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Mutation and Selection During the Secondary Response to 2-Phenyloxazolone (Hypermutation/Memory B Cells/Igk-Voxl) CRISTINA RADA, SATISH K

Mutation and Selection During the Secondary Response to 2-Phenyloxazolone (Hypermutation/Memory B Cells/Igk-Voxl) CRISTINA RADA, SATISH K

Proc. Natl. Acad. Sci. USA Vol. 88, pp. 5508-5512, July 1991 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 , 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). response to the 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 in the Igk-VOxl gene increased in sequences chain joining region) rearrangement of anti-PhOx 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- 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 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.

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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 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 (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 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). The fre- day 5 and day 7. Both His-34 and Phe-36 are residues in quency of Pro-95-Pro-96 sequences decreased, on average, contact with the hapten, as revealed by the x-ray structure of by a factor of 10 among antigen-binding cells. Thus, we the NQ10/12.5-hapten complex (19). Only one clone was estimate the enrichment to be in the order of 10-fold. isolated carrying a replacement mutation in one of the other The frequency of base substitutions in Igk-VOxl from contact residues (Tyr-32, Gln-89, Trp-91, Leu-96, and Phe- nonimmune animals was within experimental background, 98) (Table 3). This result further supports the view that whereas most clones isolated from animals at days 14 and 21 replacement mutations in these residues (unlike the His-34 -* primary response or day 3,5, and 7 in the secondary response Asn-34/Gln-34 and/or Tyr-36 Phe-36 mutations), if they were hypermutated (Table 1). The unmutated sequences occur, are selected against because they decrease binding ranged from 14% to 41% of all clones, and there was no affinity (19). Mutations at residues Ser-26, Ser-28, Met-33, evidence of them disappearing during the maturation of the and Ser-77 (all residues not directly involved in contacts with response. These sequences therefore represent a background the hapten) became common late in the secondary response of nonmutating cells. No hypermutated VKOx1/JK5 rear- (Fig. 1), but whether some or all of these substitutions are rangements were recovered at day 45 in the primary re- associated with increased binding affinity is not yet known. sponse, a result that confirms the low frequency of memory In contrast, the frequency of mutations in framework 1 B cells in spleen at that stage of the antibody response (17). clearly decreased between day 3 and days 5-7, suggesting Hypermutation Is Active in the Early Secondary Response. that clones carrying such mutations were selected against and The average number of mutations per clone increased steadily in both primary and secondary responses (Table 2). Table 2. Accumulation of mutations in canonical Hypermutation continued from day 14 to day 21 in the (Pro-95-Leu-96) and mutated VKOx1/JK5 primary response at an apparent rate of 2.2 x 10-4 bp per clones from antigen-selected cells generation, assuming a division time of 10 hr. We stress that Mutations/ the choice of a division time of 10 hr is rather arbitrary because we have no direct evidence that hypermutation Total 1000 bases Coding/ occurs in the fast-dividing cell population in germinal centers mutations Total Silent silent (18). Between days 3 and 7 in the secondary response, the Primary response apparent rate of mutation was also high (2.3 and 1.3 x 10-4 Day 14 42 8.3 3.0 1.8 bp per generation between days 3-5 and 5-7, respectively). Day 21 46 13.6 4.1 2.3 These rates were calculated by using both replacement and Secondary response silent mutations and therefore are affected by selective Day 3 82 16.5 4.2 2.9 factors. The apparent rates of silent mutation was 1.3 x 10-4 Day 5 114 18.4 4.5 3.1 bp per generation from both days 14 to 21 in the primary Day 7 157 19.2 3.7 4.2 response as well as from days 3 to 5 in the secondary Unmutated sequences are excluded from the calculations. Fre- response. Silent mutations did not increase, but rather de- quency of base substitutions due to nucleotide misincorporation by creased, between day 5 and day 7 in the secondary response. Taq polymerase for this set of experiments is 0.7 per 1000 bases. Downloaded by guest on October 2, 2021 5510 Immunology: Rada et al Proc. Nati. Acad. Sci. USA 88 (1991)

