Abstract

Article1 June 1997free access The extent of affinity maturation differs between the memory and antibody-forming cell compartments in the primary immune response Kenneth G. C. Smith Kenneth G. C. Smith Search for more papers by this author Amanda Light Amanda Light The Walter and Eliza Hall Institute of Medical Research, PO Royal Melbourne Hospital, Victoria, 3050 Australia Search for more papers by this author G. J. V. Nossal G. J. V. Nossal Search for more papers by this author David M. Tarlinton Corresponding Author David M. Tarlinton The Walter and Eliza Hall Institute of Medical Research, PO Royal Melbourne Hospital, Victoria, 3050 Australia Search for more papers by this author Kenneth G. C. Smith Kenneth G. C. Smith Search for more papers by this author Amanda Light Amanda Light The Walter and Eliza Hall Institute of Medical Research, PO Royal Melbourne Hospital, Victoria, 3050 Australia Search for more papers by this author G. J. V. Nossal G. J. V. Nossal Search for more papers by this author David M. Tarlinton Corresponding Author David M. Tarlinton The Walter and Eliza Hall Institute of Medical Research, PO Royal Melbourne Hospital, Victoria, 3050 Australia Search for more papers by this author Author Information Kenneth G. C. Smith2, Amanda Light1, G. J. V. Nossal3 and David M. Tarlinton 1 1The Walter and Eliza Hall Institute of Medical Research, PO Royal Melbourne Hospital, Victoria, 3050 Australia 2Department of Medicine, University of Cambridge School of Clinical Medicine, Addenbrooke's Hospital, Hills Road, Cambridge, UK 3Department of Pathology, The University of Melbourne, Parkville, Victoria, 3052 Australia *Corresponding author. E-mail: [email protected] The EMBO Journal (1997)16:2996-3006https://doi.org/10.1093/emboj/16.11.2996 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Immunization with protein-containing antigens results in two types of antigen-specific B cell: antibody forming cells (AFCs) producing antibody of progressively higher affinity and memory lymphocytes capable of producing high affinity antibody upon re-exposure to antigen. The issue of the inter-relationship between affinity maturation of memory B cells and AFCs was addressed through analysis of single, antigen-specific B cells from the memory and AFC compartments during the primary response to a model antigen. Only 65% of splenic memory B cells were found capable of producing high affinity antibody, meaning that low affinity cells persist into this compartment. In contrast, by 28 days after immunization all AFCs produced high affinity antibody. We identified a unique, persistent sub-population of bone marrow AFCs containing few somatic mutations, suggesting they arose early in the response, yet highly enriched for an identical affinity-enhancing amino acid exchange, suggesting strong selection. Our results imply that affinity maturation of a primary immune response occurs by the early selective differentiation of high affinity variants into AFCs which subsequently persist in the bone marrow. In contrast, the memory B-cell population contains few, if any, cells from the early response and is less stringently selected. Introduction A feature of T-cell-dependent antibody responses in higher vertebrates is an improvement in affinity of antibody for antigen with time—a process known as affinity maturation. Affinity maturation is apparent both in the higher average affinity of serum immunoglobulin for antigen late in the primary response (Eisen and Siskind, 1964) and also in the ability of memory B-cells upon challenge to produce a response of higher affinity than the early primary response (Steiner and Eisen, 1967; Siskind and Benaceraff, 1969). While substantial information has been gathered in relation to affinity maturation in the generation of B-cell memory (Gray, 1994; Rajewsky, 1996), comparatively little is known concerning the affinity maturation of serum antibodies which also occurs during the course of the primary response. Antigen-specific antibody is initially produced by foci of antibody forming cells which develop during the first week of the response and which are associated with the T-cell areas along the periarteriolar lymphoid sheath (Van Rooijen et al., 1986; Jacob et al., 1991). The AFC of the foci first secrete IgM but subsequently switch in an almost synchronous manner to a downstream immunoglobulin isotype such as IgG1 (Nossal and Reidel, 1989; Jacob et al., 1991). Direct analysis of the AFCs comprising these foci reveals that they secrete low affinity antibody (Lalor et al. 1992) encoded by V gene segments that have not been altered by somatic hypermutation (Jacob et al., 1993; McHeyzer-Williams et al., 1993). During the second week of the response the foci involute (Jacob et al., 1991; McHeyzer-Williams et al., 1993), presumably due to death in situ as there is extensive