Two waves of memory B-cell generation in the primary immune response
Ayako Inamine1,2,
Yoshimasa Takahashi1,
Nobue Baba1,
Kensuke Miyake3,
Takeshi Tokuhisa4,
Toshitada Takemori1 and
Ryo Abe2,5
1 Department of Immunology, National Institute of Infectious Diseases, 1-23-1 Toyama, Shinjuku-ku, Tokyo 162-8640, Japan
2 Division of Immunobiology, Research Institute for Biological Science and 5 Genome and Drug Research Center, Tokyo University of Science, 2669 Yamazaki, Noda, Chiba 278-0022, Japan
3 Division of Infectious Genetics, Institute of Medical Science, University of Tokyo, Tokyo 108-0071, Japan
4 Department of Developmental Genetics (H2) Graduate School of Medicine, Chiba University, Chiba 260-8670, Japan
Correspondence to: T. Takemori; E-mail: ttoshi{at}nih.go.jp
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Abstract
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Memory B cells can be generated independently of germinal center (GC) formation and affinity maturation in Bcl-6-deficient mice, but the contribution of the GC-independent pathway for memory B-cell generation in normal mice remains unknown. To examine this, we administrated anti-inducible co-stimulator (ICOS) mAbs into mice at the onset of GC formation in the primary response. This manipulation affected the generation of GC B cells in the spleen, but neither IgG1 memory B cell nor production of IgG1 long-term antibody was affected. In ICOS-manipulated mice, GC B cells accumulated somatic mutations in the IgVH genes and underwent affinity maturation; however, memory B cells scarcely accumulated mutations and reconstituted the secondary response by low affinity, supporting the notion that low-affinity memory B cells are generated in a GC-independent manner. Thus, it appears that memory B cells are established by two different pathways, associated with or without GC reaction and affinity maturation. The generation and long-term persistence of low-affinity IgG1 memory B cells and antibodies in ICOS-manipulated mice support the idea that low-affinity memory B cells may give rise to long-term antibody-forming cells.
Keywords: affinity maturation, blocking antibody, germinal center, ICOS, somatic mutations
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Introduction
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The white pulp cord of the spleen is histologically divided into the B-cell follicle and T-cell area [the periarteriolar lymphocytic sheath (PALS)], where chemokines B Lymphocyte chemoattractant (BLC) and secondary lymphoid chemokine (SLC)/EBV-induced molecule 1 ligand chemokine (ELC) are synthesized by stromal cells, respectively (1). BLC is a chemoattractant to B cells via CXCR5 (25), whereas SLC and ELC are chemoattractants to T cells through CCR7 (68). Upon T-cell-dependent antigen exposure, B cells increase expression of CCR7 and increase responsiveness to the CCR7 ligands, resulting in a migration towards the T-cell area (6). On the other hand, T cells are stimulated with antigens presented by dendritic cells in the inner PALS, followed by expression of CXCR5, which may, in turn, result in T-cell migration towards the B-cell area (9, 10). Such dynamics upon an antigenic challenge enable B cells and T cells to mutually activate at the boundary of the B-cell follicle and T-cell area (11) through interactions with co-stimulatory ligandreceptor pairs expressed on B and T cells, respectively, such as the CD40CD40 ligand (CD40CD154), B7.1 (CD80)/B7.2 (CD86)CD28 and inducible co-stimulator (ICOS)/activation-inducible lymphocyte immunomodulatory (AILIM)ICOS/AILIM ligands (1217).
After TB interaction at the boundary of the B-cell follicle and T-cell area, activated B cells increase expression of lymphotoxin
, which in turn induces the maturation of follicular dendritic cells in the B-cell follicle (18). Mature follicular dendritic cells then secrete BLC and attract activated B cells to form germinal centers (GCs) (19). GC B cells express activation-induced cytidine deaminase and accumulate somatic mutations in their Ig genes, resulting in the generation of high-affinity variants (20). Although it has been widely believed that the precursors for memory B cells are generated in GCs, the accumulation of B cells without somatic mutations in the memory compartment of normal and GC-deficient mice suggests that memory B-cell development itself starts to operate at the early immune response, probably without a GC environment and the affinity maturation of B cell receptor (BCR) (21, 22). Supporting this idea, it has been observed that memory phenotype B cells already appeared at day 7 post-immunization before the improvement of BCR affinity within GCs (23). Moreover, transgenic B cells expressing low-affinity BCR can differentiate into memory B cells even without the improvement of BCR affinity (24). Thus, it might be possible that low-affinity memory B cells can be generated through a GC-independent pathway in the early primary response as well as a GC-dependent pathway at a later stage for high-affinity memory B cells.
