T15-Idiotype-Negative B Cells Dominate the Phosphocholine Binding Cells in the Preimmune Repertoire of T15i Knockin Mice

Phosphocholine (PC)³ is a major epitope present on a variety of microorganisms that infect man, including Streptococcus pneumoniae (1). PC functions in the pathogenesis of S. pneumoniae by facilitating transport of the bacterium across epithelial and endothelial membranes (2, 3), and PC is also a major target of the immune response to S. pneumoniae (4, 5). Anti-PC Abs are highly protective against lethal pneumococcal infection both in vivo and in passive transfer assays (4, 5, 6, 7, 8). In mice, the primary immune response to PC is dominated by a single clone of B cells expressing the germline T15H chain (V1 gene) and κ22–33 L chains (9, 10, 11, 12, 13, 14). These T15-Id⁺ anti-PC Abs have been shown to provide optimal protection against infection with S. pneumoniae (15). Additional families of anti-PC Abs (M603, M167/M511, and D16) have been characterized (16, 17, 18, 19, 20). The H chain variable region of all these Abs is encoded by the V1 gene (14), but each variant expresses a different amino acid at its V:D junction (14, 16, 17, 18, 19, 20, 21). In addition, each H chain pairs with a different L chain to produce a PC-binding Ab. The T15 (Asp⁹⁵H at V:D junction) H chain associates with a κ22–33 L chain, the M603 (Asn⁹⁵H) H chain associates with a κ8–28 L chain, the M167 (Ala⁹⁶H) H chain pairs with a κ24 L chain, and the D16 (Gly⁹⁵H) H chain associates with a κ1 L chain. The germline T15H chain can also associate with the κ8–28 L chain to create a low-affinity anti-PC Ab (21, 22); however, expression of this clone is not seen in the anti-PC response of normal mice. A T15H:κ8–28L hybridoma was cloned from anti-T15-Id suppressed mice (22), and several anti-PC hybridomas from terminal deoxynucleotide transferase (TdT) transgenic mice express T15:κ8–28 H and L chains (23).

We have previously analyzed the development of PC-specific B cells in transgenic mice expressing M167H, M603H, or T15H transgenes (24). In the M167H mice, PC-specific B cells expressing κ24 L chains are present at a level 100–500 times higher than that expected from random association of the transgene-encoded M167 H chain with all possible L chains. These M167-Id⁺, PC-specific B cells were selectively amplified by an Ag-driven, receptor-mediated process. PC-specific B cells expressing the M167 H chain in association with L chains other than κ24 were not detected (24, 25). In contrast, analysis of PC-specific B cells in homozygous T15i knockin (KI) mice showed that 5–10% of the V_H1-Id⁺ B cells bound PC but only 20% of these PC-specific B cells expressed the κ22-dependent T15-Id (24). Thus, in this T15H chain transgenic mouse, T15-Id⁺ B cells do not dominate the preimmune PC-binding B cell population. It was possible that the T15-Id⁻, PC-specific B cells represented a single dominant clone such as the low-affinity T15H:κ8–28L clone, or that this population was multiclonal. To elucidate the identity and number of L chains expressed in the T15-Id⁻, PC-specific B cell population, individual PC-dextran (dex)-binding B cells were sorted by FACS and grown in culture, and the L chains were amplified by RT-PCR of extracted RNA and then sequenced. Because T15-Id⁺ B cells dominate the primary immune response in most mice, it was also of interest to determine whether or not the T15-Id⁻, PC-specific B cells were functional in vivo. T15i KI mice were immunized with 6-(O-PC) hydroxyhexanoate (EPC)-conjugated keyhole limpet hemocyanin (KLH) and the Ab-secreting cells (ASC) characterized with respect to their idiotype.

