Affinity maturation of secreted IgM pentamers on B cells

Takeyuki Shimizu1, Yuko Kozono1,2, Haruo Kozono1, Masayuki Oda3 and Takachika Azuma1

1 Division of Biosignaling, Research Institute for Biological Sciences, Tokyo University of Science, Noda, Chiba 278-0022, Japan 2 Present address: Clock Cell Biology, IMCB, National Institute of Advanced Industrial Science and Technology, Ibaragi 305-8566, Japan 3 Present address: Department of Cellular Macromolecule Chemistry, Graduate School of Agriculture, Kyoto Prefectural University, Kyoto 606-8522, Japan

The first two authors contributed equally to this work
Correspondence to: T. Azuma; E-mail: tazuma{at}rs.noda.tus.ac.jp
Transmitting editor: K. Okumura


    Abstract
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
We prepared a series of hapten-BSA conjugates with varying ratios of biotin to measure ligand–receptor interactions on B cells by flow cytometry using avidin for detection. Surface plasmon resonance measurements of the interaction with a monoclonal anti-(4-hydroxy-3-nitrophenyl)acetyl (NP) antibody suggested that NP5-BSA or NP7-BSA harboring 29 or 23 biotin molecules (NP5-BSA-bio29 or NP7-BSA-bio23) would be suitably sensitive for flow cytometric analysis. By using NP-BSA-bio, we analyzed NP-binding cells in immunized mice. Unexpectedly, 30–40% of spleen cells expressing IgM could bind to NP5-BSA or NP7-BSA after immunization of mice with NP40-chicken {gamma}-globulin. The proteins binding to NP7-BSA-bio23 on the cell surface were analyzed by immunoprecipitation and western blotting. Surprisingly, most of the proteins binding NP-BSA-bio on the cell surface were not the membrane form of IgM monomer, but a secreted IgM pentamer. It is likely that the IgM pentamer bound through Fc receptors for polymeric IgA or IgM and contributed to antigen binding. Comparison of the binding ratio of NP0.9-BSA:NP5-BSA between B cells of primary and secondary immunization suggested that the affinity of IgM matured during immunization.

Keywords: antibody, B lymphocyte, Fc receptor, memory


    Introduction
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Hapten-conjugated proteins have long been used as T cell-dependent antigens for studies on the immune response, especially in the investigation of antibody affinity maturation (17). Although these studies have employed various conjugates which differed in the number of haptens or in the size of carrier proteins, such as ovalbumin, chicken serum albumin, chicken {gamma}-globulin (CGG) or keyhole limpet hemocyanin, little is known about the effects of these factors on the magnitude of antibody production or memory cell formation during the immune response. Recently, we have examined these issues by means of surface plasmon resonance (SPR) measurements and found that the apparent affinity (avidity) largely depended on both the valence of the haptens and the size of the carrier proteins (8). The valence of haptens in particular would be expected to affect the magnitude of signals through BCR, since the retention time or occupancy of the bound antigens as well as the degree of BCR cross-linking depend largely on hapten valence (9,10). Differences in the signal strength of antigens with different hapten valences would alter the quality as well as the intensity of the immune response.

It has been well established that after stimulation with T cell-dependent antigens, B cells within the periateriolar lymphoid sheaths enter the follicles, and acquire a centroblast and centrocyte phenotype in the germinal center (1116). Somatic hypermutation (SHM) takes place through these B cell stages and B cells with mutated BCR are continuously selected on the basis of antigen specificity as well as affinity. It was shown that some of these selected B cells move into the bone marrow (BM) and remain there as long-lived plasma cells (17,18). Memory B cells were characterized as having higher affinity than naive B cells due to SHM and selection by antigens (19,20). BCR of these cells were sometimes believed to have undergone class switching to isotypes other than IgM (21,22). Therefore, in previous studies, memory B cells were characterized as hapten-binding, IgMIgG1+CD38 cells. However, isotype switching was not necessarily required for establishing memory B cells (23,24). Therefore, increased affinity as a result of SHM and antigen selection would provide the most reliable condition for memory B cell generation.

Although the affinity maturation of secreted IgG has been shown by ELISA (25), it is rather difficult to measure the affinity of BCR on the cell surface. We attempted to develop a method for measuring the relative affinity/avidity of BCR by flow cytometry by employing a series of (4-hydroxy-3-nitrophenyl)acetyl (NP)-BSA-biotin conjugates (NP-BSA-bio) with various valences of NP and biotin. The specificity of the binding of these conjugates to anti-NP mAb and the sensitivity of detection of NP-BSA-bio bound to BCR by streptavidin (SA) were tested with the BIAcore system, which utilizes SPR as a means of detection, as well as with flow cytometry using K46µ, a B cell transfectant with a BCR of anti-NP binding specificity (26).

Through the use of NP-BSA-bio in combination with SA–phycoerythrin (PE), we studied the binding of NP-BSA to B cells obtained from mice immunized with NP-CGG of various NP valences. Unexpectedly, we observed that a massive number of B cells capable of binding with NP5-BSA appeared transiently with a peak at around day 7 following primary immunization with NP40-CGG or NP13-CGG. By analyzing the proteins responsible for this phenomenon, it became evident that a secreted form of IgM pentamer and not the membrane form of IgM, i.e. BCR, was involved in the NP binding. By comparing the binding activity to NP0.9-BSA-bio and NP5-BSA-bio, the affinity maturation of IgM on B cells was investigated.


    Methods
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Reagents
NP was prepared from 4-hydroxyphenylacetic acid as described previously (27). The hydroxysuccinimide ester of NP was prepared as described (28). 2,4,6-Trinitrobenzene sulfonic acid (Wako, Tokyo, Japan) and ethoxymethylene-2-phenyl-2-oxazolin-5-one (Sigma-Aldrich, St Louis, MO) were purchased for use in preparing trinitrophenyl (TNP)- or phenyloxazolone (phOx)-conjugated proteins respectively. For preparing NP-BSA-bio and phOx-BSA-bio, biotin was first conjugated to BSA with NHS-biotin and this was followed by haptenylation with the respective reagents. The number of conjugated biotin molecules was estimated using HABA reagents (Pierce, Rockville, MD) after treatment with pronase for 16 h according to the manufacturer’s instructions. The numbers of hapten moieties introduced were determined spectrophotometrically using the molar extinction coefficients of NP (4.23 x 103 at 430 nm) and phOx (3.2 x 104 at 348 nm) (29). Protein concentrations were estimated using the BCA assay (Pierce).

