Silent development of memory progenitor B cells

Abstract

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.

Figure 1

Soluble antigen 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 4–5 mice. Lower concentration indicates higher affinity Ab. Standard errors 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.

Figure 2

Injection of soluble antigen extends GC longevity. A, Immunization schedule for the analysis of GC size. Mice received either 6 (300 μg/ml, 100μg/ml or 20 μg/ml), 2 (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 experimental procedures), enumerated and plotted as % of total for each class size. C, Vanishing GC response on day 21 in mice without soluble antigen injection (0b). Arrows point to residual GCs. D, Prolonged GC reaction on day 21 in mice after 6 injections with 300 μg Ars-KLH (6b300). Spleen section shows large germinal centers (PNA = blue) with anti-idiotypic staining (mAbE4 = brown).

Figure 3

Counter-inhibition 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).

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 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 counter-inhibition assay as in Figure 3. Affinity-purified canonical antibody accounted for ~14% of all canonical antibody in the sample, as assessed with mAbE4.

Figure 5

Visualizing Sulf-binding memory B cells. Mice were initially immunized with Ars₁₅-KLH (100 μg) i.p. in IFA. 3 mice were given serial injections of Sulf-KLH on days 7, 9, 12, 14, 17, 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 germinal center response. Consecutive spleen sections were pre-incubated with the indicated competitors (Ars-BSA, Sulf-BSA) or with no competitor, before and during a subsequent stain with fluorescently labeled mAbE4 (green) and anti-B220 (blue). A, Representative example of GC with Sulf-binding E4+ B cells, as revealed by inhibition of mAbE4-staining with Sulf-BSA. B, Percentages of E4+ GC in which a majority of B cells bound Sulf-BSA, as assessed by inhibition of mAbE4 stain.

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 antigen. B, Extensive affinity maturation in memory progenitor cells without AFC formation. This model is consistent with data from mice that received serial injections of antigen during the primary immune response. A pause in antigenic stimulation is required for memory progenitors to finalize their development into competent memory cells.