T15i KI mice (26) were obtained from Dr. K. Rajewsky (University of Cologne, Cologne, Germany) through Dr. H. Gu (National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD). They were backcrossed to C57BL/6 mice for 10 generations and then inbred to obtain homozygous T15i/T15i mice (T15i KI). κ22–33 transgenic mice were produced by injection of cloned κ22–33 L chain DNA (27) into oocytes from B6C3HF2 mice and transgene-positive mice were detected by PCR analysis of tail DNA using 5′ V_κ22 and 3′ J_κ5 primers. A single transgene-positive founder, which expressed κ22–33 L on peripheral blood B cells, was selected by staining with anti-T15-Id Ab from clone T139.2 (28). This mouse was backcrossed to C57BL/6 mice to establish the κ22–33 L line. κ8–28 transgenic mice were obtained from J. L. Claflin (University of Michigan, Ann Arbor, MI) and were produced by the University of Michigan Transgenic Animal Model Core (Ann Arbor, MI). One founder expressing four to five copies of the transgene was backcrossed to BALB/c mice. Both the κ22–33 and κ8–28 L chain transgenic mice were crossed to T15i KI mice and F₁ mice carrying both the H and L transgenes selected by staining peripheral blood B cells with PE-conjugated anti-V_H1-Id Ab (hybridoma T68.3) (28) and FITC-conjugated PC-dex. F₁ H and L chain mice were crossed to obtain progeny homozygous for the T15H chain that also expressed a transgene-encoded L chain. These T15i × κ22 and T15i × κ8 mice were inbred with selection of progeny producing PC-dex-binding B cells at each generation.

Single spleen cell suspensions were prepared and stained as previously described (25, 29). PE conjugation of anti-V_H1-Id and anti-T15-Id was conducted by Molecular Probes (Eugene, OR). FITC-conjugated PC-dex was a gift of Dr. H. Dintzis (The Johns Hopkins University, Baltimore, MD) and was used at a final concentration of 2.5 μg/10⁶ cells. The synthesis of PC-dex was as previously described (30). Stained cells were analyzed on a FACScan flow cytometer using CellQuest software (BD Immunocytometry Systems, San Jose, CA).

Spleen cells for FACS sorting were prepared from T15i KI mice and stained with FITC-PC-dex. These cells were subsequently reacted with anti-FITC-paramagnetic colloidal particles (Miltenyi Biotec, Bergisch Gladbach, Germany) and enriched on a magnetic selection column. The eluted cells were then stained with PE-anti-T15-Id Ab. Cell sorting was performed using the FACStar^Plus outfitted with an automatic cell deposition unit (BD Biosciences). Single cells were directly sorted into 96-well plates containing EL-4 cells (see EL-4-based B cell culture). The two populations obtained with this technique were T15-Id⁺PC-dex⁺ and T15-Id⁻PC-dex⁺ B cells.

The EL-4-based human B cell culture system (31, 32, 33, 34, 35) was modified by Q.-S. Mi (data not shown) and used for the generation of single PC-specific B cell clones derived from T15i KI mice. Briefly, individual wells of 96-well flat-bottom plates were set up with 50,000 irradiated (5,000 cGy) murine EL-4 thymoma cells (clone B5; kindly provided by Dr. J. Crowe, Vanderbilt University, Nashville, TN, with the permission of R. H. Zubler, Geneva University Hospital Geneva, Switzerland) in 200 μl of RPMI 1640 medium supplemented with 10% FCS, 10⁻⁵ M 2-ME , 25 mM HEPES buffer, penicillin (100 U/ml), streptomycin (100 μg/ml), 10 μg/ml LPS, and 10% supernatant from culture of J774A.1 macrophage cells (no. TIB-67; American Type Culture Collection, Manassas, VA). Single PC-specific B cells were sorted by FACS directly onto a feeder layer of irradiated EL-4 cells and cultured at 37°C in 5% CO₂. The culture supernatants were collected on day 10 and tested for anti-PC Ab production and total IgM by ELISA. Those wells containing PC-binding Abs were tested for the presence of T15-Id⁺ Abs.

Anti-PC Abs produced by single B cell clones in EL-4 B cell cultures were measured by an ELISA assay as described previously (7). In brief, each culture supernatant was added to duplicate wells (50 μl/well) of microtiter plates which had been coated with 100 μl of EPC-BSA (5 μg/ml) and blocked with 5% Norland Hi-pure liquid gelatin (Norland Products, New Brunswick, NJ) in PBS with 0.1% NaN₃. The plates were incubated overnight at room temperature. After washing three times with TBS plus Tween 20, PC-BSA-bound Abs were detected using biotinylated goat anti-mouse IgG and IgM (American Type Culture Collection and Fisher Biotech (Silver Spring, MD)) or biotinylated anti-T15-Id (25) followed by streptavidin-conjugated alkaline phosphatase (Calbiochem, La Jolla, CA.). After addition of 100 μl of p-nitrophenyl phosphate (1 mg/ml; Sigma-Aldrich, Saint Louis, MO) in 1 M diethanolamine buffer (pH 9.8), the plates were read on a Bio-Tek ELx800 ELISA reader (Bio-Tek Instruments, Winooski, VT) at 405 nm and Ab values were determined from internal IgM anti-PC standards included on each plate.

