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The Journal of Immunology, 2007, 179, 5181 -5190
Copyright © 2007 by The American Association of Immunologists, Inc.
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Silent Development of Memory Progenitor B Cells1

Katja Aviszus*, Xianghua Zhang{dagger} and Lawrence J. Wysocki2,*

* Integrated Department of Immunology, National Jewish Medical and Research Center and University of Colorado School of Medicine, Denver, CO 80206; and {dagger} Isogenis, Denver, CO 80206


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
T cell-dependent immune responses generate long-lived plasma cells and memory B cells, both of which express hypermutated Ab genes. The relationship between these cell types is not entirely understood. Both appear to emanate from the germinal center reaction, but it is unclear whether memory cells evolve while obligatorily generating plasma cells by siblings under all circumstances. In the experiments we report, plasma cell development was functionally segregated from memory cell development by a series of closely spaced injections of Ag delivered during the period of germinal center development. The injection series elevated serum Ab of low affinity, supporting the idea that a strong Ag signal drives plasma cell development. At the same time, the injection series produced a distinct population of affinity/specificity matured memory B cells that were functionally silent, as manifested by an absence of corresponding serum Ab. These cells could be driven by a final booster injection to develop into Ab-forming cells. This recall response required that a rest period precede the final booster injection, but a pause of only 4 days was sufficient. Our results support a model of memory B cell development in which extensive affinity/specificity maturation can take place within a B cell clone under some circumstances in which a concomitant generation of Ab-forming cells by siblings does not take place.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
At least three pathways contribute to Ab formation in T cell-dependent immune responses: a short-term Ab-forming cells (AFC)3 response in extrafollicular regions of secondary lymphoid tissue, a long-lived AFC response in the bone marrow by germinal center (GC) emigrants, and a memory B cell response that provides recall immunity for the life of the animal (1, 2, 3, 4, 5, 6, 7). The short-term extrafollicular response occurs within a few days of immunization and is characterized in the spleen by clusters of AFC in the peri-arteriolar-lymphoid sheath regions and by Ab products of low affinity, many of which are likely derived from marginal zone B cells and encoded by unmutated V region genes (8, 9, 10). The bulk of this reaction is concluded by 2 wk, although some plasma cells persist for longer periods (6, 7, 11). Interestingly, this short-term AFC component is absent from the A/J immune response to the hapten p-azophenylarsonate (Ars) (12). The reason for this is not known. Perhaps Ars-specific precursors to the early extrafollicular AFC are missing in this strain (12, 13). Alternatively, the diazonium bond linking Ars to the carrier protein may be unstable in the extrafollicular environment. Regardless of the reason, the absence of the extrafollicular pathway simplifies serological studies of the long-term AFC and memory cell components of humoral immunity.

The long-lived AFC and memory responses are each derived from GC B cells that hypermutate their Ab V region genes (8, 14, 15, 16, 17, 18, 19, 20, 21). In a well-defined immune response to the hapten, 4-hydroxy-3-nitrophenyl acetyl, the earliest bone marrow AFC are seen at ~2 wk following immunization (20, 21). Although these early bone marrow AFC carry few somatic mutations, a high frequency of the mutations are those that enhance affinity for 4-hydroxy-3-nitrophenyl acetyl (20). Tarlinton and Smith (22) have taken this as an indication that an early strong signal through the BCR favors development of GC B cells along the bone marrow AFC pathway. Additional evidence for this idea comes from studies in bcl-2 transgenic (Tg) mice, in which affinity maturation is perturbed in GC but not in bone marrow AFC (23), and from studies in which an increased AFC response was observed following multiple injections of soluble Ag during the primary immune response (24). Most recently, Paus et al. (25) extended this finding using hen egg lysozyme-Tg mice.

The population dynamics behind memory B cell development have proved to be the most difficult to define. Based on genealogical analyses, it is clear that hypermutation in memory precursors occurs over multiple rounds of DNA replication and presumably cell division (26, 27, 28, 29). There is also good evidence for competition among developing memory lineage cells, a process that may occur locally between adjacent clones rather than globally among all participating clones (30, 31). But much less is known about how memory lineage B cells in GC sense and respond to variations in Ag dose and the relationship between memory lineage cells and long-term AFC. Smith et al. (20) observed a continuous improvement in the affinity of Ab produced during the primary immune response and continuous seeding of bone marrow AFC by GC B cells. These results imply that AFC and memory B cells may continuously generated by common lineages during a primary immune response. However, Decker et al. (32) used a splenic fragment culture system to show that some memory progenitors mutate their Ab genes and generate higher order memory cells that do not produce AFC siblings until they encounter Ag sometime later. Their results suggest that a pause in antigenic stimulation is required to render some memory cells competent for differentiation into AFC and that a subsequent challenge with Ag after the pause is required to induce differentiation into AFC. To our knowledge, in vivo evidence for this scenario of memory development has not been reported.