Cell lines
The K46µ lymphoma line (26) was kindly provided by Dr J. C. Cambier (Denver, CO), and cultured in Iscove’s modified Dulbecco’s medium supplemented with 10% FCS, 100 U/ml penicillin, 100 µg/ml streptomycin, 2 mM glutamine and 50 µM ß-mercaptoethanol. A hybridoma secreting the anti-NP mAb, N1G9, was donated by Dr K. Rajewsky (30,31).

SPR measurements
The BIAcore biosensor system, BIAcore 2000 (BIAcore, Uppsala, Sweden), was used to measure real-time interactions between NP-CGG or NP-BSA-bio and the anti-NP mAb, N1G9, that was bound to 6000 RU of rabbit anti-mouse IgG Fc (Pierce) coupled to a CM5 sensor chip. An isotype-matched mAb was used to discount non-specific binding.

Mice and immunization
Mice were purchased from KWL (Wakayama, Japan) or SLC (Shizuoka, Japan) and housed in our institutional animal facility. The experiments were carried out under the Guidelines for Animal Protocols of our institution. Groups of C57BL/6 mice were immunized with a single i.p. injection of 100 µg of NP4-, NP13-, NP40- or phOX35-TNP40-CGG with alum. A booster immunization was carried out using 100 µg NP40-CGG in PBS. Splenocytes were isolated as previously described (32).

Flow cytometric analysis
FITC–anti-IgM (R6-60.2 or II-41), -Ig{kappa} (187.1) and -IgMa (DS-1), biotin–anti-Ig{lambda} (R26-46) and -IgMb (AF6-78), and SA–PE were purchased from BD Bioscience PharMingen (San Diego, CA). FITC–goat anti-mouse IgM was purchased from Southern Biotechnology Associates (Birmingham, AL). Splenocytes were stained with 1 µg/ml of NP-BSA-bio or phOx-BSA-bio in the combination with FITC–anti-IgM, followed by SA–PE staining. To minimize non-specific staining, anti-Fc{gamma}RII-specific mAb, 2.4G2, were added. For pre-incubation with sera, spleen cells from non-immunized mice were incubated with serum for 30 min on ice followed by washing and staining with NP-BSA-bio and FITC–anti-IgM. Data were acquired by a FACSCalibur or a FACSVantage and analyzed with CellQuest software (BD Bioscience, Mountain View, CA). Lymphocytes were gated for each figure.

ELISA
Seven days after immunization with NP4-CGG, NP13-CGG, or NP40-CGG with alum, antisera were collected from the immunized mice. Mice were then sacrificed and tissue samples were obtained. For measuring anti-NP antibody production, plastic plates coated with NP9-BSA were used. Peroxidase-conjugated goat anti-mouse IgM or IgG (Southern Biotechnology Associates) was used for detection of bound antibodies. Antisera from hyper-immunized mice were used as standard.

Single-cell sorting and RT-PCR
Spleen cells of normal or immunized mice were stained with FITC–anti-Ig{kappa} plus biotin–anti-Ig{lambda} followed by SA–PE. Single cells were sorted on a FACSVantage into a 96-well plate containing RT buffer. cDNA was synthesized by SuperScript II reverse transcriptase with oligo-dT primer (Invitrogen, Carlsbad, CA). Ig{lambda}1 cDNA was amplified with primers specific to V{lambda} (5'-ATGGC CTGGA TTTCA CTTAT ACTCT CTC) and C{lambda}1 (5'-CTTTC CCATG CTCTT GCTGT CAGG). A small aliquot of amplified cDNA was re-amplified with internal primers specific to J{lambda}1 (5'-GTGTT CGGTG GAGGA ACCAA ACTG) and C{lambda}1 (5'-GTTGC TCTGT TTGGA AGGCT GGGT). Ig{lambda}2 and Ig{lambda}3 messages were amplified using the same sets of primers, which were V{lambda} and C{lambda}2 and 3 (5'-TGAGA TCTCC ACTGG TCCGA TGTC) for the first round of PCR, and J{lambda}2 and 3 (5'-RTTTT CGGCR GTGGA ACCAA GGTC) and internal C{lambda}2 and 3 (5'-GTTGY CCTCT TTGGT GGGAT TTGA) for the second round. V{kappa} cDNA was amplified using the universal V{kappa} primer (5'-GGCTG CAGST TCAGT GGCAG TGGRT CWGGR AC) (33) in combination with a C{kappa} primer (5'-GTCGT TCATA CTCGT CCTTG GTCAA C). The second round of PCR was performed using a mixture of J{kappa} primers (J{kappa}1: 5'-GTTCG GTGGA GGCAC CAAGC TGGA; J{kappa}2: 5'-GTTCG GAGGG GGGAC CAAGC TGGA; J{kappa}4: 5'-GTTCG GCTCG GGGAC AAAGT TGGA; J{kappa}5: 5'-GTTCG GTGCT GGGAC CAAGC TGGA) with an internal C{kappa} primer (5'-GTTCA GGACG CCATT TTGTC GTTCA C). The PCR products were analyzed by agarose-gel electrophoresis and stained with ethidium bromide. In order to determine the V–J junctional sequences, amplified cDNA was re-amplified with VL and internal CL primers, and amplified fragments were sequenced by Beckman Coulter (Fullerton, CA) ACE2000-XLE DNA sequencer.

Western blotting
One week after immunization with NP40-CGG/alum or 5 days after booster immunization, cell suspensions were prepared from spleen cells of normal or immunized mice. Cells were incubated with 1 µg/ml of NP7-BSA-bio23 in PBS containing 1% BSA. After washing the unbound NP-BSA-bio, cells were suspended in lysis buffer for 30 min on ice. The lysis buffer contained 1% NP-40, 50mM Tris–HCl (pH 7.5), 150 mM NaCl, 1 mM sodium orthovanadate, 1 mM PMSF, aprotinin, leupeptin and pepstatin A. The detergent-soluble fraction was collected after centrifugation and proteins bound to NP7-BSA-bio23 were precipitated with SA–agarose beads (Pierce). The serum was prepared and reacted with NP7-BSA-bio23 in the lysis buffer and antibodies bound to NP-BSA-bio were precipitated with SA–agarose. The pellet was suspended in sample loading buffer containing 20 mM Tris–HCl (pH 7.0), 30% glycerol, 1% SDS and bromophenol blue. In order to prepare the total-cell lysate, cells were incubated in the lysis buffer, and the detergent soluble fraction was collected and diluted in the sample loading buffer. Samples were subjected to 3% polyacrylamide–0.5% agarose gel electrophoresis (34) and the protein was transferred to a PVDF membrane (Millipore, Bedford, MA). The blot was probed with peroxidase-labeled goat anti-mouse IgM or IgG, followed by detection with ECL reagent (Pharmacia, Piscataway, NJ).