Mice were immunized i.p. with 100 μg of EPC-KLH in CFA. Five days postimmunization, single spleen cell suspensions were prepared and assayed for anti-PC ASC using an ELISPOT assay (36). In brief, cells were adjusted to 4 × 10⁶/ml in RPMI 1640 medium, and four 1–10 serial dilutions were made. Fifty microliters of cells at each dilution were put into duplicate wells of flat-bottom 96-well plates that had been coated with 100 μl of EPC-BSA (5 μg/ml) and blocked with BSA. Plates were incubated at 37°C for 4–5 h in a 5% CO₂ incubator, washed five times with PBS containing 3% BSA and 0.05% Tween 20, and developed by the addition of biotin-labeled Abs for 1 h at room temperature, followed by avidin-alkaline phosphatase for 1 h at room temperature. Final color development was achieved using 5-bromo-4-chloro-3-indolyl phosphate (Sigma-Aldrich) in 2-amino-2-methyl-1-propanol buffer according to the manufacturer’s recommendations.

Colonies of PC-specific B cells were expanded from single cells by in vitro culture as described above. The cells were pelleted and frozen at −70°C until used. Total RNA was extracted from the frozen pellets using the RNeasy Mini kit (Qiagen, Valencia, CA) according to the manufacturer’s recommended procedure. The RNA was eluted in a final volume of 60 μl of diethyl pyrocarbonate-treated water. cDNA was prepared and V_κ genes were simultaneously amplified using the SuperScript One Step RT-PCR System (Life Technologies, Gaithersburg, MD). The RT-PCR was composed of 20 μl of purified RNA template, 25 μl of 2× reaction mix, 10 pmol each of primers MsV_κM and M13-MsC_κ2 (M13-MsC_κ2 is comprised of sequences homologous to the V_κ-constant region and −21 M13 primer subsequently used for direct sequencing of the amplified cDNA (37)), 1 μl of SuperScript RT/Taq Mix and diethyl pyrocarbonate-treated water to a final volume of 50 μl. RT-PCR were performed with a TouchDown thermocycler (Thermo Hybaid, Middlesex, U.K.) equipped with a 96-well block and heated lid. The cycling conditions were 50°C for 30 min and 94°C for 2 min for the reverse transcription reaction followed by 40 cycles of 90°C for 30 s, 50°C for 1 min and 72°C for 1 min with a final extension at 72°C for 10 min. The 50-μl RT-PCR were purified using the Strata-Prep PCR purification kit (Stratagene, La Jolla, CA) as described in the accompanying instructions and the PCR product was eluted with 50 μl of 10 mM Tris (pH 8.5). The presence of the expected size product was confirmed by ethidium bromide staining of gels, and one-tenth of the eluted sample (5 μl) was sequenced using the ABI PRISM Dye Terminator Cycle Sequencing Ready Reaction kit (PE Applied Biosystems, Foster City, CA) and 3.2 pmol of the −21 M13 primer. Excess dye terminators were removed by passing the samples through Centri-Sep columns (Princeton Separations, Adelphia, NJ). The purified sequencing reactions were electrophoresed and sequencing data were collected using an ABI 373A DNA Sequencer (PE Applied Biosystems).

Sequence data were imported into the Vector NTI Suite (InforMax, Bethesda, MD). Comparison of the sequences to each other and to the GenBank European Molecular Biology Laboratory databases identified three homology groups. In addition, comparison of both the individual sequences and derived consensus sequence from the aligned homology groups to GenBank European Molecular Biology Laboratory allowed us to identify the V_κ gene family to which the individual sequences and derived consensus sequences were members. Sequences within each of the groups were aligned and the original histograms were reexamined to identify and resolve disparate nucleotides. At those few sites where ambiguous or conflicting nucleotides were observed, the sequences were compared with the corresponding germline sequences. If histogram peaks consistent with the germline sequence were observed at that base, the sequence was adjusted to match the germline sequence.