CDRN1 CDR 2 CDR 3 Vk~x1. QIVLTQSPAIMSASPGEKVTMTC SASSSVSYMH WYQQKSGTSPKNWIY DTSKLAS GVPARFSGSGSGTSYSLTISSMFEAEDAATYYC QQWSSNP LTFG Primary DRYI 14 40SNQP2..-- -I . S.------40SNQP3 ....--- A.------40SNQP5...... -- -TT -__ 40SNQP7. ...------E .-- -F.-- - 40SNQPS.------N--N.-- - - - 40SNQP9..------V-

------40SNQP12 - ---.-- -N -F ..--- -.-- 40SNQP13..-- --N -.------40SNQP15. ...------V ....------I----A.------

------40SNQP23 - -.K.--- 40SNQP24..-- -I. -- - - 40SNQP26. ...-- -N.-- -.- -.------40SNQP27..-- -G - --.- - 40SNQP28S...... --NR -T.--- - 40SNQP29...... --- -V.---- 40SNQP30. . .--- --L.-- -N.- - DAY 21 43SNQP4. ...N.---R- -Y ---G- --..-- -V.- 10 43SNQP5..------N--N .---D---E...... ----A------S .-- 43SNQP7. -V------T-N--N ....------NT------43SNQPS. R .-- -.-- --- 43SNQP15. ...------N ..------T .------43SNQP17...... --- -N.-- -S . 43SNQP19..-- -N.-- 43SNQP25. ...---- -N.- F.------43SNQP26. . .. --R ....-- - -T .- - 43SNQP27...... ------T ..------TT.------T .-- Secondary DAY 3 44SNQP2..N.------44SNQPS..------R------10-44SNQP9.------N--- R------G------R------44SNQP10-O ------R.------L .-- -R--N -F--R ....------T .-- 44SNQP14 ..------K- .-R---N -----N------44SNQP17-7..VQ -F .-- V.-- 44SNQP20O. . .-- -F.-- -GF-N -F----D.------F.-- 44SNQP23. ...-- -N .-- -V ...--- -T.-- - 44SNQP24. ..-- -R..-- -P ...--- -R.------44SNQP25..------44sNQP26. --A .--V---V .- - L-----R--N -F ....------S . --HN 44SNQP27..------R--N ------44SNQP29.------V-V------N--N ....------R .--- 44SNQP31..------R.------44SNQP32...... -- .--- 44SNQP33. - -N-RN ------.------.N---NF------44SNQP34. .- -N ....N-----N.-- -.-- - DRY 5 1047SNQP1. --A------R-N-- --N-R-VN -F--NA------D------T------*N.- 47SNOP3..-- - -I.- - - 47SNQP7. . .------F--R-N .------VN -F .....------V------0 47SNQPS. ...------T-LQ -F ....----P------s - 47SNQP9..------N------47SNQP10O...... ------T.---- 47SNQP14. ..--- P.------

47SNQP17...... ------N.---

47SNQP18----..-- -- -N.------47SNQP19. ---I ....------R---N ------P ..------TN------N---R-- 47SNQP20O. ...N------IN -FR-R.------V .---S------T .- 47SNQP21...... ------NN--T--

47SNQP23...... ------T .- 47SNQP21 --- 0 -F .-- -A-- -. -V.-- -H -D . 47SNQP28S. ...-- -I- .-- -V ..-- -R .- 47SNQP32 ...-- -T.-- -T ... -.-

