Canonical germinal center B cells may not dominate the memory response to antigenic challenge
Yi-Feng Lu,
Mallika Singh and
Jan Cerny
Department of Microbiology and Immunology, University of Maryland School of Medicine, 655 West Baltimore Street, BRB 13-15, Baltimore, MD 21201, USA
Correspondence to:
J. Cerny
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Abstract
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Spleen and bone marrow (BM) are the major sites of antibody production and anamnestic response in systemically immunized mice. We examined the VDJ segment repertoire of antibody plaque-forming cells (APFC) in those two sites in the course of antibody responses to the hapten nitrophenyl (NP). Individual IgG APFC expressed any one of 10 VH segments of the V186.2/V3 (J558) gene family: 186.2, 102, 23, C1H4, 165.l, CH10, 3, 593.3, 24.8 and 671.5. The majority of cells in both spleen and BM expressed the V186.2 gene joined to a D segment with Tyr95. During a 2-month period after a single immunization, the V186.2+ APFC in BM accumulated 3 times as many somatic mutations than splenic APFC (average 8.5 versus 3 mutations/VH); this process was Th dependent as shown by in vivo depletion of CD4+ lymphocytes. However, the V186.2+ APFC in both spleen and BM shared a recurrent W33L replacement, indicating their common origin from germinal centers. The APFC expressing the other (analogue) VH segments were evenly represented in the spleen and BM, but they accumulated few, if any, mutations. The anamnestic V186.2+ APFC were highly mutated both in the spleen and BM; they represented a new and unexpected clonotype. The V/D segments were joined by Gly95 instead of Tyr95, the W33L was absent and a new shared K58R replacement appeared. The APFC expressing the `analogue' VH genes comprised ~20% of the anamnestic response and did not accumulate more mutations, but their affinities were in the range of the memory V186.2+ cells. These data suggest that the late primary and secondary responses to a hapten may be born by different B cell lineages, and that some clonotypes may reach the memory pool without an extensive mutation and expansion.
Keywords: antibody-forming cells, bone marrow, somatic hypermutation, spleen, VH genes
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Introduction
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Following primary immunization with a T-dependent antigen, antibody-forming cells and memory B cells evolve in different compartments of lymphatic tissues (1), and perhaps from separate precursor cell lineages (2). The antibody-secreting plasmacytes, which produce the bulk of primary antibody, differentiate from B cells in the T cell-rich areas of periarteriolar lymphatic sheaths, whereas memory B cells originate in germinal centers (GC) that form transiently after the immunization (1). It is now widely accepted that the GC B cells, which undergo rounds of somatic hypermutations in their rearranged BCR genes followed by affinity-based selection, are progenitors of both cells that produce antibody with increasing affinity during the advancement of immune response and the memory B cells that give rise to the rapid anamnestic antibody response upon antigen recall (3). This model provides a rational explanation for the finding, made in several laboratories, that the primary antibody responses and the anamnestic response are dominated by different clonotypes (48). This scenario also predicts that the repertoire of memory antibody response reflects that of the GC B cells (911); however, this proposal deserves further scrutiny.
The repertoire of progenitor memory B cells has been studied extensively in splenic GC during the primary response to a hapten, (4-hydroxy-3-nitrophenyl)acetyl (NP), in mice with IgHb allotype. The heavy chain V regions (VH) of NP-binding Ig molecules in these animals are encoded by several germline segments of the V186.2/V3 subfamily of the J558 family (1217). The repertoire of NP-reactive GC B cells can be summarized as follows:
- The majority of expressed BCR are encoded by VH186.2/DFL16.1/JH2 rearrangements (10,1820) with canonical Tyr95 that is considered critical for NP binding (10).
- In most studies (18,20), but not all (10), the CDR3 appear to have little junctional diversity.
- The V186.2+ GC B accumulate somatic point mutations to an average of 35 mutations/VH (10,1820). This includes a recurrent point mutation in VH position 33 that replaces Trp (W) with Leu (L) in a majority of recovered cells (10,19). This mutation alone increases the affinity of V186.2DFL16.1JH2(V
1) antibody for binding to NP by 10-fold (21) indicating a selection of GC B cells into the higher-affinity memory cell population.