We have recently shown that T15-Id⁻ B cells dominate the preimmune PC-binding population of B cells in the spleens of homozygous T15i KI mice (24). Because the T15H chain expressed in these mice can form a PC-binding B cell with either a κ22–33 or κ8–28 L chain, T15i KI spleen cell FACS profiles were compared with those of control T15i × κ22 and T15i × κ8 transgenic mice (Fig. 1,A). Analysis of spleen cells for surface IgM (Fig. 1, AA–AC) and V_H1-Id (Fig. 1, AD–AF) expression showed that 25–30% of cells in all three types of transgenic mice were surface IgM⁺ and V_H1-Id⁺. When the V_H1-Id⁺ B cells were analyzed for their ability to bind PC, 2.3% of the spleen cells in the T15i KI mice bound PC-dex (Fig. 1,AD). As expected, all theV_H1-Id⁺ B cells in T15i × κ22 (Fig. 1,AE) and T15i × κ8 (Fig. 1,AF) mice bound PC-dex. Further analysis of the PC-dex-binding B cells for the presence of the κ22-dependent T15-Id showed that 13% of the PC-specific B cells in T15i KI mice were T15-Id⁺ (Fig. 1,AG). In T15H × κ22 mice, 100% of the PC-dex-binding cells carried the T15-Id (Fig. 1,AH), whereas none of the cells in T15H × κ8 mice displayed this Id (Fig. 1,AI). The absolute number of PC-specific and the absolute number of T15-Id⁺, PC-dex-binding B cells present in each of the three transgenic mice is shown in Fig. 1 B. The above data strongly suggest that PC-specific receptors in T15i KI mice are being produced by the T15H chain in association with L chains other than κ22–33.

To directly assess the usage of L chains by the T15-Id⁻, PC-specific B cells in T15i KI mice, an in vitro B cell culture system was used to generate single B cell clones. The FACS profile for the enriched FITC-PC-dex-binding B cells is shown in Fig. 2,A. Single T15-Id⁻ and T15-Id⁺, PC-specific B cells were sorted into individual wells and cultured, and supernatants were analyzed by ELISA for the presence of IgM Abs and for PC-specific Abs. A total of 211 of the 768 wells (27%) from sorted T15-Id⁻ cells were positive for IgM Abs (Fig. 2,B), and 107 of these IgM⁺ wells (51%) contained anti-PC Abs. Fourteen percent of the total wells produced anti-PC Abs (Fig. 2,B). Fifty-one PC-specific clones were selected for idiotype analysis and L chain amplification. Three of these PC-binding clones expressed the T15-Id. Analysis of cultures from sorted T15-Id⁺, PC-specific B cells revealed that 153 of the 384 wells (40%) tested were positive for IgM Abs (Fig. 2,B) and 95 of these wells (62%) produced anti-PC Abs (Fig. 2,B). Twenty-five percent of the total wells made anti-PC Abs (Fig. 2 B). Of the 52 anti-PC clones selected for idiotype and L chain analysis, 49 (94%) were T15-Id⁺. The mean value for the amount of anti-PC Ab produced by T15-Id⁺ colonies was higher than that produced in T15-Id⁻ wells (1.7 ± 1.1 vs 1 ± 0.85 OD), but this difference was not statistically significant. There was no difference in the total IgM produced by T15-Id⁺ and T15-Id⁻ colonies.

