Contrasting the In Situ Behavior of a Memory During Primary and Secondary Immune Responses

This information is current as Kalpit A. Vora, Kathleen Tumas-Brundage and Tim Manser of October 2, 2021. J Immunol 1999; 163:4315-4327; ; http://www.jimmunol.org/content/163/8/4315

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 1999 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Contrasting the In Situ Behavior of a Clone During Primary and Secondary Immune Responses1

Kalpit A. Vora, Kathleen Tumas-Brundage, and Tim Manser2

Whether memory B cells possess altered differentiative potentials and respond in a qualitatively distinct fashion to extrinsic signals as compared with their naive precursors is a current subject of debate. We have investigated this issue by examining the par- ticipation of a predominant anti-arsonate clonotype in the primary and secondary responses in the of A/J mice. While this clonotype gives rise to few Ab-forming cells (AFC) in the primary response, shortly after secondary immunization its memory cell progeny produce a massive splenic IgG AFC response, largely in the red pulp. Extensive clonal expansion and migration take place during the secondary AFC response but Ab V region is not reinduced. The primary and secondary germinal center (GC) responses of this clonotype are both characterized by ongoing V gene hypermutation and phenotypic selection, little or no inter-GC migration, and derivation of multiple, spatially distinct GCs from a single progenitor. However, the kinetics of these responses differ, with V genes containing a high frequency of total as well as affinity-enhancing mutations Downloaded from appearing rapidly in secondary GCs, suggesting either recruitment of memory cells into this response, or accelerated rates of hypermutation and selection. In contrast, the frequency of mutation observed per V gene does not increase monotonically during the primary GC response of this clonotype, suggesting ongoing emigration of B cells that have sustained affinity- and specificity- enhancing mutations. The Journal of Immunology, 1999, 163: 4315–4327.

uring a primary to -dependent After this period, at least two differentiative pathways are available http://www.jimmunol.org/ Ags in mammals, B cells undergo extensive prolifera- to a B cell, proliferation and differentiation to an Ab-forming cell tion and differentiation in the microenvironment of the (AFC) outside of follicles or migration to a follicle and D 3 germinal center (GC) (1–6). This process ultimately results in the participation in the GC/memory B cell pathway. In addition, recent formation of the memory B cell compartment (7, 8). It is now well data (30–33) have supported past assertions (34) that B cells par- established that GCs are the primary locale in which Ab V gene ticipating in the primary immune response are capable of differ- somatic hypermutation takes place (9, 10). In addition, a number of entiation to long-lived AFCs that reside in the bone marrow. How previous studies have suggested that both positive selection based these lineage and migratory decisions are made is currently un- on increased affinity for Ag (9–14) and negative selection against

clear. It has been suggested that the nature of naive B cells initially by guest on October 2, 2021 autoreactive V regions (15–18) also occurs in this microenviron- recruited into the response (35, 36), the affinity and specificity of ment. GCs also form during secondary and higher order immune the B cell Ag receptor (32, 33, 37, 38), the type of Ag driving the responses. These anamnestic GC responses appear to differ quan- response (19), and levels of T cell help (19, 38, 39) can all influ- titatively from the primary GC reaction (19). However, whether ence these decisions. Moreover, whether all these pathways of dif- qualitative differences in V gene hypermutation, phenotypic selec- ferentiation are also available to memory B cells participating in tion, and participation of memory B cell clones distinguish the anamnestic responses is a subject of debate (21–23, 34). Investi- secondary GC reaction from the primary GC reaction is a current gation of these issues is complicated by the fact that most immune area of controversy (20–26). responses to even simple are clonally diverse and often Before initiation of the primary GC reaction, Ag-specific B cells exhibit substantial response to response variation regarding the are thought to undergo activation via cognate interaction with helper T cells in the T cell-rich periarteriolar lymphoid sheath participation of individual clonotypes. In addition, in many im- (PALS) of the and the paracortex (6, 27–29). mune responses clonotypes that dominate the primary immune re- sponse may not effectively seed the memory compartment, making the identification of B cells destined for this compartment during the primary response difficult (40–43). Kimmel Cancer Institute and Department of Microbiology and Immunology, Jeffer- We (44) and others (45, 46) have extensively characterized the son Medical College, Philadelphia, PA 19107 T cell-dependent B cell response of A/J mice to the arson- Received for publication April 27, 1999. Accepted for publication August 3, 1999. ate (Ars). As assessed by serological and hydridoma studies, the 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 primary response to Ars is clonally diverse, but during the latter with 18 U.S.C. Section 1734 solely to indicate this fact. stages of this response a single B cell clonotype reproducibly 1 This work was supported by a grant from the National Institutes of Health emerges and dominates subsequent anamnestic responses. This (AI23739) to T.M., and by National Institutes of Health Training Grants CA09678 clonotype expresses a V region encoded by a single combination (K.A.V.) and CA09683 (K.T.B.). of V gene segments, with variation before hypermutation only at 2 Address correspondence and reprint requests to Dr. Tim Manser, Kimmel Cancer Institute, BLSB 708, 233 South 10th Street, Philadelphia, PA 19107. E-mail address: VH-D and D-JH junctions (47, 48). We term this V region and the [email protected] clones that express it “canonical.” Canonical clonotypes undergo 3 Abbreviations used in this paper: GC, germinal center; PALS, periarteriolar lym- extensive V region somatic hypermutation and both positive and phoid sheath; AFC, Ab-forming cell; Ars, arsonate; KLH, keyhole limpet hemocya- nin; PNA, peanut agglutinin; CDR, complementarity-determining region; FWR, negative phenotypic selection during the primary response (16, framework region; R, replacement; S, silent; FDC, follicular . 44). The secondary serum Ab response is largely composed of Abs