    Results
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Characterization of antigens for immunization and binding assays
In this study, CGG and BSA were used as carrier proteins to which hapten and/or biotin were conjugated. Average numbers of hapten or biotin molecules per carrier protein are expressed with subscripts (e.g. NP5-BSA-bio29 or NP40-CGG).

NP4-CGG, NP13-CGG, and NP40-CGG were used as immunogens, and binding of these to a primary anti-NP mAb, N1G9, was examined by SPR measurement. Since this mAb was shown to have an affinity to NP-Cap of 5 x 105 M–1, and to lack SHM in VH and VL regions (30,31), it was considered to be useful as a model of BCR on naive B cells. N1G9 was immobilized on BIAcore sensor chips through rabbit anti-mouse IgG Fc antibodies and the interactions of NP-CGG with this chip-coupled mAb were used to mimic antigen–BCR interactions on the cell surface. The results were analyzed using Scatchard plots (Fig. 1A). Since the binding of NP-CGG to N1G9 was characterized in terms of multivalent–divalent interactions, the plots would not be expected to fall in a linear fashion. However, within a limited range of NP-CGG concentrations, the plots generate straight lines and the apparent association constant (Ka) estimated from the slopes was 1.4 x 107 M–1 for NP4-CGG, 9.3 x 107 M–1 for NP13-CGG and 2.4 x 108 M–1 for NP40-CGG. It is clear that the binding of NP-CGG increased with an increase in the NP valence from 5 to 40.



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Fig. 1. Binding of NP-CGG or NP-BSA-bio to the NP-specific mAb, N1G9 (A and B), and binding of SA to NP-BSA-bio in the antigen–antibody complex (C). Scatchard plots for interactions with NP-CGG (A) and NP-BSA-bio (B) to N1G9 immobilized on the chips through rabbit anti-mouse IgG Fc. The apparent association constants were estimated from the slope of the plots. For analysis of the interaction between SA and NP-BSA-bio in the antigen–antibody complexes in (C), the initiation point of the reaction of 500 nM NP-BSA-bio with N1G9 on the chip is shown by an arrow. After washing with buffer, 500 nM SA was added to react with NP-BSA-bio in the pre-formed antigen–antibody complex at the point indicated by the arrow. Non-specific binding of SA has been removed from the sensorgrams. The binding of NP5-CGG, NP13-CGG, and NP40-CGG are analyzed in (A), and binding of NP2.3-BSA-bio1.8 (a), NP11-BSA-bio1.8 (b), NP1.6-BSA-bio14 (c), NP8.4-BSA-bio14 (d), NP0.9-BSA-bio29 (e) and NP5-BSA-bio29 (f) are shown in (B) and (C).

 
We used NP-BSA for the binding analyses of antigen–BCR interactions on the cell surface. For these analyses, we needed to employ the biotin–SA system for the detection by flow cytometry. First, we analyzed the interaction of N1G9 with biotinylated NP-BSA (NP-BSA-bio) by SPR measurement in order to examine the effects of NP valence as well as biotinylation on the interaction (Fig. 1B). NP0.9-BSA-bio29, NP1.6-BSA-bio14 and NP2.3-BSA-bio1.8 showed marginal binding (Ka < 1 x 106 M–1), while NP-BSA-bio with an NP valence >5, i.e. NP5-BSA-bio29, NP8.4-BSA-bio14 and NP11-BSA-bio1.8, bound to N1G9 efficiently with Ka values of 1.6 x 107, 4.2 x 107 and 6.3 x 107 M–1 respectively. Since similar NP valence-dependent binding was observed using NP-BSA with no biotinylation (8), it was concluded that NP5-BSA bound to BCR with high avidity and an affinity as low as 105 M–1, whereas NP0.9-BSA did not, and that biotinylation provided little inhibition of the antigen–antibody interactions. Next, we examined the interaction between SA and NP-BSA-bio in the chip-coupled antigen–antibody complex (Fig. 1C). Considering the high affinity between biotin and SA, it was rather surprising that the sensitivity depended largely on the number of biotin moieties. The binding of SA to NP2.3-BSA-bio1.8, NP11-BSA-bio1.8, NP1.6-BSA-bio14 or NP8.4-BSA-bio14 was inefficient compared with the binding to NP0.9-BSA-bio29 or NP5-BSA-bio29.

The binding of NP-BSA-bio to BCR was examined using a B cell transfectant, K46µ, which expresses B1-8 µ chains and {lambda}1 chains, and possesses anti-NP binding activity (35). Since the amino acid sequences of VH and VL regions of N1G9 and B1-8 are identical (30,36), binding data on N1G9 obtained from SPR measurements can be applied to those of K46µ. As expected from the SPR results, essentially no binding was detected with either NP2.3-BSA-bio1.8 or NP11-BSA-bio1.8 (Fig. 2). An increase in the number of biotin molecules to 14 (NP8.4-BSA-bio14) improved the binding significantly. Finally, we found that NP-BSA with 23–29 biotin molecules had maximum sensitivity for K46µ, but showed little non-specific binding to the parent K46 cells (data not shown) and, therefore, used these higher-valence conjugates for further flow cytometry analysis of antigen–BCR interactions on B cells.



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Fig. 2. Flow cytometric profiles for the binding of NP-BSA-bio to BCR on K46µ. The percentages of NP-BSA-bio binding cells in the total cell population were compared. NP-BSA-bio used for staining is indicated for each figure. Negative control was stained with BSA-bio29.

 
It has been shown previously that the binding ratio of antibodies to low and high hapten number conjugates, as determined by ELISA, is a measure of antibody affinity (25). Results of SPR measurement also provided information on the relative affinity of sensor chip-immobilized antibodies. As shown in Fig. 1(C), N1G9 exhibited an RU of ~60 in the binding of NP0.9-BSA-bio29 and an RU of ~350 in binding to NP5-BSA-bio29. In this case, the ratio of binding of NP0.9-BSA-bio29 and NP5-BSA-bio29 was ~0.2. Therefore, the primary immature antibodies are likely to have a ratio of ~0.2. The mature mAb, C6, however, had a value of close to 1 (data not shown). The relative affinity of BCR on the cell surface was examined by flow cytometry (Fig. 2). In the case of BCR on K46µ, the ratio of binding to NP0.9-BSA-bio29 and NP5-BSA-bio29 was 0.4–0.5. Although this value was higher than that for N1G9 from results obtained from SPR measurements, we considered that the binding ratio to NP0.9-BSA-bio and NP5-BSA-bio corresponded to BCR affinity.