The expressed V_κ L chains in PC-specific B cells from the single cell cultures were determined by RT-PCR amplification of extracted RNA and direct sequencing of the amplified products. The V_κ primer used for L chain amplification has been described previously and was demonstrated to amplify V_κ genes from 14 different V_κ gene families representative of seven different V_κ gene subgroups (37). RNA was extracted from 52 colonies sorted as T15-Id⁺ and 44 colonies from the T15-Id⁻ PC-binding cells. One-step RT-PCR amplification of the extracted RNA yielded an appropriately sized band on ethidium bromide-stained gels from all of the colonies examined. Table I provides a summary of the frequency and identity of the V_κ genes expressed in the selected clones from the T15-Id⁺- and T15-Id⁻-sorted cells. Sequence data were obtained from 47 of 52 T15-Id⁺-sorted colonies and 39 of 44 T15-Id⁻-sorted colonies. Forty-four of the 47 colonies from the T15-Id⁺-sorted cells expressed transcripts displaying 100% or near 100% identity with the germline V_κ22–33 gene (GenBank accession no. MMU235965) (38). The remaining three colonies expressed transcripts displaying 98% identity with the germline V_κ19–15 gene (GenBank accession no. MMY15976) (39). V_κ gene transcripts in 25 of the 39 colonies from T15-Id⁻-sorted cells displayed 100% identity to the germline V_κ8–28 gene (GenBank accession no. MMU235947) (38). Thirteen of the 39 colonies from T15-Id⁻-sorted cells expressed transcripts with 98% identity to the germline V_κ19–15 gene. The remaining colony examined expressed a germline V_κ22–33 gene. The single V_κ22–33 L chain-expressing colony identified among the colonies sorted as T15-Id⁻ was determined to be T15-Id⁺ by ELISA and most likely represents a sorting error. The three colonies which were sorted as T15-Id⁺, whose supernatants were determined to contain T15-Id⁺ Ab but were found to express the V_κ19–15, can be explained if one assumes that the initial cell(s) sorted into the well expressed both the V_κ22–33 and V_κ19–15 genes. This would result in the supernatants being T15-Id⁺ by ELISA. If the V_κ22–33-expressing component were subsequently lost during culture, then direct sequencing of cDNA from the colony would identify the remaining V_κ gene, which in these cases were V_κ19–15. The few clones not included in the V_κ gene analysis were excluded because the resulting sequencing histograms showed evidence of more than one sequence in the amplified cDNA. With one exception, V_κ gene transcripts from all of the PC-specific colonies, regardless of Id or V_κ gene expressed, were spliced to the J_κ5 gene and the observed J_κ5 sequences displayed 100% identity to the germline J_κ5 gene. The single exception, a T15-Id⁺ colony, had its V_κ22–33 gene spliced to the J_κ2 gene. The spliced J_κ2 gene displayed 100% identity to the germline J_κ2 gene.

Although T15-Id⁻ B cells dominate the PC-binding cell population in T15i KI mice, it was of interest to determine whether or not these T15H:κ8–28L and T15H:κ19–15L B cells would dominate the immune response to PC in vivo. To analyze the immune response to PC in T15i KI mice, mice were immunized i.p. with 100 μg of EPC-KLH in CFA. T15i × κ22 and T15i × κ8 mice were also immunized and used as controls for T15-Id⁺ and T15-Id⁻ B cell responses. Five days after immunization, spleen cells were analyzed in ELISPOT for the number of B cells secreting Abs specific for PC and for the number of B cells secreting Abs bearing V_H1 and T15 Id (Table II). The T15i × κ22 control mice produced the largest response with 7.5 × 10⁵ ASC/spleen; ∼2% of all B cells were secreting V_H1-Id⁺ Abs. As expected, there was no statistical difference (p = 0.62) between the number of V_H1-Id⁺ and T15-Id⁺ ASC in these double-transgenic mice. The T15i × κ8 control mice produced a very low response, having only 1.8 × 10⁴ ASC or 0.03% of the PC-specific B cells secreting V_H1-Id⁺ Abs. This response is 3-fold lower than the response seen in the normal C57BL/6 control (Table II) and only double that seen in an unimmunized T15i × κ8 mouse. None of these ASC produced T15-Id⁺ Abs. When the immune response was analyzed in T15i KI mice, ∼1.8 × 10⁵ ASC/spleen were detected, and these ASC produced T15-Id⁺ Abs. There was no indication that the T15-Id⁻ PC-specific B cells were giving rise to any ASC following immunization with EPC-KLH. However, it was possible that the T15-Id⁻, PC-dex-binding B cells were undergoing clonal expansion without terminally differentiating into an ASC. Spleen cells from immune and nonimmune T15i KI, T15i × κ22, T15i × κ8, and C57BL/6 control mice were stained with PC-dex and anti-T15-Id Abs to determine whether or not T15-Id⁻, PC-specific B cells had expanded following immunization (Fig. 3). The data in Fig. 3, A and E, show that in T15i KI mice T15-Id⁺, PC-specific B cells expand 6-fold following immunization, whereas there is no change in the number of T15-Id⁻, PC-specific B cells. There was also no change in PC-binding cells seen in either T15i × κ22 or T15i × κ8 mice, while a detectable increase in PC-specific B cells was seen in the C57BL/6 control mice (Fig. 3, D and H).

