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Kimmel Cancer Institute and Department of Microbiology and Immunology, Jefferson Medical College, Philadelphia, PA 19107
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Before initiation of the primary GC reaction, Ag-specific B cells are thought to undergo activation via cognate interaction with helper T cells in the T cell-rich periarteriolar lymphoid sheath (PALS) of the spleen and the lymph node paracortex (6, 27, 28, 29). After this period, at least two differentiative pathways are available to a B cell, proliferation and differentiation to an Ab-forming cell (AFC) phenotype outside of follicles or migration to a follicle and participation in the GC/memory B cell pathway. In addition, recent data (30, 31, 32, 33) have supported past assertions (34) that B cells participating in the primary immune response are capable of differentiation to long-lived AFCs that reside in the bone marrow. How these lineage and migratory decisions are made is currently unclear. It has been suggested that the nature of naive B cells initially recruited into the response (35, 36), the affinity and specificity of the B cell Ag receptor (32, 33, 37, 38), the type of Ag driving the response (19), and levels of T cell help (19, 38, 39) can all influence these decisions. Moreover, whether all these pathways of differentiation are also available to memory B cells participating in anamnestic responses is a subject of debate (21, 22, 23, 34). Investigation of these issues is complicated by the fact that most immune responses to even simple epitopes are clonally diverse and often exhibit substantial response to response variation regarding the participation of individual clonotypes. In addition, in many immune responses clonotypes that dominate the primary immune response may not effectively seed the memory compartment, making the identification of B cells destined for this compartment during the primary response difficult (40, 41, 42, 43).
We (44) and others (45, 46) have extensively characterized the T cell-dependent B cell response of A/J mice to the hapten arsonate (Ars). As assessed by serological and hydridoma studies, the primary response to Ars is clonally diverse, but during the latter stages of this response a single B cell clonotype reproducibly emerges and dominates subsequent anamnestic responses. This clonotype expresses a V region encoded by a single combination of V gene segments, with variation before hypermutation only at VH-D and D-JH junctions (47, 48). We term this V region and the clones that express it "canonical." Canonical clonotypes undergo extensive V region somatic hypermutation and both positive and negative phenotypic selection during the primary response (16, 44). The secondary serum Ab response is largely composed of Abs derived from this clonotype that bear characteristic VH region amino acid substitutions due to somatic hypermutation. These recurrent mutations have been shown to independently and additively confer increased affinity for Ars (49). Thus, the canonical 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 memory compartment after undergoing extensive V gene hypermutation and selection. Here, we have exploited this experimental system to determine how the primary and secondary AFC and GC responses of a single B cell clonotype may differ.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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A/J mice 8–12 wk of age at the time of first immunization were purchased from The Jackson Laboratory (Bar Harbor, ME) and were housed in a specific pathogen-free rodent facility and given autoclaved food and water.
Immunizations
For primary GC analysis, A/J mice were immunized intraperitoneally (i.p.) with 100 µg Ars-keyhole limpet hemocyanin (KLH) precipitated in alum. Nine to 15 days after immunization, mice were sacrificed and their spleens were removed and prepared for cryosectioning as previously described (29, 37). For secondary GC and secondary AFC "cluster" analysis, mice were immunized i.p. with 100 µg Ars-KLH in alum or CFA, rested for at least 6 wk, and then boosted with 100 µg Ars-KLH in PBS i.p. After boosting, spleens were removed at various times and prepared for cryosectioning as described above.
Immunohistochemistry
Frozen spleens were sectioned and three 5- to 6-µm thick parallel sections were placed on each slide. The slides were fixed in 100% cold acetone and stored at -80°C. The procedures used for immunohistochemical staining of the sections have been described in detail previously (37). Briefly, parallel sections were stained with biotinylated Ars-BSA or the anti-idiotypic Abs E4 or 5Ci followed by streptavidin coupled to alkaline phosphatase (Dako, Carpinteria, CA). All sections were also stained with peanut agglutinin (PNA) coupled to HRP (Sigma, St. Louis, MO) to elaborate GC. Some sections were also stained with HRP-conjugated donkey anti-mouse 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).