47SNQP33. ...-- -N -FF .-- -A.-- - --

47SNQP36.- .R-- ....------T .-

47SNQP3S. ...------IN -F ..---- -V ..------T-- 47SNQP39..------H 1047SNOP40. ... LQ -F..-- - .------P. - ---S 47SNQP42.. . T. ....---N------L-T-- -- DAY 7 10 4SSNQP5.------*N------A------V------48SNQP6. --I ..N------VN -F ...------F-----G-X------TT.- 48SNQP9..------v------H----- 48SNQP10------. -N-R-P--N -F N.--. -R -- - 48SNQP18. ...--- -N -F--F.-RN - ..TT .-

48SNQP19. ...-- -N------48SNQP21l------T-N----Q- -F--R ------V------N------T----- 48SNQP22. ...---- -N -F ....-- -N.---V.-- 48SNQP23. . .-- -C.--- -P-IN -F ....------T.-- -Y .- 48SNQP27.------N-N-R--N -F---- .--. ------I------T---V------4SSNQP28 ------S--- T-N---IQ -F------V----N----- 1048SNQP32 -. -I.-- - - 10,48SNQP102 ------. - -- - -KS 4SSNQP104.--I ..N------VN -F ...------F-- -G.------TT-- 48SNQP107.------T.------48SN QP110 O- -- -I( -F ....--- - - N .- 48SNQP113. ...------N -F-F.--- -V . ..-- ---. N .-- 48SNQP115. ...------T---D-N-IQ -F ..------V ..--- -N.------48SNQP117..------48SNQP118 -I-- .- --. T ..-- N - . 48SNQP119..-- - -T.-- - 48SNQP121 -- --. -N-R--N -F ..------T.--- -- 48SNQP123..-- -I.------48SNQP128 -.. P --N------R--H-N -F-----S------N------V------T----- 48SNQP132.------G-N--N -F------Q------* 48SNQP136..------T------S ---- 48SNQP138.------T------

FIG. 1. (Legend appears at the bottom of the opposite page.) Downloaded by guest on October 2, 2021 Immunology: Rada et al. Proc. Natl. Acad. Sci. USA 88 (1991) 5511