apoptosis occurring in these areas during this time, a wave of AFC emigrants is not detected in other locations (Smith et al., 1996) and blocking apoptosis with transgenic BCL-2 prevents involution (Smith et al., 1994). Together these results indicate that the AFCs of the foci are not responsible for producing high affinity antibody as the primary response progresses. The involution of the foci during the second week means that the continued production of high affinity antibody after this time must be due to AFCs in other locations. In some primary immune responses antigen-specific AFCs have been detected in bone marrow shortly after immunization (Zachau et al., 1989; Smith et al., 1996). The bone marrow has also been shown to be the location of long-term antibody production in certain responses (Bachmann et al., 1994; Hyland et al., 1994; Slifka et al., 1995) and to become a major site of IgG synthesis as animals age (Benner et al., 1981). These data suggest the bone marrow is an important location for the long-term production of high affinity antibody in the T-cell-dependent response to antigen. Identification, isolation and analysis of high affinity AFCs during the course of a primary immune response would add to our understanding of the mechanism underlying affinity maturation of serum immunoglobulin. Generation of a high affinity memory B-cell population occurs in most instances within germinal centers (GCs). GCs are histologically discrete structures which develop in the B-cell follicles during the first week of the response and usually persist for a further three to four weeks. Within the GC, the processes of V gene somatic hypermutation, B-cell proliferation and selection are considered to act in concert to enrich efficiently for those cells bearing surface immunoglobulin with increased affinity for the immunizing antigen (Nossal 1992; MacLennan, 1994; Kelsoe, 1996). During the first two weeks after immunization, the number of antigen-specific B cells in the GC increases substantially before entering a period of decline (Jacob et al., 1991; McHeyzer-Williams et al., 1993; Smith et al., 1994; Liu et al., 1996). During the period of expansion, the frequency of V gene mutation increases as does the clonal restriction of the GC B cells and the frequency of amino acid exchanges associated with enhanced affinity for the immunizing antigen (Weiss et al., 1992; Jacob et al., 1993). The decline in GC B-cell number after the second week leading to the establishment of the stable, recirculating memory B-cell pool is substantial; fewer than 5% of the peak GC B-cell number persist in the memory population that remains in the spleen more than 10 weeks after immunization (Smith et al., 1994). It is thus possible that the memory B-cell population is established by the selective recruitment from among GC cells of high affinity variants. The end result would be a memory B-cell population of uniformly high affinity. Whether or not this is the manner in which the memory B-cell population is actually formed is yet to be determined. While these features of the immune response adequately explain the early production of low affinity antibodies and the improved affinity of memory B cells, the basis of the increasing affinity of serum antibody for antigen during the primary response and the mechanism for establishing the memory B-cell population remain to be clarified. In this report we examine affinity maturation of antigen-specific B cells during the primary response to the hapten (4-hydroxy-3-nitrophenyl)acetyl (NP) in C57BL/6 mice. Individual NP-specific IgG1+ B cells of either a GC/memory B-cell phenotype or an AFC phenotype were purified by flow cytometry (McHeyzer-Williams et al., 1991; Smith et al., 1996) and examined for the extent of VH gene somatic mutation, the presence of a particular affinity-enhancing amino acid exchange in the VH gene, and the affinity of the antibody expressed by the cell. Our analyses show that as the primary response progresses, the cells of the GC produce antibody of increasing affinity, correlated with an increasing frequency of VH gene somatic mutation. The GC B-cell population, however, does not mature to a point of being uniformly high affinity and, furthermore, the same distribution of low and high affinity B cells persists into the memory population. In contrast, a population of uniform high affinity AFCs is identified in the bone marrow and these cells appear to be responsible for long-term production of high affinity antibody after primary immunization. Analysis of the molecular basis of affinity maturation in these AFCs reveals that although they contain few somatic mutations, they are highly selected for the single affinity-enhancing amino acid exchange in the first complementarity determining region (CDR1) of the VH segment characteristic of the anti-NP response