Memory B-cell development is completely dependent on helper signals from T cells, so the blockade of CD40CD154, CD80/CD86CD28 and ICOS/AILIMICOS/AILIM ligand (B7h/B7RP-1/GL50) interactions impairs GC formation and the memory B-cell response (2532). Injection of antibodies for CD154 and CD86 at the onset of GC formation has been used to block GC reaction and following memory B-cell development without inhibiting the foci of antibody-forming cells (AFCs) or an early serum antibody response (33). Thus, injection for blocking antibodies at the onset of GC formation allows us to elucidate the role of GC reaction in the following B-cell response.
ICOS/AILIMICOS/AILIM ligand interaction has been shown to be involved in the primary B-cell response by IgG antibody production, GC B-cell expansion and memory B-cell generation (34). In gene-targeted ICOS/AILIM- or ICOS/AILIM ligand-deficient mice (ICOS/ or ICOS-L/ mice), the IgG antibody response as well as the size and mean number of GCs in the spleen were impaired in the primary response to T-cell-dependent antigens (31, 32, 35), although the effect on affinity maturation was unknown. The secondary IgG antibody response was reduced in ICOS/ mice compared with wild type (WT) mice (35), suggesting a reduction in the number of memory B cells or a defect in their functions.
To investigate whether or not memory B-cell development occurs at the early immune response in the absence of a GC environment in normal mice, the mice were injected with anti-ICOS/AILIM mAb at day 5 post-immunization when GC formation begins. ICOS/AILIM is expressed on activated T cells within GCs (10); therefore, we expected that the injection of anti-ICOS/AILIM antibody at the onset of GC formation would lead to the selective blockade of a GC-dependent pathway for memory B-cell generation. We show here that the blockade of ICOS/AILIMICOS/AILIM ligand interaction within GCs impairs the recruitment of high-affinity B cells into the memory compartment, whereas low-affinity memory B cells are generated and maintained for a long period. Taken together with previous findings, we propose that low-affinity memory B cells can be generated without GCs and maintained for a long period when higher affinity competitors are reduced.
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Methods
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Mice and immunization
Female C57BL/6 mice and C57BL/6 mice with deficient recombination-activating gene (Rag-1/) were obtained from SLC Inc. (Hamamatsu, Japan) and the Jackson Laboratory (Bar Harbor, ME, USA), respectively. All mice were maintained in a specific pathogen-free condition. C57BL/6 mice were intra-peritoneally (i.p.) immunized with 100 µg of (4-hydroxy-3-nitrophenyl) acetyl (NP) coupled on chicken gamma globulin (NP16-CG) in alum as previously described (21). At days 5, 6 and 7 post-immunization, the mice were intravenously injected with 100 µg of anti-ICOS/AILIM mAb (B10.5; 36) or isotype-matched rat IgG2a mAb against phenyloxazolone (G18) as the control mAb. All mice were used in accordance with National Institute of Infectious Diseases Institutional Animal Care and Use Committee guidelines.
Cell staining and sorting
A single-cell suspension was prepared from spleens and bone marrows (BMs) as described previously (21). Cells were incubated with 10 µg ml1 of anti-Fc
RII/III (2.4G2; BD Biosciences, San Jose, CA, USA) prior to staining with antibodies. To track the generation of GC B cells, splenocytes were stained with PE-conjugated (4-hydroxy-5-iodo-3-nitrophenyl) acetyl (NIP)34-BSA (NIP-BSAPE), Texas red-coupled anti-B220 (B220TX), allophycocyanin (APC)-conjugated anti-CD38 (CS/2, CD38APC) and FITC-coupled peanut agglutinin (PNA) at 1 : 100 dilutions (PNAFITC; Biomeda Vector Laboratories, Burlingame, CA, USA). As a control, cells were stained with BSAPE, instead of NIP-BSAPE. To monitor the generation of memory B cells, cells were incubated with a mixture of biotinylated antibodies against IgM, IgD, CD90, CD5, CD43 and CD11b (BD Biosciences) to exclude naive B cells, B-1 cells, plasma cells and T cells from the analysis. After washing, cells were stained for 30 min with a mixture of NIP-BSAPE, IgG1FITC, AlexaFluor 647-coupled anti-CD38 and AlexaFluor 594-coupled anti-B220 mAbs. After washing, cells were incubated with Tricolor (TC)-conjugated streptavidine (streptavidineTC; Caltag Laboratories, Burlingame, CA, USA) and propidium iodide (Sigma, St Louis, MO, USA) to exclude dead cells from the analysis.
To purify GC and memory B cells, splenocytes were incubated with 10 µg ml1 of anti-Fc
RII/III mAb for 20 min, followed by a 30-min incubation with biotinylated mAbs against IgM, IgD, CD90, CD5, CD43 and CD11b. After washing, cells were incubated with streptavidine-coated microbeads (MiltenyiBiotec, Glad Bach, Germany) and IgM/IgD B cells were enriched by a MACS column (MiltenyiBiotec) according to the manufacturer's instructions. Enriched cells were provided for staining as described above. After washing, dead cells were excluded from the population and NIP-binding (NIP-bdg) GC B cells (IgG1dull/CD38dull/B220+/IgM/IgD/CD43/CD5) and memory B cells (IgG1high/CD38+/B220+/IgM/IgD/CD43/CD5) were sorted by FACSVantage SE (Becton Dickinson, Mountain View, CA, USA), as described previously (21).
