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Role of MHC Class II on Memory B Cells in Post-Germinal Center B Cell Homeostasis and Memory Response¹

Michiko Shimoda^2,*,, Tao Li^*, Jeanene P. S. Pihkala^* and Pandelakis A. Koni^2,*,

^* Program in Molecular Immunology, Immunotherapy Center, Department of Pathology, and Department of Medicine, Medical College of Georgia, Augusta, GA 30912

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

 
We investigated the role of B cell Ag presentation in homeostasis of the memory B cell compartment in a mouse model where a conditional allele for the -chain of MHC class II (MHC-II) is deleted in the vast majority of all B cells by cd19 promoter-mediated expression of Cre recombinase (IA-B mice). Upon T cell-dependent immunization, a small number of MHC-II⁺ B cells in IA-B mice dramatically expanded and restored normal albeit delayed levels of germinal center (GC) B cells with an affinity-enhancing somatic mutation to Ag. IA-B mice also established normal levels of MHC-II⁺ memory B cells, which, however, subsequently lost MHC-II expression by ongoing deletion of the conditional iab allele without significant loss in their number. Furthermore, in vivo Ag restimulation of MHC-II^– memory B cells of IA-B mice failed to cause differentiation into plasma cells (PCs), even in the presence of Ag-specific CD4⁺ T cells. In addition, both numbers and Ag-specific affinity of long-lived PCs during the late post-GC phase, as well as post-GC serum affinity maturation, were significantly reduced in IA-B mice. These results support a notion that MHC-II-dependent T cell help during post-GC phase is not absolutely required for the maintenance of memory B cell frequency but is important for their differentiation into PCs and for the establishment of the long-lived PC compartment.

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

 
Major histocompatibility complex class II (MHC-II)³-restricted B cell Ag presentation is essential for the induction of Ag-specific humoral immune response and generation of immunological memory. Upon infection or immunization with T cell-dependent (TD) Ag, Ag-specific B cells present Ag to Ag-specific CD4⁺ T cells primed by dendritic cells (DCs), and this cognate T cell-B cell interaction promotes B cell proliferation and differentiation either into extrafollicular plasma cells (PCs) or germinal center (GC) B cells (1, 2). During ensuing massive proliferation, GC B cells undergo somatic mutations in the V region of their Ig BCR, which can change the affinity of the BCR to Ag. High-affinity B cells survive by preferential interaction with follicular DCs and T cells, while low-affinity B cells die by apoptosis (3). This GC selection process is responsible for the generation of "non-Ab-secreting" memory B cells and "Ab-secreting" long-lived PCs in bone marrow (BM) (4), both of which are necessary for long-term immunological memory and protection (4, 5, 6).

In contrast to the extensive studies on GC B cell differentiation and selection, the mechanisms delineating memory B cells and long-lived PCs from GC B cells and the maintenance of these two B cell populations long after their generation are largely unknown. Previous studies revealed that both memory B cells (7) and long-lived PCs are long-lived (8, 9) and can be maintained without immunizing Ag persistence (10, 11, 12). Other studies also suggested that memory B cells were maintained in mice deprived of CD4⁺ T cells (13) or in mice deficient in complement receptor 2 (Cr2) (14). Furthermore, recent studies have shown that polyclonal stimulation such as CpG DNA or bystander T cell help can provide proliferation and even differentiation of memory B cells (11, 12). In addition, virus-specific memory B cells can be activated and differentiated into PCs in the absence of CD4⁺ T cells (15). Thus, these studies support a notion that established memory B cells are mainly maintained independent of Ag itself or Ag-specific T cell help, and their differentiation into PCs is CD4⁺ T cell independent. In contrast, there are also findings suggesting that even long-lived PCs are replenished by differentiation of high-affinity precursors during the post-GC phase, reflected in post-GC serum Ig affinity maturation, which itself is dependent on Ag-deposition (5, 16, 17, 18, 19, 20). Irradiated Cr2^–/– chimeric mice reconstituted with Ag-primed B cells and T cells showed reduced frequency of memory B cells and Ab-secreting cells (ASCs) (19). In addition, post-GC B cells failed to induce Blimp-1, XBP-1, and Bcl-2, which resulted in failure to generate the precursor population of long-lived PCs (14). These results strongly suggest that there is an Ag-dependent mechanism for the maintenance of long-lived PC compartment by differentiation of precursors, which is required for the maximum retention of long-term protective humoral immunity.

Classically, Ag-specific B220⁺ memory B cells are defined as class-switched IgD^–IgM^– B cells (21, 22) and highly express CD38 (23, 24). However, several other studies now also show that Ag restimulation promotes B220⁺CD138^– memory B cells to generate B220^–CD138^– preplasma memory B cells that are also maintained in the post-GC phase and differentiate into PCs upon Ag recall (6, 25, 26). In addition, by using a transgenic BCR model, precursors for long-lived PCs were identified in BM, which proliferate and differentiate into PCs in the absence of Ag (18). It is not clear at present whether memory B cells and long-lived PCs are largely maintained independently as a separate compartment once derived from GC B cells (4) or whether, in fact, these B cell subsets in the post-GC B cell compartment sequentially or independently act as precursors of long-lived PCs (6). To support the latter, it has been suggested that virus-specific long-term Ab memory is provided by Ag-driven and CD4⁺ TD continuous differentiation of memory B cells into short-lived PCs (20). Therefore, these post-GC B cells are considered to be points of selection of high-affinity clones responsible for post-GC serum affinity maturation, and their terminal differentiation into PCs is controlled by certain mechanisms to achieve maximal affinity in the long-lived PC compartment.