PCR amplification of V genes
VH genes in genomic DNA present in processed microdissected tissues was subjected to two rounds of PCR amplification using Ampli-Taq DNA polymerase (Perkin-Elmer, Foster City, CA) and various combinations of nested oligonucleotide primers. Each round of PCR consisted of 40 cycles of 95°C/1 min, 58°C/30 s, and 72°C/3 min ending with one cycle of 72°C/6 min. The 5' primers were specific for sequences in the 5' region of the canonical VHIdCR gene segment and were either hybridized in the promoter region of this gene or just 5' of the translation initiation codon in the leader intron. The 3' primers either hybridized between the JH3 and JH4 gene segments or to a region of common homology in the JH2, JH3 and JH4 gene segments. The second round amplification was performed on 1.5–2 µl of the first round reaction (50 µl total). All of the primers contained restriction sites to allow cloning of the products. PCR products of 0.5–1.3 kbp were obtained (depending on the primer combinations used), processed, cloned, and their inserts subjected to nucleotide sequencing as described previously (37).
When genomic DNA from microdissected tissues was amplified by PCR, controls containing either no tissue or processed tissue microdissected from a region of the spleen that did not stain with E4 or 5Ci were always included. The no tissue controls never yielded a PCR product, but the unstained tissue controls sometimes did. This was not unexpected, as the primer combinations used would amplify any VH gene partially encoded by the VhIdCR gene segment, irrespective of D or JH use. When these "control" PCR products were cloned and sequenced, they invariably were found to be noncanonical, being composed of VhIdCR or a closely related germline VH gene segment, in combination with a noncanonical D or JH segment. These clones also were never found to contain somatic mutations, indicative of their derivation from the naive B cell compartment.
Dendrogram construction and shape assignment
As detailed in Results, VH clones obtained from single spleens were judged to have been derived from a common precursor on the basis of sharing of their VH-D and D-JH junctional nucleotides. The sequences of groups of related clones containing three or more members were used to construct geneological dendrograms, essentially as has been described by us and others (10, 50, 51, 52). During this exercise, we assumed that a mutation shared by several clones had been sustained once in vivo, and then inherited by daughter cells that then each gave rise to a single PCR clone. Exceptions were sometimes made to this rule of "mutational parsimony" in the case of mutations in five VH codons: three (10, 31, and 105) that appear to be mutational "hotspots" (53) and overlap RGYW consensus hotspot sequences (54); and the CDR2 codon 58 and 59 affinity-enhancing mutations (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 whose sequences could easily be explained by PCR recombination events (11, 55) were observed infrequently (<5% of clones) in our studies. However, when the locations of a clone in the dendrogram as defined by mutations in its 5' half, as compared with mutations in its 3' half, were discordant, it was assumed that the clone had been generated via PCR recombination and it was excluded from further analysis.
During the construction of dendrograms, we also assumed that any given lineage or sublineage was founded by a single (hypothetical) B cell. These assumptions were based on: 1) previous studies showing that GC are formed by a limited number of precursors (1, 2, 3, 4, 5); and, 2) the fact that sublineages were defined by combinations of somatic mutations that, given the nature of the hypermutation process, were unlikely to have taken place independently multiple times.
The dendrograms thus constructed could often be assigned one of three "shapes": bush, tree, or trunk. A "bush" was defined by two or more sublineages emanating from a single, hypothetical founder cell. A "tree" consisted of a single geneological "trunk" from a common founder cell, and extensive "branching" emanating from the top of this trunk. This "trunk" was defined by three or more mutations that were shared by all of the clones. A "trunk" was defined as described for a "tree," except it lacked sublineage branches defined by two or more mutations. In some cases, the dendrograms did not fit into one of these three specific categories, and so were assigned mixed names.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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We previously reported the results of an immunohistochemical analysis of the participation of canonical clonotypes in the primary anti-Ars response in the spleens of A/J mice (37). This study exploited the anti-idiotypic mAbs E4 and 5Ci, which are highly specific for canonical V regions (56, 57, 58). We found that canonical B cells did not participate in the early PALS AFC response, but initially appeared infrequently and in a small number of GCs 6 days after immunization. While the number of E4, 5Ci+ GCs remained low throughout the primary response (four to five E4, 5Ci+ GCs per spleen on average), the frequency of canonical B cells per GC increased after day 6 and was maximal between days 12 and 14, after which this response waned. In this time frame, all of the E4, 5Ci+ GCs also stained positive with anti-IgG but not anti-IgM, consistent with previous hybridoma studies showing that H chain class switching takes place early in the anti-Ars primary response (44, 52). While the kinetics of this GC response are similar to those previously reported for other hapten-protein conjugates (9, 13, 14, 29), the magnitude of this response is far less, perhaps reflecting an extremely low precursor frequency of canonical clonotypes.