ELISPOT and ELISA assay
NP-specific AFCs in BM were detected by enzyme-linked immunospot (ELISPOT) with the aid of HRP-conjugated goat antibodies against mouse IgM or IgG1 (Southern Biotechnology Associates, Birmingham, AL, USA) as developing reagents as previously described (36). The total number of anti-NP AFCs was measured by using NP18-BSA and high-affinity ones were measured by NP2-BSA as coating reagents (37).
Immunohistochemistry
Spleens were obtained from mice that had been injected with anti-AILIM/ICOS mAb or control mAb. Spleens were embedded in O.C.T. compound (Miles, Elkhart, IN, USA), frozen in liquid nitrogen for 30 s and stored at 80°C. Frozen sections (5 µm thick) were fixed in acetone on ice for 10 min. After washing with a staining buffer (0.1 M Tris, pH 8.0, 0.1 M NaCl, 0.1% Tween 20, 1% BSA, 30% heat-inactivated normal goat serum), the sections were pre-incubated with a staining buffer containing anti-Fc
RII/IIImAb (2.4G2) (100 µg ml1) or purified MOPC21 (1 mg ml1) or both for 4 h at room temperature or overnight at 4°C. All reagents for staining were centrifuged at 15 000 revolutions per minute for 20 min before use. To investigate the formation of small foci of NP-specific B cells in the spleen, the sections were stained with a mixture of NIP-BSAPE at 1 : 10 dilution and APC-conjugated anti-B220 (anti-B220APC; 2 µg ml1, BD Biosciences) and PNAFITC at 1 : 100 dilution. As a negative control, BSAPE was used for staining, instead of NIP-BSAPE. To investigate the expression of Bcl-6, the sections were stained with a mixture of biotinylated PNA (1 : 50, Biomeda Vector), anti-B220APC and purified rabbit anti-Bcl-6 (20 µg ml1) at the first step, followed by PE-coupled ultra-avidin at 1 : 100 dilution (Leinco Technologies Inc. St Louis, MO, USA) and FITC-coupled affinity-purified (Fab')2 goat anti-rabbit IgG at 1 : 200 dilution (CAPPEL, West Chester, PA, USA), which was pre-absorbed with murine and rat Igs. Anti-Bcl-6 antibody was affinity purified from the immune sera of rabbits that had been immunized with glutathione-S-transferase-murine Bcl-6 fusion protein (38). In some experiments, instead of NIP-BSAPE, anti-CD4PE (12 µg ml1; BD Biosciences) was used for staining in combination with PNAFITC and anti-B220APC. The samples were examined by confocal laser microscopy (LSM510 Laser Scan Microscope; Carl Zeiss, Oberkochen, Germany) with the aid of dual Argon laser (488 nm)/Helium Neon laser (543 and 433 nm) systems.
Sequence analysis of VH gene
NIP-bdg GC and memory B cells were directly sorted into 400 µl of proteinase K solution (500 µg ml1 proteinase K in PBS) and incubated overnight at 37°C. The crude lysate was subjected to two rounds of nested PCR for amplifying the VH186.2 gene. PCR products were size fractionated by agarose gel electrophoresis and the purified fragments were cloned into the PCR-ScriptTM Amp cloning vector (Stratagene, La Jolla, CA, USA). The sequence of the variable (V), diversity (D) and joining (J) segment in a plasmid vector was determined by automated sequencing using an ABI PRISM 337 DNA sequencer (PE Biosystems, Foster City, CA, USA).
Cell transfer
C57BL/6 mice were immunized with NP-CG in alum, followed by injection with anti-AILIM/ICOS mAb or control Ig or immunized with CG (100 µg per head) in alum. Sixty to seventy days after immunization, a single-cell suspension was prepared from the pooled spleens of NP-primed and ICOS/AILIM-manipulated mice. For B-cell purification, splenocytes were incubated with anti-Fc
RII/III antibody and with a mixture of biotinylated mAbs against CD3, CD8, CD11b, TER-119, Gr-1 and CD43. After washing, B cells were negatively enriched by a MACS system (>95% B220+). T cells were purified from the pooled spleens of CG-primed C57BL/6 mice (>90% CD3+) by a MACS system after incubation with mAb against B220, IgM, IgD, CD11b, TER-119 and Gr-1. Purified B cells (3 x 107) and T cells (1.5 x 107) were mixed and adoptively transferred into Rag-1/ mice by intravenous injection. Twenty-four hours after the transfer, the recipient mice were i.p. challenged with 50 µg of soluble NP-CG.