Immunization of NP-CGG and appearance of IgM+NP+ B cells
In order to examine the relationship between the hapten valence of CGG and the immune response, C57BL/6 mice were immunized with NP-CGG and the spleen cells collected on day 7 were analyzed by flow cytometry using FITC–anti-IgM antibodies and NP7-BSA-bio21 (Fig. 3A). FITC–anti-IgM antibodies stained ~50% of unimmunized spleen cells in which NP-binding cells were under the detection level. Fewer NP7-BSA-bio21-binding cells were detectable after immunization with NP4-CGG. Immunization with NP13-CGG caused 76% of IgM+ B cells to acquire the ability to bind to NP7-BSA-bio21. A further increase in the valence of NP (NP40-CGG) resulted in no increase in the number of NP7-BSA-bio21-binding IgM+ cells (65%). CD4+ or CD8+ T cells did not show any NP binding (data not shown). We referred to these cells as IgM+NP+ B cells.



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Fig. 3. Appearance of hapten-binding B cells in spleens of mice immunized with hapten-conjugated CGG. (A) Binding of NP7-BSA-bio21 cells was analyzed using a single-cell suspension of spleen cells obtained from mice on day 7 after immunization with NP-CGG having different NP valences or from non-immunized control mice. The amounts of bound NP-BSA-bio and of IgM BCR expressed on the cell surface were measured by flow cytometry and the percentages of each in the total lymphocytes are shown. (B) Mice were immunized with NP-CGG and sera were prepared on day 7. Titers of anti-NP IgM (top) and IgG1 (bottom) in sera were measured by ELISA, and plotted against the number of NP haptens on NP-CGG. Serum from non-immunized mice was used as a control (n.i). (C) Mice were immunized with antigens indicated at the top, and spleen cells were stained with anti-IgM and NP7-BSA-bio23 or phOx10-BSA-bio23 in combination with FITC–anti-IgM.

 
The titers of anti-NP IgM and IgG in serum were measured by ELISA (Fig. 3B). After immunization with NP4-CGG, a slight increase in anti-NP antibodies was observed. On the other hand, NP13-CGG and NP40-CGG caused a clear immune response of IgM and IgG1 production, and titers were indistinguishable between sera prepared from mice immunized with these two immunogens. We analyzed the number of antibody-forming cells in spleen by the ELISPOT assay and found a smaller number of anti-NP IgM- or IgG1-producing cells in mice immunized with NP4-CGG, but indistinguishable numbers in NP13-CGG or NP40-CGG immunized mice (data not shown). These results suggested that the appearance of IgM+NP+ B cells correlated with the increase in anti-NP antibodies in serum.

We examined whether the large number of hapten-binding IgM+ B cells which appeared on day 7 after the primary immunization was also observed with other hapten systems. After immunization of C57BL/6 mice with phOx38-CGG, spleen cells were stained with phOx10-BSA-bio23. We found IgM+NP+ and IgM+phOx+ B cells in NP-CGG- and phOx-CGG- immunized mice respectively (Fig. 3C). The hapten-binding activity depended on the antigens used for immunization, indicating that the binding was specific. We also found TNP binding to B cells after immunization with TNP40-CGG (data not shown). Therefore, the appearance of a large number of hapten-bound cells seems to be a phenomenon common to hapten systems.

Characterization of proteins responsible for NP binding
The spleen cells from NP40-CGG-immunized mice obtained on day 7 were analyzed by flow cytometry using anti-Ig{lambda} and anti-Ig{kappa}, since anti-NP antibodies of the primary response were known to bear {lambda}1 chains. A large fraction of B cells was sorted as {kappa}+{lambda}dull with minor single {lambda}+ and single {kappa}+ fractions (Fig. 4). This {kappa}+{lambda}dull fraction was absent in B cells from unimmunized mice and was considered to be responsible for NP binding. In order to determine the type of L chains synthesized by these cells, single-cell analysis was performed by RT-PCR using primers specific to respective L chains (Table 1). Although expression of {kappa} chains was identified, no {lambda}1 chains were detected in the {kappa}+{lambda}dull fraction. On the other hand, {lambda} chains were detected in all B cells of the single {lambda}+ fraction. These results suggested that the B cells in the {kappa}+{lambda}dull fraction synthesized only {kappa} chains, but not {lambda} chains, and that the {lambda} chains detected were derived from Ig bound to the B cell surface.



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Fig. 4. Flow cytometric analysis using anti-{kappa} or anti-{lambda} antibodies. A single-cell suspension of spleen cells obtained from mice on day 7 post-immunization or from non-immunized control mice was analyzed using a FACSVantage. The fractions indicated were sorted and analyzed by single-cell RT-PCR (Table 1).

 

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Table 1. Ig L gene expression in different populations of spleen B cells
 
Next, we analyzed the materials responsible for NP binding. Spleen B cells from NP40-CGG-immunized mice were isolated and reacted with NP7-BSA-bio23 followed by solubilization with a buffer containing NP-40. Materials forming a complex with NP7-BSA-bio23 in detergent lysate were precipitated with SA–agarose and analyzed by non-reducing SDS–PAGE followed by western blotting (Fig. 5A). The NP-binding proteins of cell lysates from NP40-CGG-immunized mice showed a broad band corresponding to IgM pentamers, which was almost absent in those from non-immunized mice (Fig. 5A, NP-binding cell lysate). These results suggested that a secreted form of the IgM pentamer with anti-NP specificity existed on the B cell surface. On the other hand, we did not recover any NP-binding BCR, i.e. monomer IgM or secreted IgG, in spite of the existence of a large amount of NP-specific IgG in the sera (Fig. 5A, NP-binding sera). These results indicated that the BCR from most of the IgM+NP+ B cells was not specific to NP and that the binding of secreted IgM enabled B cells to bind NP. When B cells from non-immunized mice were solubilized with lysis buffer and analyzed by SDS–PAGE without reacting with NP7-BSA-bio23 and SA–agarose, bands corresponding to both pentamer and monomer IgM were observed (Fig. 5A, total cell lysate), indicating that B cells possess two types of IgM irrespective of immunization; a monomer type that functions as a BCR and a pentamer type that might bind to the cell surface through an unidentified mechanism. Comparison of the band intensity provided an approximate ratio of pentamer relative to monomer that was <0.1.