Copyright © 1999 by The American Association of Immunologists 0022-1767/99/$02.00 4316 CONTRASTING PRIMARY AND SECONDARY MEMORY B CELL RESPONSES

derived from this clonotype that bear characteristic VH region were found to be noncanonical, being composed of VhIdCR or a closely amino acid substitutions due to somatic hypermutation. These re- related germline VH gene segment, in combination with a noncanonical D current mutations have been shown to independently and addi- or JH segment. These clones also were never found to contain somatic mutations, indicative of their derivation from the naive B cell tively confer increased affinity for Ars (49). Thus, the canonical compartment. anti-Ars clonotype provides a paradigm for a true memory B cell clonotype, initially expressing a nearly invariant Ag receptor with known affinity and specificity, and reproducibly seeding the mem- Dendrogram construction and shape assignment ory compartment after undergoing extensive V gene hypermuta- As detailed in Results,VH clones obtained from single spleens were judged tion and selection. Here, we have exploited this experimental sys- to have been derived from a common precursor on the basis of sharing of

tem to determine how the primary and secondary AFC and GC their VH-D and D-JH junctional nucleotides. The sequences of groups of responses of a single B cell clonotype may differ. related clones containing three or more members were used to construct geneological dendrograms, essentially as has been described by us and others (10, 50–52). During this exercise, we assumed that a mutation shared by several clones had been sustained once in vivo, and then inher- Materials and Methods ited by daughter cells that then each gave rise to a single PCR clone. Mice Exceptions were sometimes made to this rule of “mutational parsimony” in A/J mice 8–12 wk of age at the time of first immunization were purchased the case of mutations in five VH codons: three (10, 31, and 105) that appear from The Jackson Laboratory (Bar Harbor, ME) and were housed in a to be mutational “hotspots” (53) and overlap RGYW consensus hotspot specific pathogen-free rodent facility and given autoclaved food and water. sequences (54); and the CDR2 codon 58 and 59 affinity-enhancing muta- tions (see Results). Artifactual base changes introduced via the PCR were

assumed to be present at a frequency of one per clone or less. VH clones Downloaded from Immunizations whose sequences could easily be explained by PCR recombination events (11, 55) were observed infrequently (Ͻ5% of clones) in our studies. How- For primary GC analysis, A/J mice were immunized intraperitoneally (i.p.) ever, when the locations of a clone in the dendrogram as defined by mu- with 100 ␮g Ars-keyhole limpet hemocyanin (KLH) precipitated in alum. tations in its 5Ј half, as compared with mutations in its 3Ј half, were dis- Nine to 15 days after immunization, mice were sacrificed and their spleens cordant, it was assumed that the clone had been generated via PCR were removed and prepared for cryosectioning as previously described (29, recombination and it was excluded from further analysis. 37). For secondary GC and secondary AFC “cluster” analysis, mice were During the construction of dendrograms, we also assumed that any ␮ immunized i.p. with 100 g Ars-KLH in alum or CFA, rested for at least given lineage or sublineage was founded by a single (hypothetical) B cell. http://www.jimmunol.org/ ␮ 6 wk, and then boosted with 100 g Ars-KLH in PBS i.p. After boosting, These assumptions were based on: 1) previous studies showing that GC are spleens were removed at various times and prepared for cryosectioning as formed by a limited number of precursors (1–5); and, 2) the fact that sub- described above. lineages were defined by combinations of somatic mutations that, given the nature of the hypermutation process, were unlikely to have taken place Immunohistochemistry independently multiple times. The dendrograms thus constructed could often be assigned one of three Frozen spleens were sectioned and three 5- to 6-␮m thick parallel sections “shapes”: bush, tree, or trunk. A “bush” was defined by two or more sub- were placed on each slide. The slides were fixed in 100% cold acetone and lineages emanating from a single, hypothetical founder cell. A “tree” con- stored at Ϫ80°C. The procedures used for immunohistochemical staining sisted of a single geneological “trunk” from a common founder cell, and

of the sections have been described in detail previously (37). Briefly, par- extensive “branching” emanating from the top of this trunk. This “trunk” by guest on October 2, 2021 allel sections were stained with biotinylated Ars-BSA or the anti-idiotypic was defined by three or more mutations that were shared by all of the Abs E4 or 5Ci followed by streptavidin coupled to alkaline phosphatase clones. A “trunk” was defined as described for a “tree,” except it lacked (Dako, Carpinteria, CA). All sections were also stained with peanut ag- sublineage branches defined by two or more mutations. In some cases, the glutinin (PNA) coupled to HRP (Sigma, St. Louis, MO) to elaborate GC. dendrograms did not fit into one of these three specific categories, and so Some sections were also stained with HRP-conjugated donkey anti-mouse were assigned mixed names. IgM or donkey anti-mouse IgG preparations (Jackson ImmunoResearch, West Grove, PA). Ars binding and idiotope-positive AFCs and GCs were microdissected from stained sections using a micromanipulator-controlled micropipette, and tissue was processed as previously described (10, 11). Results The pattern and extent of hypermutation in canonical VH genes derived from individual GCs at various stages of the primary PCR amplification of V genes anti-Ars response VH genes in genomic DNA present in processed microdissected tissues was subjected to two rounds of PCR amplification using Ampli-Taq DNA poly- We previously reported the results of an immunohistochemical merase (Perkin-Elmer, Foster City, CA) and various combinations of analysis of the participation of canonical clonotypes in the primary nested oligonucleotide primers. Each round of PCR consisted of 40 cycles anti-Ars response in the spleens of A/J mice (37). This study ex- of 95°C/1 min, 58°C/30 s, and 72°C/3 min ending with one cycle of 72°C/6 ploited the anti-idiotypic mAbs E4 and 5Ci, which are highly spe- min. The 5Ј primers were specific for sequences in the 5Ј region of the cific for canonical V regions (56–58). We found that canonical B canonical VHIdCR gene segment and were either hybridized in the pro- moter region of this gene or just 5Ј of the translation initiation codon in the cells did not participate in the early PALS AFC response, but Ј leader intron. The 3 primers either hybridized between the JH3 and JH4 initially appeared infrequently and in a small number of GCs 6 ϩ gene segments or to a region of common homology in the JH2, JH3 and JH4 days after immunization. While the number of E4, 5Ci GCs re- gene segments. The second round amplification was performed on 1.5–2 ␮l ϩ of the first round reaction (50 ␮l total). All of the primers contained re- mained low throughout the primary response (four to five E4, 5Ci striction sites to allow of the products. PCR products of 0.5–1.3 GCs per spleen on average), the frequency of canonical B cells per kbp were obtained (depending on the primer combinations used), pro- GC increased after day 6 and was maximal between days 12 and cessed, cloned, and their inserts subjected to nucleotide sequencing as de- 14, after which this response waned. In this time frame, all of the scribed previously (37). ϩ When genomic DNA from microdissected tissues was amplified by E4, 5Ci GCs also stained positive with anti-IgG but not anti-IgM, PCR, controls containing either no tissue or processed tissue microdis- consistent with previous hybridoma studies showing that H chain sected from a region of the spleen that did not stain with E4 or 5Ci were class switching takes place early in the anti-Ars primary response always included. The no tissue controls never yielded a PCR product, but (44, 52). While the kinetics of this GC response are similar to those the unstained tissue controls sometimes did. This was not unexpected, as previously reported for other hapten-protein conjugates (9, 13, 14, the primer combinations used would amplify any V gene partially en- H 29), the magnitude of this response is far less, perhaps reflecting an coded by the VhIdCR gene segment, irrespective of D or JH use. When these “control” PCR products were cloned and sequenced, they invariably extremely low precursor frequency of canonical clonotypes. The Journal of Immunology 4317 Downloaded from

