Proc. Natl. Acad. Sci. USA Vol. 88, pp. 5508-5512, July 1991 Immunology Mutation and selection during the secondary response to 2-phenyloxazolone (hypermutation/memory B cells/Igk-VOxl) CRISTINA RADA, SATISH K. GUPTA, ERMANNO GHERARDI, AND CUSAR MILSTEIN Medical Research Council Laboratory of Molecular Biology, Hills Road, Cambridge CB2 2QH, United Kingdom Contributed by Cesar Milstein, March 15, 1991 ABSTRACT The most characteristic feature of the mouse failed to hypermutate upon new antigenic stimulation (10). antibody response to the hapten 2-phenyloxazolone is the Other results-e.g., ref. 11-have often been interpreted in recurrent expression of the light-chain variable region Igk- support of the latter view. VOxi gene in its germ-line or mutated configuration. The We have now investigated this problem by a different analysis ofsomatic mutants oftheIgk-VOxl gene reported here approach. We have isolated PhOx-specific B cells at different indicates that, as found during the primary response, hyper- stages ofthe primary and secondary response and recovered mutation is also activated during the secondary response. the canonical (VKOX1 light chain variable region/JK5 light Somatic mutations in the Igk-VOxl gene increased in sequences chain joining region) rearrangement of anti-PhOx antibodies obtained at 14 21 in by DNA amplification with the PCR. Sequence analysis of day and day the primary response and the clones provides strong evidence in favor of our earlier again in the secondary response at days 3, 5, and 7. The ratio conclusion that the memory B-cell pool can reenter hyper- of replacement to silent mutations also increased, particularly mutation. Thus, new rounds ofmutation and selection appear between days 5 and 7, suggesting that a stage of negative to be induced by each successive antigenic challenge. selection operates on new somatic mutants generated in the secondary response. Most Igk-VOxl mutants isolated in the secondary response had the features ofselected memory clones MATERIALS AND METHODS (i.e., they carried mutations known to increase binding affinity Cell Purification. BALB/c mice were immunized i.p. with for the hapten). However, some clones had chain-termination 100 gg of oxazolone-chicken serum albumin/alum precipi- codons, and others had mutations predicting a nonfunctional tate plus 109 Bordetella pertussis. For secondary responses, light chain. At least three and possibly five of these clones also animals were boosted i.v. with 100 gg of oxazolone-chicken expressed the mutation characteristic of the memory response serum albumin. Spleen white cells (2 x 108; obtained from to 2-phenyloxazolone (His-34 -- Asn-34/Gln-34). We conclude pools of at least three animals) were incubated at 40C with that after a second antigenic challenge, new somatic variants, Dynabeads M-450 (Dynal, Merseyside, U.K.) coated with including some leading to the loss of antigen binding, are oxazolone-bovine serum albumin at a ratio of 1:3 (cells per generated by hypermutation of cells derived from the memory beads) for 30 min, washed in cold 20% fetal calf serum in pool. Dulbecco's minimal Eagle's medium six times, and the cell pellet was stored at -70'C. DNA Amplification. Genomic DNA was extracted from Work in several laboratories over the last decade has shown 107 total spleen cells or from =106 antigen-specific cells that somatic hypermutation plays a dominant role in the using standard procedures (12). The DNA equivalent to 1-5 maturation of the antibody response. Antibodies encoded by x 104 cells was used for PCR amplification. Amplification unmutated light- and heavy-chain genes are a feature of early was carried out for 30 cycles (1 min at 920C, 2 min at 550C, primary responses, whereas the vast majority ofhigh-affinity and 2 min at 720C) in 10 mM Tris hydrochloride, pH 8.3/ antibodies isolated at subsequent stages are encoded by gelatin at 2 g/liter/2 mM Mg2+/0.5 mM dNTPs/0.5 uM immunoglobulin genes that have accumulated extensive mu- primers/2.5 units of Taq polymerase (Cetus). The primers tations (1, 2). Somatic hypermutation of antibody genes is were as follows: JK5FOR (5'-CGTTAGATCTCCAGCTTG- triggered only after antigen-induced B-cell proliferation (3) GTCCCAAG-3'), which primes the JK5-encoding segment and is confined to the variable-(diversity)-joining [V(D)J] and carries a Bgl II site, and VKOXBACK (5'-CCGGG- region and the adjacent noncoding DNA segments (4). Point GAATTCTCAGCTTCCTGCTAATCA-3'), which primes mutations occur at a rate of 10-3 to 10-4 base pairs (bp) per the leader sequence of the Igk-VOxl gene and contains an generation (5, 6), which is 104-105 times higher than the rate EcoRI site. Amplified DNA was restricted, purified, and of mutation of antibody genes in myelomas in culture (7). cloned into M13mpl8. VKOx1 clones were selected by hy- Although some evidence exists that hypermutation may bridization to oligonucleotide VKOX149 (5'-GTGTCAT- occur in the germinal centers (8), the B-cell subpopulations AAATCCATCT-3'), complementary to nucleotides 132-149 capable of undergoing somatic hypermutation are insuffi- of the Igk-VOxl gene (13), and sequenced (14). In control ciently defined. Somatic hypermutation is generally accepted experiments, the frequency of base substitutions due to to operate on cells derived from the antigen-stimulated pri- nucleotide misincorporation by Taq polymerase was 1/ mary B-cell pool. That somatic hypermutation also occurs in 1500. the memory B-cell pool is suggested by the accumulation of both silent and total mutations in primary, secondary, and tertiary anti-2-phenyloxazolone (anti-PhOx) antibodies (6, 9). RESULTS This conclusion, however, has been challenged by the results Unselected B Cells. The antibody response ofBALB/c mice of adoptive transfer experiments, in which memory B cells to the hapten PhOx is characterized by the VKOx1 chain The publication costs of this article were defrayed in part by page charge Abbreviations: CDR, complementarity-determining region; PhOx, payment. This article must therefore be hereby marked "advertisement" 2-phenyloxazolone; JK5S K light-chain joining region 5; VKOX1, K in accordance with 18 U.S.C. §1734 solely to indicate this fact. light-chain variable-region of canonical anti-PhOx antibodies. 5508 Downloaded by guest on October 2, 2021 Immunology: Rada et al. Proc. Natl. Acad. Sci. USA 88 (1991) 5509 rearranged to the JK5 segment, which encodes Leu-96 (15). At Table 1. Percent frequency of point mutations in canonical least three different heavy-chain variable-region genes are (Pro-95-Leu-%) VKOX1/JKS clones from antigen-selected cells used in combination with the Igk-VOxl gene. Thus, accu- No. mutations per clone mulated mutations in the Igk-VOxl gene reflect the muta- tional drift in the maturation of the anti-PhOx response. Clones, no. 0 1 2-4 5-7 -8 We used PCR to amplify and clone VKOxl/JK5 rearrange- Nonimmune 8 63 37 ments with a pair of JK5 and VKOx1 leader primers. VKOx1 Primary response clones were selected (to >90%) by hybridization to the Day 14 21 14 33 48 5 VKOX149 oligonucleotide and identified by their sequence. Day 21 16 28 24 24 18 6 Sequence analysis indicated that the clones fell into three Day 45 13 85 15 major categories: (i) the productive, canonical VKOxl/JK5 Secondary response rearrangement, leading to the Pro-95-Leu-96 sequence found Day 3 24 26 21 25 12 16 in anti-PhOx antibodies; (ii) a second productive VKOxl/JK5 Day 5 34 30 17 22 16 15 rearrangement leading to the Pro-95-Pro-96 sequence very Day 7 49 41 12 14 16 16 rarely found in anti-PhOx antibodies (3, 15, 16); and (iit) a group of various nonproductive rearrangements. Here we The Changing Pattern of Mutations During Secondary Re- report results from clones in category i only. sponse to PhOx. That antigen selection influences the The frequency of nucleotide substitutions in Pro-95- pattern Leu-96 clones isolated from unselected B cells of nonimmune of mutations is indicated by two considerations: (i) the animals was progressive increase of replacement vs. silent mutations within the background associated with the (Table 2) and (it) the nature of the observed mutations and, experimental procedure. The frequency of base substitutions in the was higher in immunized animals, and the increase was particular, progressive accumulation of mutations confined, as expected, to canonical (Pro-95-Leu-96) VKOx1/ characteristic ofthe affinity maturation (Fig. 1). The increase JK5 rearrangements (results not shown). However, the pro- in the ratio of replacement vs. silent mutations obtained at portion of clones with two or more mutations was low. This day 7 and the decrease in the rate ofaccumulation of somatic high frequency of unmutated VKOx1/JK5 clones in unselected mutants indicate that selection plays a dominant role at this spleen B cells is most likely due to a high background of stage in the secondary response. Thus, the results indicate Igk-VOxl genes that do not participate in the anti-PhOx that somatic mutation operates at least up to day 5 from response. reimmunization and that antigen selection subsequently dom- Cells Selected by Antigen. When genomic DNA from cells inates the pattern of somatic mutants (see below). selected by binding to antigen-coated beads was isolated, A large proportion of the VKOX1 mutants isolated at day 3 amplified, and cloned, the proportion of VKOx1/JK5 clones in the secondary response carried mutations at His-34 (Fig. from nonimmune animals was higher than in unselected B 1 and Table 3). A lower proportion ofclones (about one-fifth) cells (-20% compared to 5-1o) and increased further in carried the Tyr-36 to Phe-36 substitution, but the frequency immunized animals (40-45% at day 14 in the primary re- of this mutation increased considerably in cells recovered at sponse and at day 3 in the secondary response).