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Fig. 5. Existence of IgM pentamer on spleen B cells. (A) Detergent lysates (NP-binding cell lysates) were prepared after reacting with NP7-BSA-bio23. Sera (NP-binding sera) were reacted with NP7-BSA-bio23. NP-binding proteins were precipitated with SA–agarose and analyzed on western blots using peroxidase-labeled anti-IgM (top) or anti-IgG (bottom). Lysates not reacting with NP7-BSA-bio23 and SA–agarose (total lysates) were also analyzed to show the BCR position. The marker lane contained a mixture of purified IgM pentamer and IgG. The total lysate of K46µ cells shows the position of IgM BCR on the cell surface. The positions of IgM pentamer (IgM-p), IgM BCR (BCR) and IgG (IgG) are indicated on the right. NC, serum or lysates from a non-immunized mouse; 1st, from an NP40-CGG immunized mouse after primary immunization; 2nd, from a mouse 5 days after booster immunization. (B) Spleen cells of immunized mice were pre-incubated without (left), with non-immunized serum (middle) or with NP40-CGG-immunized serum (right), followed by staining with NP-BSA-bio and FITC–anti-IgM. (C) Mixtures of spleen cells of C57BL/6 and BALB/c mice or a CBF1 mouse were stained with anti-IgMa and -IgMb antibodies.

 
In order to determine that the anti-NP antibodies in serum can provide B cells with NP-binding ability, spleen cells from non-immunized mice were incubated with serum from immunized mice and analyzed by flow cytometry (Fig. 5B). When the spleen cells were pre-incubated with non- immunized serum, no NP-binding activity was observed, while after treatment with serum of NP40-CGG immunized mice, IgM+NP+ B cells appeared. These results suggested that spleen B cells acquired NP-binding activity by absorbing the secreted anti-NP IgM on the cell surface.

The detection of IgM on B cells was rather surprising because IgM pentamers absorbed by these cells would result in more than one IgM on the cell surface. In order to examine whether this phenomenon occurs in normal situations, we analyzed the IgM allotype on B cells of C57BL/6 (IgMb allotype), BALB/c (IgMa allotype) and CBF1 mice. Splenic B cells expressed either IgMb or IgMa on the cell surface in C57BL/6 or BALB/c mice respectively (data not shown). By mixing an equal number of cells from C57BL/6 and BALB/c mice and staining with anti-IgMa and -IgMb antibodies, B cells were found to express only one of two allotypes and not both at once on the cell surface. Although B cells of CBF1 mice were also expected to express either IgMa or IgMb because of allelic exclusion, they provided populations of IgMa and IgMb cells which were weakly, but significantly, positive for IgMb and IgMa (referred as IgMaIgMb/dull and IgMa/dullIgMb cells respectively) (Fig. 5C). These results suggested that B cells expressed two forms of IgM, one which existed as a BCR and the other which was present as an adsorbed form, although the amount of the latter was less than that of the former.

Affinity maturation of IgM+NP+ B cells
The binding of NP0.9-BSA or NP5-BSA to spleen cells and changes in the number of IgM+NP+ B cells were examined after immunization with NP40-CGG. The primary IgM+NP+ B cells were characterized by weak binding of NP0.9-BSA (Fig. 6A). These cells were absent on day 4, but started to appear on day 5. On day 7, their number reached a maximum followed by a gradual decrease with time to day 70 (Fig. 6B). Following a booster immunization with NP40-CGG, IgM+NP+ B cells reappeared as early as day 4 and reached a maximum on day 5 (Fig. 6C and D). The binding ratios of NP0.9-BSA:NP5-BSA were 0.2–0.6 for primary B cells, whereas this ratio of NP0.9-BSA:NP5-BSA for the secondary B cells was close to 1 (Fig. 6E). These results indicated that the IgM+NP+ B cells of the primary immunization possessed an affinity similar to that of B1-8 and that those which reappeared following the secondary immunization had an elevated affinity to NP.



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Fig. 6. Affinity maturation of IgM+NP+ B cells during the immune response. (A) Spleen cells were obtained 7 days after immunization with NP40-CGG and stained with either NP5-BSA-bio29 + SA–PE or NP0.9-BSA-bio29 + SA–PE, followed by staining with FITC–anti-IgM antibodies. Percentages of each fractions are indicated. (B) The kinetics of the appearance of IgM+NP+ B cells in primary immunization is shown. The numbers represent average percentages obtained from a total of 60 mice used. No IgM+NP+ B cells existed in mice on day 70 post-immunization with NP40-CGG. (C) On day 70 after primary immunization (d0), mice were immunized with NP40-CGG and 5 days later, spleen cells were isolated and analyzed. (D) The kinetics of the appearance of IgM+NP+ B cells in secondary immunization of NP40-CGG. The numbers represent average percentages obtained from a total of 30 mice used for secondary immunization. (E) The binding ratio of NP0.9-BSA-bio29 relative to NP5-BSA-bio29 is shown. The ratio was calculated based on the percentage of NP0.9-BSA-bio29 binding cells and that of NP5-BSA-bio29 binding cells on days 6–8 after primary immunization and days 5–8 after secondary immunization.

 

    Discussion
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
The avidity of N1G9 to NP-CGG increased with an increase in the NP valence (Fig. 1A). We examined this effect on the immune response at the cellular level. The biotin–SA system has been widely used for flow cytometric detection of antigens or antibodies bound to the cell surface. Since the level of detection would depend on the number of SA molecules bound to the biotin conjugates, an increase in the number of biotin molecules would raise this level. In the case of antibody biotinylation, relatively low numbers of biotin molecules were conjugated to antibodies in order to avoid inhibition by biotin introduced into the antibody combining sites. In interactions between NP-BSA-bio and N1G9, however, essentially no inhibition by biotin was observed (Fig. 1B). Thus, an increase in the number of biotin molecules resulted in an increase in the number of SA molecules bound to NP-BSA-bio (Fig. 1C). This brought about an enhanced sensitivity in detection of NP-BSA-bio bound to BCR on K46µ and enabled us to estimate the number of NP binding cells by flow cytometry using SA–PE. Recently, we have shown that the relative affinity of BCR on the cell surface could also be estimated by the binding ratio of NP1-BSA-bio and NP5-BSA-bio (37). In addition, due to the high specificity of the biotin–SA interaction, no non-specific binding was observed either with SPR measurement or flow cytometry, in spite of the high numbers of biotin molecules in the conjugates (Fig. 2).