To determine whether memory cells can evolve in vivo without secreting long-term AFC generation, we exploited several advantageous features of the anti-Ars immune response. In addition to having no early extrafollicular component, this immune response embodies a memory pathway in which approximately half of the B cell participants express one set of Ab V gene segments with a common V{kappa}-J{kappa} junction and a VHCDR3 that only varies at two boundary codons, mutations excepted (33, 34). A highly specific anti-clonotypic Ab called mAb E4 can be used to identify this canonical Ab structure. Most importantly, rare mutants of the canonical Ab acquire specificity for a related hapten sulfanilic acid (Sulf) and can be recruited into the response when Sulf is provided secondarily as an immunogen (35, 36, 37). This switch in specificity from Ars to Sulf can be used as a qualitative indicator of "affinity-maturation" and memory B cell development.

Using this model in conjunction with variations in immunogen, dose and tempo, we demonstrate the existence of memory progenitor cells with a stem cell-like property (38). They can be driven by Ag to evolve higher affinity and specificity for Ag without significant differentiation into AFC. We also show that a pause in antigenic stimulation of only 4 days is needed to render memory progenitors competent for AFC differentiation upon a subsequent antigenic challenge. Finally, we show that Ag pulsing can have opposite effects on memory progenitors and AFC with respect to the affinity of the Ab product.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Animals and immunization protocols for serological studies

A/J mice were purchased from The Jackson Laboratory and housed in the Biological Resource Center at National Jewish Medical and Research Center. All animals were used according to an institutional animal care and use committee-approved animal protocol. In all cases, 6- to 8-wk-old animals were used, and immunizations were i.p. as specified.

ELISA, europium (Eu), and radioimmunoassays

In direct binding assays, 96-well trays were coated overnight with Ars5-BSA or Ars15-BSA (10 µg/ml in 100 µl). After washing, plates were incubated with blocking buffer (2% BSA/1% gelatin/0.05% Tween 20/0.04% thimerosal in PBS) for 1 h at room temperature (RT). Serial dilutions of sera and standard anti-Ars Ab, mAb 36-65 (17, 39), were incubated for 1 h at RT. The plates were washed and incubated with biotin-rat-anti-mouse {kappa} (mAb 187.1) (40) (bio-rat-anti-M{kappa}, 0.5 µg/ml) for 1 h. After washing, the trays were incubated for 30 min with streptavidin-coupled HRP (SA-HRP, 1 µg/ml; Vector Laboratories) or SA-Eu (0.05 µg/ml; Wallac) and developed with 2'2'-azino-bis(3-ethylbenz-thiazoline-6-sulfonic acid) (ABTS; Sigma-Aldrich) or enhancement solution (100 µl; Wallac). Results were quantified using a Wallac Victor 2 fluorometer using a photometry setting of 450 nm for ABTS absorbance and excitation and emission settings of 340 and 615 nm, respectively, for Eu fluorescence (41).

Relative affinity of total serum Ab was determined based on the method of Herzenberg et al. (42). Plates were coated with Ars5-BSA or Ars15-BSA (10 µg/ml, 4°C overnight), blocked, and incubated with sera at dilutions that gave ~90% of maximum binding and developed as above in the direct binding assay. Dilutions that gave 50% of maximum binding were determined using Microsoft Excel 2001 using linear regression.

To determine relative affinity by hapten inhibition, plates were coated overnight with Ars5-BSA, blocked, and incubated with sera at dilutions that afforded ~90% of maximum binding. Sera were mixed with Ars-N-acetyl-L-tyrosine (Ars-Tyr) at concentrations ranging from 10–3–10–9 M. Plates were developed with 125I-labeled rat anti-mouse {kappa} (40 ng/ml), and bound radioactivity was measured in a gamma 5500B counter (Beckman Instruments). Values at the 50% inhibition point (I50) were calculated using Jmp 2.3.6 (SAS Institute).

Canonical Ab were quantified in a competition assay (43). Trays were coated with mAb E4 (44) blocked and incubated with serial dilutions of sera or standard mAb 36–65 (Id+) premixed with bio-mAb 36–65 (17) (0.2 µg/ml). The assays were developed with SA-HRP and ABTS as above.

A counterinhibition assay was used to detect canonical Abs in serum with specificity for Sulf among a mixture including others with specificity for Ars (37). Trays were first coated with mAb E4, blocked as above, and incubated for 1 h with competing sera, together with a biotin-labeled Ab called mAb 3A4 (70 ng/ml), which has no capacity to bind Ars or Sulf (37, 43) and Ars-Tyr or Sulf-N-acetyl-L-tyrosine (Sulf-Tyr) (2.5 x 10–4 M). Test sera were used at a dilution that inhibited mAb E4 binding to mAb 3A4 by 90%. Plates were developed with SA-Eu as above. Canonical Ab in sera inhibits the binding of bio-mAb 3A4 to mAb E4-coated wells in the absence of hapten, but this is counterinhibited by hapten if it binds to the serum Ab.

In one experiment (see Fig. 4B), sera were pooled from four control animals which were not given the final booster injection with Sulf-keyhole limpet hemocyanin (KLH). The pooled sample was affinity purified on a Sulf-BSA Sepharose column. Bound Abs were eluted with 3 M potassium thiocyanate, dialyzed, and tested in the counterinhibition assay.