FIGURE 1. Somatic mutation in canonical VH genes cloned from individual primary GCs. Data from individual GCs are shown on separate lines. Data from GCs microdissected from spleen sections obtained from different mice are separated by bold lines, and those obtained from different sections of the /http://www.jimmunol.org ء same spleen are separated by light lines. , For VH clones obtained from individual GCs in a single spleen, identity at junctional nucleotides is indicated by a “J” preceded by a number indicating a particular unique combination of these nucleotides. Also indicated is whether GCs were found in the same follicle (“F”) or whether they were present in adjacent regions (“A”) of the spleen. #, Average mutation frequency per V gene base pair was calculated by dividing the total number of base pairs sequenced in all VH clones obtained from a given time interval (e.g., days 9 and 10) by the total number of somatic mutations observed in these same clones. The R/S mutation ratios in CDRs and FWRs were calculated in a similar manner, except only mutations in the mature VH region coding sequences were considered. $, Two of the clones obtained from this GC had apparent somatic mutations in the first two nucleotides of the VH-D junction region. &, A few of the VH clones obtained were not completely “canonical” because they lacked D regions of the canonical length and derived from the DFL16.1 gene segment. Two of these had D regions derived from the Sp2 D segment family, and one had a 1-aa D region characteristic of the CRI-D anti-Ars V region family (45). However, these three VH genes did use the canonical JH2 gene segment. @, The number of unique somatic by guest on October 2, 2021 mutations per GC was evaluated by counting mutations shared by two or more VH clones only once. NA, Not applicable.

To extend these in situ studies to an analysis of canonical V As mentioned above, canonical VH regions differ before hyper- region hypermutation and selection, an immunohistochemical- mutation only at VH-D and D-JH junctions (47, 48). The consensus microdissection-PCR approach analogous to that pioneered by sequence in these regions is VHIdCR-TCN-NNN-D FL16.1-NNN-

Kelsoe and colleagues (10, 11) was employed. A/J mice were im- JH2, where N indicates nucleotides often resulting from N region munized with Ars-KLH in alum, sacrificed at various times after addition (the first “TC” nucleotides appear to result from P nucle- immunization, and their spleens processed for histology. Ex- otide addition). Thus, at least seven of the nine canonical VH junc- pressed canonical VH region genes and their flanking sequences tional nucleotides are added in a template-independent fashion and were recovered from genomic DNA via PCR from 17 microdis- ϩ therefore can be used as markers of common clonal origin. We and sected E4, 5Ci GCs representative of various stages of the pri- others have previously shown that it is improbable for canonical V mary anti-Ars response, and the PCR products were cloned. Mi- genes derived from different A/J mice to share all these junctional crodissected regions within the GC (as defined by PNA staining) nucleotides (47, 60). consisted of 50–100 cells. Care was taken to avoid the inclusion of Fig. 1 summarizes the results obtained from the analysis of pri- PNAϪ cells in the surrounding mantle zone. Multiple plasmid mary GCs, and Fig. 2 provides a representative example of the clones obtained from each GC were then sequenced. Clones con- sequence data obtained from an individual day 10 primary GC. taining hypermutated versions of the canonical V IdCR V gene H H Canonical V clones derived from the GCs of single mice were were easily distinguished from clones that might have been de- H rived from other germline genes (obtained only rarely in this anal- judged to have originated from the same clonal precursor on ysis) because most of these related J558 family members have the basis of sharing of VH-D and D-JH junctional nucleotides. The been characterized (59). Kelsoe and colleagues have previously majority of the VH clones obtained were canonical, but three of measured the PCR error frequency under similar conditions, and the GCs gave rise to clones representative of other families of have demonstrated that this frequency follows a Poisson distribu- anti-Ars V regions that differ from canonical VH genes in their D tion (11), making it extremely unlikely that when two or more regions. Fig 1 also catalogues the presence of the recurrent, affin- ity-enhancing mutations at complementarity-determining region mutations are observed per VH gene, that all of these mutations could have resulted from PCR error. Moreover, the results of the (CDR)-2 positions 58 and 59, as well as a mutation in codon 105 studies reported here suggest that, in our hands, the PCR error in the D region at an intrinsic mutational hotspot (RGYW; Refs. 53 frequency is even lower, perhaps only 0.2 mutation(s) per VH and 54). All of the GCs analyzed gave rise to V gene clones con- gene, on average (see below). taining multiple mutations at a frequency higher than expected for 4318 CONTRASTING PRIMARY AND SECONDARY MEMORY B CELL RESPONSES

FIGURE 2. Representative examples of canonical Downloaded from VH sequence data obtained from GCs and AFC “clus- ters” at various stages of the anti-Ars response. The se- quences of multiple VH PCR product clones obtained from the events indicated at the beginning of each se- quence are shown compared with the consensus se- quence of canonical VH genes. The consensus sequence is composed of the germline VhIdCR gene segment and 5Ј flanking sequences in the leader intron region, the A/J http://www.jimmunol.org/

DFL16.1 and JH2 segments, and regions in the V-C in- tron flanking JH2. Within the D region, the highly vari- able nucleotides often resulting from N region addition are indicated by an “N.” The sequences of only the CDR1, 2, and D (CDR3) regions are shown completely, as well as codons or 10-bp sections of flanking sequence in which a somatic mutation was observed. The codon that encodes the mature amino terminus is indicated by a “1,” and the position of all other codons not in CDRs by guest on October 2, 2021 and some CDR codons relative to this codon are indi- cated by numbers. In the clone sequences, identity with the consensus sequence is indicated by a dash, and nu- cleotide differences are shown explicitly.