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Results
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GC B cells were detected in the spleen at day 5 post-immunization
Murine B cells express CD38 at a resting state, whereas the expression is down-regulated in GC B cells and plasma cells (39). To analyze the generation of GC B cells upon stimulation with T-cell-dependent antigen, we enumerated NP-specific CD38dull B cells in the spleen of C57BL/6 mice after immunization with alum-precipitated NP-CG. As shown in Fig. 1(A), a small number of NIP-bdg/CD38dull B cells was detected in the spleen at day 5 post-immunization by FACS analysis, followed by a rapid expansion up to day 6 post-immunization. NIP-bdg/CD38dull cells consisted of B220+ or B220dull B cells that bound high or low levels of PNA, respectively. NIP-bdg/CD38dull/PNAhigh B cells were of GC origin (40) and detected in the spleen at day 5 post-immunization at a low level but increased in number from 1 x 104 to 2 x 103 per splenocyte at day 6 post-immunization.

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Fig. 1. GC B cells are generated in the spleen from day 4 to day 6 post-immunization. Splenocytes were obtained from NP-CG-primed C57BL/6 mice at the indicated periods and incubated with anti-Fc RII/III mAb, followed by APC-conjugated anti-CD38, B220TX, PNAFITC and NIP-BSAPE at the first step and propidium iodide at the second step. (A) Viable NIP-bdg B cells were selected under a lymphocyte gate on forward and light scatter (data not shown). Thereafter, CD38dull-negative cells were gated (top) and the number of PNA+/B220+ cells was determined (bottom, boxed). (B) The mean number (open columns) of NIP-bdg/CD38dull-negative/PNA+/B220+ cells in individual mice (closed circle) is shown.
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GC reaction starts at the T-cell and B-cell boundary from day 4 to day 6 post-immunization
We analyzed the histological localization of NIP-bdg B cells in the spleen of C57BL/6 mice from day 4 to day 6 post-immunization with alum-precipitated NP-CG. As shown in Fig. 2, microscopic examinations with low (x20) and high magnifications (x40) revealed that NIP-bdg B cells formed various sizes of clusters in the white pulp cord (Fig. 2A and C) as well as the red pulp (Fig. 2C) of the spleen at day 5.5 post-immunization. Clusters consisting of B220 cells with a high level of Ig in the red pulp (Fig. 2C) or in the T-cell area (Fig. 2D) may represent plasma blast foci, whereas NIP-bdg B cells with a low level of Ig formed small foci along the B-cell follicle in the vicinity of the T-cell-rich inner PALS at day 5.5 post-immunization (Fig. 2AD). As shown in Fig. 2(B and D), these cells bound to a high level of PNA and expressed cytoplasmic Bcl-6 (Fig. 2F), which indicated that they were GC B cells. The foci developed in size towards the B-cell area at day 6 post-immunization (Fig. 2E and G), which could be recognized as a typical GC structure. In contrast, Bcl-6+/PNA+ cells were barely detected in the PALS or within the B-cell area of the spleen in non-immunized C57BL/6 mice and immunized and non-immunized CD40-deficient mice (data not shown). Together, these results suggest that antigen-activated B cells may histologically develop into GC B cells from day 4 to day 6 post-immunization at the border of the B-cell follicle and T-cell area.

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Fig. 2. NIP-bdg GC B cells form a small cluster in the spleen from day 5 to day 6 post-immunization. Cryosections were prepared from spleens of C57BL/6 mice immunized with NP-CG/alum at day 5.5 (A, B, C, D and F) and day 6 (E and G) post-immunization. In (AE), they were stained with NIP-BSAPE (green), anti-B220APC (blue) and PNAFITC (red). Pink, yellow or white staining represents NIPB220+PNA+ cells, NIP+B220PNA+ cells or NIP+B220+PNA+ cells, respectively. In (F and G) sections were stained with biotinylated PNA, anti-B220APC (blue) and rabbit anti-Bcl-6 antibodies at the first step and developed by ultra-avidinPE (red) and anti-rabbit IgGFITC (green) at the second step. Pink or yellow staining represents B220+PNA+ cells or Bcl-6+B220PNA+ cells, respectively. Co-localization of Bcl-6 and PNA was observed in a small number of cells for unknown reasons. The NIP-BSAPE-positive signals were diminished by pre-incubation with NIP-keyhole limpet hemocyanin (KLH) at a dose 10 times that of NIP-BSA, but neither with BSA nor KLH.