Figure 4
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  		FIGURE 4. A, Defining the minimal period of rest required before eliciting a recall response by Sulf-switch mutants. Mice were immunized as shown. Initial injection was with Ars15-KLH (100 µg) i.p. in IFA. Serial injections and booster injections were with 100 µg of Ag i.p. in PBS. B, Pooled sera from control group (last row, Table IV) were affinity purified on Sulf-BSA Sepharose and tested in the counterinhibition assay as in Fig. 3. Affinity-purified canonical Ab accounted for ~14% of all canonical Ab in the sample, as assessed with mAb E4.

 
Hybridoma studies

One Ars-5 mouse (45), carrying multiple copies of a canonical VHIdCR-Dfl16.2-JH2-IgM transgene on a C57BL/6 genetic background, was immunized i.p. with 300 µg of Ars15-KLH in CFA. Serial injections with Sulf-KLH i.p. (300 µg) were delivered on days 7, 10, 12, 14, 17, and 20. The mouse was injected i.p. with 100 µg of Sulf-KLH in IFA at day 45 and bled on day 70. Sera were tested in a counterinhibition assay for Abs with bearing the E4 Id that bound Sulf-Tyr more strongly than Ars-Tyr. It was given a booster injection with Sulf-KLH (100 µg) on day 200 and sacrificed for hybridoma formation with SP2/0 cells on day 203 (46). Selected hybridomas were cloned three times by limiting dilution.

Constructing an unmutated correlate of the Sulf-binding mAb

A 3.7-kb EcoRI- HindIII fragment, containing the assembled H chain V gene of mAb R16.7 (origin of the VH transgene in Ars-5 mice) was cloned into a plasmid vector for expression in context of an IgG2b constant gene (47). This genetic construction was transfected into a cell line (36–65{kappa}) expressing an unmutated version of the {kappa} gene encoding the canonical L chain of the dominant anti-Ars Id (34). Secreted Abs were purified from ascites fluid by affinity chromatography with Ars-BSA Sepharose (43).

V gene sequencing

Canonical L chain V genes were amplified from hybridoma DNA (100 ng) by a nested PCR strategy and directly sequenced, as described (48, 49). cDNA encoding Ig H chains was prepared by the method of Chomczynski (50) using primers specific for IgG1: 5'-CCAGGGTCACCATGGAGTTAGT or IgG2b: 5'-GGATCCAGAGTTCCAAGTCACA. Hybridoma cDNA was initially amplified through 30 cycles using cDNA synthesis primers in conjunction with a primer that hybridized to the leader exon in the VHIdCR gene: 5'-GGATGGAGCTTCATCTTTCTCT. A second round of amplification (15 cycles) was performed with nested primers for IgG1: 5'-GAGTTAGT TTGGGCAGCAGATC or IgG2b: 5'-CCAGGCACCCAGAGGTCACGGA, in conjunction with the same 5' leader primer.

Histology

Spleens were quick-frozen in Tissue-Tek OCT. Serial sections of 6–8 µm were fixed in acetone and stored at –80°C. Sections were thawed at RT and blocked with 5% normal goat serum in PBS for 10 min at RT. For immunohistochemistry analyses of GCs, frozen sections were incubated with bio-mAb E4, and bio-peanut lectin agglutinin (PNA) and developed using Vectastain ABC alkaline phosphatase and Vectastain ABC Elite kits (both Vector Laboratories) followed by the 5-bromo-4-chloro-3-indolyl phosphate/NBT alkaline phosphatase substrate kit IV or the diaminobenzidine peroxidase substrate kit, respectively (both Vector Laboratories), according to the manufacturer’s protocol. Sections were photographed using a Nikon Diaphot coupled to a Photometrix CCD camera and analyzed using IPLab Spectrum Version 3.1a. GC sizes were determined as "small" being <2000 pixels, "medium" 2000–4000 pixels, and "large" >4000 pixels.

For immunofluorescent analyses of GCs, spleen sections were preincubated for 30 min with one of two competitors (Ars-BSA or Sulf-BSA at 10 µg/ml) in staining buffer (2% FCS in PBS, 0.1 NaN3). Sections were then stained for 30 min with biotinylated or FITC-coupled PNA (Vector Laboratories) and E4 and allophycocyanin-B220 (clone RA3-6B2; eBioscience) in the presence of the competitors. Biotinylated stains were resolved with streptavidin-Cy3. Slides were photographed using the Marianas system (Intelligent Imaging Innovations) and analyzed using Slidebook 4.0.2. Pictures of an entire section were assembled in a montage. Sections were renormalized keeping the settings for the different channels constant across the sections. Numbers of total and E4+ GCs were determined, and the frequency of E4+ GCs in each section was calculated. The proportions of E4+ GC binding Ars and Sulf were determined from the frequencies of E4+ GC in three consecutive sections, incubated with either no hapten or the competitors Ars-BSA or Sulf-BSA.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Differential effects of serial Ag injections on late primary and secondary immune responses

To determine how repeated antigenic stimulation during an ongoing primary immune response influences development of long-lived plasma cells and memory cells, we took advantage of the observation that the immune response elicited in strain A/J mice by Ars lacks an early extrafollicular AFC component (12). This permitted us to examine the long-term AFC and memory responses within the same animal by assaying serum Ab at different time points following immunization.