PCR error. In addition, canonical VH clones obtained from indi- assumption that each plasmid clone represented a distinct GC B vidual GCs nearly always shared all junctional nucleotides. At- cell, which seems reasonable given that DNA from numerous E4, tempts to reconstruct the hypermutation and clonal expansion 5Ciϩ cells was amplified in each PCR reaction. These theoretical events that took place in each GC were undertaken using the pre- constructs rely on the doctrine of mutational parsimony, which viously established procedure of building geneological dendro- assumes that if a mutation is shared by several clones, it took place grams (10, 50–52). During this exercise, we made the simplifying only once in a and was inherited by daughter cells. The Journal of Immunology 4319 Downloaded from http://www.jimmunol.org/

FIGURE 3. During the primary response, B cells of a common clonal origin but resident in different GC undergo independent hypermutation and clonal expansion. The upper right panel shows two GCs present in a spleen section from an A/J mouse immunized 15 days earlier with Ars-KLH. The section was stained with PNA (red) and E4 (blue). Original magnification was ϫ100. PNA staining of the marginal zone region indicates that the two GCs were ϩ by guest on October 2, 2021 present in adjacent follicles. The PNA areas of the GCs were microdissected, and canonical VH genes were recovered and multiple clones sequenced, as described in Materials and Methods. The sequence data obtained were used to construct dendrograms, as described in Results and Materials and Methods. The lower panel schematizes the histology data shown in the upper panel, with inclusion of the dendrograms. In these dendrograms, numbered circles illustrate the hypothetical B cells from which each VH clone was derived, and circles labeled with an “N” indicate hypothetical “node” cells that were intermediates in the hypermutation-clonal expansion process. The mutations that define each branch in the dendrograms are listed according to the number of the VH codon or other location where they took place. When multiple mutations were observed in the same codon or location, this is indicated by the number of the mutation in parentheses after the codon number or location. Both of the GCs shown, as well as a third GC located in a different region in a different section obtained from the same spleen, gave rise to canonical VH clones with shared VH-D and D-JH junctional nucleotides, a strong indication of common clonal origin. The sequence of this region is shown in the upper left panel, with the junctional nucleotides labeled in red. The “third GC” is also illustrated in the upper left panel. All of the individual GCs gave rise to clones that shared subsets of somatic mutations, allowing the construction of the dendrograms. However, only the affinity-enhancing CDR2 position 58 mutation (see text) was shared by groups of clones isolated from the two GCs shown, but not the “third GC.” As illustrated, this suggests that a common precursor expressing an unmutated VH gene (illustrated by a green circle labeled with a “P”) gave rise to the three GCs after clonal expansion and migration. Subsequently, the offspring of this precursor underwent hypermutation, clonal expansion, and phenotypic selection that were confined to the individual GCs. ca, central arteriole; RP, red pulp.

This assumption is subject to caveats due to the nonrandom nature clones from a common, mutated precursor during a period in of hypermutation (61) and the possibility that any given base which the rate of mutation was similar or greater than that of B cell change may have resulted from a PCR misincorporation or recom- division. The criteria we used to assign dendrograms to these three bination event (11, 55). However, when appropriate caution is ap- categories are described in Materials and Methods. plied, the utility of these theoretical constructs for the analysis of As can be seen in Fig. 1, dendrograms of all of these types could data obtained from individual GCs is well established (10, 11). be generated from the clones isolated from individual GCs during One dendrogram form—the “trunk”—results when most or all the 9–10, 11–12, and 13–15 day time frames. In addition, the mutations are shared by all clones. This indicates isolation of V average frequency of mutation per V region base pair at these time gene clones during a period in which the rate of B cell division was points was indistinguishable. The Ars affinity-enhancing mutation much greater than the rate of V gene mutation per division. At the at CDR2 position 58 was reproducible observed after days 9–10, other extreme are dendrogram “bushes” in which many mutation- but the analogous position 59 mutation was observed in only one ally distinct clones emanate from a common, often unmutated pre- GC from day 12. Another measure of phenotypic selection of V cursor. This indicates a period in which the rate of V gene mutation regions, the ratio of mutations causing amino acid substitutions (R, was comparable or greater than the rate of B cell division. Between replacement) to those that do not (S, silent) in CDR vs framework these extremes is the “tree,” which suggests the derivation of regions (FWR) (25) was also evaluated. These ratios did not reveal 4320 CONTRASTING PRIMARY AND SECONDARY MEMORY B CELL RESPONSES Downloaded from http://www.jimmunol.org/ by guest on October 2, 2021 FIGURE 4. Time course of the secondary canonical anti-Ars B cell response in the spleen. A/J mice were primed with Ars-KLH, rested, and boosted with Ars-KLH as described in Materials and Methods. Multiple mice were then sacrificed at 1- to 2-day intervals and their spleens processed for histology. Spleen sections were stained with PNA (red) and E4 (blue), also as described in Materials and Methods. Representative results of this analysis are shown. Just before boosting (day 0, time point indicated at the upper left of each panel), rare E4ϩ cells were observed throughout the spleen with an apparent concentration in the marginal zones. At day 2, note the increased level of E4ϩ cells throughout the follicles and red pulp, but not in GCs. At day 4, note the large and abundant clusters of intensely staining cells (most probably AFCs) in the red pulp and the follicular borders, but the few E4ϩ cells in follicular areas, including GCs. At day 6, note the numerous E4ϩ cells in GCs (in this panel, two of the GCs are of this type), with small numbers of intensely staining cells also in the red pulp, some adjacent to the red pulp sinuses. The day 8 panel illustrates the continuation of the E4ϩ GC reaction, but at this time the E4ϩ cells in the red pulp were infrequent. At day 12, note that E4ϩ staining characteristic of surface Igϩ B cells or AFCs is not observed, but most GCs stain in a punctate, reticular pattern characteristic of immune complexes deposited on the surface of FDC. The distinction between the asymmetric, reticular staining characteristic of immune complexes on FDC and the individual, circular cell staining characteristic of Id-expressing B cells is illustrated in the insert panels. These GC images were taken at an original magnification of ϫ400 to maximize clarity. The original magnification of all other images was ϫ100. a progressive pattern of change from days 9 to 15. Nonetheless, tations were rarely observed among clones derived from different mutations resulting in translation termination codons, or replace- GCs, save for the affinity-enhancing position 58 mutation. Two of ment mutations in codons know to encode Ars contact residues(62) the GCs from the triplet group were directly adjacent to one an- or highly conserved FWR residues (63), were only rarely observed other (day 15) and a pair of GCs in another spleen was present in (data not shown). the same follicle (day 9). The adjacent pair of GCs that are mem- bers of the GC triplet isolated from the day 15 mouse is shown in Fig. Migration and expansion of canonical clones before and during 3, as well as a schematic diagram illustrating how we interpret mi- the primary GC response gration, clonal expansion, and hypermutation to have taken place in The data obtained from GCs microdissected from the spleens this clone. of individual mice also provided information regarding clonal migration and expansion before and during the GC reaction. In the The early secondary anti-Ars response is characterized by a case of the spleens of four mice, three pairs (groups 1J, 2J, and 3J massive memory AFC response during which V region in Fig. 1) and one triplet (group 8J) of GCs gave rise to V gene hypermutation does not take place clones that shared junctional nucleotides, indicating a common Approaches identical with those used for the analysis of the pri- clonal origin. Thus, canonical clones must often undergo expan- mary anti-Ars response were used to analyze the secondary re- sion before the nucleation of multiple GCs. Strikingly, however, in sponse of canonical clonotypes in the spleen. Mice were primed the case of each of these four groups of GCs, shared somatic mu- with Ars-KLH in alum or CFA and rested for at least 6 wk. Spleens The Journal of Immunology 4321