The data presented in this manuscript confirm and extend our previous observation that the PC-specific B cells present in unimmunized homozygous T15i KI mice are predominantly T15-Id⁻ (24). Less than 20% of the PC-dex-binding cells in these H chain-only transgenic mice expressed receptors bearing the T15-Id. Analysis of the L chains expressed in the T15-Id⁻, PC-binding B cells revealed that two L chains, V_κ8–28 and V_κ19–15, are present in this population. Failure to identify V_κ24-expressing colonies is not unexpected, because while the V_κ24 L chain can associate with the T15H chain, the resulting Ab does not bind PC (21). Anti-PC Abs using the V_κ8–28 L chain dominate the immune response to PC on the LPS of Proteus morganii (16, 17), and V_κ8–28-bearing anti-PC Abs are induced in xid mice in response to EPC-KLH (8). In these Abs, the V_κ8–28 L chain is associated with the M603-like (Asn⁹⁵H) variant of the V1 H chain. PC-specific Abs expressing the V_κ8–28 L chain in conjunction with the germline T15 (Asp⁹⁵H) H chain have only been observed in T15-Id-suppressed mice (22) and in S. pneumoniae R36A-immunized mice constitutively expressing TdT (23). PC-specific Abs using a V_κ19–15 L chain have not been previously described.

To better understand our observations regarding PC binding and the lack of a response of T15-Id⁻, PC-binding cells in vivo to EPC-KLH we have analyzed the aligned derived amino acid sequences for the three V_κ genes expressed in the PC-binding B cells of T15i KI mice (Fig. 4). This analysis identifies regions that share a high degree of sequence identity as well as regions that do not. The biological significance of some of these regions becomes more obvious when considered in the context of 1) specific V_κ amino acid residues that interact with the hapten PC, 2) the interplay between V_κ and V_H chain residues and how this interplay can alter the required properties of the pocket within which PC is bound, and 3) other domains whose interaction with determinants of the carrier molecule can alter the avidity of the Ab/Ag complex. To help visualize these interactions, Fig. 5 presents a model of the regions of the V_κ8–28 gene we will discuss and their relationship to the bound PC. To reduce the complexity of the overall image we have extracted the regions of interest from the complete three-dimensional structure of the McPC603 Fab-PC complex (Brookhaven Protein Data Bank accession no. 2 MCP).

The first observation that we believe can be explained by examining the structure of PC-binding Abs is that, with one exception, all of the PC-binding clones analyzed in this study and virtually all other anti-PC-binding Abs use J_κ5, regardless of which V_κ gene is being used. The overwhelming use of J_κ5 in PC-binding Abs can be understood when we consider that J_κ5 is the only J_κ region that can provide the invariant leucine residue observed at the V_κ-J_κ junction. Mutagenesis studies have demonstrated that this Leu¹⁰², observed at the bottom of the PC-binding pocket in Fig. 5, is essential for PC binding and that substitution of a Tyr or Phe for the Leu resulted in a loss of Ab binding to PC affinity columns (40). Additional support for the requirement of a Leu at the V_κ-J_κ junction of PC-binding Abs is that in the two examples of PC-binding Abs that do not use J_κ5 (a V_κ22-J_κ2 (this study) and V_κ22-J_κ1 (41)) a codon coding for Leu is observed at the V_κ-J_κ junction. The leucines in these two examples are most likely coded for by the CTC 3′ of the Pro codon of V_κ22–33.