To extend these in situ studies to an analysis of canonical V region hypermutation and selection, an immunohistochemical-microdissection-PCR approach analogous to that pioneered by Kelsoe and colleagues (10, 11) was employed. A/J mice were immunized with Ars-KLH in alum, sacrificed at various times after immunization, and their spleens processed for histology. Expressed canonical VH region genes and their flanking sequences were recovered from genomic DNA via PCR from 17 microdissected E4, 5Ci+ GCs representative of various stages of the primary anti-Ars response, and the PCR products were cloned. Microdissected regions within the GC (as defined by PNA staining) consisted of 50–100 cells. Care was taken to avoid the inclusion of PNA- cells in the surrounding mantle zone. Multiple plasmid clones obtained from each GC were then sequenced. Clones containing hypermutated versions of the canonical VHIdCR VH gene were easily distinguished from clones that might have been derived from other germline genes (obtained only rarely in this analysis) because most of these related J558 family members have been characterized (59). Kelsoe and colleagues have previously measured the PCR error frequency under similar conditions, and have demonstrated that this frequency follows a Poisson distribution (11), making it extremely unlikely that when two or more mutations are observed per VH gene, that all of these mutations could have resulted from PCR error. Moreover, the results of the studies reported here suggest that, in our hands, the PCR error frequency is even lower, perhaps only 0.2 mutation(s) per VH gene, on average (see below).
As mentioned above, canonical VH regions differ before hypermutation only at VH-D and D-JH junctions (47, 48). The consensus sequence in these regions is VHIdCR-TCN-NNN-D FL16.1-NNN-JH2, where N indicates nucleotides often resulting from N region addition (the first "TC" nucleotides appear to result from P nucleotide addition). Thus, at least seven of the nine canonical VH junctional nucleotides are added in a template-independent fashion and therefore can be used as markers of common clonal origin. We and others have previously shown that it is improbable for canonical V genes derived from different A/J mice to share all these junctional nucleotides (47, 60).
Fig. 1
summarizes the results obtained
from the analysis of primary GCs, and Fig. 2 provides a representative example of
the sequence data obtained from an individual day 10 primary GC.
Canonical VH clones derived from the GCs of
single mice were judged to have originated from the same clonal
precursor on the basis of sharing of VH-D
and D-JH junctional nucleotides. The majority of
the VH clones obtained were canonical, but three
of the GCs gave rise to clones representative of other families of
anti-Ars V regions that differ from canonical
VH genes in their D regions. Fig 1 also
catalogues the presence of the recurrent, affinity-enhancing mutations
at complementarity-determining region (CDR)-2 positions 58 and 59, as
well as a mutation in codon 105 in the D region at an intrinsic
mutational hotspot (RGYW; Refs. 53 and 54).
All of the GCs analyzed gave rise to V gene clones containing multiple
mutations at a frequency higher than expected for PCR error. In
addition, canonical VH clones obtained from
individual GCs nearly always shared all junctional nucleotides.