FIGURE 5. Somatic mutation in the canonical VH genes cloned from individual secondary AFC “clusters.” The data are presented as described in Fig. 1, except no dendrogram information is shown, as mutations shared by subsets of clones derived from single AFC “clusters” were never observed (see text). ء , This cluster was adjacent to a day 6 GC (indicated by “AGC”) from which canonical VH clones were also obtained. This GC is indicated by “Acluster” in Fig. 6. However, the clones obtained from the AFC cluster and the GC did not have identical junctional sequences. #, A Lys3Thr mutation at codon Downloaded from 59 has been shown to increase affinity of canonical V regions ϳ3-fold (49). Mutations to Ser, Asn, and several other amino acids are also recurrently observed at this position, but have not been individually tested for their affects of affinity for Ars. NA, Not applicable. from two mice that had been rested for 6 wk were processed for cells were observed at a high frequency but were scattered histology and stained for E4, 5Ciϩ cells as described above. E4, throughout both the red and white pulp. By day 4, a massive http://www.jimmunol.org/ 5Ciϩ cells were observed at a low frequency (75–100 per section) E4,5Ciϩ cellular response had developed, consisting largely of scattered throughout the red and white pulp, with an apparent con- loose clusters of cells confined to the red pulp. The intensity and centration in the follicular and marginal zone areas (Fig. 4). Be- asymmetric pattern of staining of these cells was characteristic of cause such cells are undetectable in the spleens of naive A/J mice AFCs. These cells also labeled intensely with Ars-BSA. Most of (K.A.V. and K.T.B., unpublished observations), this indicates a these AFCs were embedded in clusters of cells that stained in- substantial increase in canonical B cell precursor frequency in the tensely with anti-IgG. Only scattered IgMϩ AFCs were found in spleen due to primary Ars immunization. The remaining mice were these same locales (data not shown). At day 6, most of the AFCs boosted with Ars-KLH in PBS and spleens taken at 1- to 2-day were found surrounding the red pulp sinuses. This response grad- ϩ intervals up to day 12 and processed for histology. As illustrated in ually waned and was not detectable at day 10. At day 6, E4, 5Ci by guest on October 2, 2021 Fig. 4 with E4 staining, at day 2 canonical V region-expressing B GCs were first observed, in greater abundance than seen at day 6

ء FIGURE 6. Somatic mutation in canonical VH genes cloned from individual secondary GCs. The data are presented as described in Figs. 1 and 5. , VH clones were obtained from an AFC cluster adjacent to this GC (indicated by “Acluster”). See Fig. 5 for information on this cluster. #, VH clones derived from different B cell precursors were isolated from a single GC (indicated by “GC”). Only one set of clones was canonical. @, Clones with canonical VH and JH2 segments but noncanonical D segments. NA, Not applicable. 4322 CONTRASTING PRIMARY AND SECONDARY MEMORY B CELL RESPONSES Downloaded from http://www.jimmunol.org/ FIGURE 7. During the secondary immune response, a single B cell clone can give rise to numerous GCs throughout the spleen in which hypermutation and clonal expansion take place rapidly and independently. The data obtained from the analysis of five GCs in the spleen of an A/J mouse boosted 6 days earlier is schematically illustrated, essentially as described in Fig. 2. However, in this figure, the individual mutations that define each dendrogram branch are not shown. The five GCs analyzed were present in three spatially distinct regions of the spleen. The left pair of GCs were located in adjacent regions of the white pulp (indicated by a solid line surrounding the GCs) but apparently in different follicles (indicated with a dotted line). The other three GCs were present in a different spleen section, Ն180 ␮M away from that containing the left pair of GCs. This large distance is illustrated with a thick solid line. The middle two GCs were present in adjacent regions of the white pulp, but apparently in different follicles. The right GC was present in the same section as these latter two, but was located at the opposite end of the spleen. This distance is indicated by a thin solid line. The VH clones obtained from these individual GCs all had identical VH-D and D-JH junctional nucleotides (shown in bold at the top of the figure), indicating derivation from a common by guest on October 2, 2021 precursor. VH clones obtained from individual GCs also shared numerous somatic mutations among themselves, allowing the construction of the dendro- grams illustrated in each GC. This suggests that hypermutation, phenotypic selection, and clonal expansion was confined to individual GCs after they were formed. Because the time course data illustrated in Fig. 3 indicate that GCs containing canonical B cell clones are absent from the spleen at day 4 after boosting, the data summarized here suggest an extremely rapid period of intra-GC hypermutation and clonal expansion between days 4 and 6 of the secondary response. VH clones derived from three of the GCs also shared three mutations in common, indicating that a small amount of mutational diversification and clonal expansion took place before the nucleation of the five GCs. These mutations were: a G3C in codon 10 giving rise to a Glu3Asp mutation; and two G3A changes in codon 56 giving rise to a Gly3Asn mutation. None of these mutations are associated with increased affinity for Ars. This early mutation period is illustrated by a shaded box, in which the hypothetical immediate GC precursor (“N1”) is shown. Also shown is the hypothetical unmutated parental (“P”) B cell, which apparently directly gave rise to the other GCs. While it cannot be determined whether this initial period of expansion and mutation took place at an anatomical site distinct from all of the GCs, this is illustrated for the sake of simplicity. One of the GCs gave rise to clones that appeared to have been derived from both “P” and “N1” precursors (see text).

of the primary response. These GCs persisted through at least day previously been observed among canonical VH genes expressed by 8 (Fig. 4) and most were stained by anti-IgG, but not anti-IgM secondary hybridomas. Of the 14 clones and ϳ10 kbp sequenced (data not shown). By days 10 and 12, nearly all GCs stained pos- from these clusters, only 6 unshared mutations were observed, itive with E4 and 5Ci, but with an intensity and in a pattern sug- with each seen only in one clone. These scattered base changes, gestive of Ag-Ab complexes deposited on the surface of follicular which were present at a frequency of 0.06%, most likely resulted dendritic cells (FDC) (see insets in Fig. 4). from PCR error. Therefore, these data are consistent with the gen-