A/J mice were given an initial injection of Ars-KLH in IFA. Groups of these mice then received either none, two, or six injections of Ars-KLH in PBS during the period of GC development (49), as illustrated in Fig. 1A. The mice were bled at day 41 (late primary response), and the relative quantities of high-affinity and low-affinity anti-Ars Ab in sera were determined by ELISA using trays coated with Ars5-BSA (bound by high-affinity Ab) or Ars15-BSA (bound by high- plus low-affinity Ab). Fig. 1B illustrates the results of this assay, expressed as ratios of Ab titers on the two forms of Ag (Fig. 1B, {blacksquare}). The titers were indistinguishable in the presence of 2-ME (10 µM), indicating little or no IgM Ab (data not shown). The lack of a strong primary IgM response is not surprising considering the absence of a short-term extrafollicular AFC response to Ars in this strain (9, 12). Groups that received any injection series produced a higher proportion of lower affinity Ab at day 41 than the group that did not, and the proportion of low-affinity Ab generally increased with the dose of Ag. Moreover the titers of serum Ab increased in groups receiving the soluble injections (Fig. 1D). These data are consistent with those of Pulendran et al. (24) in supporting the idea that higher doses of Ag drive lower affinity GC B cells into the long-term bone marrow AFC pathway.


Figure 1
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  		FIGURE 1. Soluble Ag differentially affects primary and secondary immune responses. A, Immunization schedule. The initial immunization was with 100 µg of Ars15-KLH i.p. in IFA (n = 4–5 mice/group). The primary injection series on days 7–20 was in PBS. B, Differential effects of primary injection series on overall affinity of Ab in the late primary and secondary immune responses. Dilution points at 50% of maximum binding were determined and used to calculate the ratios shown. High-affinity Ab selectively bind Ars5-BSA Ab, while both high- and low-affinity Ab bind Ars15-BSA. C, Relative affinities among higher affinity Ab in late primary and secondary immune response sera. Trays were coated with Ars5-BSA. Sera were used a dilution giving 90% of maximal binding on Ars5-BSA. Ordinate indicates Ars-Tyr concentrations at which binding to Ars5-BSA was inhibited by 50% (I50). Each point is average of log10 I50 for four to five mice. Lower concentration indicates higher affinity Ab. SEs indicated. D, Anti-Ars titers defined as dilution at 50% of maximal binding on Ars-BSA for day 41 sera. E, Fold increase in anti-Ars titers between days 230 and 244 as assessed on Ars5-BSA.

 
To examine the effects of the injection series on the memory immune response, mice were rested until day 230 and given a final booster injection of Ars-KLH in PBS. Sera were taken 14 days later and tested for titers of high- and low-affinity Ab on Ars5-BSA and Ars15-BSA. Total serum Ab titers rose from 4- to 15-fold between day 230 and 14 days after the booster injection (Fig. 1E), indicating that secondary serum Ab included only a minor amount of Ab from the primary response. Results of the test (Fig. 1B, {square}) revealed that the injection series did not have the same effect on Ab affinity at this point as it did in the late primary immune response. Instead, the injections resulted in a consistent but modest overall improvement in the affinity of the recall response Ab. Collectively, these results indicate that the injection series had opposite effects on the affinities of the primary long-term AFC response and the memory response. This dichotomy suggested that the injection series might have generated a silent population of high-affinity memory precursor cells that, during their development, did not generate AFC.

Evidence for affinity maturation in a silent memory-precursor population

To more conclusively test whether the injection series with Ars-KLH drove the development and affinity maturation of a silent memory population, we determined relative affinities of serum Ab in late primary and secondary response sera, using a hapten-inhibition assay with Ars-Tyr. Importantly, to focus selectively on the highest affinity Ab, this assay was conducted using trays coated with Ars5-BSA. In this test, Ab of higher affinity are inhibited by lower concentrations of Ars-Tyr. The I50 values, shown in Fig. 1C, reveal that the injection series resulted in the generation of high-affinity Ab in the secondary immune response that was not detected in day 41 sera. This affinity maturation effect was generally greatest in mice receiving lower doses of Ag. The injection series extended GC longevity, as determined by GC size distributions observed at day 21 (Fig. 2). These results support the idea that injection of Ag during the primary immune response drives development and affinity maturation of a population of memory precursor cells, which do not generate significant long-term AFC along the way. However, following a rest period, these cells are rendered competent to produce a recall AFC response upon a secondary challenge with Ag.


Figure 2
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  		FIGURE 2. Injection of soluble Ag extends GC longevity. A, Immunization schedule for the analysis of GC size. Mice received either six (300, 100, or 20 µg/ml), two (300 µg/ml), or no injections of indicated quantities of Ars-KLH in PBS. B, Distribution of GC sizes following immunization. Sections of spleens were stained with PNA to identify GC, which were measured (see Materials and Methods), enumerated and plotted as the percent of total for each class size. C, Vanishing GC response on day 21 in mice without soluble Ag injection (0b). Arrows point to residual GCs. D, Prolonged GC reaction on day 21 in mice after six injections with 300 µg of Ars-KLH (6b300). Spleen section shows large GCs (PNA = blue) with anti-idiotypic staining (mAb E4 = brown).