- In addition to the V186.2 clonotypes, the NP-specific GC contained a variable portion of B cells expressing other VH segments of the V186.2/V3 gene family, such as V102, V23, VC1H4, V165.1, CH10, V3, V24.8 and V593 (17,19,20). These have been dubbed `analogue' genes (20) due to their sequence homology to the dominantly expressed V186.2 segment (22). The analogue gene rearrangements collectively account for ~65% of all clones recovered from GC at 6 days post-immunization, but they became rare by the second week (20).
Does the repertoire of memory anti-NP antibody mirror that of GC B cells? The answer may depend on the experimental design and method. Studies (14,16) have shown that B cell hybridomas generated from mice undergoing secondary responses bear certain characteristics of GC cells: the majority of the clones expressed the VH186.2 gene segments rearranged to DFL16.1 genes that contained, on average, 45 mutations/VH including the W33L replacement. Nevertheless, the secondary clones tended to have different CDR3 rearrangements in which the nucleic acid deletions at the DJ boundary were more extensive (14,16) compared to those in the GC B cells (10,1820). This repertoire pattern suggests that precursors of memory B cells represented only a small fraction of the GC cell population. However, these studies used complicated experimental designs that could bias the results. The secondary responses were elicited respectively in mice treated neonatally with an anti-idiotypic antibody (16) and in a single mouse that received two consecutive antigen boosts within 3 weeks after the priming (14). In contrast, Siekevitz et al. (23) generated secondary anti-NP hybridomas upon an adoptive transfer of antigen-primed cells followed by challenge with a anti-idiotype as a surrogate antigen. The secondary clones expressing the VH186.2 gene had fewer average mutations (~1.5 mutations/VH) than would be expected if they originated from GC, but many of them had nucleotide deletions in D segments similar to the aforementioned hybridomas from other studies (14,16). Neither of these studies assessed the role of GC analogue B cells (20) in the secondary response.
The aim of the present study was to examine the repertoire of NP-specific anamnestic responses elicited in primed, intact animals with a low dose of soluble antigen boost. Within 2 days the memory B cells (but not the naive lymphocytes) differentiate into effector IgG antibody-producing cells (24,25) that are readily identified in lymphocyte suspension ex vivo, using a modified antibody plaque-forming cell (APFC) assay (26). The rearranged Ig genes from individual APFC can be amplified and sequenced without the need for either cell fusion and hybridoma selection or FACS that may introduce a sampling bias. Using this approach, we aimed to analyze the repertoire of memory effector cells in comparison with GC lineage cells that produce high-affinity antibody during the advancement of primary response. Furthermore, memory responses were examined not only in the spleen, but also in bone marrow (BM), because the latter is a major site of rapid anamnestic responses (2729). The antigen boost may have initiated a rapid migration of activated memory cells from peripheral lymphatics to BM, where they differentiate into plasmacytes (29). Another hypothesis is that BM may be colonized by memory B cells and/or their progenitors which give rise to antibody-forming cells in situ after secondary immunization (27,28). The latter scenario is consistent with the observation that GC B cells home to BM upon adoptive transfer into naive recipients (30). Finally, the question arises whether compartmentalization of memory cells is a random or selective process. If it is selective, the memory responses in the spleen and BM could have different repertoires.
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Methods
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Mice, antigens and immunization
C57BL/6 and C57BL/6-scid mice (age 68 weeks) were purchased from the Jackson Laboratory (Bar Harbor, ME) or the Charles River Laboratory (Wilmington, MA) and maintained in sterile microisolator cages (Lab Products, Maywood, NJ). NP or its analogue (4-hydroxy-5-iodo-3-nitrophenyl)acetyl (NIP) (Cambridge Research Biochemical, Cambridge, UK) were conjugated at various substitution ratios to keyhole limpet hemocyanin, chicken
-globulin (CGG) (both from Sigma, St Louis, MO) or BSA (Amersham Life Science, Cleveland, OH) as described (31). Mice were immunized with a single i.p. injection of 10 µg of an antigen in alum and challenged with 10 µg of a soluble antigen in PBS, i.p.
T cell depletion in in vivo
Anti-CD4 mAb GK1.5 (ATCC, Rockville, MD) was produced as ascitic fluid and the IgG was purified on a HiTrap Protein G column (Amersham Pharmacia Biotech, Piscataway, NJ). Mice were injected i.v. with 12 mg IgG in 0.25 ml of PBS on days 18, 23, 32 and 42 after the immunization with NP-CGG; control animals received 1 mg of purified rat IgG (Sigma ) on the same days .
