T15i knockin (KI) mice express a H chain that is encoded by a rearranged T15 VDJ transgene which has been inserted into the JH region of chromosome 12. This T15H chain combines with a κ22–33 L chain to produce a T15-Id+ Ab having specificity for phosphocholine (PC). Inasmuch as T15-Id+ Abs dominate the primary immune response to PC in normal mice, it was surprising to find that 80% of the PC-dextran-binding B cells in unimmunized homozygous T15i KI mice were T15-Id. Analysis of L chains expressed in these T15-Id, PC-specific B cells revealed that two L chains, κ8–28 and κ19–15, were expressed in this population. The Vκ region of these L chains was recombined to Jκ5, which is typical of L chains present in PC-specific Abs. When T15i KI mice were immunized with PC Ag, T15-Id+ B cells expanded 6-fold and differentiated into Ab-secreting cells. There was no indication that the T15-Id B cells either proliferated or differentiated into Ab-secreting cells following immunization. Thus, T15-Id B cells dominate the PC-binding population, but they fail to compete with T15-Id+ B cells during a functional immune response. Structural analysis of T15H:κ8–28L and T15H:κ19–15L Abs revealed L chain differences from the κ22–33 L chain which could account for the lower affinity and/or avidity of these Abs for PC or PC carrier compared with the T15-Id+ T15H:κ22–33L Ab.

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 VH1-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′ Vκ22 and 3′ Jκ5 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-VH1-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-VH1-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 FACStarPlus 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-IdPC-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 Vκ 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 MsVκM and M13-MsCκ2 (M13-MsCκ2 is comprised of sequences homologous to the Vκ-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 Vκ 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, AAAC) and VH1-Id (Fig. 1, ADAF) expression showed that 25–30% of cells in all three types of transgenic mice were surface IgM+ and VH1-Id+. When the VH1-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 theVH1-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.

FIGURE 1.

T15-Id B cells dominate the PC-specific B cell repertoire in the spleen of T15i KI mice. A, Idiotype analysis of spleen cells in T15i KI (AA, AD, and AG), T15-Id × κ22 (AB, AE, and AH) and T15i × κ8 (AC, AF, and AI) mice. Spleen cells from the three T15i KI lines were stained with a combination of 1) FITC-anti-IgM and PE-anti-VH1 (AAAC); 2) FITC-PC-dex and PE-anti-VH1 (ADAF); or 3) FITC-PC-dex and PE-anti-T15-Id Abs (AGAI) and analyzed by flow cytometry as described in Materials and Methods. The percentages of cells within the boxed regions are based on the total number of cells analyzed. One representative experiment of five is shown. B, Absolute number of T15-Id+ and PC-binding B cells in the spleen of T15i KI, T15i × κ22, and T15i × κ8 mice. The calculated numbers of Id+ cells are based on total cell counts and the percentages of Id+ cells were determined by FACS analysis. Data shown are expressed as mean ± SD of 5–10 mice of each strain.

FIGURE 1.

T15-Id B cells dominate the PC-specific B cell repertoire in the spleen of T15i KI mice. A, Idiotype analysis of spleen cells in T15i KI (AA, AD, and AG), T15-Id × κ22 (AB, AE, and AH) and T15i × κ8 (AC, AF, and AI) mice. Spleen cells from the three T15i KI lines were stained with a combination of 1) FITC-anti-IgM and PE-anti-VH1 (AAAC); 2) FITC-PC-dex and PE-anti-VH1 (ADAF); or 3) FITC-PC-dex and PE-anti-T15-Id Abs (AGAI) and analyzed by flow cytometry as described in Materials and Methods. The percentages of cells within the boxed regions are based on the total number of cells analyzed. One representative experiment of five is shown. B, Absolute number of T15-Id+ and PC-binding B cells in the spleen of T15i KI, T15i × κ22, and T15i × κ8 mice. The calculated numbers of Id+ cells are based on total cell counts and the percentages of Id+ cells were determined by FACS analysis. Data shown are expressed as mean ± SD of 5–10 mice of each strain.

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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.

FIGURE 2.