Canonical VH genes were recovered by PCR from several of the eration of a major fraction of the canonical clusters in the spleen day 4 and 6 E4, 5Ciϩ AFC clusters and multiple clones sequenced. from members of a single memory B cell clone that underwent Fig. 5 summarizes the data obtained, and Fig. 2 presents represen- extensive migration and expansion without further hypermutation. tative sequence data from a day 4 AFC cluster. The most striking The data obtained from the analysis of day 6 E4, 5Ciϩ AFC results were obtained from a single mouse at day 4, in which three clusters were consistent with these results in revealing canonical of the five clusters analyzed appeared to have been derived from VH clones that shared nearly all somatic mutations, sometimes the same clonal precursor based on sharing of all junctional nu- including the affinity-enhancing 58 mutation. The average muta- cleotides. Two of these clusters were adjacent to each other and tion frequency per VH clone bp in all of the day 4 and 6 cluster one was present in a spatially distinct region of the spleen. More- clones analyzed was 1.5%, not greatly different from the 1.1% over, all of the clones isolated from these three clusters shared observed in VH clones derived from primary GCs. In total, the nearly all of their somatic mutations, including the affinity-enhanc- analysis of extrafollicular AFC foci revealed a response derived ing CDR2 position 58 mutation and a position 59 mutation that has from canonical memory B cells that appeared to have undergone The Journal of Immunology 4323 hypermutation and affinity-based selection during the primary approach (64). In general, the data we obtained from our immu- response. nohistochemistry/microdissection/PCR studies confirm and extend the conclusions of these investigators. We observed a low fre- Induction of hypermutation and rapid phenotypic selection take ϩ quency of E4, 5Ci GCs, consistent with their estimates that even place during the secondary GC response of canonical clones at the height of the anti-Ars primary response only 40,000 GC B A total of 13 GCs were analyzed, as described above, from mice cells of this clonotype are present on average per spleen. Despite that had been primed with Ars-KLH in alum or CFA at least 6 wk this minor participation, our data show that multiple GCs that are earlier and boosted 6 or 8 days before spleens were taken. The distinct with respect to somatic mutation are often derived from the ϩ extensive apparent deposition of E4,5Ci immune complexes on offspring of a single canonical precursor. This demonstrates that FDCs after day 8 (see Fig. 4) precluded the unambiguous identi- cell division and extensive migration in the absence of hypermu- fication of GCs that contained canonical V region-expressing B tation usually take place in canonical clones before entry into the cells at later time points. Fig. 6 summarizes the data obtained, and primary GC reaction. Because we have previously shown that Fig. 2 presents representative sequence data from individual day 6 PALS-associated “foci” do not form during the anti-Ars primary and day 8 GCs. Eleven of the 13 GCs gave rise to only canonical response (37), the site of this pre-GC phase of clonal expansion

VH clones and two gave rise to clones representative of other anti- remains to be identified. Nonetheless, these data provide further Ars V region families. One of these GCs also gave rise to canon- evidence that extensive focal proliferation of Ag-activated B cells ical VH clones—the only example of a “mixed clone” GC obtained in the PALS is not a prerequisite for the generation of precursors in our entire analysis. At day 6, all GCs gave rise to clones whose to the GC/memory pathway (27, 37). sequences suggested ongoing hypermutation (resulting in dendro- Liu et al. also documented a modest average frequency of V gram “trees” or “bushy trees”), but the average frequency of mu- region hypermutation in canonical GC B cells isolated from days Downloaded from tation in these GCs (1.3%) was similar to what had been observed 10 and 12 of the primary anti-Ars response (0.5–1%), as compared in the primary response (1.1%). In addition, only two of the GCs with hybridomas expressing canonical V regions isolated from an- produced clones with mutations at CDR2 positions 58 or 59 that amnestic anti-Ars responses (2.5–3%; Ref. 24), yet Ͼ80% of the might be affinity enhancing (see Fig. 5). E4ϩ, PNAϩ B cells they analyzed at day 10 expressed hypermu- The data obtained from the analysis of day 8 secondary GCs tated V genes (64). Our data corroborate these findings, and also differed somewhat, revealing a higher average mutation frequency indicate that this modest frequency per V region base pair remains http://www.jimmunol.org/ per VH clone bp (3.0%) and a greater prevalence of affinity-en- rather constant through at least day 15 of the GC response. In hancing mutations at positions 58 and 59. In addition, the general addition, all of the primary GCs we analyzed showed evidence of shape of the dendrograms was somewhat different from day 6, now ongoing hypermutation. Analogous studies using other hapten sys- including “trees” with much longer “trunks.” While detailed com- tems have indicated that clonal expansion in primary GCs can take parison of the day 6 and day 8 secondary GC data are difficult due place without induction of hypermutation, even during the inter- to the sample size, collectively they indicate that canonical B cells mediate stages of the response (14). Whether these differences in in the day 8 GCs had often undergone more extensive hypermu- hypermutation frequency are reflective of changes in the nature of tation as compared with that observed at day 6, as well as intense the GC response driven by different Ags, or variations in the po- by guest on October 2, 2021 phenotypic selection. Nonetheless, analysis of the R/S ratio of mu- tential of distinct B cell clonotypes to undergo hypermutation tations in CDRs and FWRs did not reveal obvious differences be- awaits further investigation. tween these two time points (Fig. 6). Among VH clones obtained from primary day 11 through 15 GCs, we did observe a high frequency of the CDR2 position 58 A single canonical clone that has undergone extensive affinity-enhancing mutation. However, we did not detect an in- expansion and migration can give rise to multiple GCs during creasing frequency of the CDR2 position 59 affinity-enhancing the secondary response, followed by a period of clonal mutation. Moreover, dendrogram “trunks” or “trees” also did not expansion and hypermutation that is confined to individual GCs increase with time. Either would have been indicative of prefer- The most striking secondary GC data were obtained from a single ential clonal expansion of hypermutated subclones that had ac- ϩ mouse analyzed at day 6, from which six E4, 5Ci GCs were quired increased affinity for Ag. This discrepancy indicates that the analyzed. Of these GCs, five gave rise to canonical VH clones, position 58 mutation may be a “hot event,” resulting from both a which all appeared to have been derived from a common precursor high frequency of occurrence and phenotypic selection. Indeed, its based on identity of VH junctional nucleotides. All of the VH frequency of observation was only slightly higher than a docu- clones obtained from these GCs were hypermutated, but only a few mented intrinsic hotspot mutation at a consensus RGYW motif of the somatic mutations in clones isolated from different GCs (53), resulting in a translationally silent mutational change at po- were shared. Some of these GCs were adjacent to one another, sition 105 in the D region (see Fig. 1). Although a much larger while others were present in different locales of the spleen. As sample size would be required for unequivocal conclusions to be illustrated in Fig. 7, the data obtained from this analysis suggest drawn, we also did not observe progressive increases in the R/S that a common, unmutated precursor (labeled “P”), which initially mutation ratio in CDRs, or a decrease in this ratio in FWRs. In underwent clonal expansion and extensive migration while sus- contrast, we obtained only a few VH clones with translation ter- taining only a few mutations (giving rise to the “N1” GC precursor mination codons or replacement mutations in codons know to en- cell), gave rise to these five GCs. After entering GCs, the progeny code Ars contact residues in CDRs or highly conserved FWR of this clone appear to have undergone extensive hypermutation amino acids (data not shown). Thus, these data suggest that there and expansion with little or no inter-GC migration (see Fig. 7 for is a strong selection for maintenance of Ag binding capacity during details). the primary GC reaction, but not necessarily for iterative increases in affinity for Ag. Discussion These findings appear inconsistent with previous conclusions The previous studies of Liu et al. were the first to attempt to di- that V gene mutations accumulate monotonically and that V region rectly examine the participation of canonical anti-Ars B cell clono- phenotypic selection occurs progressively during the primary GC types in the primary GC response, using a FACS single-cell PCR response (5, 11). A means to reconcile our data with previous 4324 CONTRASTING PRIMARY AND SECONDARY MEMORY B CELL RESPONSES Downloaded from