Attempts to reconstruct the hypermutation and clonal expansion
events that took place in each GC were undertaken using the
previously established procedure of building geneological dendrograms
(10, 50, 51, 52). During this exercise, we made the
simplifying assumption that each plasmid clone represented a distinct
GC B cell, which seems reasonable given that DNA from numerous E4,
5Ci+ cells was amplified in each PCR reaction. These
theoretical constructs rely on the doctrine of mutational parsimony,
which assumes that if a mutation is shared by several clones, it took
place only once in a precursor cell and was inherited by daughter
cells. This assumption is subject to caveats due to the nonrandom
nature of hypermutation (61) and the possibility that any
given base change may have resulted from a PCR misincorporation or
recombination event (11, 55). However, when appropriate
caution is applied, the utility of these theoretical constructs for the
analysis of data obtained from individual GCs is well established
(10, 11).
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As can be seen in Fig. 1
, dendrograms of all of these types could be
generated from the clones isolated from individual GCs during the
9–10, 11–12, and 13–15 day time frames. In addition, the average
frequency of mutation per V region base pair at these time points was
indistinguishable. The Ars affinity-enhancing mutation at CDR2 position
58 was reproducible observed after days 9–10, but the analogous
position 59 mutation was observed in only one GC from day 12. Another
measure of phenotypic selection of V regions, the ratio of mutations
causing amino acid substitutions (R, replacement) to those that do not
(S, silent) in CDR vs framework regions (FWR) (25) was
also evaluated. These ratios did not reveal a progressive pattern of
change from days 9 to 15. Nonetheless, mutations resulting in
translation termination codons, or replacement mutations in codons know
to encode Ars contact residues(62) or highly conserved FWR
residues (63), were only rarely observed (data not
shown).
Migration and expansion of canonical clones before and during the primary GC response
The data obtained from GCs microdissected from the spleens
of individual mice also provided information regarding clonal
migration and expansion before and during the GC reaction. In the
case of the spleens of four mice, three pairs (groups 1J, 2J, and 3J in
Fig. 1) and one triplet (group 8J) of GCs gave rise to V gene clones
that shared junctional nucleotides, indicating a common clonal origin.
Thus, canonical clones must often undergo expansion before the
nucleation of multiple GCs. Strikingly, however, in the case of each of
these four groups of GCs, shared somatic mutations were rarely observed
among clones derived from different GCs, save for the
affinity-enhancing position 58 mutation. Two of the GCs from the
triplet group were directly adjacent to one another (day 15) and a pair
of GCs in another spleen was present in the same follicle (day 9). The
adjacent pair of GCs that are members of the GC triplet isolated
from the day 15 mouse is shown in Fig. 3,
as well as a schematic diagram illustrating how we interpret migration,
clonal expansion, and hypermutation to have taken place in this
clone.
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Approaches identical with those used for the analysis of the
primary anti-Ars response were used to analyze the secondary
response of canonical clonotypes in the spleen. Mice were primed with
Ars-KLH in alum or CFA and rested for at least 6 wk. Spleens from two
mice that had been rested for 6 wk were processed for histology and
stained for E4, 5Ci+ cells as described above.
E4, 5Ci+ cells were observed at a low frequency
(75–100 per section) scattered throughout the red and white pulp, with
an apparent concentration in the follicular and marginal zone areas
(Fig. 4). Because such cells are
undetectable in the spleens of naive A/J mice (K.A.V. and K.T.B.,
unpublished observations), this indicates a substantial increase in
canonical B cell precursor frequency in the spleen due to primary Ars
immunization. The remaining mice were boosted with Ars-KLH in PBS and
spleens taken at 1- to 2-day intervals up to day 12 and processed for
histology. As illustrated in Fig. 4 with E4 staining, at day 2
canonical V region-expressing B cells were observed at a high frequency
but were scattered throughout both the red and white pulp. By day 4, a
massive E4,5Ci+ cellular response had developed,
consisting largely of loose clusters of cells confined to the red pulp.