in IgHb allotype mice (Cumano and Rajewsky, 1986). This enrichment is not seen to the same degree in the long-term memory B-cell population. In addition, such AFCs persist for several weeks in the bone marrow. Our results, which suggest that selection operates to different extents in the development of memory and effector B cells in the primary response, are discussed in the context of a model for B-cell selection in the T-cell-dependent humoral immunity. Results Somatic mutation and antibody affinity progressively increase in GC B cells during the primary response The kinetics of the splenic GC/memory B-cell response to the hapten NP coupled to a protein carrier were established using a six parameter flow cytometry system. Antigen-specific GC B cells, defined as B220+IgM−IgD−IgG1+ and NP-binding (hereafter referred to as NP+), reached a maximum frequency of 0.3% of splenocytes at day 14 post immunization before declining such that by day 240 they represented 0.002% (Figure 1A). Single B cells of this phenotype were isolated at various times 6–56 days after immunization, cDNA was made and VH186.2-Cγ1 gene rearrangements amplified by PCR. The VH186.2 gene segment in association with Vλ1 dominates the response to NP in C57BL/6 mice (Bothwell et al., 1981; Cumano and Rajewsky, 1985). The PCR product was sequenced directly, meaning that the background frequency of mutation due to reverse transcriptase and/or Taq polymerase was essentially nil (Table I). In broad agreement with previous reports (Weiss et al., 1992; Jacob et al., 1993) few somatic mutations per VH gene were seen at day 6, more substantial numbers by day 10, with the average number of mutations continuing to increase over time until reaching near maximum levels at day 17 (Table I and Figure 1B). The Trp to Leu exchange at amino acid position 33 of the VH chain, which alone confers a 10-fold increase in antibody affinity for NP (Allen et al., 1988), was absent at day 6 and rare at day 10 despite a substantial increase in the average frequency of mutations per VH gene during this time. By day 14, however, this particular substitution was present in over half of the VH genes analysed despite there being only a slight increase in the frequency of mutation, suggesting that most of this increase was due to selection on the basis of affinity. Beyond day 14 little change in the frequency of the position 33 mutation in the GC and memory populations was observed (Table I and Figure 1C). As the GC reaction proceeded the ratio of amino acid replacement (R) to silent (S) mutations in the CDRs and framework regions of the VH genes of NP-specific B cells changed in a manner consistent with selection on the basis of improved antigen binding, namely increasing in the former and decreasing in the latter (Table I). These results confirm the progressive accumulation of somatic mutations and conserved amino acid exchanges in antigen-specific GC B cells thought to be consistent with the generation of a high affinity memory population. Figure 1.Kinetics of the primary B-cell response to NP–KLH. (A) Frequency of antigen-specific GC/memory B cells in the spleen at various times after immunization. Each point is the average of three animals and is the percentage of splenocytes having the phenotype B220+IgM−IgD−IgG1+NP-binding. (B) Average frequency of mutations in VH186.2 genes PCR amplified from single antigen-specific GC/memory B cells at the indicated times. Cells were sorted from a pool of three spleens. See Table I and Materials and methods for further details. (C) Percentage of the VH186.2 sequences shown in (B) containing the affinity enhancing Trp to Leu exchange at amino acid position 33. (D) Affinity distribution of NP-binding IgG1 produced by sorted splenic antigen-specific GC and memory B cells after in vitro culture. The ratios of NP2/NP15 binding of the antibodies have been segregated into three groups on the basis of the binding properties of NP-specific hybridomas: 0–0.4 (low affinity), 0.5–0.8 (intermediate affinity) and >0.8 (high affinity). See Table II for details. (E) Affinity maturation of NP-specific IgG1 in serum after primary immunization. Serum samples from a cohort of five immunized mice were assayed for NP2-binding IgG1 by direct ELISA. Optical density was converted into arbitrary units by means of a high affinity standard common to all assays. Each dot represents the measurement from a single mouse. Download figure Download PowerPoint Table 1. VH186.2 gene sequence summary of NP-binding IgG1+ B cells Cell type Day post immunization Number of V genes sequenced Sequences mutated (%) Mutations per V gene average (range) Position 33 Trp→Leu (%) R/S ratio CDR 1 and 2 R/S ratio FWR 1–3 Splenic 6 21 25 0.4 (0–2) 0 0 2.3 Germinal center 10 15 100 4.5 (2–6) 6.7 4.4 3 Memory B cell 14 24 96 4.7 (0–11) 54 14 2.4 14a 30 100 5.1 (1–12) 50 