Table 3. Percent frequency of replacement mutations in residues bacteria provide an alternative and efficient way to recover in contact with antigen in canonical (Pro-95-Leu-96) and mutated and analyze antibody genes in their germ-line or rearranged VKOX1/JK5 clones from antigen-selected cells configuration (20-22). This approach is very suitable for Primary Secondary repertoire analysis (23), but it has its own limitations because response response of the technical aspects of the DNA amplification step itself, which introduces point mutations at a measurable rate and Day Day Day Day Day because the and are as 14 21 3 5 heavy- light-chain genes not recovered 7 pairs. Trp-91, Tyr-32, Gln-89, In this study we have used this approach to readdress the Leu-95, and Phe-98 0 0 0 0 1 question of whether PhOx memory B cells may or may not His-34 Asn/Gln 22 33 56 45 55 reenter hypermutation after new antigenic stimulation. Pre- Tyr-36 Phe 11 0 22 37 52 vious analysis of hybridomas showed that both total and His-34 - Asn/Gln silent mutations progressively increased in the secondary and and Tyr-36 Phe 6 0 22 37 48 tertiary responses in the Igh-VOxl and Igk-VOxl genes, suggesting new rounds of hypermutation and selection after replaced by VKOx1 mutants carrying predominantly comple- new antigenic stimulation (6, 9). On the other hand, the mentarity-determining region (CDR) 1 mutations' (Fig. 1). results of adoptive transfer experiments in which memory B Even so, the overall number of mutations increased during cells were found incapable of further mutation, as shown in the secondary response. the classic experiments of Askonas et al. (24) and more Deleterious Mutations. Further evidence demonstrating the recently by Siekevitz et al. (10) and other studies, are often reoccurrence of hypermutation in cells derived from memory taken as evidence that somatic hypermutation does not occur B cells came from the finding of somatic mutants with in secondary and later responses. putative impaired' ability to bind antigen in the secondary The results of the present study provide further evidence response (Fig. 1, arrowheads). Of 66 clones analyzed at days for the reoccurrence of somatic hypermutation in secondary 3, 5, and 7, there were 2 with stop codons and 6 with changes antibody responses. We found that both total and silent in the'critical Trp-35, Cys-88, or Pro-95 residues. These mutations increased during the secondary response at least changes predict either a'truncated or nonfunctional light up to day 5 after the antigen boost. In addition, and most chain. A third codon was found in a stop clone isolated at day revealingly, approximately one in eight of the VKOx1 se- 21 in the The primary response (Fig. 1). three truncated quences isolated during the secondary response carried del- mutants and two of the mutants carrying deleterious substi- eterious mutations (Fig. 1, arrowhead). The frequency of tutions also carried one or multiple mutations characteristic these mutations is well above that expected from artifacts in of antigen-selected memory B cells (His-34-- Asn-34/Gln-34 the amplification or cloning step. We did not isolate a single and/or Tyr-36 -- Phe-36). Sequences 47SNQP8 and 47S- clone carrying such mutations of 151 clones sequenced from NQP40 (day 5 secondary response) have two replacement unselected B cells. The base changes in these clones that led mutations characteristic of the mature response (His-34 to either the stop codons or the deleterious mutations also Gln-34, Tyr-36 -_ Phe-36) and appear to be clonally related. argue against artifacts. They were as follows: T -* C (2), C Both sequences display a Pro-95 -* Ser-95 replacement, a -* -- change that may disrupt the structure of CDR3. One of the T (3), G -* A, A T, T -* A, and C -- A. No A -* G substitution was two clones had a further mutation in Ser-31, which was found, although it is the most common artifact produced by the PCR (ref. 25; C.M., E.G., and therefore introduced in the clonal progeny after the Pro-95 -* Ser-95 substitution, unless it was due to a PCR artifact. J. Jarvis, unpublished results). In at least three and possibly Deleterious mutations were also found in four clones that did five cases, deleterious mutations occurred in clones carrying the most not carry the selected His-34 -* Gln-34 substitution. One of typical mutation known to be associated with them was isolated at day 5, and three were isolated at day 7 increased binding affinity (His-34 -- Asn-34/Gln-34). These in the secondary response. The low number of mutations' in sequences thus carry the hallmark of memory B cells, and the these clones (Fig. 1) suggests that they originate from hyper- presence in these clones of additional mutations that lead to mutation of a virgin B-cell subpopulation. It is difficult, the production oftruncated and/or nonfunctional light chains however, to escape the conclusion that at least some of the can only be explained by further mutation after antigen deleterious mutations found in the secondary response in restimulation. clones carrying mutations characteristic of the memory B The presence ofdeleterious mutants among a population of cells are the result of a new round of mutations derived from antigen-binding cells deserves comment. We think this is due cells in the memory pool. to the time window in which new mutations have not yet replaced the pre-existing immunoglobulin receptor. There are precedents for such intermediates-e.g., B cells express- DISCUSSION ing intracellular IgG and membrane IgM (26). Unfortunately Extensive studies on the generation of somatic mutants of the turnover of surface IgG in cells derived from memory B antibody genes were made possible by the combination ofthe cells restimulated with antigen is not known, but experiments hybridoma technology and fast procedures for direct se- on Epstein-Barr virus-transformed B-cell lines indicate that quencing of immunoglobulin mRNAs (15). There are limita- the half-life of membrane-bound IgG is in the order of 15-20 tions, however, with this approach. In particular, predomi- hr in the absence of antigen and -3 hr in the presence of nantly (if not only) B-cell blasts are immortalized by fusion, antigen (27). If the turnover time of surface IgG in memory and thus the panel of mutants emerging may be significantly cells is comparable to that of the Epstein-Barr virus trans- biased. DNA amplification by PCR and then cloning in formed lines, clearly cells carrying a recent deleterious

