibody feedback is the mechanism responsible for the effects of antibody observed in vivo. Affinity-dependent variation in antigen uptake is an important factor because it determines the fitness of B cells to interact with T cells in silico (not depicted). To test in vivo whether competition from High-affinity antibody leads to reduced B cell–T cell interaction, we analyzed IgG1 heavy chain germline transcription, which is a good indicator of T-dependent B cell activation (Toellner et al., 1996). Indeed, within 6 h, injection of High-affinity antibody led to reduced production of IgG1 heavy chain germline transcripts (Fig. 3 j). This confirms that antibody-dependent restriction of antigen access and uptake inhibits downstream T cell–B cell interaction, ultimately leading to death by neglect (Fig. 3 j). Upstream processes such as expression of activation-induced cytidine deaminase (AID), essential for hypermutation, were not influenced (Fig. 3 j).

Several mechanisms for the preferential selection of affinity-matured GC B cells have been proposed. T cells in GCs help the B cells that have been most efficient in taking up and presenting antigen (Meyer-Hermann et al., 2006; Allen et al., 2007; Victora et al., 2010). Although BCR ligation has been shown to induce B cell activation and antigen presentation to T cells in an affinity-dependent way, this affinity dependence acts only over a relatively small affinity range (Fleire et al., 2006). As GC B cell affinity evolves, one would expect a mechanism that raises the selection pressure in line with the evolution of B cell clones (Tarlinton and Smith, 2000). During infections, large amounts of antigen are produced over a prolonged time. Therefore, direct competition of B cells for antigen or consumption of antigen is an unlikely mechanism to keep stringent selection pressure over a prolonged response. We show here that, dependent on its affinity, secreted antibody enters GCs and, over time, limits antigen access. As GC B cell affinity evolves, the selection pressure rises, gradually restricting access to and uptake of antigen. This may complement direct affinity-dependent B cell activation (Fleire et al., 2006) and lead to efficient directional selection of B cells over a large affinity range. Indeed, mice deficient in soluble IgM have delayed affinity maturation (Boes et al., 1998; Ehrenstein et al., 1998). This study focused on the analysis of effects of IgM antibodies on affinity maturation. Ig class switching may provide another layer of regulation, reducing antibody avidity once the response has advanced and providing a range of additional signals through the Ig heavy chain (Song et al., 1998; Hjelm et al., 2006).

One of the conundrums of GC biology has been whether responses in different GCs interact. Entry and exit of B cells from GCs have been observed, but these are typically naive B cells (Hauser et al., 2007; Schwickert et al., 2007), and different GCs usually show separate genealogies (Jacob and Kelsoe, 1992). The antibody-dependent selection mechanism demonstrated here makes inter-GC B cell migration dispensable, as soluble antibody produces a systemic selection threshold. At some stage it can be expected that the restriction of antigen access is too strong to allow for B cell survival, ending the GC reaction. Indeed, in silico experiments predict a natural end to the GC reaction once antibody affinity is sufficiently high (Fig. 1, b and c), and mice without antibody feedback have prolonged GC responses (Fig. 1 d).

Artificially adding exogenous antibody of moderate avidity may be a way to manipulate vaccine responses. In silico modeling indicates that antibodies of low affinity added at the start of a reaction will accelerate the early stages of affinity maturation, and preliminary experiments using IgM in vivo show that this strategy may be effective.

Antibody feedback with a dynamic selection threshold accelerates optimization of Ig variable region genes during the development of an antibody response. Changes in selection pressure caused by consumption of limited resources or ecological changes induced by evolving species have been described as factors shaping evolution (Schoener, 2011). In the case of the GC, B cells produce their own selective environment, bootstrapping themselves by producing a mediator that tunes their own selection and provides adequate selection pressure throughout the entire environment. This maximizes the speed of evolution of B cell clones systemically, which surely is an evolutionary adaptation to the enormous selection pressure caused by our continuous fight to adapt to new pathogens.

MATERIALS AND METHODS

Acknowledgments

Footnotes

References