5.5 3.2 17 15 100 7.7 (5–14) 73 11.5 2.1 42a 19 95 8.7 (0–20) 58 7.7 1.2 Splenic AFC 14 17 76 1.5 (0–4) 41 >17 1.2 Bone marrow AFCs 14a 31 74 1.9 (0–5) 71 >38 2.2 35a 21 95 4.8 (0–11) 62 9.8 1.3 GSX2.1b – 29 0 0 (–) – – – a Data from these experiments are shown in Figures 3 and 4. b GSX2.1 is an IgG1b NP-specific hybridoma utilizing VH186.2 and made on day 7 of a primary response. To determine the extent of the correlation between the accumulation of mutations and increasing affinity of antibody, purified IgG1+ NP-specific GC and memory cells were used to establish clonal, in vitro cultures in a time course equivalent to that of the VH gene sequencing described above. Cells were cultured on a feeder layer of fibroblasts transfected with CD40 ligand in the presence of IL-4 and IL-5. After 7 days supernatants were assayed by ELISA for the presence of NP-binding IgG1. Approximately 10% of antigen-specific GC/memory B cells secreted detectable levels of antibody under such conditions (Table II). The affinity of the antibody in positive wells was determined by differential binding to substrates with either low (NP2–BSA) or high (NP15–BSA) haptenation ratios (Herzenberg et al., 1980). Antibody of high affinity will bind at both high and low haptenation ratios while low affinity antibody will bind only at high haptenation ratios. The proportion of sorted B cells making antibody with improved affinity increased from 3% at day 6, to 40% at day 14, 50% at day 17 and 65% at day 21, thereby confirming the relationship between mutation and increasing affinity on a single cell level (Table II and Figure 1D. Surprisingly, of the day 50 antigen-specific memory B cells stimulated in vitro to produce antibody, 65% were found able to bind NP with enhanced affinity. Thus no increase in mutation frequency, affinity enhancing mutations or frequency of GC cells capable of producing high affinity antibody occurred after day 21 of the primary response. This resulted in a memory B-cell population of mixed rather than uniformly high affinity (Tables I and II and Figure 1D). Table 2. Clonal cultures of NP-specific germinal center and memory B cells Day post immunization %Cultures secreting NP-binding IgG1 Numbers of NP+ cultures analyzed %Cultures with NP2:NP15 >0.8 IgG1 secreted per clone (ng/ml) 6 9.3 37 3 7.0 ± 4.6 10 11.0 25 12 6.7 ± 3.5 14 2.5 30 35 52.0 ± 4.3 17 8.0 43 43 12.5 ± 5.6 21 8.0 49 55 9.5 ± 3.2 56 9.0 34 58 9.1 ± 5.0 Supernatants from clonal cultures stimulated with CD40 ligand, IL-4 and IL-5 were titrated on and BSA plate coats in an ELISA. The NP2:NP15 ratio for IgG1 was determined from the optical density at a non-saturating point on the titration curve. Ratios are presented in full in Figure 1D. Affinity maturation of serum immunoglobulin Having established the kinetics and extent of affinity maturation in the GC B-cell population, we next determined to what degree this was reflected in serum Ig. Serum was collected from a cohort of animals at regular intervals after primary immunization and the titre of total and high affinity NP-binding IgG1 determined. High affinity NP-specific IgG1 was first detected in the day 7 samples with the titre increasing thereafter to plateau at around day 21 (Figure 1E). The most rapid increase in high affinity IgG1 occurred between days 7 and 14, the same time period in which somatic hypermutation was first observed in GC cells and during which the frequency of position 33 mutations seen in the VH genes of these GC cells increased substantially (Figure 1B and C). No further increase in the titre of high affinity NP-binding serum IgG1 was observed after day 21 indicating that affinity maturation was complete at this time for this dose of antigen. Identification of high affinity AFCs in the bone marrow The apparent concordance between affinity maturation of serum antibodies and GC B cells prompted us to isolate the cells responsible for antibody secretion after the decline of the splenic foci. A number of previous experiments suggested bone marrow as one location for high affinity AFCs. We therefore attempted to isolate primary response NP-specific IgG1+ AFCs from the bone marrow with the flow cytometry system used to isolate such cells in the spleen. A small population of IgG1+ AFCs was identified (Figure 2) which had a phenotype similar to that described for AFCs in the spleen (Lalor et al., 1992; Smith et al., 1996). ELIspot assays were used to measure the in situ secretion capabilities of these cells and showed that all AFCs making anti-NP IgG1 were negative for surface IgM, IgD and GR-1 but positive for syndecan (Table III). Within the syndecan+ compartment, 90% of NP-specific AFCs could be further partitioned on the basis of cell surface binding of NP (in the form of an APC conjugate) and anti-IgG1 (Figure 2). Such NP+IgG1+ AFCs could be reliably sorted to 80–100% purity