Preparation of NIP-red blood cells
Sheep blood cells in Alsever's solution (Colorado Serum, Denver, CO) were washed in DPBS and resuspended to 10% (v/v) in 0.1 M bicarbonate buffer, pH 9.4. Succinimide ester of NIP (Cambridge), dissolved in dimethylformamide (Sigma), was added to a final concentration of 25 µg/ml of sheep red blood cells and the mixture was incubated at room temperature for 3060 min. Cells were then washed and resuspended to 20% (v/v) in DPBS.
APFC assay
Lymphocytes producing the NP-specific IgG antibody were detected with a modified hemolytic plaque assay (26) using sheep red blood cells coupled with hapten. Antibodies produced early in the response to NP bind more avidly to NIP, whereas the secondary antibodies bind to NP and NIP with similar affinities (32,33). In order to capture cells producing both types of antibody molecules, we performed the plaque assay with sheep red blood cells conjugated to NIP. Petri dishes (10 cm diameter) were pre-coated with 5 ml of 0.5% agarose (Gibco, Grand Island, NY). A mixture containing 2.5 ml of 0.3% agarose in Basal Medium Eagle (Gibco) kept in a 46°C waterbath, 100 µl of lymphocyte suspension and 100 µl of NIP-red blood cells, with or without 25 µl of antibody mixture containing goat anti-mouse IgM (Southern Biotechnology Associates, Birmingham, AL) and rabbit anti-mouse IgG (Sigma), was poured on the solid agarose base and the dishes were incubated at 37°C/5% CO2 for 2 h. The plates were then covered with 3 ml of diluted guinea pig complement (Gibco) and incubated for an additional 2 h to make the hemolytic plaques visible. The hemolytic plaque assay readily detects the IgM-producing cells; however, the IgG-producing cells can be detected with an optimal concentration of exogenous anti-IgG antibody (26). Our assay was calibrated for the detection of IgG using stable cells lines B1-8 (34) and B1-8
1 (35) that produce anti-NP antibody of IgM and IgG1 isotype respectively. The agarose plates contained a mixture of anti-mouse Ig antibodies that inhibited the IgM-producing cells and facilitated the IgG producers (Table 1
).
For enumeration of APFC, 820x105 cells were plated per dish in duplicate. For the isolation of a single APFC, the cell suspension was highly diluted in order to increase the probability of sampling one cell. We plated 1x104 splenocytes (estimated to be 5x103 B cells) and 34x104 BM cells (estimated as 34x103 B cells), which yielded
1 plaque per dish. The center of each plaque, containing a cell clearly visible under a dissecting microscope, was aspirated in a volume of ~0.4 mm3 of agarose. From the total number of cells plated in 2.5 ml volume, we calculate that each sample contained approximately one B cell. On average, 3060% of samples yielded a PCR product using the specific primers (see below). In contrast, no products were obtained from random sampling of the red blood cells lawn.
Hybridomas and affinity measurements
Spleen cells were harvested from mice at day 4 after the secondary challenge with NP-CGG and fused with an SP2/0 myeloma cell line (ATCC), using polyethylene glycol (average mol. wt 1450) (Sigma) according to standard procedures. Supernatants from fusion wells were screened by ELISA for binding activity to both NP-BSA and NIP-BSA. Hybridomas were cloned by limiting cell dilution at 0.3 cell/well and the positive clones were expanded. mAb were purified from culture supernatants using Protein ASepharose columns (Pharmacia Biotech, Uppsala, Sweden); their H chain and L chain isotypes were determined by standard ELISA with NP-BSA and NIP-BSA as antigens in solid phase. The plates were probed with goat anti-mouse Ig isotype-specific antibodies, and anti-
and anti-
chain antibodies (all from Southern Biotechnology Associates). The VDJ segments from the clones were characterized by PCR amplification and sequencing as described below.
The affinities of mAb were estimated in two ways. Their Ka values for NP and NIP haptens were measured by fluorescence quenching in a Shimadzu RF-5301 fluorospectrophotometer using monovalent haptens NP- and NIP-aminocaproric acid (Cambridge Research) as described (35,36). Relative affinities were also determined by the binding of mAb to NP-BSA conjugates substituted with different amounts of haptenic groups (37). Purified mAb at concentrations from 0.3 l to 3 µg/ml were tested by ELISA for binding to NP3-BSA and NP18-BSA. Increasing ratio of binding calculated as OD450NP3-BSA/OD450NP18-BSA was indicative of increasing affinity of mAb to NP. A similar ELISA protocol was used to determine the heteroclicity of the NP-induced mAb, i.e. their preferential binding to NIP (32). The mAb were allowed to react with NP18-BSA and NIP24-BSA and the binding reaction = OD450NIP/OD450NP was calculated. Ratios ~1.0 indicate a lack of heteroclitic reactivity.