Next we consider the observation that from the FACS data it is clear that the T15H:V_κ8–28L and T15H:V_κ19–15L B cells dominate the PC-binding population in the spleens of preimmune T15i KI mice. However, when these mice were immunized with EPC-KLH, only T15-Id⁺ ASC were detected. Thus, T15-Id⁺ B cells dominated the functional primary immune response in T15i KI mice. These data are consistent with the description of the PC response in adult mice as examined using the splenic fragment assay (13, 42, 43). However, because the idiotype of PC-binding cells from unstimulated splenocytes has not been analyzed in normal mice, we cannot compare the T15-Id⁺:T15-Id⁻ ratio seen in the T15i KI mice to that of a normal C57BL/6 mouse. The failure of the T15H:V_κ8–28L and T15H:V_κ19–15L B cells to respond to immunization with EPC-KLH might be because they have been tolerized. However, this seems unlikely because 1) they were able to expand and differentiate into ASC when stimulated in vitro in an Ag-independent culture system; 2) we observed a small ASC response following EPC-KLH immunization of T15i × V_κ8 mice; and 3) in T15-Id-suppressed mice (22) and TdT transgenic mice (23) (both of which lack T15-Id⁺ B cells) B cells expressing T15H:V_κ8–28 L chains have been observed after immunization with S. pneumoniae R36A. A more likely explanation for the lack of an in vivo response by T15H:V_κ8–28 and T15H:V_κ19–15 B cells would be that the low relative affinity of these Abs for PC or the low avidity for the PC plus carrier determinants would not allow them to compete with the T15H:V_κ22L-expressing B cells. We can understand the role of the specific V_κ gene in determining the affinity/avidity of the PC-binding Abs for PC and PC conjugates by examining the PC binding pocket shown in Fig. 5. Fig. 5 presents the structure of the V_κ8L chain from the McPC603 Ab and it reveals a ring of amino acid residues surrounding the trimethyl ammonium group of PC. The aligned sequences for the V_κ genes in Fig. 4 reveal that these amino acids (DHSYP for V_κ8–28) are the terminal V_κ residues contributing to the V_κ complementarity determining region (CDR)3. Of particular interest is the Asp residue at 97L which sits at the bottom of the PC binding pocket and is found in V_κ8–28L but is absent in both the V_κ22–33L and V_κ19–15L PC-binding Abs. This residue illustrates the complementary nature of residues contributed by V_H and V_L to the PC-hapten binding pocket. For the V_κ8L chain in the McPC603 Ab, this Asp interacts electrostatically with the nitrogen of the choline moiety of PC and hydrogen bonds to the Asn at 95H (44, 45, 46). PC-binding Abs that use the V_κ8–28 L chain are generally found associated with the M603H variant of the V1 gene. One difference between the germline T15H chain (expressed by all of the B cells in the T15i KI mice) and the M603H chain is an Asp/Asn substitution at the D_H:J_H junction. In PC-binding Abs that do not use the V_κ8–28 L chain, this T15H-encoded Asp⁹⁵H residue sits in the PC binding pocket and interacts similarly to the Asp⁹⁷L of V_κ8–28 L with the nitrogen of the PC moiety. Mutagenesis studies have demonstrated the necessity for a single Asp group in the PC binding pocket (40), which can be contributed by either the V_H or V_κ chains. However, the T15H:V_κ8–28L combination places two negatively charged Asp residues within the PC binding pocket. The presence of an Asp residue at both 95H and 97L destabilizes the choline binding pocket and results in a dramatic decrease in the relative affinity and avidity of these Abs for PC and PC conjugates (21, 22, 40, 47, 48). So for the T15H:V_κ8–28L B cells, the inability to respond to immunization with EPC-KLH or to compete with B cells expressing T15H:V_κ22–33L receptors would be due to the >10-fold lower affinity of their binding sites for PC than the binding sites on T15H:V_κ22–33 B cells.