 
Specificity maturation in a silent memory-precursor population

Up to this point, our interpretations were based solely on quantitative differences in Ab affinity, resulting from different immunization schedules. To qualitatively test the silent memory concept, we exploited a special feature of the anti-Ars immune response. During hypermutation, rare mutants of the predominant canonical Ars clonotype (33, 34) arise that acquire an enhanced affinity for a related hapten, Sulf, while losing or severely reducing their affinity for Ars (35, 36, 37). Somatic mutations at VH codon 35 are responsible for this change in antigenic specificity. These mutants can be selected by injecting Sulf-KLH during a primary immune response initiated by Ars-KLH. The initial Ars injection is required because Sulf does not bind to the unmutated canonical BCR with sufficient affinity to drive a primary immune response (35, 51). We used this system to determine whether a Sulf-KLH injection series, delivered during the primary immune response, would select and expand a silent population of Sulf-binding canonical memory precursor B cells.

In this experiment, mice were first injected with Ars-KLH in CFA. This was followed by three injections on consecutive days with Sulf-KLH in PBS. In different groups of mice, the Sulf-KLH injections were initiated on different days, starting on day 6 for the first group and on day 15 for the last. A control group received a series of injections with Ars-KLH starting on day 11. Late primary response sera were collected, and the mice were given a booster injection with Sulf-KLH in IFA, both on day 41. Sera sampled on day 57 were used to assess the secondary immune response. A hapten counterinhibition assay, described in Fig. 3 (37), was used to test for the presence of canonical Ab that bound to Sulf with greater affinity than to Ars (switched specificity mutants) as revealed by superior counterinhibition achieved by Sulf-Tyr relative to Ars-Tyr. The canonical Id was identified in this assay with an anti-clonotypic Ab called mAb E4, which is highly specific for unique features of canonical Ab including the appropriate H and L chain V regions and VHCDR3 (44, 52). The results, shown in Table I, revealed that serum Ab with switched antigenic specificity could only be detected in mice that had received the serial injections of Sulf-KLH. The number of mice demonstrating Sulf-switched Ab was highest in those receiving the injections that ended at day 10 or later. Only one in four mice injected on or before day 9 showed evidence for the switch, possibly reflecting the immaturity of GCs at early time points. This result indicated that the Sulf-KLH injections selected and expanded rare mutant B cells producing this Ab of switched antigenic specificity. However, Sulf-switched Ab was only observed in secondary immune response sera. In view of its absence in the late primary sera (day 41), this indicates that Sulf-KLH injection series drove the expansion of mutant Sulf-specific memory progenitors that apparently did not produce significant AFC during their development.


Figure 3
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  		FIGURE 3. Counterinhibition assay used to identify canonical Ab of switched specificity. A, Binding of biotin-labeled Ab 3A4 to anti-Id E4 is inhibited by canonical E4+ Id in serum. If serum Id binds hapten, inhibition is reversed. Note, 3A4 does not bind Ars-Tyr or Sulf-Tyr. B, Representative results for two sera, one of which contains E4+ Id that binds more strongly to Sulf-Tyr than Ars-Tyr (switched specificity, representative of group 5, Table IV), and the other of which behaves conversely (not switched, representative of control group, Table IV).

 

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  		Table I. Recruiting memory B cells with a new mutation-generated Ag specificity

 
Structural correlates of specificity maturation

To determine the structural consequences of multiple Ag injections during the primary immune response, we produced hybridomas from a mouse that was given a series of six injections with Sulf-KLH during a primary immune response to Ars-KLH (see Materials and Methods). This experiment was performed in an Ars-5-Tg mouse carrying a µ H chain transgene encoding a canonical anti-Ars Ab (45). This animal was used so that we could unambiguously identify somatic mutations in the event that the VH gene was extensively altered. From this animal, we recovered three canonical Sulf-binding Ab reactive with the mAb E4. Sequences of their V genes confirmed that the Ab were encoded by the Tg VH. This was most evident by the presence of four somatic mutations that were present in the original transgene. The L chain was encoded by an endogenously derived canonical V{kappa}10.1-J{kappa}1 rearrangement with the invariant junctional Arg codon (43, 53, 54). The Ab were of the IgG class, which is consistent with a transchromosomal recombination event resulting in a switched isotype as described previously in this mouse (37, 45, 55).

The three mAb were very heavily mutated, more so by far than any of the numerous conventionally elicited canonical anti-Ars Ab examined to date. Their V genes carried between 39 and 53 nucleotide changes, producing between 24- and 32-aa replacements (Table II). Notably, all three carried mutations producing amino acid replacements at the critical VH35 codon in CDR-1, which determines Ars/Sulf specificity (35, 36, 56).