Generation of single T15-Id and T15-Id+ PC-specific B cell clones using an EL-4 B cell culture system. A, Isolation of single T15-Id and T15-Id+ PC-specific B cells. Spleen cells from T15i KI mice were enriched for PC-binding B cells by magnetic cell separation and subsequently stained for T15-Id expression. Single T15-Id+ and T15-Id PC-binding B cells within the respective gates shown were directly sorted into individual wells of 96-well plates. Each well had 200 μl of medium containing 50,000 irradiated EL-4 cells, 10 μg/ml LPS, and 10% macrophage supernatant. B, Percentage of wells which contain IgM Abs or PC-specific Abs in the EL-4 cultures.

FIGURE 2.

Generation of single T15-Id and T15-Id+ PC-specific B cell clones using an EL-4 B cell culture system. A, Isolation of single T15-Id and T15-Id+ PC-specific B cells. Spleen cells from T15i KI mice were enriched for PC-binding B cells by magnetic cell separation and subsequently stained for T15-Id expression. Single T15-Id+ and T15-Id PC-binding B cells within the respective gates shown were directly sorted into individual wells of 96-well plates. Each well had 200 μl of medium containing 50,000 irradiated EL-4 cells, 10 μg/ml LPS, and 10% macrophage supernatant. B, Percentage of wells which contain IgM Abs or PC-specific Abs in the EL-4 cultures.

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The expressed Vκ 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 Vκ primer used for L chain amplification has been described previously and was demonstrated to amplify Vκ genes from 14 different Vκ gene families representative of seven different Vκ 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 Vκ 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 Vκ22–33 gene (GenBank accession no. MMU235965) (38). The remaining three colonies expressed transcripts displaying 98% identity with the germline Vκ19–15 gene (GenBank accession no. MMY15976) (39). Vκ gene transcripts in 25 of the 39 colonies from T15-Id-sorted cells displayed 100% identity to the germline Vκ8–28 gene (GenBank accession no. MMU235947) (38). Thirteen of the 39 colonies from T15-Id-sorted cells expressed transcripts with 98% identity to the germline Vκ19–15 gene. The remaining colony examined expressed a germline Vκ22–33 gene. The single Vκ22–33 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 Vκ19–15, can be explained if one assumes that the initial cell(s) sorted into the well expressed both the Vκ22–33 and Vκ19–15 genes. This would result in the supernatants being T15-Id+ by ELISA. If the Vκ22–33-expressing component were subsequently lost during culture, then direct sequencing of cDNA from the colony would identify the remaining Vκ gene, which in these cases were Vκ19–15. The few clones not included in the Vκ gene analysis were excluded because the resulting sequencing histograms showed evidence of more than one sequence in the amplified cDNA. With one exception, Vκ gene transcripts from all of the PC-specific colonies, regardless of Id or Vκ gene expressed, were spliced to the Jκ5 gene and the observed Jκ5 sequences displayed 100% identity to the germline Jκ5 gene. The single exception, a T15-Id+ colony, had its Vκ22–33 gene spliced to the Jκ2 gene. The spliced Jκ2 gene displayed 100% identity to the germline Jκ2 gene.

Table I.

Frequency of VκL chain gene usage in PC-specific splenic B cells from T15i KI mice

Vκ GenesSingle Anti-PC Ab-Secreting B Cell Clonesa
T15-IdT15-Id+
Vκ22 1 (3) 44 (94) 
Vκ25 (64) 0 (0) 
Vκ19 13 (33) 3 (6) 
Vκ GenesSingle Anti-PC Ab-Secreting B Cell Clonesa
T15-IdT15-Id+
Vκ22 1 (3) 44 (94) 
Vκ25 (64) 0 (0) 
Vκ19 13 (33) 3 (6) 
a

RNA from 39 clones derived from T15-Id PC-dex+-sorted cells and 47 clones derived from T15-Id+ PC-dex+-sorted cells was amplified by RT-PCR and sequenced as described in Materials and Methods. The percentage of total clones is listed in parentheses.

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 VH1 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 VH1-Id+ Abs. As expected, there was no statistical difference (p = 0.62) between the number of VH1-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 VH1-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).

Table II.