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Memory B cells were generated independent of the formation of GCs
The mice deficient for ICOS/AILIMICOS/AILIM ligand interaction show an impaired memory B-cell response (31, 32, 35), suggesting that this interaction is required for both GC-dependent and -independent pathways of memory B-cell generation. To selectively inhibit memory B-cell generation through GCs, anti-ICOS/AILIM mAb was administered into mice at the onset of GC formation (at day 5 post-immunization). C57BL/6 mice were immunized with alum-precipitated NP-CG and injected with either rat anti-ICOS/AILIM mAb or control rat IgG antibody at days 5, 6 and 7 post-immunization. As shown in Fig. 3(A), administration of anti-ICOS/AILIM mAb reduced the size of each GC at day 10 post-immunization as has been observed in ICOS/AILIM- or ICOS/AILIM ligand-deficient mice (31, 32, 35).

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Fig. 3. ICOS/AILIM-manipulated mice with a reduced number of GC B cells, but not memory B cells in their spleens. Mice were immunized with NP-CG in alum and treated with control antibody or anti-ICOS/AILIM mAb at days 5, 6 and 7 post-immunization. (A) Splenic cryosections were prepared from the mice at day 10 post-immunization and stained with PNAFITC (green), B220APC (blue) and CD4PE (red). The size of each GC per section was estimated by LSM510 software. (B) Anti-ICOS mAb (closed circle) or control Ig (open circle) was administered into C57BL/6 mice (n > 5) at days 5, 6 and 7 post-immunization with NP-CG in alum. The number of GC B cells and memory B cells in the spleen of individual mice was analyzed by FACS at days 10, 30 and 70 post-immunization.
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As shown in Fig. 3(B), FACS analysis revealed that NP-specific IgG1+ memory B cells with phenotype IgG1high/CD38+/B220+/IgM/IgD/CD43/CD5 were detected in the spleens of ICOS/AILIM-manipulated mice at days 10, 30 and 70 post-immunization, comparable to the level found in control mice. In contrast, as expected from histological analysis, the number of NP-specific IgG1+ GC B cells with phenotype IgG1dull/CD38dull/B220+/IgM/IgD/CD43/CD5 was significantly reduced in anti-ICOS/AILIM-treated mice approximately five to six times below the level found in control mice at day 10 post-immunization (P = 0.0004), whereas the effect was marginal at day 30 post-immunization (P = 0.048), probably owing to the temporal effect of anti-ICOS/AILIM mAb. These results suggest that anti-ICOS/AILIM antibody treatment at the onset of GC formation blocked the full maturation of GCs, but it did not affect the number of memory B cells retained over a long period.
Administration of anti-ICOS/AILIM mAb reduced the frequency of memory B cell accumulating mutations and impaired affinity maturation of IgG1 antibodies
Within GCs, B cells accumulate somatic mutations and generate high-affinity variants, which are eventually selected as the precursors of either memory B cells or long-lived AFCs (41, 42). Thus, memory B cells generated through the GC-dependent pathway carry somatically mutated V genes with improved affinity, while those generated through the GC-independent pathway do not carry them. To gain an insight into the origin of memory B cells in anti-ICOS/AILIM antibody-treated mice, NIP-bdg GC and memory B cells were purified from mice that had been treated with anti-ICOS/AILIM mAb or control IgG (each group, n = 15). The rearranged VH186.2 gene in purified B cells was amplified by PCR and sequenced as previously described (21). As shown in Fig. 4, NIP-bdg GC B cells accumulated both somatic mutations in the VH186.2 gene and a nucleotide substitution from tryptophan to leucine at amino acid position 33 (Leu 33) at days 10 and 30 post-immunization, as efficiently as did GC B cells in normal mice. The Leu 33 substitution generally results in a 10-fold increase in affinity of the germ line gene product (43), supporting the notion that the anti-ICOS/AILIM antibody treatment does not affect the clonal selection for high-affinity B cells within GCs.

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Fig. 4. Manipulation by anti-ICOS/AILIM mAb reduced the frequency of memory B cell bearing affinity-enhancing mutation. NIP-bdg GC B cells and memory B cells were purified from the pooled spleens of C57BL/6 mice (each group: n = 15), which had been treated with control antibody or anti-ICOS/AILIM antibody. The rearranged VH genes were amplified by two rounds of nested PCR. Genes encoded by VH186.2 were dominant among the recovered clones (>90%) and selected for further analysis. Open circles represent the number of mutations in individual VH186.2 genes and closed circles indicate the number of mutations in individual genes carrying a Trp to Leu substitution at position 33 (Leu 33) together with Tyr at position 99 (Tyr99). Complete sequence data are available from DDBJ/EMBL/GenBank under accession numbers AB187273AB187380 (GC B cells) and AB187381AB187502 (memory B cells).
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In contrast, the frequency of mutations in NIP-bdg memory B cells was significantly reduced in anti-ICOS/AILIM-manipulated mice at days 30 and 70 post-immunization (P = 0.002 and P < 0.0001; Fig. 4C and D). In particular, non-mutated cells were dominant in the memory compartment of ICOS/AILIM-manipulated mice at day 70 post-immunization, whereas the majority of the memory compartment of control mice consisted of mutated ones. These results suggest that the majority of memory B cells detected in ICOS/AILIM-manipulated mice is not of a GC origin.