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  		Table II. Amino acid replacements (somatic mutations) in V genes of Sulf-switch mutant canonical mAba

 
To determine how the somatic mutations altered Ab affinity and specificity, we measured their affinities for Ars-Tyr and Sulf-Tyr by fluorescence quenching (57, 58). For comparative purposes, it was necessary to generate an unmutated correlate (mAb T-H12) of the precursor to these Abs (see Materials and Methods). The T-H12 Ab bound Ars with a modest affinity, but its affinity for Sulf was barely measurable (<1/50th of that for Ars). In contrast, the three mAbs had dramatically increased affinities for Sulf that ranged from 375- to 1,300-fold over that of mAb T-H12. At the same time their affinities for Ars were reduced from 25- to >100-fold relative to mAb T-H12 (Table III). Thus, somatic mutations in these Abs produced changes in the ratios of affinities with which the two haptens were bound that ranged from 25,000- to 40,000-fold relative to the precursor Ab in the Ars-5 mouse. Collectively, these results reveal the influence of the Sulf-KLH injection series on the evolution and affinity maturation of a silent memory precursor population.


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  		Table III. Affinities of canonical mAb for Ars-Tyr and Sulf-Tyr

 
Only a brief pause is required to render memory precursors competent for recall responses

Up to this point, our results indicated that injections of Ag during the primary immune response drove the development of a memory precursor population that is silent, as assessed by a lack of corresponding serum Ab. After a pause in antigenic stimulation, however, the cells attained competence to differentiate into AFC upon further antigenic challenge. To define the minimal pause required to render the memory precursors competent, we immunized mice with Ars-KLH in IFA and delivered a series of three Sulf-KLH injections in PBS on days 8, 10, and 12, as shown in Fig. 4A. The mice were rested for various periods of time and then given a final booster injection of Sulf-KLH in PBS. One group did not receive the final booster injection. All mice were bled 2 wk following the final booster injection, except for the control, which was bled at a point corresponding to the latest bleed (experimental group no. 5). Sera were tested in the counterinhibition assay for the presence of Ab reactive with mAb E4 that bound more strongly to Sulf-Tyr than to Ars-Tyr. The results in Table IV show that, as before, the control mice that did not receive the final booster injection had no detectable canonical Ab of switched antigenic specificity. In contrast, such Ab was observed in four of five groups of mice that did receive the booster injection. The results show that a rest period of only 4 days was sufficient to render the memory progenitors competent to generate a serum Ab response upon subsequent antigenic challenge.


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  		Table IV. Determining rest period required for a successful recall response

 
As seen in Table IV, Sulf-Tyr afforded some counterinhibition of sera from the control group (last row) that received the soluble injections of Sulf-KLH but not the final booster injection. For this reason, we examined control sera more closely to distinguish Ars-binding canonical Ab that cross-react weakly with Sulf from potentially genuine Sulf-switched canonical Ab. To this end, Sulf-binding Abs were affinity purified from the pooled sera of the control group, represented by the last row in Table IV and tested in the counterinhibition assay. If the small amount of inhibition by Sulf-Tyr (11%) was due to genuine Sulf-switched Ab, as opposed to cross-reactive Ab, then we would expect Sulf-switched Ab to be enriched in the affinity-purified fraction. However, this was not the case. The affinity-purified fraction was again much more strongly inhibited by Ars-Tyr than Sulf-Tyr (Fig. 4B). Thus, we cannot detect Sulf-switched Ab in control sera of mice that did not receive a final booster injection, even when the canonical Abs are affinity purified on Sulf.

Silent memory progenitors in GC

To look for direct evidence of silent memory progenitor cells, we immunized mice with Ars-KLH in IFA and delivered a series of six Sulf-KLH injections (100 µg) according to the protocol shown in Fig. 2A. Mice were sacrificed on days 27. An additional group of control animals receiving the Sulf-KLH injection series were given a final booster injection with Sulf-KLH on day 27 and bled on day 41 to confirm the presence of canonical Ab with a switched specificity (data not shown). Consecutive spleen sections were then stained with mAb E4 in the presence or absence of competing Ag in the form of Ars-BSA or Sulf-BSA to look for developing E4+ memory B cells that bound Sulf more strongly than Ars.

The result of this experiment is illustrated in Fig. 5. Sulf-BSA reduced the staining of B cells by mAb E4 in 30–70% of all E4+ GCs of experimental mice. In contrast, Sulf-BSA did not reduce E4 staining of control GC from an animal immunized only with Ars, even though Ars-BSA was an effective competitor in the same GC. This result demonstrates the physical presence in GC of E4+ Sulf-binding B cells generated by the Sulf-KLH injection series despite an absence of corresponding serum Ab.