In this study, ∼30% of the PC-dex binding cells expressed the V_κ19–15L chain. Why haven’t B cells expressing the T15H:V_κ19–15L Ab been observed among PC-binding Ab before, and why didn’t the T15H:V_κ19–15L-expressing B cells respond to immunization with EPC-KLH? A possible answer to these questions may be found by comparing the derived amino acid sequence of V_κ19–15L chain to that of V_κ8–28L and V_κ22–33L then extrapolating the differences onto the three-dimensional structure of the McPC603 Ab and considering those differences in the context of the two-component site concept proposed by Andres et al. (49). A comparison of the CDR3 sequences for the V_κ19–15L and V_κ22–33L Abs reveals that they are very similar and it would be predicted that the relative affinity of these two Abs for PC should be similar. If we assume that the homology within the PC binding pocket observed between the V_κ19–15 and V_κ22–33 proteins reflects an equivalent affinity for the PC moiety itself, then the difference in response to a particular PC carrier conjugate must come from somewhere else. Several studies have demonstrated that changes in the V_H CDR2 loop of anti-PC Abs can alter their avidity for PC conjugates by affecting their interaction with the carrier molecule (17, 50, 51). A similar study with phenylphosphocholine-specific Abs has shown that carrier determinants interacting with both the V_H and V_L chains contribute to the overall avidity of the Ab/PC conjugate complex (52). In addition, these authors and others have proposed that the increased avidity that interaction with carrier molecules would provide could generate changes in the selected B cell repertoire and even allow for initiation of a mature, high-affinity immune response from populations of cells that originally have very low or undetectable affinities for the simple hapten (47, 48, 52). Examination of the aligned derived amino acid sequences in Fig. 4 and the model presented in Fig. 5 reveals that V_κ19–15L lacks a string of six amino acids (amino acids 33–38). This string of amino acids forms an exposed loop for the V_κ8–28 L chain and presumably also for the V_κ22–33 L chain (51). If this exposed loop of the V_κ chain was necessary for interaction with the carrier molecule of PC-conjugates, then V_κ19–15L would not be able to form these additional bonds and therefore would have a significantly lower overall avidity for PC conjugates. Therefore, even though V_κ19–15 is able to pair with the germline T15H and the T15H:V_κ19–15L Ab could bind PC, B cells expressing T15H:V_κ19–15L would not be able to compete with T15-Id⁺ B cells in a normal response to EPC-KLH.

Analysis of the frequency of V_κ gene usage in the PC-dex binding cells was performed assuming that V_H and V_L chain association is stochastic as suggested by Kaushik et al. (53). A conservative estimate of frequencies of gene usage in our experiments was made by multiplying the observed percentage of cells expressing a particular V_κ gene by the frequency of PC-positive cells observed in the EL-4 cultures (0.62 for T15-Id⁺-sorted cells and 0.51 for the T15-Id⁻-sorted cells). Of the total B cells present in T15i mice, PC-specific cells expressing V_κ22–33, V_κ8–28, or V_κ19–15 genes represent 0.74, 2.5, and 1.4%, respectively. In LPS-stimulated B cells from normal mice, the frequency of expression of these V_κ genes is calculated to be 0.18, 0.95, and 0.96%, respectively (53, 54). Therefore, among the PC-binding cells, usage of these specific V_κ genes is 1.5- to 4.1-fold greater than that observed in the total B cell population. However, virtually all of the PC-dex binding colonies, irrespective of the V_κ gene used, were rearranged to J_κ5, whereas, in LPS-activated B cells, 20% of the colonies express J_κ5 (53, 54). This means that the V_κ19–15:J_κ5, V_κ8–28:J_κ5, and V_κ22–33:J_κ5 genes are in fact expressed at a 7-, 13-, and 21-fold higher frequency in PC-binding cells than expected in the total B cell population. These data strongly suggest that the PC-specific B cells are being clonally expanded by an environmental or endogenous PC-containing Ag as seen previously in transgenic mice expressing the M167 and M603 H chains (24).

We have described V_κ gene usage among PC-binding B cells in the T15i KI mouse. Surprisingly, we observed that B cells expressing the T15H:V_κ22–33L were not the dominant population in the spleen of unimmunized T15i KI mice. We also identified a population expressing T15H:V_κ19–15L which has not previously been described. An examination and comparison of the V_κ L chains used identified correlations between structural determinants of the V_κ L chains and the relative affinity of these Abs for PC and/or avidity for the EPC-KLH conjugate. Finally, in the unimmunized T15i KI mice, there appears to be a strong positive selection of specific structural determinants, particularly at the V_κ-J_κ junction of the V_κ L chains, into the PC-binding B cell population. This selection is most likely being driven by interaction with some self or ubiquitous environmental Ag.