We detected a large fraction of IgM+NP+ B cells in spleens after immunization with NP-CGG, but none in pre-immune spleens (Fig. 3A) or in mice immunized with unrelated hapten conjugates or CGG alone (Fig. 3C and data not shown). The IgM+NP+ B cells were evident after immunization with NP13-CGG or NP40-CGG, comprising 30–40% of the total number of spleen cells, although they were hardly detected after immunization with NP4-CGG. We examined whether the appearance of IgM+NP+ B cells was related to the amount of anti-NP IgM in the sera and found that NP13-CGG or NP40-CGG induced a similar amount of IgM production, while that of NP4-CGG was rather weak (Fig. 3B). Such a parallel relationship in the ability of NP-CGG between induction of anti-NP IgM secretion and of IgM+NP+ B cells suggested that the appearance of IgM+NP+ B cells was closely related with the amount of secreted anti-NP IgM in the blood. Immunization with NP40-CGG also resulted in a large fraction of {kappa}+{lambda}dull B cells (Fig. 4). RT-PCR analysis revealed that these B cells synthesized only {kappa} chains and not {lambda} chains (Table 1). It has been shown previously that immunization of C57BL/6 mice with NP-CGG induced a predominant secretion of antibodies bearing {lambda}1 chains (38). Therefore, it is suggested that predominant production of anti-NP IgM bearing {lambda}1 chains resulted in the appearance of {kappa}+{lambda}dull B cells possessing NP-binding activity.

We analyzed the molecules responsible for the NP binding. The NP-binding proteins recovered from the detergent-soluble fraction of immunized B cells were a secreted form of IgM pentamer (Fig. 5A). The monomer IgM of BCR was not recovered in the NP-binding material. In addition, although the immune sera contained both anti-NP IgM and anti-NP IgG, only secreted IgM could be detected. This indicated that IgM molecules on the B cell surface were not in the form of immune complexes with NP-CGG, since these complexes should contain both anti-NP IgM and anti-NP IgG. It was also suggested that IgG, which could be bound to Fc{gamma}RII on B cells, did not contribute to the NP-binding capacity of B cells. B cells from non-immunized mice had no NP-binding activity, but did acquire it after treatment with immune sera from NP40-CGG-immunized mice (Fig. 5B). All these results suggested that secreted IgM bound to B cells. Recently, Shibuya et al. (39) reported the presence of Fc{alpha}/µR on B220+ cells and it is possible that the IgM pentamers bound to the B cells through Fc{alpha}/µR. However, a question was raised as to the binding of the secreted IgM, since not all B cells were capable of NP binding. This may be related to the broad expression of Fc{alpha}/µR on B220+ cells (39). Among splenic B cells, NP binding was detected in IgMdullIgD+ mature B cells, but not in IgMhiIgD immature B cells (data not shown), which corresponded with the Fc{alpha}/µR expression level (40). It is likely that B cells with weak expression of Fc{alpha}/µR could be sorted as NP since they would fall below the detection level of flow cytometry analysis. We are currently examining the relationship between Fc{alpha}/µR and appearance of IgM+NP+ cells.

If the binding of secreted IgM to B cells really occurred, it was predicted that F1 mice from parents of different IgM allotypes should have B cells that are double positive in terms of IgM allotype, although such a fraction had not been reported previously. However, careful analysis of the flow cytometry pattern showed that the expression of IgM allotypes of CBF1 B cells was different from that of a mixture of BALB/c and C57BL/6 B cells. It was evident that a significant amount of IgMaIgMb/dull or IgMa/dullIgMb B cells existed (Fig. 5C). Since the sera from CBF1 mice contain both secreted IgMa and IgMb, these IgM would compete for B cell binding sites. In addition, the band intensity in the western blots suggested that IgM pentamers represent <10% of IgM BCR (Fig. 5A). Therefore, the IgM pentamers bound to the B cells induced only a slight shift in allotype expression, which was observed as IgMaIgMb/dull or IgMa/dullIgMb. Our sensitive assay using NP-BSA-bio enabled us to detect such a low amount of NP-specific IgM pentamer on B cells.

In primary immunization, after a rapid appearance, which peaked on day 7, the percentage of IgM+NP+ B cells decreased gradually and disappeared by day 70 (Fig. 6B). These B cells reappeared after booster immunization with NP40-CGG and reached a maximum number on day 5 (Fig. 6D). The binding ratio of NP0.9-BSA-bio29:NP5-BSA-bio29 was in the range of 0.2–0.6 for primary IgM+NP+B cells on day 7, but it increased to 1.0–1.1 with secondary immunization (Fig. 6E), suggesting that the secondary IgM possessed enhanced affinity, i.e. that affinity maturation proceeded in IgM antibodies as it does in IgG antibodies. Although amino acid replacements of IgM by SHM, typical for high-affinity anti-NP antibody, were observed (41), affinity maturation of IgM antibodies has not been shown during immunization by an ELISA method similar to that used for IgG (25). This was explained in terms of high avidity of IgM. In fact, the binding ratio of NP1-BSA:NP10-BSA was ~1, even in the case of low-affinity IgM (Shimizu, unpublished results). Recently, Tobita et al. (42) reported that IgM immobilized on a biosensor chip behaved similarly to IgG in the interaction of NP-BSA with different hapten valence, suggesting that the present method using NP-BSA-bio was applicable to both IgM and IgG, when these antibodies were immobilized on a biosensor chip through anti-Fc antibodies. This situation may be similar to IgM pentamer bound on B cells through Fc{alpha}/µR, suggesting that affinity of IgM could be estimated by binding of NP-BSA-bio with different NP valency. In the present experiment, we were able to show affinity maturation of B cell-bound IgM. Since matured IgM antibodies can only be secreted after secondary stimulation with NP-CGG, maturation proceeded to the stage of generation of memory B cells in possession of IgM BCR. Recently, the presence of IgM+ memory cells was demonstrated in response to phOx, although direct binding to phOx was not shown (43). These IgM+ memory B cells would later convert to IgM-producing plasma cells as a result of booster immunization.

It is likely that the immune system responds to antigens more dynamically than was previously thought. The high sensitivity of the reagents used for flow cytometry analysis allows for the detection of virus-specific T cells and massive expansion of virus-specific CD8+ T cells has been reported (4447). In the present study, we showed that B cells were coated with IgM pentamers, most likely through Fc{alpha}/µR, which caused a massive increase in antigen-binding B cells. The immunological significance of the appearance of these IgM-coated B cells remains unresolved and is currently under investigation.