Administration of anti-ICOS/AILIM mAb impaired affinity maturation of IgG1 antibodies, but not the amount of antibodies
Figure 5 shows that NP-specific IgG1 antibodies were comparable between ICOS-manipulated mice and control mice; however, the level of high-affinity NP-specific IgG1 antibodies was reduced in anti-ICOS/AILIM-treated mice, three to four times below the level of the control mice at day 20 (P = 0.008), day 40 (P = 0.0018) and day 70 (P = 0.003) post-immunization (Fig. 5B). This resulted in a significant reduction in the ratio of high-affinity to total IgG1 antibodies (NP2/NP18) in the anti-ICOS/AILIM-treated mice throughout the immune response (Fig. 5C). These results suggest that the injection of anti-ICOS/AILIM mAb at the onset of GC formation had a long-lasting effect on the affinity maturation of NP-specific IgG1 antibodies.

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Fig. 5. Affinity maturation in the anti-NP IgG1 antibody response was impaired by injection of anti-ICOS/AILIM mAb at day 5 post-immunization. Mice were immunized with NP-CG in alum and injected intravenously with control rat Ig (open circle; n = 8) or rat anti-ICOS/AILIM mAb (closed circle; n = 8) at days 5, 6 and 7 post-immunization. The level of NP-specific IgG1 antibody in individual serum was estimated at days 10, 20, 40 and 70 post-immunization by ELISA with the aid of NP18-BSA and NP2-BSA as coated antigens for estimation of the total (A) and high-affinity (B) NP-specific IgG1 antibodies. (C) The ratios of total to high-affinity antibodies were calculated. The data were statistically evaluated by the MannWhitney non-parametric test (two-tailed). The data are representative of three independent experiments.
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Memory B cells in anti-ICOS/AILIM mAb-treated mice reduced the affinity of the secondary IgG1 response
According to the VH gene sequence, it appeared that memory B cells in ICOS/AILIM-manipulated mice mostly consisted of low-affinity cells. To evaluate the validity of this, NP-primed B cells were purified from the pooled spleens (n = 11) of ICOS-manipulated or control mice at day 70 post-immunization. B cells were mixed with or without CG-primed T cells of normal mice and adoptively transferred into Rag-1/ mice, followed by challenge with soluble NP-CG 24 h after transfer. As shown in Fig. 6, NP-primed B cells of ICOS/AILIM-manipulated mice and control mice efficiently reconstituted an anti-NP IgG secondary response in adoptive recipients at similar levels; however, the affinity of serum antibodies was low in the hosts repopulated with NP-primed B cells of ICOS/AILIM-manipulated mice compared with the serum antibody affinity of the control mice (data not shown). Likewise, the frequency of NP-specific IgG1 AFCs was comparable between the control and ICOS/AILIM-manipulated mice (Fig. 6A), but the ratios of high-affinity NP-specific IgG AFCs were significantly reduced in ICOS/AILIM-manipulated mice (Fig. 6B; P = 0.0032). Taken together, these results suggest that the blockade of ICOS/AILIMICOS/AILIM ligand interaction at the onset of GC formation severely impaired the recruitment of high-affinity B cells into the memory compartment.

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Fig. 6. Anti-NP secondary response in Rag-1/ mice reconstituted with B cells from anti-ICOS/AILIM mAb-treated mice. B cells were purified from the pooled spleens (n = 11) of mice that had been immunized with NP-CG in alum 70 days before or non-immunized mice (naive). These mice were treated with either control antibody or anti-ICOS/AILIM mAb at days 5, 6 and 7 post-immunization. Purified B cells were mixed with T cells purified from the pooled spleens of CG-immunized mice and transferred into RAG-1-deficient mice. Recipient mice were challenged with (+) or without () 50 µg of NP-CG 1 day after transfer. Anti-NP AFCs in the BM were estimated at day 10 post-immunization by ELISPOT with the use of NP2-BSA or NP18-BSA as a coating antigen. (A) Circles represent the number of NP18-specific IgG1 AFCs in individual mice (open circle, control Ig; closed circle, anti-ICOS/AILIM). (B) The ratios of NP2/NP18-specific IgG1 AFCs were plotted. The data were statistically evaluated by the MannWhitney non-parametric test (two-tailed). The data are representative of three independent experiments.
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Discussion
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B cells and T cells interact at the border of the B-cell follicle and T-cell area from day 2 to day 3 post-immunization (11). The present results raise the possibility that activated B cells progress to GC B cells and start to form small foci at the boundary of the B-cell follicle and T-cell area in the spleen at day 5 post-immunization. These foci developed in size vigorously from day 5.5 towards the B-cell area; however, the development was inhibited by anti-ICOS/AILIM mAbs given at day 5 post-immunization, suggesting that the ICOS/AILIMICOS/AILIM ligand interaction is required for either the generation (3032) or full maturation of GCs, although they are not mutually exclusive.