Figure 5
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  		FIGURE 5. Visualizing Sulf-binding memory B cells. Mice were initially immunized with Ars15-KLH (100 µg) i.p. in IFA. Three mice were given serial injections of Sulf-KLH on days 7, 9, 12, 14, 17, and 20. Mice were sacrificed on day 27. Control mouse ("Ars injection only") received only the initial injection in IFA and were sacrificed on day 13 at the height of the GC response. Consecutive spleen sections were preincubated with the indicated competitors (Ars-BSA, Sulf-BSA) or with no competitor, before and during a subsequent stain with fluorescently labeled mAb E4 (green) and anti-B220 (blue). A, Representative example of GC with Sulf-binding E4+ B cells, as revealed by inhibition of mAb E4 staining with Sulf-BSA. B, Percentages of E4+ GC in which a majority of B cells bound Sulf-BSA, as assessed by inhibition of mAb E4 stain.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
In this report, we demonstrated that Ag injected during the primary immune response drove the development and affinity/specificity maturation of a silent pool of memory precursor B cells. Their silence is inferred from a lack of corresponding serum Ab that only appeared if a pause in antigenic stimulation occurred before a final antigenic challenge. The pause is apparently required for the memory precursors to develop competency for a secondary AFC response. In the model we examined, a pause of only 4 days was required for the development of competent memory cells.

The interpretation of silent memory derives from two experimental approaches that took advantage of an extensively studied model immune response to the hapten Ars in strain A mice. First, the absence of an immediate extrafollicular AFC response to Ars immunization permitted us to assay serum Ab from the long-term AFC component and to compare its affinity to that of Ab produced by memory B cells driven in a recall response, all in the same animal (12). Second, rare mutants of a canonical Ars-associated clonotype lose affinity for Ars while acquiring affinity for another hapten, Sulf (35, 36, 37, 56). These can be recruited with Sulf, and their serum Ab identified with a monoclonal anti-Id. Using this system, we qualitatively assessed the long-term primary AFC response and the memory response for these mutant Ab products with switched antigenic specificity. Results of both approaches led to the same conclusion: that antigenic pulses during the primary immune response drove mutation and clonal selection in memory precursor cells without a detectable AFC response by immediate siblings. This generated memory progenitors with V regions that had undergone affinity/specificity maturation, as revealed by serology and hybridoma sampling studies performed after a final recall challenge and by immunohistology performed on GC immediately following the antigenic pulses. The mutants that we isolated in the form of hybridomas increased their Sulf/Ars affinity ratios by >20,000-fold relative to the starting germline Ab. Although the simplest interpretation of our experiments is that memory B cell development with affinity maturation was achieved during the entire series of soluble Ag injections, it is possible that memory development is inhibited by high concentrations of soluble Ag and only takes place following the last injection, as the concentration of soluble Ag decreases.

We found that injecting low to modest doses of Ag during the primary response resulted in an overall reduction in Ab affinity for primary response Abs. A similar effect of soluble Ag was reported by Nossal et al. (59), who found that it enhanced the magnitude of the splenic AFC precursor frequency when injected from 8 to 10 days following an initial immunization. These and related data support a model in which GC cells receiving the strongest signals through the BCR develop along the AFC pathway, while those that receive weaker signals continue to mutate their Ig V genes and develop along the memory pathway in GC (22).

Although critical transcription factors that differentially guide memory vs plasma cell differentiation have been defined, the cellular events that induce expression of these in vivo are not very well understood. In their analysis of the immune response to a multideterminant synthetic polypeptide, Press and Giorgetti (60) reported evidence of silent memory B cell development to a particular side chain determinant. They proposed a model in which B cell clones that possessed low-affinity receptors could receive an adequate signal for memory development but inadequate with respect to AFC differentiation because low-affinity receptors gathered insufficient Ag to recruit the necessary quantity or quality of T cell help. A simpler model is that AFC differentiation is favored by a strong B cell-intrinsic signal initiated by highly aggregated BCR. Both models invoke BCR aggregation, one directly and the other indirectly (for Ag uptake and processing), as key determinants in the fate of Ag-stimulated B cells.

In our studies, the relative degree to which the BCR is aggregated could explain why soluble injections of Ars-KLH induced differentiation of the primary Ars-specific B cells into AFC, while soluble injections of Sulf-KLH did not induce differentiation of Sulf-switch mutants. In the first instance, it was the low-affinity component of the anti-Ars response that was enhanced. In the second case, high-affinity, Sulf-binding canonical mutants remained silent. If soluble Ag reaches the follicles as reported by Pape et al. (61), high-affinity Sulf-switch clones will engage disproportionally more BCR in a monovalent nonstimulatory manner relative to low-affinity primary Ars-specific clones. This "prozone" effect would limit both BCR-initiated signaling and Ag presentation to T cells and thereby favor development of memory cells according to both variations of the hypothesis. In the extreme case, very high doses of soluble Ag could lead to B cell death for lack of adequate BCR aggregation. This is consistent with the observation that high doses of soluble Ag increase the frequency of apoptotic B cells in GC and, in extreme cases, induce GC dissolution. Notably, in studies of Ag-induced B cell death, considerably more soluble Ag (milligrams) was injected than in the work reported here (62, 63, 64). In addition, the BCR in these studies have much higher affinities than those of primary anti-Ars B cells (58).