Recovery and molecular analysis of GC B cells
Spleens were removed from mice on days 12 after primary immunization, frozen sections were prepared for immunohistochemistry, and NP-reactive GC B cells were identified by dual staining with peanut agglutinin and NIP-BSA as previously described (38). Cells (~100) from individual GC were microdissected (20,38) and transferred into microcentrifuge tubes for PCR DNA amplification. The first-round PCR product was re-amplified with the nested primers shown below, with additional recognition sequences for restriction enzymes (17). The PCR product was digested with the restriction enzymes, ligated to a plasmid, cloned in competent Escherichia coli and sequenced as described previously (17).
Amplification and sequencing of VDJ DNA recovered from individual APFC
The individual NIP+, IgG APFC were picked using 50-µl disposable micropipettes (Fisher Scientific, Pittsburgh, PA) and transferred into a 0.2 ml microcentrifuge tube with 2.5 µl of 10xPCR buffer, 0.5 µl of 20 mg/ml proteinase K (Boehringer Mannheim, Mannheim, Germany), 0.5 µl of 5% Tween 20 and 21.5 µl of dH2O. The tubes were incubated at 56°C for 1 h and the proteinase K was then inactivated at 96°C for 10 min. DNA amplification was carried out by two rounds of PCR using pairs of nested primers. The initial round of amplification used primers 5'-CCTGACCCAGATGTCCCTTCTTCTCCAGCAGG-3' and 5'-GGGTCTAGAGGTGTCCCTAGTCCTTCATGACC-3', corresponding to the V186.2 genomic DNA 5' transcription start site sequence and to the intron JH2 sequence respectively. Using the Expand High Fidelity PCR kit (Boehringer Mannheim), the crude cell lysate was mixed in a 50 µl reaction volume with 350 µM dNTP, 0.4 µM of each primer and 1.0 µl of Expand High Fidelity polymerase. It was then amplified by one cycle of 95°C 2 min/55°C 4 min/72°C 2 min and then 40 cycles of 95°C 1 min/55°C 1 min/72°C 2 min in a GeneAmp 2400 PCR System (Perkin Elmer). Reaction mixture (2 µl) from the first-round PCR was amplified for an additional 40 cycles (94°C 30 s/55°C 1 min/68°C 1 min) using the nested primers 5'-CTAGAATTCAGGTCCAACTGCAGCAGCC-3', complementary to the initial 20 nucleotides of the V186.2/V3 gene family; and 3' primer 5'-GGGTCTAGAGGTGTCCCTAGTCCTTCATGACC-3' complementary to the JH2 gene segment. The second reaction mixture (5 µl) was electrophoresed on a 1% agarose gel for identification of amplified DNA (~350 bp fragment). The PCR product, isolated with the QIAquick gel extraction kit (Qiagen, Hilden, Germany), was directly sequenced by the Biopolymer Laboratory of the University of Maryland School of Medicine using an automatic DNA sequencing system (Applied Biosynthesis). DNA codons are numbered according to Kabat et al. (39).
Assignment of DNA sequences to germline VH genes
Germline sequences of individual VH segments of V186.2/V3 gene family differ from one another by 230 nucleotide residues (22). The APFC that utilized V segments 102, 23, C1H4, 165.1, CH10, 3, 593.3, 24.8 and 671.5 contained few, if any, somatic mutations (see Results), and, therefore, the sequences were easily matched to their respective germline counterparts. In contrast, the sequences representing the V186.2 gene differed from their presumptive germline counterpart by up to 20 nucleotides indicating that the V segments were either somatically mutated or that they were derived from a different germline gene. Distinction was possible because the nucleotide differences between the recovered sequences and the V186.2 germline sequence typically fell into the positions that are shared by members of the V186.2/V3 gene family and spared those positions that characterize individual germline genes. Thus codons nos 11, 20, 43, 50, 74 and 91 (and others) that distinguish the 186.2 gene were never mutated. Moreover, when mutation did occur in a codon that defines the germline V186.2 gene it spared the key residue. An example is given with codon 58 which is AAG in V186.2 and AAC in all other members of this gene family. Most 186.2 sequences from anamnestic response shared the point mutation AAG
AGG, whereas the third germline G residue was never changed. These patterns allowed an unambiguous classification of V186.2 mutants.