It has been widely believed that memory B cells are generated after the affinity maturation of BCR within GCs (41); however, recent studies using Bcl-6-deficient mice and transgenic mice expressing low-affinity BCR revealed that memory B cells can be generated independent of GCs and affinity maturation of BCR (22, 24). To exclude the secondary effect of gene modification, we produced GC-deficient mice by injecting anti-ICOS mAb into normal mice at the onset of GC formation and monitored the kinetics of GC and memory B cells in response to NP-CG in alum. We observed that the number of memory B cells in the primary immune response in the ICOS-manipulated mice is comparable to that of control immunized mice. The majority of memory B cells in the ICOS-manipulated mice did not accumulate somatic mutations and these cells reconstituted the secondary response in adoptive hosts by low-affinity IgG1 antibodies, suggesting that in the normal primary response non-mutated and low-affinity memory B cells are generated without GC formation. The origin of GC-independent and GC-dependent memory B cells is unknown, but it is conceivable that the majority of memory B cells generated at the early immune response may enter into the GC and convert into high-affinity ones by the accumulation of somatic mutations at later time points. Alternatively, non-mutated memory B cells are generated from naive B cells at the initial immune response, whereas mutated ones are differentiated from GC B cells upon antigen-driven selection. In addition, considering that marginal zone B cells promptly respond to T-cell-dependent antigens (44), we could not exclude another possibility that marginal zone and follicular B cells generate memory cells with different kinetics after immunization.
The number of memory B cells in the ICOS-manipulated mice was retained at a level similar to that of normal mice until day 70 post-immunization, suggesting that low-affinity memory B cells are maintained as long as those generated in GCs, regardless of their reduced BCR affinity. The memory compartment mostly consisted of non-mutated cells in the ICOS-manipulated mice, whereas non-mutated ones only exist at a level of 1020% in normal memory B cells (21, 37). Despite the different cellular component, both the ICOS-manipulated mice and normal mice retained a comparable number of memory cells in the spleen over a long period after immunization, suggesting that the size of the memory compartment in the immune response is limited. Memory B cells migrate into the marginal zone in the spleen after selection (45, 46), which may provide the limiting space for their persistence. Therefore, a reduction in the frequency of non-mutated cells in the normal memory compartment, in parallel to the generation of mutated and high-affinity memory cells, could be attributable to the competition between low- and high-affinity memory B cells in the resident place, if an antigen is involved in the efficient persistence of memory cells (45, 46). Alternatively, as discussed above, involvement of non-mutated memory B cells in the GC reaction could be associated with a reduction in the frequency of non-mutated cells in the normal memory compartment. Further analysis is needed to clarify this issue.
Previous reports suggest that the memory B-cell response is impaired in mice deficient in ICOS or ICOS-L, probably owing to the malfunction of T and B cells in the primary response (31, 32, 35). Consistently, affinity maturation in serum IgG1 antibodies was impaired in tertiary immunized ICOS-L/ mice (35). ICOS-L is constitutively expressed in naive B cells at a low level, whereas T cells express ICOS upon T-cell antigen receptor stimulation (47), thereby raising the possibility that ICOS-ICOS-L co-stimulation is involved in the early phase of B- and T-cell interaction upon antigen stimulation. It has been reported that B cells in the spleen of normal mice that had been immunized with NP-ovalbumin peptide (OVA) in CFA 10 days before reconstitute the secondary response in adoptive hosts upon stimulation with NP-OVA in incomplete Freund's adjuvant (IFA), whereas B cells of ICOS-L-deficient mice do not (35). The present view is that ICOSICOS-L co-stimulation may play a role in GC-independent memory B-cell generation at the early immune response as well as a GC-dependent one at the later immune response. Together, the present results suggest that memory B cells are generated by two different pathways, independent of and dependent on GC formation and affinity maturation, whereas both pathways may require ICOSICOS-L co-stimulation.
Expansion of GC B cells peaks at day 7 to day 10 post-immunization (21, 23). The frequency of GC B cells rapidly declined at day 20 and finally reached a level
10 times above the level of non-immunized mice, although GC B cells continuously accumulated somatic mutations in the rearranged VH genes over long periods following immunization (21). As expected from the results of the ICOS-deficient mice (31, 32, 35), administration of anti-ICOS mAb at the onset of GC formation (at day 5 post-immunization) caused a five- to six-time reduction in GC response at day 10 post-immunization, compared with the level of the control mice (P = 0.0004), whereas it did not affect the accumulation of somatic mutations and subsequent affinity maturation in GC B cells. ICOS manipulation caused a 2-fold reduction in the number of GC B cells relative to that of control mice at day 30 post-immunization (P = 0.048), in which the remaining GC B cells increased the number of mutations per VH genes and underwent affinity maturation, as efficiently as did control GC B cells. Given that GC B cells accumulate somatic mutations with proliferation (48), the results may imply that GC B cells may proliferate in ICOS-manipulated mice as efficiently as do GC B cells in control mice but give rise to fewer survivor cells at each round of proliferation. ICOSICOS-L signaling causes T-cell proliferation and Th2 cytokine production, including IL-4, which may support GC B-cell development and survival (49, 50). Further analysis is needed to clarify this issue.