FIGURE 8. Numerous shared somatic mutations among VH clones derived from individual secondary GCs may not be indicative of derivation from a common B cell precursor that underwent V region hypermutation during the primary response. Data obtained from two GCs, one present in the spleen of an A/J mouse 12 days after primary immunization (A) and one present in the spleen of an A/J mouse 8 days after secondary immunization (B), are illustrated in dendrogram form as described in Fig. 2. The four clones isolated from the primary GC shared 10 mutations, including both CDR2 position 58 and 59 affinity-enhancing mutations, as well as six distinguishing mutations. The four clones isolated from the secondary GC shared 22 mutations, including both Ј CDR2 position 58 and 59 affinity-enhancing mutations, as well as five distinguishing mutations. Sequence in the 3 flanking region of the VH genes was http://www.jimmunol.org/ obtained in the case of the day 8 secondary GC clones but not in the case of the day 12 primary GC clones. Therefore, the differences in the number of mutations defining the “trunks” of both “trees” does not indicate an overall difference in the frequency of mutation in these clones. studies has recently been provided by Tarlinton, Kelsoe, and their One interpretation is that these mutations took place in the primary colleagues, analyzing affinity maturation during the the primary response in a memory cell precursor, whose progeny were subse- response to (4-hydroxy-3-nitrophenyl)acetyl (31, 32, 65). Their quently recruited into the secondary GC reaction where they un- findings suggest that while hypermutation may be restricted to derwent further hypermutation. This scenario is illustrated for a GCs, the phenotypic selection of mutant V regions takes place not group of VH clones isolated from a day 8 secondary GC in den- by guest on October 2, 2021 only in the GC, but in other microenvironmental locales as well. drogram form in Fig. 8B. However, similar data were sometimes During the anti-Ars primary GC response, B cells whose V regions obtained from VH clones isolated from primary GCs, also as il- have sustained combinations of mutations that result in substantial lustrated in dendrogram form for a day 12 primary GC in Fig. 8A. increases in Ars affinity may be preferentially recruited into an Because all canonical VHIdCR hypermutation takes place after Ars extrafollicular, and perhaps extrasplenic maturation pathway. immunization (54), such primary GC data reinforce the conclusion Given this scenario, B cells that had recently entered the GC and that V gene mutation occurs at a very high rate in the GC micro- therefore had undergone only the initial stages of hypermutation environment (9–11, 13) and that majority representation of only a and phenotypic selection would be preferentially sampled by bulk few of the enormous number of V region variants generated by microdissection. Examination of the kinetics and extent to which mutation can also take place rapidly (11, 14). Thus, the shared canonical B cell clones populate extrasplenic compartments such mutations observed among V clones derived from secondary GCs as the bone marrow, as well as the patterns and frequency of so- H might well have taken place during the early stages of the second- matic mutations in the V genes these canonical clones express, will ary GC reaction, in B cells directly recruited from the naive B cell be required to further test this idea. compartment. Indeed, if the rates of V gene hypermutation and There have been limited studies of V gene hypermutation and selection are accelerated in anamnestic GCs (see below), then na- selection during the secondary GC reaction, and whether this re- ive B cells recruited into this response could rapidly give rise to sponse differs qualitatively from the primary GC response is a subject of controversy. Cerny and colleagues suggested that B cells progeny expressing V regions with frequencies and patterns of that have sustained V region hypermutation in the primary re- mutation thought previously to be characteristic of only memory B sponse often undergo extensive expansion without hypermutation cell clones that had undergone multiple GC reactions (20, 26). In in secondary GCs (39). In contrast, Milstein, Berek, and coworkers this regard, we obtained no VH clones containing known affinity- have argued that memory B cells re-enter GCs during secondary enhancing 58 and 59 mutations from day 6 secondary GCs, while and higher order responses, where they sustain further rounds of such clones were often obtained from day 4 and 6 secondary AFC hypermutation and phenotypic selection (20, 26). Adding to the “clusters.” Moreover, the scarcity of affinity-enhancing 58 and 59 confusion are conflicting results regarding the ability of purified mutations in day 6 secondary GCs indicates that even low-affinity memory B cells to undergo further hypermutation ex vivo (21, 66). B cells can enter the secondary GC reaction, as previously shown The data we have obtained from the analysis of E4, 5Ciϩ sec- for the primary GC reaction (67). Clearly, to unambiguously ad- ondary GCs do not allow an unequivocal resolution of this issue. dress the question of whether memory B cells can re-enter the

We did isolate several groups of canonical VH clones from indi- hypermutation-selection pathway in the secondary response will vidual day 8 secondary GCs that shared many somatic mutations, require a reproducible method for tracking these cells in situ that including both position 58 and 59 affinity-enhancing mutations. is independent of the structure and function of their Ag receptors. The Journal of Immunology 4325