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Results
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Distinctive repertoires of late APFC in the spleen and bone marrow
The kinetics of appearance of hapten-specific, IgG-producing APFC in the spleen and bone marrow in mice immunized systematically (i.p.) with NP-CGG in alum is shown in Fig. 1
. This pattern of antibody-producing cells is familiar from several recent studies (4042). The splenic APFC reached the peak at days 710 after the immunization and then declined to a low, steady level between days 35 and 65. In contrast, specific APFC did not appear in bone marrow until the second week after priming and their number increased through the 2-month period of observation (Fig. 1
). Cells persisting in the spleen and bone marrow for 2 months after the immunization will be referred to as `late' APFC.
VDJ rearrangements in a small sample of early splenic APFC (day 7) involved predominately the V186.2 segment without somatic mutations (Table 2
). At the later stage of the response (days 3565), about half of the splenic APFC used the V186.2 gene and the remaining cells expressed nine different analogue genes that were evenly represented (Table 2
). (See Methods for description of V gene assignment.) Nucleotide substitutions were present in 3040% of VH genes with an average of 3.0 mutations/V186.2 gene and 1.8 mutations/analogue V gene. The frequency of mutants and the average mutations in the rearranged segments of the V186.2/V3 gene family in the late splenic APFC are thus very similar to that found in the splenic GC B cells in NP-CGG-immunized mice (Table 2
, column 2) (10,17,18,20). However, the late APFC in bone marrow showed a different repertoire (Table 2
). In a sample that was comprised of 12 distinct clonotypes from six animals (Table 2
, footnote), it was found that 90% of V186.2+ APFC were mutated with an average of 8.5 mutations/V gene, which is nearly a 3-fold increase over the mutation frequency found either in GC B cells or in late splenic APFC. The dramatic difference between the distribution of mutations among the V186.2+ APFC in the spleen and those in the bone marrow is shown in Fig. 2
(a). Most of the V186.2+ APFC mutants in the spleen and BM carried the W33L and used the canonical CDR3 with Tyr95 (see Figs 6a and 7a
), which affirms their relatedness to the GC B cell lineage, yet the APFC in BM accumulated more somatic mutations.

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Fig. 2. Distribution of somatic mutations in the rearranged VH186.2 genes among hapten-specific APFC in the spleen (empty bars) and BM (stripped bars) at the (a) late primary responses (days 3565) after immunization and (b) anamnestic response (day 4) after the antigen boost).
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Fig. 6. Nucleotide sequences in CDR1 and CDR2 of mutated VH186.2 genes recovered from individual APFC in the spleen and bone marrow during the late primary response to NP-CGG/alum (a) and in the secondary response to soluble NP-CGG (b). Germline-encoded VH186.2 sequence is shown at the top; the positions of nucleotide changes in the sequences from individual APFC (code numbers at the left) are shown. The sequences were grouped together according to mutations in position 33 and 58. The splenic APFC 23-18 (a, sixth sequence from the top) contained somatic mutations in other regions of the VH186.2 gene.
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Fig. 7. Structures of CDR3 of rearranged VH186.2 genes from Fig. 4 . The names of corresponding D genes are shown in the right column.
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Despite the high mutation frequency in the V186.2+ cells, the BM APFC expressing the analogue VH genes were either unmutated or contained only 13 mutations/gene (Table 2
, V102 and V23), a frequency comparable to that of GC B cells and the late splenic APFC. Thus the high number of mutations in BM APFC is peculiar to the dominant V186.2 clonotype.
Somatic repertoire of late APFC in BM is T cell dependent
Inasmuch as somatic hypermutation is driven by Th cells (43,44), it was of interest whether the appearance of the highly mutated BM APFC was T cell dependent. CD4+ lymphocytes were depleted with repeated injections of anti-CD4 mAb GK1.5 beginning on days 18 after the immunization with NP-CGG, i.e. at the time of diminishing GC reaction (42,45). The treatment reduced the numbers of CD3+CD4+ lymphocytes to <0.1% in both spleen and BM until days 35, when they slowly begun to rise (Fig. 3
). As shown in Table 3
, the repertoire of the late NP-specific IgG APFC in the BM from Th-depleted mice was comparable to the control group in respect to the dominance of V186.2 gene rearrangements, prevalence of W33L replacement and high ratios of R/S mutations in CDR1 + 2; however, the average frequency of mutations/VH was reduced by half, to the levels found in GC B cells. Indeed, the Th cell depletion selectively decreased BM APFC with >7 mutations/VH, whereas the cells with 16 mutations/VH were equally frequent in both Th-depleted and control animals (Fig. 4
).