FIG. 1 (on opposite page). Protein translation of VKOX1 mutants isolated at different stages of the antibody response to PhOx. The top line shows the sequence of the protein encoded by the germ-line Igk-VOxl gene segment (13) and the first four residues of the JK5 segment. The sequence ofresidues 5-13 of the JKSsegment is not shown because it was dictated by the primerJK5FOR. The clones marked with an arrowhead contain either in-frame stop codons (*) or replacement mutations at residues Trp-35, Cys-88, or Pro-95. These mutations are thought to disrupt the light-chain folding or the structure of CDR3 and may lead to loss of antigen binding, although direct evidence for this is not yet available. Downloaded by guest on October 2, 2021 5512 Immunology: Rada et al. Proc. Natl. Acad. Sci. USA 88 (1991) mutation may be isolated on the ground of the preexisting We gratefully acknowledge the many contributions that J. Jarvis pool of receptor antibody. and R. Pannell made to this study. C.R. was supported by a grant The reoccurrence of somatic hypermutation in memory B from the Ministry of Education of Spain, and S.K.G. was supported cells in the secondary response strengthens the conclusion by the Department of Biotechnology, Government of India. we had reached in earlier studies that memory cells reenter 1. Moller, G., ed. (1987) Immunol. Rev. 96, 5-162. hypermutation after antigenic challenge. This fact does not 2. Moller, G., ed. (1988) Immunol. Rev. 105, 5-159. mean that the net contribution of hypermutation in the 3. Griffiths, G. M., Berek, C., Kaartinen, M. & Milstein, C. (1984) generation ofhigher affinity antibodies is identical at different Nature (London) 312, 271-275. stages of the antibody response. Once clones with a high 4. Lebecque, S. G. & Gearhart, P. J. (1989) J. Exp. Med. 172, number of replacement mutations have been selected, it is 1717-1727. less and less likely that new rounds of mutations would lead 5. McKean, D., Huppi, K., Bell, M., Staudt, L., Gerhard, W. & Weigert, M. (1984) Proc. Natl. Acad. Sci. USA 81, 3180-3184. to further improvement in binding affinity. Indeed, the strat- 6. Berek, C. & Milstein, C. (1987) Immunol. Rev. 96, 23-41. egy that the animal employs in the maturation ofthe antibody 7. Adetugbo, K. & Milstein, C. (1977) Nature (London) 265, response is complex, and hypermutation is only one compo- 299-304. nent of it, however important. Analysis of hybridomas from 8. Apel, M. & Berek, C. (1990) Int. Immunol. 2, 813-819. secondary and tertiary responses to the hapten PhOx has 9. Berek, C. & Milstein, C. (1988) Immunol. Rev. 105, 5-23. clearly indicated that the repertoire shift to alternative heavy- 10. Siekevitz, M., Kocks, C., Rajewsky, K. & Dildrop, R. (1987) and light-chain gene combinations, and the reappearance of Cell 48, 757-770. 11. Clafin, J. C., Berry, J., Flarerty, D. & Dunnick, W. (1987) J. antibodies encoded by unmutated genes (for example in Immunol. 138, 3060-3068. tertiary responses) are important components of the overall 12. Sambrook, J., Fritsch, E. F. & Maniatis, T. (1989) Molecular maturation process (for reviews, see refs. 6 and 9). Cloning:A Laboratory Manual (Cold Spring Harbor Lab., Cold The presence ofunmutated VKOx1 sequences along with a Spring Harbor, NY), 2nd Ed. majority of somatic mutants (Table 2) is not easily explained 13. Even, J., Griffiths, G. M., Berek, C. & Milstein, C. (1985) by contamination with nonantigen-binding cells. Purification EMBO J. 4, 3439-3445. on antigen-coated beads gave an enrichment of 