It has often been suggested that hypermutation and phenotypic the AFC pathway. Canonical AFCs are infrequent and appear as selection of V region structure take place in an iterative fashion (3, isolated cells during the primary response in the spleen. In marked 4, 11, 25, 40, 51). Indeed, the temporal efficiency with which mul- contrast, the early secondary response is characterized by a mas- tiple affinity-enhancing mutations are fixed in a single B cell clone sive canonical IgG AFC response in which memory cells recruited is inconsistent with one-step generation and subsequent outgrowth into this pathway undergo extensive migration and proliferation of a small number of cells that bear all these mutations (68, 69). and are ultimately organized in loose clusters, predominantly in the One possible mechanism whereby hypermutation and selection red pulp. During this response, V region hypermutation does not could take place in an iterative fashion is via the sequential for- take place, analogous to the situation in PALS “foci” characteristic mation of GCs (and, thus, discrete periods of hypermutation and of the primary anti-(4-hydroxy-3-nitrophenyl)acetyl response of selection) by the progeny of a single B cell clone. In this scenario, C57BL/6 mice (28). Interestingly, our previous hybridoma analy- if the stringency of selection was based on the affinity or specificity sis of the early stages of the “original antigenic sin” response of of the V region expressed by the GC founder B cell (70), then canonical memory clones to a cross-reactive hapten indicated that sequential GC reactions would lead to progressive intraclonal in- during the “sin” response these clones underwent extensive expan- creases in affinity or specificity. Testing this idea was one of the sion without hypermutation (71). In total, our data demonstrate ϩ reasons that we chose to microdissect E4,5Ci GCs present in the that canonical clonotypes are incompetent to give rise to PALS same splenic locales. The data we and others (10, 28) have ob- AFC foci during the primary response, but during the primary GC tained do not provide support for this hypothesis. None of the reaction they acquire the potential to subsequently undergo vigor- clonally related groups of VH genes recovered from distinct GCs ous bursts of hypermutation-free expansion and Ab production that we analyzed revealed evidence of a mutational precursor-

during secondary responses. However, apparently only a minor Downloaded from product relationship (for example, see Fig. 3). The data derived fraction of all canonical B cells produced during the primary GC from the five clonally related secondary GCs illustrated in Fig. 7 response acquire this potential, as revealed by the mutational ho- did suggest a brief period of mutation in a single clone, before the mogeneity of the VH genes expressed by secondary AFC foci de- nucleation of multiple GCs, but none of the few mutations appar- rived from single progenitors. This oligoclonal nature of the early ently sustained during this initial period are associated with in- secondary response has been extensively documented by hybrid- creased affinity for Ars (see Fig. 7). In total, these data further oma technology (50, 71, 72). We have previously suggested that reinforce the previous suggestions by us (70) and others (28, 29) http://www.jimmunol.org/ this extreme “repertoire narrowing” is due to “specificity matura- that primary GCs are “independent islands of clonal hypermutation tion”—a combination of stringent negative selection of V regions and phenotypic selection,” and extend this conclusion to secondary with cross-reactivity for self-Ags and positive selection for V re- GCs. Testing models that propose that iterative cycles of hyper- gions with increased affinity for the driving foreign Ag (16). mutation and selection take place in single GCs, perhaps via “cy- Whether the hypermutation-free growth that takes place during clic re-entry” of phenotypically selected B cells into a hypermu- the secondary AFC response results in maintenance or regenera- tating pool (69), must await methods that will allow dissection of tion of a canonical memory AFC precursor population or leads the kinetics of B cell differentiation in various subcompartments of only to terminal differentiation remains to be determined. How- individual GCs. ever, in this regard several previous studies of the memory B cell by guest on October 2, 2021 Somewhat surprisingly, the primary and secondary GC reactions response using adoptive transfers and hybridoma technology have were qualitatively similar, suggesting a lack of substantial mech- indicated that memory cells can undergo extensive clonal expan- anistic differences in the nature of these responses. Nonetheless, comparison of the data obtained from them revealed some distinc- sion and persist for prolonged periods without induction of V gene tions. As expected from previous studies (19), the secondary GC hypermutation (73, 74). The data we present here suggest that response developed more rapidly, and while V gene mutation fre- these past studies preferentially sampled B cells in an anamnestic quencies similar to those observed throughout the primary GC re- AFC pathway, rather than in an anamnestic GC pathway. sponse were also observed at day 6, by day 8 we observed 2- to Unfortunately, the near lack of overlap of the secondary canon- 3-fold higher frequencies, although substantial GC to GC variation ical AFC and GC responses thwarted attempts to determine was apparent, as was the case among primary GCs. Moreover, whether a precursor-product relationship might exist between ca- several day 8 secondary GCs gave rise to clones containing both nonical AFC clusters and canonical GCs. By day 6, when such CDR2 58 and 59 affinity-enhancing mutations. Taken together GCs are first detected, the size of canonical AFC clusters has de- with the primary GC data, these data indicate that phenotypic se- creased substantially from what is observed at day 4, and most day lection may take place more rapidly in secondary GCs, consistent 6 clusters surround the red pulp sinuses (Fig. 4), suggesting mi- with the accelerated rate of this response overall. gration of the cells in these clusters from their site of origin. These Unfortunately, whether affinity maturation beyond the fixation observations are consistent with previous observations that most of the 58 and 59 mutations or emigration of these high-affinity splenic AFCs have a very short half (75). Due to this situation, ϩ clones from the GC, as appears to take place in the primary re- E4, 5Ci AFC clusters are usually not observed adjacent to E4, ϩ sponse, took place at later times could not be determined due to the 5Ci GCs at this time. We did obtained PCR VH clones from a ϩ extensive apparent deposition of E4, 5Ciϩ immune complexes in single day 6 E4, 5Ci GC/AFC “cluster” pair (see Figs. 5 and 6). all GCs after day 8. The extent of canonical clone expansion, mi- Sequencing of these clones revealed canonical VH genes with dis- gration, and GC nucleation also appears more robust during the tinct junctional nucleotides and no shared somatic mutations be- secondary response, as exemplified by comparing the data sum- tween the two groups of clones, strongly suggesting derivation marized in Figs. 3 and 7. Whether these quantitative and kinetic from distinct progenitors. We are now pursuing alternative ap- differences result from an increased precursor frequency of canon- proaches to address this important issue. Indeed, current data re- ical clones, increased levels of carrier specific T cell help (19, 39), garding the relationship of B cells capable of giving rise to primary increased levels of Ag deposited on FDCs (34), or a combination and anamnestic AFC and GC responses have not allowed agree- of these factors will require further investigation. ment on whether the precursors to these pathways of differentiation The most remarkable difference between the behavior of canon- are members of distinct developmentally committed lineages or ical clonotypes in the primary and secondary responses occurs in not (28, 35, 36). 4326 CONTRASTING PRIMARY AND SECONDARY MEMORY B CELL RESPONSES

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