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Fig. 3. Depletion of CD4+ T cells in the spleen () and bone marrow ( ). Mice that were immunized with NP-CGG/alum (day 0) received four injections of anti-CD4 mAb (12 mg each, i.v.) on days 18, 24, 32 and 41 (arrows). CD3+CD4+ lymphocytes were enumerated by flow cytometry (two animals/interval). Horizontal bars indicate the percentages of CD3+/4+ lymphocytes in the spleen (solid) and BM (dashed) of control animals injected with normal rat IgG (average from four mice with individual range shown by the vertical bars).
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Fig. 4. Distribution of somatic mutations in VH186.2 genes in bone marrow APFC from CD4 T cell-depleted (hatched bars) and control (empty bars) mice.
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Repertoire shift in V186.2+ memory APFC
Next we examined whether the repertoire detected in an anamnestic response to NP is similar to that of GC memory B cells and their late APFC progeny. Cohorts of mice were challenged with 10 µg of soluble NP-CGG on days 35 and 65 after the priming, and NP-specific IgG APFC were sampled 4 days after the boost (Fig. 1
). The data from both animal groups (days 35 and 65) are presented together because their anamnestic responses were comparable. Eighty percent of secondary APFC expressed the V186.2 gene while the remaining 20% used various analogue VH genes (Table 4
). Somatic mutations were found in all V186.2+ cells in both spleen and BM, with an average of 11 mutations/VH (Table 4
, see the footnote for details on sampling). In contrast to the late primary APFC, there was no difference in the distribution of mutations among the secondary APFC in the two anatomic sites (Fig. 2b
). To our surprise the secondary APFC population was dominated by a clonotype that has been rarely detected before in the GC B cell lineage. Typical NP-reactive V186.2+ B cells that emerge from the primary GC have the W33L replacement (10,19), and this motif is also found in ~70% of late primary APFC in both spleen and BM (Figs 5 and 6
). Among the memory APFC, however, this mutation declined to ~30% while a new recurrent mutation appeared that replaced lysine (K) with arginine (R) in CDR2 position 58 in up to 80% of cells (Figs. 5 and 6
). Remarkably, the K58R mutants always carried Gly95, which joined the V186.2 gene to either DFL16-1 or DSP2-5/7 segments (Fig. 7
). The use of Gly95 instead of Tyr95 must have developed from junctional diversity during VDJ rearrangements. The V186.2 sequence contains a mutational hot-spot consensus motif AAGTA (46,47) in position 5859; however, that position 58 mutation is always A
G (Fig. 6
) argues for antigen selection of the mutated cells. Thus the patterns of recurrent mutations and VD junctions identify two functionally distinct populations of V186.2+ memory B cells: (i) Leu33/Tyr95 (Lys58) GC B cells and progenitors of late APFC, and (ii) Arg58/Gly95 (Trp33) progenitors of anamnestic APFC.

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Fig. 5. Frequencies of W33L (plain bars) and K58R (hatched bars) mutations in VH186.2 gene in late primary (1o) and in secondary (2o) responses to NP-CGG. Each bar represents the proportion (%) of APFC carrying the respective mutation in the spleen (light bars) and BM (dark bars).
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We noted with interest that, without exception, the W33L and K58R mutations in V186.2 were exclusive of one another among 70 APFC analyzed in this study (Fig. 6
). We speculate that there are either structural constraints imposed on the NP-binding antibody molecule that preclude the co-existence of both mutations in V186.2, or that the two clonotypes, Leu33/Tyr95 and Arg58/Gly95, represent two separate pathways of memory maturation and selection.
Repertoire of analogue VH genes in memory anti-NP response.