A high-affinity IgG response persists over a long period of post-immunization, as a result of the generation of long-lived IgG plasma cells by antigen-driven selection in GCs (51, 52). In ICOS/ or ICOS-L/ mice, the IgG1 antibody response is diminished, although the level of the reduction varied depending on the antigens and adjuvants used for the immunization (31, 32). For example, immunization with NP-OVA in CFA caused only a marginal reduction in IgG1 antibody response in ICOS/ mice, whereas the response to keyhole limpet hemocyanin in IFA was profoundly reduced in the mutant animals (31). In this context, we observed that the administration of anti-ICOS mAb at the onset of GC formation had little effect, if any, on IgG antibody response in serum to NP-CG in alum, although it impaired the production of high-affinity antibodies by day 70 post-immunization. High-affinity long-lived AFCs may arise early in the GC reaction (51). Therefore, the reduced level of high-affinity IgG antibodies in the ICOS-manipulated mice may reflect a loss of precursors early in the response, as reflected by impairment of the GC reaction. If so, what is the origin of the long-lasting IgG antibodies in the sera of the ICOS-manipulated mice? Considering the recent observation that high-affinity memory B cells may give rise to long-term AFCs (53), we propose that low-affinity memory B cells also give rise to long-lasting AFCs in the immune response. Like ICOS-manipulated mice, Rag-1/ mice reconstituted with BM cells of Bcl-6-deficient mice (Bcl-6/ RM mice) produce serum IgG1 antibodies with low affinity in the primary response at a comparable level to that of littermate controls (22). Although the response in Bcl-6/ RM mice terminates by unknown reasons at day 70 post-immunization, earlier than in control mice or ICOS-manipulated mice, Bcl-6/ RM mice and ICOS-manipulated mice have a common feature in the generation and persistence of low-affinity IgG1 memory cells and antibodies in the primary response. Therefore, it is worthwhile to analyze whether or not low-affinity memory cells progress to long-lasting low-affinity AFCs in the immune response.
In summary, our studies shed light on the cellular dynamics of the memory B-cell response emphasizing that there are two waves of memory B-cell generation within a limited period in the primary immune response, associated with or without a GC reaction, resulting in the construction of the memory compartment by low- and high-affinity cells. They are functionally intact, retain longevity and give rise to a prompt secondary immune response. Whether these two waves of memory B-cell generation reflect the different developmental pathways of low- and high-affinity memory B cells or the conversion from low- to high-affinity memory B cells remains to be elucidated.
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Acknowledgements
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We thank JT Pharmaceutical Frontier Research Laboratories, Inc. (Kanagawa, Japan) for kindly providing anti-ICOS/AILIM mAb and Hideo Yagita at Juntendo University for discussion. We also thank Miyashita, Y. Nakano and E. Watanabe at National Institute of Infectious Diseases for their technical help. This work was supported by a Special Coordination Fund for Promoting Science and Technology and by the Ministry of Education, Culture, Sports, Science and Technology, Japan.
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Abbreviations
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AFC | antibody-forming cell |
AILIM | activation-inducible lymphocyte immunomodulatory |
anti-B220APC | APC-conjugated anti-B220 |
APC | allophycocyania |
BCR | B cell receptor |
BLC | B Lymphocyte chemoattractant |
BMs | bone marrows |
CG | chicken gamma globulin |
ELISPOT | enzyme-linked immunospot |
GC | germinal center |
ICOS | inducible co-stimulator |
IFA | incomplete Freund's adjuvant |
i.p. | intra-peritoneally |
NIP | (4-hydroxy-5-iodo-3-nitrophenyl) acetyl |
NIP-bdg | NIP-binding |
NIP-BSAPE | PE-conjugated NIP-BSA |
NP | (4-hydroxy-3-nitrophenyl) acetyl |
OVA | ovalbumin |
PALS | periarteriolar lymphocytic sheath |
PNA | peanut agglutinin |
PNAFITC | FITC-coupled peanut agglutinin |
SLC/ELC | secondary lymphoid chemokine/EBV-induced molecule 1 ligand chemokine |
TC | Tricolor |
WT | wild type |
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Notes
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Transmitting editor: T. Watanabe
Received 10 September 2004,
accepted 15 February 2005.
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