using these criteria, despite representing 1–2 per 100 000 nucleated BM cells (Table III). We were thus able to isolate antigen-specific AFCs from the bone marrow for further analysis. Figure 2.Identification of NP-specific AFCs in the bone marrow during the primary immune response. (A) Antibody forming cells in the bone marrow of mice immunized 14 days previously with NP15–KLH were identified by cell surface expression of syndecan in the absence of IgM, IgD and Gr-1. (B) This population was further partitioned into cells expressing IgG1 and able to bind the hapten NP, this time coupled to the fluorescent protein allophycocyanin (APC). In the example shown here, the NP-specific AFC population represents 0.002% of nucleated BM cells. (C) and (D) are as for (A) and (B) respectively except that the bone marrow was from mice immunized with the carrier protein alone. Boxes indicate the criteria used for sorting cells for in vitro analysis. Download figure Download PowerPoint Table 3. Isolation of NP-specific IgG1 bone marrow AFCs Phenotype of sorted cells Number of cells plated Anti-NP15 IgG+ AFCs (IgM IgD Gr-1 PI)+ 5×105 0 (IgM IgD Gr-1 PI)− syndecan− 4×105 0 NP− 3×105 2 syndecan+NP+IgG1+ 14 12 This result is representative of six other experiments. The cumulative results of these six experiments show that for 102 syndecan+NP+IgG1+ cells plated, 97 anti-NP IgG1 ELIspots were observed (a sensitivity of 95%). At different times after immunization the relative affinity of antibody produced by individual sorted AFCs was assessed in both spleen and bone marrow (Table IV). This was done by comparing the frequency of AFCs producing anti-NP IgG1 of any affinity with that producing only high affinity antibody, again using differential binding to high and low haptenation plate coats. At early time points in the primary response all bone marrow and splenic AFCs produced antibody of low affinity. By day 28 splenic AFCs were present in very low numbers and, while those few cells seen were of high affinity, it was impractical to sort 50 at each time point so they are not shown in Table IV. In the bone marrow the proportion of AFCs secreting high affinity antibody increased progressively to reach 100% by day 28 and remained at this level thereafter (Table IV). This observation of affinity maturation of bone marrow AFCs at the cellular level recapitulates the phenomenon described for serum immunoglobulin (Figure 1E), and indicates bone marrow AFCs contribute to the long-term production of high affinity antibody during the primary response. The eventual uniform high affinity of the bone marrow AFCs is in contrast to the mixed affinity of GC/memory B cells at similar time points, indicating that the processes which select for increased antibody affinity operate to different extents in the memory and AFC populations. Table 4. Affinity maturation of NP-specific bone marrow AFCs High affinity AFCs (NP2/NP15×100%) Response Day Spleen Bone marrow Primary 8 3 0 14 56 57 28 ND 100 49 ND 100 Secondary 4 100 100 At each time point at least 50 NP+IgG1+syndecan+ cells were sorted onto ELIspot plates coated with NP15– and NP2–BSA, which detect total and high affinity anti-NP antibody respectively. The ratio of anti-NP IgG1 ELIspots (NP2/NP15) allowed calculation of the percentages shown. ND, not determined: while AFCs could be detected in the spleen at this time, their scarcity made sorting and analysis of affinity impractical. VH genes of bone marrow AFCs show a distinct pattern of somatic mutation To determine the basis of the improved affinity of the antibody secreted by a fraction of day 14 bone marrow AFCs we next examined these cells for the presence and distribution of VH gene somatic mutations. For comparison, splenic GC B cells from the same time point were similarly examined. The sequences of the VH186.2 genes recovered from the bone marrow AFCs and splenic GC B cells are shown in Figure 3 and summarized in Figure 4 and Table I. Single NP-specific IgG1 bone marrow AFCs were sorted 14 days after immunization, cDNA synthesized, VH186.2-Cγ1 rearrangements were amplified by PCR and sequenced as described above. The B220+NP+IgG1+ GC B cells sorted from the spleen at the same time constitute an independent experiment to that described in Figure 1. At day 14, all antigen-specific splenic GC B cells were mutated with an average of 5.1 mutations per VH gene (range 1–12) (Figure 3B). In contrast, the 74% of bone marrow AFCs which were mutated had an average of 2.5 mutations per VH gene (range 1–5), significantly fewer than seen in the GC (Student's t-test P <0.001) and equivalent to the number of mutations seen in GC cells 8 days after immunization with NP-CGG (Weiss et al., 1992). This low number of mutations suggests that bone marrow AFCs are early products of the GC. The high affinity replacement mutation at position 