Various analogue VDJ rearrangements were found in the secondary APFC in the spleen as well as in BM but unlike the heavily mutated V186.2 genes, the analogue VH segments still contained only 13 mutations/VH (Table 4
), which represents no increase over the average mutations in GC B cells (Table 2
). The patterns of recurrent somatic mutations could not be assessed because individual analogue VH gene mutants were recovered too infrequently throughout this study, with the exception of the V102 gene (Fig. 8
). Seven out of nine V102 sequences from unrelated APFC contained a G
A transition in the GGCT mutational hot-spot (46,47) in position 26, which replaces Gly with Asp, suggesting an antigen selection process among the V102+ B cells even though the mutation is in FWR2. [A different interpretation is given to V102 position 10 where we found A instead of G in every sequence, including those that did not contain any other nucleotide substitution (Fig. 8
). We believe that A in position 10 represents the correct germline sequence of this gene].

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Fig. 8. Recurrent nucleotide substitutions in rearranged VH102 genes recovered from GC, APFC (pooled data from primary and secondary responses) and secondary hybridomas from NP-CGG-immunized mice. The nucleotide sequences in framework regions 13 and CDR2 of germline-encoded VH102 are shown on the top.
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Some of the analogue VH sequences recovered from secondary APFC were unmutated, raising the possibility that the respective APFC originated from naive B cells in the presence of carrier-primed Th cells, rather than from true memory B cells. In order to gain further insight into this problem, the repertoire of analogue VH gene-expressing B cells was examined by construction of hybridomas. Mice immunized with NP-CGG/alum were boosted 60 days later with soluble antigen and the splenocytes were immortalized at days 4 after the antigen boost. Several stable, NP-binding hybridoma clones were obtained that expressed respectively VH102, CH10 and 23 (Table 5
). The binding affinities of these mAb were comparable to the affinities of the V186.2+ secondary hybridomas and at least 1 log higher than that of the V186.2+ mAb B1-8
1 (Table 5
) that is representative of the primary germline-encoded anti-NP antibody (35). The fine specificity of the secondary analogue mAb is also comparable to that of secondary canonical mAb: they bind NP and NIP equally well, whereas the primary, unmutated V186.2+ antibodies show a preferential binding to NIP (Table 5
) (35). Although the sample is small, it is noticeable that the affinities of the analogue mAb to NP did not correlate with the extent of mutation in VH, and that even unmutated mAb may have an affinity that is typical of secondary response (cf. the V23 mAb 11-2-9 and 60-2-10 and the VCH10 mAb G6-18-6 and G6-18-10 in Table 5
).
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Discussion
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The model of antibody response to NP demonstrates how primary splenic GC spawn multiple B cell populations that proceed along distinct pathways of repertoire maturation and differentiation. The predominant cell population, which uses the VH186.2DFL16-1/2 rearrangement with the canonical Tyr95 joint and accumulates somatic mutations including the recurrent W33L, apparently migrates from GC to the spleen and BM, and differentiates into high-affinity antibody-producing cells at later stages of the immune response (42,48,49). Here we show that somatic diversification of the V186.2/Tyr95/Leu33 cell lineage is tissue specific. The APFC in BM evolved from highly mutated precursors, whereas the splenic APFC did not accumulate mutations above the level seen in the GC B cells. The primary GC in the spleen cease by 45 weeks after the immunization with antigen in alum (42,45), but perhaps some B cells continue to mutate in the post-GC phase of the response (48). The distinct repertoire of BM effector cells could indicate that only the highly mutated B cells home to BM and differentiate in to APFC. Alternatively, and more likely, the progeny of GC B cells circulate through spleen and BM, and the latter environment provides signal(s) for their continuing somatic mutation and differentiation to APFC in situ, as has been proposed for human BM (50). Such signal(s) could be generated by BM CD3+CD4+ lymphocytes, which are unusual in that the majority has a memory/activated phenotype (51). Indeed, depletion of CD4+ lymphocytes 3 weeks after the immunization selectively eliminated the highly mutated APFC in BM (Fig. 4
).
Although the principal GC B cell lineage, V186.2/Tyr95/Leu33, does contain precursor memory cells (14,16), the brisk anamnestic response to a low-dose, soluble antigen boost is dominated by a different population of effector V186.2+ APFC that have a distinct N residue Gly95, germline-encoded Trp33 and the K58R replacement. B cell clones with Gly95 have rarely been recovered from NP-specific GC (10,18,48) suggesting that the V186.2+/Gly95/Arg58 memory cell subset undergoes a post-GC expansion and maturation to become a `higher order of memory' cell (52) in the spleen and BM. Furokawa et al. (53) recently found that the V186.2/Gly95 mAb (Gly99 in their numbering system) reached higher affinities than the V186.2/Tyr95 mAb did during the affinity maturation of anti-NP response. Although the authors did not mention it, all of their V186.2/Gly95 shared the K58R and none had the W33L, as is the case of APFC in our study. We propose that K58R is a key mutation for selection of the highest affinity memory B cells in the post-GC stage of anti-NP response.