410-fold over 14. Sanger, F., Nicklen, S. & Coulson, A. R. (1977) Proc. Natl. Acad. Sci. USA 74, 5463-5467. background, and yet we found, on average, 25-40% of 15. Kaartinen, M., Griffiths, G. M., Markham, A. F. & Milstein, unmutated Igk-VOxJ sequences among antigen-binding cells. C. (1983) Nature (London) 304, 320-324. Such cells represent a pool of antigen-binding cells that 16. Berek, C., Griffiths, G. M. & Milstein, C. (1985) Nature cannot enter hypermutation (28) or recruitment and prolifer- (London) 316, 412-418. ation of antigen-specific naive cells, which begin to accumu- 17. Weiss, U. & Rajewsky, K. (1990) J. Exp. Med. 172, 1681-1689. late mutations only at a later stage in the secondary response. 18. MacLennan, I. C. M. & Gray, D. (1985) Immunol. Rev. 91, This possibility is suggested by some of the sequences 61-85. 19. Alzari, P. M., Spinelli, S., Mariuzza, R. A., Boulot, G., Poljak, obtained at day 7 in the secondary response. The average R. J., Jarvis, J. M. & Milstein, C. (1990) EMBO J. 9, 3807- number of replacement and silent mutations in clones carry- 3814. ing the advantageous His-34 -+ Asn-34/Gln-34 substitution 20. Orlandi, R., Gussow, D., Jones, P. T. & Winter, G. (1989) putatively derived from the memory B-cell pool was very Proc. Natl. Acad. Sci. USA 86, 3833-3837. high (7.9 mutations per clone). On the other hand, somatic 21. Ward, E. S., Gussow, D., Griffiths, A. D., Jones, P. T. & mutants not carrying the His-34 mutation had an average of Winter, G. (1989) Nature (London) 341, 544-546. 2.2 base substitutions per clone. The data are therefore 22. Huse, W. D., Saastry, L., Iverson, S. A., Kang, A. S., Alting- compatible with the possibility that during the secondary Mees, M., Burton, D. R., Benkovic, S. J. & Lerner, R. (1989) response, naive cells also enter the hypermutation cycle. The Science 246, 1275-1281. 23. Marks, J. D., Tristem, M., Karpas, A. & Winter, G. (1991) Eur. occurrence of both unmutated clones and clones with a low J. Immunol. 21, 985-991. number of mutations was seen earlier in hybridomas derived 24. Askonas, B. A., Wiliamson, A. R. & Wright, B. E. G. (1970) from a tertiary response to PhOx (29). Proc. Natl. Acad. Sci. USA 67, 1398-1403. We conclude that the mechanism of hypermutation is 25. Keohavong, P. & Thilly, W. G. (1989) Proc. Natl. Acad. Sci. equally available to antigen-stimulated naive or memory USA 86, 9253-9257. cells. Both in the late primary and in the secondary response, 26. Pernis, B., Forni, L. & Luzzati, A. L. (1977) Cold Spring replacement mutations continue to accumulate, probably as Harbor Symp. Quant. Biol. 41, 175-183. a result of selection ofhighly mutated clones. The increase is 27. Davidson, H. W., West, M. A. & Watts, C. (1990) J. Immunol. sharper in the secondary response (Table 3), which is prob- 144, 4101-4109. 28. Linton, P.-J., Decker, D. J. & Klinman, N. R. (1989) Cell 59, ably more synchronized than the primary response. The 1049-1059. overall process of selection appears therefore to include two 29. Berek, C., Jarvis, J. M. & Milstein, C. (1987) Eur. J. Immunol. stages: (i) a positively selected proliferation ofcells from both 17, 1121-1129. the naive and the memory pool and then (it) a negative stage 30. MacLennan, I. C. M., Liu, Y. Y., Oldfield, S., Zhang, J. & of selection of new somatic mutants, where only antigen- Lane, P. J. L. (1990) Curr. Top. Microbiol. Immunol. 159, binding cells survive (30). 37-63. Downloaded by guest on October 2, 2021

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