The intensity and asymmetric pattern of staining of these cells was
characteristic of AFCs. These cells also labeled intensely with
Ars-BSA. Most of these AFCs were embedded in clusters of cells that
stained intensely with anti-IgG. Only scattered
IgM+ AFCs were found in these same locales (data
not shown). At day 6, most of the AFCs were found surrounding the red
pulp sinuses. This response gradually waned and was not detectable at
day 10. At day 6, E4, 5Ci+ GCs were first
observed, in greater abundance than seen at day 6 of the primary
response. These GCs persisted through at least day 8 (Fig. 4) and most
were stained by anti-IgG, but not anti-IgM (data not shown). By
days 10 and 12, nearly all GCs stained positive with E4 and 5Ci, but
with an intensity and in a pattern suggestive of Ag-Ab complexes
deposited on the surface of follicular dendritic cells (FDC) (see
insets in Fig. 4).
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summarizes the data obtained, and Fig. 2 presents representative
sequence data from a day 4 AFC cluster. The most striking results were
obtained from a single mouse at day 4, in which three of the five
clusters analyzed appeared to have been derived from the same clonal
precursor based on sharing of all junctional nucleotides. Two of these
clusters were adjacent to each other and one was present in a spatially
distinct region of the spleen. Moreover, all of the clones isolated
from these three clusters shared nearly all of their somatic mutations,
including the affinity-enhancing CDR2 position 58 mutation and a
position 59 mutation that has previously been observed among canonical
VH genes expressed by secondary hybridomas. Of
the 14 clones and 
10 kbp sequenced from these clusters, only 6
unshared mutations were observed, with each seen only in one clone.
These scattered base changes, which were present at a frequency of
0.06%, most likely resulted from PCR error. Therefore, these data are
consistent with the generation of a major fraction of the canonical
clusters in the spleen from members of a single memory B cell clone
that underwent extensive migration and expansion without further
hypermutation.
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Induction of hypermutation and rapid phenotypic selection take place during the secondary GC response of canonical clones
A total of 13 GCs were analyzed, as described above, from mice
that had been primed with Ars-KLH in alum or CFA at least 6 wk earlier
and boosted 6 or 8 days before spleens were taken. The extensive
apparent deposition of E4,5Ci+ immune complexes
on FDCs after day 8 (see Fig. 4) precluded the unambiguous
identification of GCs that contained canonical V region-expressing B
cells at later time points. Fig. 6
summarizes the data obtained, and Fig. 2 presents representative
sequence data from individual day 6 and day 8 GCs. Eleven of the 13 GCs
gave rise to only canonical VH clones and two
gave rise to clones representative of other anti-Ars V region
families. One of these GCs also gave rise to canonical
VH clones—the only example of a "mixed
clone" GC obtained in our entire analysis. At day 6, all GCs gave
rise to clones whose sequences suggested ongoing hypermutation
(resulting in dendrogram "trees" or "bushy trees"), but the
average frequency of mutation in these GCs (1.3%) was similar to what
had been observed in the primary response (1.1%). In addition, only
two of the GCs produced clones with mutations at CDR2 positions 58 or
59 that might be affinity enhancing (see Fig. 5).
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). A single canonical clone that has undergone extensive expansion and migration can give rise to multiple GCs during the secondary response, followed by a period of clonal expansion and hypermutation that is confined to individual GCs
The most striking secondary GC data were obtained from a single
mouse analyzed at day 6, from which six E4, 5Ci+
GCs were analyzed. Of these GCs, five gave rise to canonical
VH clones, which all appeared to have been
derived from a common precursor based on identity of
VH junctional nucleotides. All of the
VH clones obtained from these GCs were
hypermutated, but only a few of the somatic mutations in clones
isolated from different GCs were shared. Some of these GCs were
adjacent to one another, while others were present in different locales
of the spleen. As illustrated in Fig. 7,
the data obtained from this analysis suggest that a common, unmutated
precursor (labeled "P"), which initially underwent clonal expansion
and extensive migration while sustaining only a few mutations (giving
rise to the "N1" GC precursor cell), gave rise to these five GCs.