    Acknowledgements
 
This work was supported by grants from the Ministry of Education, Science, Sports and Culture of Japan. K46m and N1G9 were kindly provided from Dr J. C. Cambier and Dr K. Rajewsky respectively. We thank Dr W. Campbell and Dr Fritz Melchers for critical reading of the manuscript.


    Abbreviations
 
CGG—chicken {gamma}-globulin

NP—(4-hydroxy-3-nitrophenyl)acetyl

NP-BSA bio—NP-BSA-biotin conjugates

PE—phycoerythrin

phOx—phenyloxazolone

SA—streptavidin

SHM—somatic hypermutation

SPR—surface plasmon resonance

TNP—trinitrophenyl


    References
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 

  1. Eisen, H. N. and Siskind G. W. 1964. Variations in affinity of antibodies during the immune response. Biochemistry 3:996.[ISI]
  2. Gearhart, P. J., Johnson N. D., Douglas R. and Hood L. 1981. IgG antibodies to phosphorylcholine exhibit more diversity than their IgM counterparts. Nature 291:29.[ISI][Medline]
  3. Griffiths, G. M., Berek, C., Kaartinen, M. and Milstein, C. 1984. Somatic mutation and the maturation of immune response to 2-phenyl oxazolone. Nature 312:271.[ISI][Medline]
  4. Berek, C. 1993. Somatic mutation and memory. Curr. Opin. Immunol. 5:218.[CrossRef][ISI][Medline]
  5. Milstein, C. and Rada, C. 1995. The maturation of the antibody response. In Hojo, T. and Alt, F. W., eds, Immunoglobulin Genes, 2nd edn, p. 57. Academic Press, New York.
  6. Rajewsky, K. 1996. Clonal selection and learning in the antibody system. Nature 381:751.[CrossRef][ISI][Medline]
  7. Furukawa, K., Akasako-Furukawa, A., Shirai, H., Nakamura, H. and Azuma, T. 1999. Junctional amino acids determine the maturation pathway of an antibody. Immunity 11:329.[ISI][Medline]
  8. Oda, M. and Azuma, T. 2000. Reevaluation of stoichiometry and affinity/avidity in interaction between anti-hapten antibodies and mono- or multivalent antigens. Mol. Immunol. 37:1111.[CrossRef][ISI][Medline]
  9. Bachmann, M. F. and Zinkernagel, R. M. 1997. Neutralizing antiviral B cell responses. Annu. Rev. Immunol. 15:235.[CrossRef][ISI][Medline]
  10. Kouskoff, V., Famiglietti, S., Lacaud, G., Lang, P., Rider, J. E., Kay, B. K., Cambier, J. C. and Nemazee, D. 1998. Antigens varying in affinity for the B cell receptor induce differential B lymphocyte responses. J. Exp. Med. 188:1453.[Abstract/Free Full Text]
  11. Butcher, E. C., Rouse, R. V., Coffman, R. L., Nottenburg, C. N., Hardy, R. R. and Weissman, I. L. 1982. Surface phenotype of Peyer’s patch germinal center cells: implications for the role of germinal centers in B cell differentiation. J. Immunol. 129:2698.[Abstract/Free Full Text]
  12. Kraal, G., Weissman, I. L. and Butcher, E. C. 1982. Germinal centre B cells: antigen specificity and changes in heavy chain class expression. Nature 298:377.[ISI][Medline]
  13. Liu, Y. J., Johnson, G. D., Gordon, J. and MacLennan, I. C. 1992. Germinal centres in T-cell-dependent antibody responses. Immunol. Today. 13:17.[ISI][Medline]
  14. Lalor, P. A., Nossal, G. J., Sanderson, R. D. and McHeyzer-Williams, M. G. 1992. Functional and molecular characterization of single, (4-hydroxy-3-nitrophenyl)acetyl (NP)-specific, IgG1+ B cells from antibody-secreting and memory B cell pathways in the C57BL/6 immune response to NP. Eur. J. Immunol. 22: 3001.
  15. MacLennan, I. C., Liu, Y. J. and Johnson, G. D. 1992. Maturation and dispersal of B-cell clones during T cell-dependent antibody responses. Immunol. Rev. 126:143.[ISI][Medline]
  16. Kelsoe, G. 1996. Life and death in germinal centers (redux). Immunity 4:107.[ISI][Medline]
  17. Smith, K. G., Light, A., Nossal, G. J. and Tarlinton, D. M. 1997. The extent of affinity maturation differs between the memory and antibody-forming cell compartments in the primary immune response. EMBO J. 16:2996.[Abstract/Free Full Text]
  18. Takahashi, Y., Dutta, P. R., Cerasoli, D. M. and Kelsoe, G. 1998. In situ studies of the primary immune response to (4-hydroxy-3-nitrophenyl)acetyl. V. Affinity maturation develops in two stages of clonal selection. J. Exp. Med. 187:885.[Abstract/Free Full Text]
  19. Rajewsky, K., Forster, I. and Cumano, A. 1987. Evolutionary and somatic selection of the antibody repertoire in the mouse. Science 238:1088.[ISI][Medline]
  20. Schittek, B. and Rajewsky, K. 1990. Maintenance of B-cell memory by long-lived cells generated from proliferating precursors. Nature 346:749.[CrossRef][ISI][Medline]
  21. Kimoto, H., Nagaoka, H., Adachi, Y., Mizuochi, T., Azuma, T., Yagi, T., Sata, T., Yonehara, S., Tsunetsugu-Yokota, Y., Taniguchi, M. and Takemori, T. 1997. Accumulation of somatic hypermutation and antigen-driven selection in rapidly cycling surface Ig+ germinal center (GC) B cells which occupy GC at a high frequency during the primary anti-hapten response in mice. Eur. J. Immunol. 27:268.[ISI][Medline]
  22. Lafrenz, D., Strober, S. and Vitetta, E. 1981. The relationship between surface immunoglobulin isotype and the immune function of murine B lymphocytes. V. High affinity secondary antibody responses are transferred by both IgD-positive and IgD-negative memory B cells. J. Immunol. 127:867.[Abstract/Free Full Text]
  23. Gray, D. 1993. Immunological memory. Annu. Rev. Immunol. 11:49.[CrossRef][ISI][Medline]
  24. Klein, U., Goossens, T., Fischer, M., Kanzler, H., Braeuninger, A., Rajewsky, K. and Kuppers, R. 1998. Somatic hypermutation in normal and transformed human B cells. Immunol. Rev. 162:261.[ISI][Medline]