Number of ASC in immunized transgenic mice expressing a T15H chaina

StrainAnti-PC ASC (×10−4)/SpleenAnti-PC ASC (×10−3)/106 B Cells
VH1T15κ-chainIgM + IgGVH1T15κ-chainIgM + IgG
T15i KI 17.9 ± 4.2b 17.4 ± 5.4 18.1 ± 5.9 18.6 ± 5.4 7.1 ± 2.1 7.0 ± 2.8 7.3 ± 3.0 6.9 ± 2.4 
 (p = 0.85)c    (p = 0.98)    
T15i× κ22 75.1 ± 9.9 69.5 ± 6.7 71.5 ± 2.1 69.2 ± 7.1 19.4 ± 3.1 18.4 ± 1.5 18.5 ± 0.6 17.9 ± 1.8 
 (p = 0.62)    (p = 0.70)    
T15i× κ8 1.8 ± 0.5 1.9 ± 0.8 1.7 ± 0.5 0.3 ± 0.1 0.3 ± 0.2 0.3 ± 0.1 
C57BL/6 5.6 ± 1.9 3.9 ± 1.9 5.1 ± 1.5 4.6 ± 1.3 1.4 ± 0.6 1.0 ± 0.6 1.3 ± 0.5 1.2 ± 0.3 
StrainAnti-PC ASC (×10−4)/SpleenAnti-PC ASC (×10−3)/106 B Cells
VH1T15κ-chainIgM + IgGVH1T15κ-chainIgM + IgG
T15i KI 17.9 ± 4.2b 17.4 ± 5.4 18.1 ± 5.9 18.6 ± 5.4 7.1 ± 2.1 7.0 ± 2.8 7.3 ± 3.0 6.9 ± 2.4 
 (p = 0.85)c    (p = 0.98)    
T15i× κ22 75.1 ± 9.9 69.5 ± 6.7 71.5 ± 2.1 69.2 ± 7.1 19.4 ± 3.1 18.4 ± 1.5 18.5 ± 0.6 17.9 ± 1.8 
 (p = 0.62)    (p = 0.70)    
T15i× κ8 1.8 ± 0.5 1.9 ± 0.8 1.7 ± 0.5 0.3 ± 0.1 0.3 ± 0.2 0.3 ± 0.1 
C57BL/6 5.6 ± 1.9 3.9 ± 1.9 5.1 ± 1.5 4.6 ± 1.3 1.4 ± 0.6 1.0 ± 0.6 1.3 ± 0.5 1.2 ± 0.3 
a

Mice were immunized with 100 μg of EPC-KLH. Five days after immunization, the spleens were analyzed by ELISPOT assay for the number of anti-PC ASC expressing VH1-Id, T15-Id, κ L chains and IgM plus IgG Abs.

b

Values shown represent the mean ± SD.

c

Values of p were derived by comparing the number of VH1-Id+ ASC to the number of T15-Id+ ASC using Student’s t test.

FIGURE 3.

Increased fraction of T15-Id+ PC-binding B cells in the spleen of EPC-KLH-immunized T15i KI mice. Spleen cells from T15i KI (A and E), T15i × κ22 (B and F), T15i × κ8 (C and G), and C57BL/6 (D and H) mice immunized with EPC-KLH were stained 5 days after immunization with PC-dex-FITC and PE-anti-T15-Id Ab and analyzed by flow cytometry as described in Materials and Methods. Numbers indicate the percentage of the total number of cells analyzed. One representative experiment of five is shown.

FIGURE 3.

Increased fraction of T15-Id+ PC-binding B cells in the spleen of EPC-KLH-immunized T15i KI mice. Spleen cells from T15i KI (A and E), T15i × κ22 (B and F), T15i × κ8 (C and G), and C57BL/6 (D and H) mice immunized with EPC-KLH were stained 5 days after immunization with PC-dex-FITC and PE-anti-T15-Id Ab and analyzed by flow cytometry as described in Materials and Methods. Numbers indicate the percentage of the total number of cells analyzed. One representative experiment of five is shown.

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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, Vκ8–28 and Vκ19–15, are present in this population. Failure to identify Vκ24-expressing colonies is not unexpected, because while the Vκ24 L chain can associate with the T15H chain, the resulting Ab does not bind PC (21). Anti-PC Abs using the Vκ8–28 L chain dominate the immune response to PC on the LPS of Proteus morganii (16, 17), and Vκ8–28-bearing anti-PC Abs are induced in xid mice in response to EPC-KLH (8). In these Abs, the Vκ8–28 L chain is associated with the M603-like (Asn95H) variant of the V1 H chain. PC-specific Abs expressing the Vκ8–28 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 Vκ19–15 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 Vκ 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 Vκ amino acid residues that interact with the hapten PC, 2) the interplay between Vκ and VH 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 Vκ8–28 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).