After entering GCs, the progeny of this clone appear to have undergone
extensive hypermutation and expansion with little or no inter-GC
migration (see Fig. 7 for details).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Liu et al. also documented a modest average frequency of V region hypermutation in canonical GC B cells isolated from days 10 and 12 of the primary anti-Ars response (0.5–1%), as compared with hybridomas expressing canonical V regions isolated from anamnestic anti-Ars responses (2.5–3%; Ref. 24), yet >80% of the E4+, PNA+ B cells they analyzed at day 10 expressed hypermutated V genes (64). Our data corroborate these findings, and also indicate that this modest frequency per V region base pair remains rather constant through at least day 15 of the GC response. In addition, all of the primary GCs we analyzed showed evidence of ongoing hypermutation. Analogous studies using other hapten systems have indicated that clonal expansion in primary GCs can take place without induction of hypermutation, even during the intermediate stages of the response (14). Whether these differences in hypermutation frequency are reflective of changes in the nature of the GC response driven by different Ags, or variations in the potential of distinct B cell clonotypes to undergo hypermutation awaits further investigation.
Among VH clones obtained from primary day 11
through 15 GCs, we did observe a high frequency of the CDR2 position 58
affinity-enhancing mutation. However, we did not detect an increasing
frequency of the CDR2 position 59 affinity-enhancing mutation.
Moreover, dendrogram "trunks" or "trees" also did not increase
with time. Either would have been indicative of preferential clonal
expansion of hypermutated subclones that had acquired increased
affinity for Ag. This discrepancy indicates that the position 58
mutation may be a "hot event," resulting from both a high frequency
of occurrence and phenotypic selection. Indeed, its frequency of
observation was only slightly higher than a documented intrinsic
hotspot mutation at a consensus RGYW motif (53), resulting
in a translationally silent mutational change at position 105 in the D
region (see Fig. 1). Although a much larger sample size would be
required for unequivocal conclusions to be drawn, we also did not
observe progressive increases in the R/S mutation ratio in CDRs, or a
decrease in this ratio in FWRs. In contrast, we obtained only a few
VH clones with translation termination codons or
replacement mutations in codons know to encode Ars contact residues in
CDRs or highly conserved FWR amino acids (data not shown). Thus, these
data suggest that there is a strong selection for maintenance of Ag
binding capacity during the primary GC reaction, but not necessarily
for iterative increases in affinity for Ag.
These findings appear inconsistent with previous conclusions that V gene mutations accumulate monotonically and that V region phenotypic selection occurs progressively during the primary GC response (5, 11). A means to reconcile our data with previous studies has recently been provided by Tarlinton, Kelsoe, and their colleagues, analyzing affinity maturation during the the primary response to (4-hydroxy-3-nitrophenyl)acetyl (31, 32, 65). Their findings suggest that while hypermutation may be restricted to GCs, the phenotypic selection of mutant V regions takes place not only in the GC, but in other microenvironmental locales as well. During the anti-Ars primary GC response, B cells whose V regions have sustained combinations of mutations that result in substantial increases in Ars affinity may be preferentially recruited into an extrafollicular, and perhaps extrasplenic maturation pathway. Given this scenario, B cells that had recently entered the GC and therefore had undergone only the initial stages of hypermutation and phenotypic selection would be preferentially sampled by bulk microdissection. Examination of the kinetics and extent to which canonical B cell clones populate extrasplenic compartments such as the bone marrow, as well as the patterns and frequency of somatic mutations in the V genes these canonical clones express, will be required to further test this idea.
There have been limited studies of V gene hypermutation and selection during the secondary GC reaction, and whether this response differs qualitatively from the primary GC response is a subject of controversy. Cerny and colleagues suggested that B cells that have sustained V region hypermutation in the primary response often undergo extensive expansion without hypermutation in secondary GCs (39). In contrast, Milstein, Berek, and coworkers have argued that memory B cells re-enter GCs during secondary and higher order responses, where they sustain further rounds of hypermutation and phenotypic selection (20, 26). Adding to the confusion are conflicting results regarding the ability of purified memory B cells to undergo further hypermutation ex vivo (21, 66).