33 was found in 50% of splenic day 14 GC B cells, in agreement with previous reports (Weiss et al., 1992). Remarkably, despite having fewer mutations, all but one of the mutated bone marrow AFCs carried the position 33 Trp to Leu exchange (Figure 3A) thereby explaining the improved affinity of a fraction of bone marrow AFCs at this time. The proportion of mutated VH genes with the position 33 exchange was higher among these AFCs than even the cells of the secondary response: 96% of mutated bone marrow sequences versus 60% of secondary response AFCs (McHeyzer-Williams et al., 1991; Cumano and Rajewsky, 1986). Figure 3.VH gene nucleotide sequences from day 14 NP-specific B cells. (A) Single NP-binding IgG1+ antibody forming cells were sorted 14 days after immunization from bone marrow. VH186.2 was the predominant VH gene in these AFCs. (B) VH186.2 gene sequences from single NP-specific IgG1 GC cells, sorted from spleen 14 days after immunization. Only those codons which differ from the germline sequence are shown and numbered according to Kabat et al. (1991). Amino acids are shown using the single letter code. DH and JH regions are compared with known germline sequences. Details of the amplification and sequencing are given in Materials and methods and Table I. Download figure Download PowerPoint Figure 4.Distribution of somatic mutations in the VH genes of IgG1+ NP-specific B cells. (A) Frequency distribution of somatic mutations in the VH genes of single NP-specific AFCs sorted 14 days after immunization from bone marrow pooled from three mice. These sequences are presented in Figure 3. The number of sequences recovered is plotted against the number of mutations per VH gene. Solid segments indicate sequences in which the amino acid at position 33 of the VH gene has been mutated to encode leucine. (B–D) As for (A) except that the starting populations were, respectively, day 14 NP-specific splenic GC B cells (sequences shown in Figure 3), day 35 NP-specific bone marrow AFCs and day 42 splenic memory B cells. Additional data on these sequences are shown in Table I. Download figure Download PowerPoint Persistence of early, affinity selected AFCs in the bone marrow The unique distribution of somatic mutations in the VH genes of day 14 bone marrow AFCs suggested that if these cells persisted in that location, they should be identifiable by virtue of their mutational pattern. For this reason VH186.2 genes were sequenced from single AFCs sorted from bone marrow at day 35 after primary immunization. Three main observations could be made by comparing sequences from day 14 and 35 AFCs (Figure 4A and C). First, the proportion of AFCs containing unmutated VH gene sequences declined from 26% at day 14 to 5% (a single sequence) at day 35, paralleling the loss of low affinity AFCs in the bone marrow between days 14 and 28 as measured by ELIspot (Table IV). Secondly, the unique population of somatically mutated AFCs seen at day 14 was still present at day 35 in at least similar absolute numbers [since the number of NP-specific AFCs in the bone marrow increases ∼3-fold between days 14 and 35 (Smith et al., 1996) while the total cellularity remains constant]. This suggests that, once selected, high affinity bone marrow AFC clones are long-lived in a fashion analogous to memory B cells. These persistent mutated AFCs may correspond to the previously observed long-lived plasma cells in the bone marrow (Ho et al., 1986). Finally, additional GC-derived cells with more mutations but less dependence on the position 33 mutation emigrate to the bone marrow after day 14, consistent with the idea that combinations of mutations other than the position 33 Trp to Leu exchange can result in a high affinity NP-specific antibody (Allen et al., 1988). The pattern of mutations seen in the antigen-specific splenic memory cell population was again distinct from that seen in the bone marrow AFC population at day 35 (Table I and Figure 4C and D), comprising cells with a higher average number of mutations per VH gene and without the subpopulation of highly selected but sparsely mutated genes seen in the AFCs of the bone marrow. Somatic mutation and selection in day 14 AFCs in the spleen Our examination of bone marrow AFCs at days 14 and 35 post immunization had revealed a unique pattern of somatic hypermutation in the VH genes of a fraction of these cells. If these cells arose as a consequence of somatic mutation and selection in the GC, they should be detectable in organs containing GCs, such as the spleen. We therefore examined the distribution of somatic mutations in the VH genes of NP-specific IgG1+ AFCs isolated from the spleen 14 days after immunization. Single AFCs were sorted and VH186.2 containing rearrangements amplified from cDNA and sequenced as before. A summary of these sequences is presented in Figure 5 and Table I. Fo