FIGURE 4.

The derived amino acid sequences for the germline Vκ22–33 (GenBank accession no. CAB46322), Vκ19–15 (GenBank accession no. CAA75915-15), and Vκ8–28 (GenBank accession no. CAB46307-28) genes were aligned using Align X, a component of the Vector NTI suite. The framework regions (FWR) and CDR are indicated and separated by spaces between them. The exposed loop generated by CDR1 and the string of residues forming the ring around PC in the PC binding pocket are in bold and are illustrated in Fig. 5. Using the Vκ22–33 sequence as a template (which is the same length as the Vκ8–28 sequence), residues are numbered to be consistent with those in the molecular model presented in Fig. 5. Therefore, the Asp acid 3′ of the leader sequence is numbered residue 1.

FIGURE 4.

The derived amino acid sequences for the germline Vκ22–33 (GenBank accession no. CAB46322), Vκ19–15 (GenBank accession no. CAA75915-15), and Vκ8–28 (GenBank accession no. CAB46307-28) genes were aligned using Align X, a component of the Vector NTI suite. The framework regions (FWR) and CDR are indicated and separated by spaces between them. The exposed loop generated by CDR1 and the string of residues forming the ring around PC in the PC binding pocket are in bold and are illustrated in Fig. 5. Using the Vκ22–33 sequence as a template (which is the same length as the Vκ8–28 sequence), residues are numbered to be consistent with those in the molecular model presented in Fig. 5. Therefore, the Asp acid 3′ of the leader sequence is numbered residue 1.

Close modal
FIGURE 5.

Molecular model illustrating the relative positions of PC to Vκ8 binding site residues and the exposed loop of CDR1. The fragments illustrated were extracted from the complete three-dimensional structure of the McPC603 Fab-PC complex (Brookhaven Protein Data Bank no. 2 MCP). PC is shown in space fill with the phosphate molecule projecting up and to the left and the carbon chain projecting into the binding pocket. Surrounding the PC moiety is the ring of amino acids contributed by the carboxyl terminal region of the Vκ8 gene starting with the variant Asp acid residue (lower center) and continuing around the back, to the left and forward, ending with the conserved Leu residue contributed by Jκ5. To the right of the PC binding pocket is illustrated the exposed loop which is generated by residues from CDR1 of the Vκ8 gene. All residues on the inside face of the exposed loop are within 15 Å of the phosphorous atom of the PC moiety. Note that the last amino acid residue coded for by the germline sequence for Vκ8–28 is a Tyr and not the Phe shown above for the Vκ8 chain of McPC603.

FIGURE 5.

Molecular model illustrating the relative positions of PC to Vκ8 binding site residues and the exposed loop of CDR1. The fragments illustrated were extracted from the complete three-dimensional structure of the McPC603 Fab-PC complex (Brookhaven Protein Data Bank no. 2 MCP). PC is shown in space fill with the phosphate molecule projecting up and to the left and the carbon chain projecting into the binding pocket. Surrounding the PC moiety is the ring of amino acids contributed by the carboxyl terminal region of the Vκ8 gene starting with the variant Asp acid residue (lower center) and continuing around the back, to the left and forward, ending with the conserved Leu residue contributed by Jκ5. To the right of the PC binding pocket is illustrated the exposed loop which is generated by residues from CDR1 of the Vκ8 gene. All residues on the inside face of the exposed loop are within 15 Å of the phosphorous atom of the PC moiety. Note that the last amino acid residue coded for by the germline sequence for Vκ8–28 is a Tyr and not the Phe shown above for the Vκ8 chain of McPC603.