The short duration of the pause in antigenic delivery required for memory B cell development was unexpected. Although the mechanism by which this pause promotes competence in memory B cells could involve follicular Th cells, cytokines, or other B cell extrinsic factors, the short period of the pause suggests that the mechanism may be intrinsic to the B cell. According to the preceding argument, if the period following the last injection of soluble Ag provides for Ag clearance by mechanisms that do not involve the BCR, we would not necessarily expect B cells to differentiate into plasma cells during this period. However, the rest period may allow for Ag clearance and for accumulation of secreted Ab by primary AFC, such that upon a later injection with the same Ag dose, a higher degree of BCR aggregation is attained due to partial blockade or removal by secreted Ab. We recognize that more complex models are possible, and propose this one only because of its minimalist nature.

The idea that B cell memory is stratified into tiers that are generated upon successive challenges with Ag was proposed by Decker et al. (32) who used a limiting-dilution splenic focus assay to assess memory development in vitro. They provided evidence that each pulse of Ag produced both AFC and memory cells that underwent further rounds of hypermutation. In vivo, the long-term AFC response may be considered one tier of memory because it emanates from the GC reaction. The silent memory progenitor cells would constitute a second tier. However, whether hypermutation and affinity maturation occur again in memory B cells that are participating in a true secondary immune response is still unresolved. Although it is clear that secondary antigenic challenge drives memory cells to undergo massive proliferation and differentiation into AFC in the red pulp of the spleen and the medullary cords of lymph nodes without hypermutation (65, 66), a secondary antigenic challenge also induces GC reactions. So, it is conceivable that these GC arise from memory B cells that mutate and develop into another tier of memory during the recall response.

It is noteworthy that in our study there was a lack of affinity improvement in the recall response of mice that did not receive the Ars-KLH injection series (Fig. 1C). In these mice, Ab of the late primary response sera (day 41) achieved affinities that were indistinguishable from those of secondary response sera (day 244). This result stands in contrast to the affinity maturation seen in mice that received the primary injection series. At the same, time it is consistent with a report by Takahashi et al. (21), who argued that the GC reaction continuously seeds the primary AFC reaction, which consequently undergoes continuous affinity maturation. We can envision two explanations for the lack of further affinity improvement in Ab of the recall response in mice that did not receive the injection series (Fig. 6). One possibility is that silent memory cells may occur routinely, even when the concentration of Ag is not increased artificially by further injections or naturally during an actual infection. According to this scenario, the failure to observe affinity maturation in mice not receiving the injection series could be due to a limited duration of Ag stimulation by the particular Ag that we used, Ars-KLH. In preceding studies and unpublished work, we noted that GC development peaks already by day 14 in mice that receive only one Ag injection (49). In contrast, the duration of GC reactions are extended in mice that receive the Ars-KLH injection series (Fig. 2). Additional Ars-KLH injections delivered during the primary immune response may promote exaggerated affinity maturation in memory precursors by extending the life of the GC reaction and the period of Ag-driven selection. Alternatively, it is possible that developing memory cells must sense an increase in Ag during an established primary immune response to develop along the silent memory pathway i.e., without significant differentiation of siblings into AFC. That is, an increase in Ag concentration may signal a novel developmental pathway.


Figure 6
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  		FIGURE 6. Models of memory cell and AFC generation. A, Continuous generation of AFC and memory cells from a common clone during affinity maturation in GC. This model is consistent with results from mice given a single injection of Ag. B, Extensive affinity maturation in memory progenitor cells without AFC formation. This model is consistent with data from mice that received serial injections of Ag during the primary immune response. A pause in antigenic stimulation is required for memory progenitors to finalize their development into competent memory cells.

 
The biological significance of silent memory is unknown, and it may be difficult to define in the context of natural infectious agents. However, it is interesting that the longevity of murine GC reactions against vesicular stomatitis virus is much greater than it is to a simple hapten protein conjugate delivered in Freund’s adjuvant (67). In this sense, delivering an Ag injection series may produce an immune response that more closely resembles a natural scenario where the concentration of Ag may rise and fall during infection. One possible advantage of silent memory is that it may inhibit the evolution of microbial resistance during infection. Thus, even if a microorganism mutated an epitope that was initially targeted by Ab secreted early in the immune response, the high-affinity Ab produced by reactivation of formerly silent memory cells might bind well enough to eliminate the infection.


   Acknowledgments
 
We thank Drs. Jacqueline Sharon and Behnaz Parhami-Seren for providing the IgG2b vector.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported by National Institutes of Health Grants AI033613 and AI048108. Back

2 Address correspondence and reprint requests to Dr. Lawrence J. Wysocki, Department of Immunology, K902a, National Jewish Medical and Research Center, 1400 Jackson Street, Denver, CO 80206. E-mail address: wysockiL{at}njc.org Back

3 Abbreviations used in this paper: AFC, Ab-forming cell; GC, germinal center; Ars, p-azophenylarsonate; Tg, transgenic; Sulf, sulfanilic acid; RT, room temperature; SA, streptavidin; ABTS, 2'2'-azino-bis(3-ethylbenz-thiazoline-6-sulfonic acid); Eu, europium; KLH, keyhole limpet hemocyanin; PNA, peanut lectin agglutinin; I50, inhibited by 50%. Back

Received for publication October 12, 2006. Accepted for publication August 8, 2007.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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