The third population of NP-reactive GC/memory B cells is composed of diverse clonotypes expressing at least 10 closely related VH analogue genes which, as a group, account for almost half of APFC in the late primary response in the spleen and BM, and a variable portion of the anamnestic response. These clonotypes were observed in earlier studies, but their potential was not appreciated. Weiss and Rajewsky (9) recovered a large number of rearranged VH analogue genes at 6 weeks after immunization with NP-CGG, many of which were said to contain somatic mutations. Others have described NP-binding hybridomas after secondary immunizations which expressed several analogue VH genes (14,23). In one study (23), the anamnestic response to NP was initiated with anti-idiotype, which led the authors to postulate that the high percentage of NP-binding hybridomas with analogue VH genes resulted from a triggering of naive B cells. A most convincing demonstration of the existence of analogue VH memory cells was provided by Decker et al. (43) in their study of secondary and tertiary responses to NP in splenic fragments. Between 20 and 50% of fragments that produced the secondary or tertiary anti-NP antibody were occupied by analogue VH gene clonotypes that contained somatic mutations, which were also recovered from APFC in our study, including V593, 24.8, 23 and 165.1. Here we show that the late primary and secondary APFC expressing analogue VH do not accumulate somatic mutations above the range found in the GC B cell progenitors, and that many of them remain unmutated. As stated earlier, we cannot rule out that the unmutated APFC were generated from naive rather than memory B cells. However, we were able to obtain secondary hybridomas that used analogue VH genes with few or no somatic mutations, which had affinities that are representative of the memory repertoire to NP (Table 5
). We hypothesize that VH analogue clones may reach the memory pool without mutation and affinity maturation, consistent with the prediction (54) that the germline-encoded affinities of certain BCR are not increased by introduction of somatic mutations.
The molecular and functional heterogeneity of memory B cells has been best demonstrated by Berek, Milstein and co-workers, in studies of hybridomas from mice that were repeatedly immunized with a hapten, oxazolone (4,55,56). The secondary and tertiary responses were dominated by distinct cell lineages, depending on immunization protocol. Berek and Milstein (56) proposed that co-evolution of functionally diverse memory B cell clones is made possible because of their compartmentalization, either by antigen presentation, anatomical separation or cell kinetics. The NP-specific memory borne by the VH186.2 clonotypes exemplifies the paradigm of repertoire maturation based on selection of higher-affinity B cell mutants during the late primary and secondary responses (55,57,58). On the other hand, the behavior of NP-reactive VH analogue clones is reminiscent of the responses in which the antibody affinities improve little (59) or not at all (60,61) after the repeated immunization. We propose that the evolution of distinct phenotypes of memory B cells may depend on the initial affinities of the respective BCR for NP and on the signals from Th cells. The latter is suggested by the observations that the expansion of NP-specific V186.2 clones is T cell-dependent: the analogue VH clonotypes become prevalent when T cell help is either decimated by advanced age (38) or by-passed altogether by immunization with a T-independent form of NP (13). We may reasonably postulate that the affinity of BCR influences the concentration of MHCpeptide ligands on the B cells, which has been shown to influence the quality of T cell help (62,63). We envision that the NP-reactive clones expressing analogue VH genes solicit a level of `help' that results in an early exit from the GC (20,30,45), and limited hypermutation and expansion. On the other hand, the V186.2+ clones may experience a prolonged interaction with Th cells leading to extensive proliferation and accumulation of somatic mutations.
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Acknowledgments
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This work was supported in part by US Public Health Service Grants AG-08193 and PO1AG10207. The authors thank Martin Flajnik for critical review and editing of the manuscript, and June Green for illustrations and manuscript preparation.
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Abbreviations
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APFC antibody plaque-forming cells |
BM bone marrow |
CGG chicken -globulin |
GC germinal center |
NIP iodonitrophenyl |
NP nitrophenyl |
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Notes
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Transmitting editor: C. Terhorst
Received 2 September 2000,
accepted 5 February 2001.
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