The data we have obtained from the analysis of E4,
5Ci+ secondary GCs do not allow an unequivocal
resolution of this issue. We did isolate several groups of canonical
VH clones from individual day 8 secondary GCs
that shared many somatic mutations, including both position 58 and 59
affinity-enhancing mutations. One interpretation is that these
mutations took place in the primary response in a memory cell
precursor, whose progeny were subsequently recruited into the secondary
GC reaction where they underwent further hypermutation. This scenario
is illustrated for a group of VH clones isolated
from a day 8 secondary GC in dendrogram form in Fig. 8B. However, similar data were
sometimes obtained from VH clones isolated from
primary GCs, also as illustrated in dendrogram form for a day 12
primary GC in Fig. 8A. Because all canonical
VHIdCR hypermutation takes place after Ars
immunization (54), such primary GC data reinforce the
conclusion that V gene mutation occurs at a very high rate in the GC
microenvironment (9, 10, 11, 13) and that majority
representation of only a few of the enormous number of V region
variants generated by mutation can also take place rapidly (11, 14). Thus, the shared mutations observed among
VH clones derived from secondary GCs might well
have taken place during the early stages of the secondary GC reaction,
in B cells directly recruited from the naive B cell compartment.
Indeed, if the rates of V gene hypermutation and selection are
accelerated in anamnestic GCs (see below), then naive B cells recruited
into this response could rapidly give rise to progeny expressing V
regions with frequencies and patterns of mutation thought previously to
be characteristic of only memory B cell clones that had undergone
multiple GC reactions (20, 26). In this regard, we
obtained no VH clones containing known
affinity-enhancing 58 and 59 mutations from day 6 secondary GCs, while
such clones were often obtained from day 4 and 6 secondary AFC
"clusters." Moreover, the scarcity of affinity-enhancing 58 and 59
mutations in day 6 secondary GCs indicates that even low-affinity B
cells can enter the secondary GC reaction, as previously shown for the
primary GC reaction (67). Clearly, to unambiguously
address the question of whether memory B cells can re-enter the
hypermutation-selection pathway in the secondary response will require
a reproducible method for tracking these cells in situ that is
independent of the structure and function of their Ag receptors.
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The data derived from the five clonally related secondary GCs
illustrated in Fig. 7 did suggest a brief period of mutation in a
single clone, before the nucleation of multiple GCs, but none of the
few mutations apparently sustained during this initial period are
associated with increased affinity for Ars (see Fig. 7). In total,
these data further reinforce the previous suggestions by us
(70) and others (28, 29) that primary GCs are
"independent islands of clonal hypermutation and phenotypic
selection," and extend this conclusion to secondary GCs. Testing
models that propose that iterative cycles of hypermutation and
selection take place in single GCs, perhaps via "cyclic re-entry"
of phenotypically selected B cells into a hypermutating pool
(69), must await methods that will allow dissection of the
kinetics of B cell differentiation in various subcompartments of
individual GCs. Somewhat surprisingly, the primary and secondary GC reactions were qualitatively similar, suggesting a lack of substantial mechanistic differences in the nature of these responses. Nonetheless, comparison of the data obtained from them revealed some distinctions. As expected from previous studies (19), the secondary GC response developed more rapidly, and while V gene mutation frequencies similar to those observed throughout the primary GC response were also observed at day 6, by day 8 we observed 2- to 3-fold higher frequencies, although substantial GC to GC variation was apparent, as was the case among primary GCs. Moreover, several day 8 secondary GCs gave rise to clones containing both CDR2 58 and 59 affinity-enhancing mutations. Taken together with the primary GC data, these data indicate that phenotypic selection may take place more rapidly in secondary GCs, consistent with the accelerated rate of this response overall.
Unfortunately, whether affinity maturation beyond the fixation of the
58 and 59 mutations or emigration of these high-affinity clones from
the GC, as appears to take place in the primary response, took place at
later times could not be determined due to the extensive apparent
deposition of E4, 5Ci+ immune complexes in all
GCs after day 8. The extent of canonical clone expansion, migration,
and GC nucleation also appears more robust during the secondary
response, as exemplified by comparing the data summarized in Figs. 3 and 7. Whether these quantitative and kinetic differences result from
an increased precursor frequency of canonical clones, increased levels
of carrier specific T cell help (19, 39), increased levels
of Ag deposited on FDCs (34), or a combination of these
factors will require further investigation.