  25. Herzenberg, L. A., Black, S. J. and Tokuhisa, T. 1980. Memory B cells at successive stages of differentiation. Affinity maturation and the role of IgD receptors. J. Exp. Med. 151:1071.[Abstract/Free Full Text]
  26. Reth, M., Petrac, E., Wiese, P., Lobel, L. and Alt, F. W. 1987. Activation of V kappa gene rearrangement in pre-B cells follows the expression of membrane-bound immunoglobulin heavy chains. EMBO J. 6:3299.[Abstract]
  27. Brownstone, A., Mitchison, N. A. and Pitt-Rivers, R. 1966. Chemical and serological studies with an iodine-containing synthetic immunological determinant 4-hydroxy-3-iodo-5-nitrophenylacetic acid (NIP) and related compounds. Immunology 10:465.[ISI][Medline]
  28. Martin, W. J., Wunderlich, J. R., Fletcher, F. and Inman, J. K. 1971. Enhanced immunogenicity of chemically-coated syngeneic tumor cells. Proc. Natl Acad. Sci. USA. 68:469.[Abstract]
  29. Makela, O., Kaartinen, M., Pelkonen, J. L. and Karjalainen, K. 1978. Inheritance of antibody specificity V. Anti-2-phenyl oxazolone in the mouse. J. Exp. Med. 146:1644.
  30. Cumano, A. and Rajewsky, K. 1986. Clonal recruitment and somatic mutation in the generation of immunological memory to the hapten NP. EMBO J. 5:2459.[Abstract]
  31. Allen, D., Simon, T., Sablitzky, F., Rajewsky, K. and Cumano, A. 1988. Antibody engineering for the affinity maturation of an anti-hapten response. EMBO J. 7:1995.[Abstract]
  32. Kozono, Y., Kotzin, B. L. and Holers, V. M. 1996. Resting B cells from New Zealand Black mice demonstrate a defect in apoptosis induction following surface IgM ligation. J. Immunol. 156:4498.[Abstract]
  33. Schlissel, M. S. and Baltimore, D. 1989. Activation of immunoglobulin {kappa} gene rearrangement correlates with induction of germline {kappa} gene transcription. Cell 58:1001.[ISI][Medline]
  34. Sørensen, V., Rasmussen, I. B., Norderhaug, L., Natvig, I., Michaelsen, T. E., Sandlie, I. 1996. Effect of IgM and IgA secretory tailpieces on polymerization and secretion of IgM and IgG. J. Immunol.156:2858.[Abstract]
  35. Justement, L. B., Wienands, J., Hombach, J., Reth, M. and Cambier, J. C. 1990. Membrane IgM and IgD molecules fail to transduce Ca2+ mobilizing signals when expressed on differentiated B lineage cells. J. Immunol. 144:3272.[Abstract/Free Full Text]
  36. Bothwell, A. L., Paskind, M., Reth, M., Imanishi-Kari, T., Rajewsky, K. and Baltimore, D. 1981. Heavy chain variable region contribution to the NPb family of antibodies: somatic mutation evident in a gamma 2a variable region. Cell 24:625.[ISI][Medline]
  37. Shimizu, T., Oda, M. and Azuma, T. 2003. Estimation of the relative affinity of B cell receptor by flow cytometry. J. Immunol. Methods 276:33.[CrossRef][ISI][Medline]
  38. Imanishi-Kari, T., Rajnavoloyi, E., Takemori, T., Jack, R. and Rajewsky, K. 1979. The effect of light chain gene expression on the inheritance of an idiotype associated with primary anti-(4-hydroxy-3-nitrophenyl)acetyl (NP) antibodies. Eur. J. Immunol. 9:324.[ISI][Medline]
  39. Shibuya, A., Sakamoto, N., Shimizu, Y., Shibuya, K., Osawa, M., Hirayama, T., Eyre, H., Sutherland, G. R., Endo, Y., Fujita, T., Miyabayashi, T., Sakano, S., Tsuji, T., Nakayama, E., Phillips, J. H., Lanier, L. L. and Nakauchi, H. 2000. Fc{alpha}/µ receptor mediates endocytosis of IgM-coated microbes. Nat. Immunol. 1:441.[CrossRef][ISI][Medline]
  40. Sakamoto, N., Shibuya, K., Shimizu, Y., Yotsumoto, K., Miyabayashi, T., Sakano, S., Tsuji, T., Nakayama, E., Nakauchi, H. and Shibuya, A. 2001. A novel Fc receptor for IgA and IgM is expressed on both hematopoietic and non-hematopoietic tissues. Eur. J. Immunol. 31:1310.[CrossRef][ISI][Medline]
  41. Toyama, H., Okada, S., Hatano, M., Takahashi, Y., Takeda, N., Ichii, H., Takemori, T., Kuroda, Y. and Tokuhisa, T. 2002. Memory B cells without somatic hypermutation are generated from Bcl6-deficient B cells. Immunity 17:329.[ISI][Medline]
  42. Tobita, T., Oda, M. and Azuma, T. 2004. Segmental flexibility and avidity of IgM in the interaction of polyvalent antigens. Mol. Immunol. 40:803.[CrossRef][ISI][Medline]
  43. White, H. and Gray, D. 2000. Analysis of immunoglobulin (Ig) isotype diversity and IgM/D memory in the response to phenyl-oxazolone. J. Exp. Med. 191:2209.[Abstract/Free Full Text]
  44. Butz, E. A. and Bevan, M. J. 1998. Massive expansion of antigen-specific CD8+ T cells during an acute virus infection. Immunity 8:167.[ISI][Medline]
  45. Murali-Krishna, K., Altman, J. D., Suresh, M., Sourdive, D. J., Zajac, A. J., Miller, J. D., Slansky, J. and Ahmed, R. 1998. Counting antigen-specific CD8 T cells: a reevaluation of bystander activation during viral infection. Immunity 8:177.[ISI][Medline]
  46. Flynn, K. J., Riberdy, J. M., Christensen, J. P., Altman, J. D. and Doherty, P. C. 1999. In vivo proliferation of naive and memory influenza-specific CD8+ T cells. Proc. Natl Acad. Sci. USA 96:8597.[Abstract/Free Full Text]
  47. Tan, L. C., Gudgeon, N., Annels, N. E., Hansasuta, P., O’Callaghan, C. A., Rowland-Jones, S., McMichael, A. J., Rickinson, A. B. and Callan, M. F. 1999. A re-evaluation of the frequency of CD8+ T cells specific for EBV in healthy virus carriers. J. Immunol. 162:1827.[Abstract/Free Full Text]




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