Close modal

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 Jκ5, regardless of which Vκ gene is being used. The overwhelming use of Jκ5 in PC-binding Abs can be understood when we consider that Jκ5 is the only Jκ region that can provide the invariant leucine residue observed at the Vκ-Jκ 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 Vκ-Jκ junction of PC-binding Abs is that in the two examples of PC-binding Abs that do not use Jκ5 (a Vκ22-Jκ2 (this study) and Vκ22-Jκ1 (41)) a codon coding for Leu is observed at the Vκ-Jκ junction. The leucines in these two examples are most likely coded for by the CTC 3′ of the Pro codon of Vκ22–33.

Next we consider the observation that from the FACS data it is clear that the T15H:Vκ8–28L and T15H:Vκ19–15L 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:Vκ8–28L and T15H:Vκ19–15L 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 × Vκ8 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:Vκ8–28 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:Vκ8–28 and T15H:Vκ19–15 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:Vκ22L-expressing B cells. We can understand the role of the specific Vκ 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 Vκ8L 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 Vκ genes in Fig. 4 reveal that these amino acids (DHSYP for Vκ8–28) are the terminal Vκ residues contributing to the Vκ 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 Vκ8–28L but is absent in both the Vκ22–33L and Vκ19–15L PC-binding Abs. This residue illustrates the complementary nature of residues contributed by VH and VL to the PC-hapten binding pocket. For the Vκ8L 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 Vκ8–28 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 DH:JH junction. In PC-binding Abs that do not use the Vκ8–28 L chain, this T15H-encoded Asp95H residue sits in the PC binding pocket and interacts similarly to the Asp97L of Vκ8–28 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 VH or Vκ chains. However, the T15H:Vκ8–28L 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:Vκ8–28L B cells, the inability to respond to immunization with EPC-KLH or to compete with B cells expressing T15H:Vκ22–33L receptors would be due to the >10-fold lower affinity of their binding sites for PC than the binding sites on T15H:Vκ22–33 B cells.

In this study, ∼30% of the PC-dex binding cells expressed the Vκ19–15L chain. Why haven’t B cells expressing the T15H:Vκ19–15L Ab been observed among PC-binding Ab before, and why didn’t the T15H:Vκ19–15L-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 Vκ19–15L chain to that of Vκ8–28L and Vκ22–33L 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 Vκ19–15L and Vκ22–33L 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 Vκ19–15 and Vκ22–33 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 VH 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 VH and VL 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 Vκ19–15L lacks a string of six amino acids (amino acids 33–38). This string of amino acids forms an exposed loop for the Vκ8–28 L chain and presumably also for the Vκ22–33 L chain (51). If this exposed loop of the Vκ chain was necessary for interaction with the carrier molecule of PC-conjugates, then Vκ19–15L would not be able to form these additional bonds and therefore would have a significantly lower overall avidity for PC conjugates. Therefore, even though Vκ19–15 is able to pair with the germline T15H and the T15H:Vκ19–15L Ab could bind PC, B cells expressing T15H:Vκ19–15L would not be able to compete with T15-Id+ B cells in a normal response to EPC-KLH.

Analysis of the frequency of Vκ gene usage in the PC-dex binding cells was performed assuming that VH and VL 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 Vκ 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 Vκ22–33, Vκ8–28, or Vκ19–15 genes represent 0.74, 2.5, and 1.4%, respectively. In LPS-stimulated B cells from normal mice, the frequency of expression of these Vκ 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 Vκ 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 Vκ gene used, were rearranged to Jκ5, whereas, in LPS-activated B cells, 20% of the colonies express Jκ5 (53, 54). This means that the Vκ19–15:Jκ5, Vκ8–28:Jκ5, and Vκ22–33:Jκ5 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 Vκ gene usage among PC-binding B cells in the T15i KI mouse. Surprisingly, we observed that B cells expressing the T15H:Vκ22–33L were not the dominant population in the spleen of unimmunized T15i KI mice. We also identified a population expressing T15H:Vκ19–15L which has not previously been described. An examination and comparison of the Vκ L chains used identified correlations between structural determinants of the Vκ 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 Vκ-Jκ junction of the Vκ 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.

3

Abbreviations used in this paper: PC, phosphocholine; ASC, Ab-secreting cell; CDR, complementarity-determining region; TdT, terminal deoxynucleotide transferase; EPC, 6-(O-PC) hydroxyhexanoate; KLH, keyhole limpet hemocyanin; KI, knockin; dex, dextran.

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