The most remarkable difference between the behavior of canonical clonotypes in the primary and secondary responses occurs in the AFC pathway. Canonical AFCs are infrequent and appear as isolated cells during the primary response in the spleen. In marked contrast, the early secondary response is characterized by a massive canonical IgG AFC response in which memory cells recruited into this pathway undergo extensive migration and proliferation and are ultimately organized in loose clusters, predominantly in the red pulp. During this response, V region hypermutation does not take place, analogous to the situation in PALS "foci" characteristic of the primary anti-(4-hydroxy-3-nitrophenyl)acetyl response of C57BL/6 mice (28). Interestingly, our previous hybridoma analysis of the early stages of the "original antigenic sin" response of canonical memory clones to a cross-reactive hapten indicated that during the "sin" response these clones underwent extensive expansion without hypermutation (71). In total, our data demonstrate that canonical clonotypes are incompetent to give rise to PALS AFC foci during the primary response, but during the primary GC reaction they acquire the potential to subsequently undergo vigorous bursts of hypermutation-free expansion and Ab production during secondary responses. However, apparently only a minor fraction of all canonical B cells produced during the primary GC response acquire this potential, as revealed by the mutational homogeneity of the VH genes expressed by secondary AFC foci derived from single progenitors. This oligoclonal nature of the early secondary response has been extensively documented by hybridoma technology (50, 71, 72). We have previously suggested that this extreme "repertoire narrowing" is due to "specificity maturation"—a combination of stringent negative selection of V regions with cross-reactivity for self-Ags and positive selection for V regions with increased affinity for the driving foreign Ag (16).
Whether the hypermutation-free growth that takes place during the secondary AFC response results in maintenance or regeneration of a canonical memory AFC precursor population or leads only to terminal differentiation remains to be determined. However, in this regard several previous studies of the memory B cell response using adoptive transfers and hybridoma technology have indicated that memory cells can undergo extensive clonal expansion and persist for prolonged periods without induction of V gene hypermutation (73, 74). The data we present here suggest that these past studies preferentially sampled B cells in an anamnestic AFC pathway, rather than in an anamnestic GC pathway.
Unfortunately, the near lack of overlap of the secondary canonical AFC
and GC responses thwarted attempts to determine whether a
precursor-product relationship might exist between canonical AFC
clusters and canonical GCs. By day 6, when such GCs are first detected,
the size of canonical AFC clusters has decreased substantially from
what is observed at day 4, and most day 6 clusters surround the red
pulp sinuses (Fig. 4), suggesting migration of the cells in these
clusters from their site of origin. These observations are consistent
with previous observations that most splenic AFCs have a very short
half life (75). Due to this situation, E4,
5Ci+ AFC clusters are usually not observed
adjacent to E4, 5Ci+ GCs at this time. We did
obtained PCR VH clones from a single day 6 E4,
5Ci+ GC/AFC "cluster" pair (see Figs. 5 and 6). Sequencing of these clones revealed canonical
VH genes with distinct junctional nucleotides and
no shared somatic mutations between the two groups of clones, strongly
suggesting derivation from distinct progenitors. We are now pursuing
alternative approaches to address this important issue. Indeed, current
data regarding the relationship of B cells capable of giving rise to
primary and anamnestic AFC and GC responses have not allowed agreement
on whether the precursors to these pathways of differentiation are
members of distinct developmentally committed lineages or not
(28, 35, 36).
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Acknowledgments |
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Footnotes |
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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:
3 Abbreviations used in this paper: GC, germinal center; PALS, periarteriolar lymphoid sheath; AFC, Ab-forming cell; Ars, arsonate; KLH, keyhole limpet hemocyanin; PNA, peanut agglutinin; CDR, complementarity-determining region; FWR, framework region; R, replacement; S, silent; FDC, follicular dendritic cell.
Received for publication April 27, 1999. Accepted for publication August 3, 1999.
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Thr mutation at codon 59 has been shown to increase affinity of
canonical V regions 
180 µM 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 precursor. VH clones
obtained from individual GCs also shared numerous somatic mutations
among themselves, allowing the construction of the dendrograms
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