In this article, we study the role of MHC-II-restricted B cell Ag presentation for memory B cell maintenance and their terminal differentiation into PCs during memory response with a new mouse model of B cell-restricted MHC-II deficiency by crossing the cd19^cre allele (27) onto an iab^neo/neo mouse background (28) (iab^neo/neocd19^cre/+: IA-B mice). The conditional iab^neo allele was generated such that loxP sites flank the promoter and the first exon of the iab gene encoding IA chain of MHC-II (28). Cre recombinase-mediated deletion of this iab^neo allele is known to cause loss of cell surface MHC-II expression (28), as is observed in the case of conventional IA chain knockout mice (29). Despite efficient MHC-II deletion from the vast majority (95%) of B cells, these IA-B mice generated normal numbers of GC B cells and memory B cells upon immunization with TD Ag by dramatic expansion of a small number of MHC-II⁺ B cells. However, these memory B cells subsequently lost MHC-II due to ongoing cd19^cre-mediated deletion of the conditional iab^neo allele without loss in number. With this unique mouse model, we demonstrate in this study the impact of MHC-II on B cells during GC differentiation and further address the requirements for MHC-II-restricted B cell Ag presentation in homeostasis of memory B cell and long-lived PCs and memory response.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

 
Animals and immunization

C57BL6/J mice were purchased from The Jackson Laboratory and were bred in our facility. Mice with a conditional loxP-targeted IA chain (iab^neo) allele were described previously (28). The deleted (null allele) version (iab) was created by crossing a male iab^neo/neo mouse with a female TIE2Cre mouse (30) and will be described elsewhere. Mice that lack MHC-II specifically from B cells were generated by interbreeding iab^neo/neo mice and iab^/ mice with cd19^cre/+ mice. Mice were genotyped for cre by PCR with primers described elsewhere (30). B cell-specific MHC-II deletion was confirmed by analyzing B cells in peripheral blood by flow cytometry at 6 wk of age. All experiments used iab^neo/neocd19^cre/+ (IA-B) mice and their cd19cre-negative littermates or iab^neo/cd19^cre/+ mice and their cd19cre-negative littermates. (4-Hydroxy-3-nitrophenyl)acetyl (NP₁₃)-chicken gammaglobulin (CGG) was prepared as described elsewhere (31). Mice 7–10 wk of age were given an i.p. challenge with 100 µl of PBS containing 5 or 50 µg of NP₁₃-CGG absorbed in alum or 50 µg of NP₂₄-Ficoll (Bioresearch Technologies). For secondary immunization, mice were given an i.p. challenge with 100 µl of PBS containing 50 µg of soluble NP₁₃-CGG or 20 µg of NP₂₄-Ficoll. Blood was collected from immunized mice by tail vein bleeding for serum Ab determination at various times. All mice were maintained under specific pathogen-free conditions and all the studies have been reviewed and approved by an appropriate institutional committee.

Flow cytometry

Single-cell suspensions were prepared from spleens by mechanical disruption of small fragments of organ between frosted glass slides followed by depletion of RBC with ACK lysing buffer (BioWhittaker). Spleen cells were then washed twice with PBS and filtered through nylon mesh in RPMI 1640/1% FCS. For general analysis, single cell suspensions of spleen were pretreated with Fc-block (2.4G2; anti-CD16/CD32; BD Biosciences) on ice for 30 min. Cells were then incubated on ice for 30 min with specific Abs to IA^b (AF6-120.1), CD4 (RM4-5), CD11c (HL3), CD11b (M1/70), CD5 (53-7.3), B220 (RA3-6B2), IgM (R6-0.2), CD21/CD35 (7G6), CD23 (B3B4), CD24 (M1/69), IgD (217-170), CD8 (53-6.7), CD90.1 (HIS51), TCR (H57-597), CD19 (1D3), CD138 (281-2), 120G8 (Ref.32 ; provided by Dr. G. Trinchieri, Schering-Plough Research Institute, Kenilworth, NJ), IA^b (Y3P; provided by our colleague Dr. L. Ignatowicz, Medical College of Georgia, Augusta, GA), and PNA-FITC (Vector Laboratories). Abs were from BD Biosciences unless otherwise indicated. NP-haptenated PE (NP₂₀-PE) was prepared as described elsewhere (31). Cells were analyzed on a FACSCalibur (BD Biosciences) with CellQuest software (BD Biosciences).

Ag-specific GC B cells and B220⁺ memory B cells were identified by five-color analysis as previously reported (33, 34). Briefly, cells were stained on ice for 45 min with a mixture of biotinylated specific Abs to IgM, IgD, CD43 (S7), CD5, Gr-1 (RB6-8C5), CD11b, CD49b (DX5), and CD90.2 (30-H12) to exclude naive B cells, PCs, B-1 cells, macrophages, NK T cells, and T cells (lineage depletion Ab mixture). For analysis of secondary response, cell-bound Igs were stripped by incubating spleen cells at 37°C for 30 min followed by washing twice with PBS/1% FCS before staining with specific Abs (35) to exclude Ab-capturing non-B cells that masquerade as memory B cells (36).

After washing, cells were stained with anti-IA^b-FITC, NP₂₀-PE, streptavidin-PE-Cy5, anti-CD38-allophycocyanin (clone 90; eBioscience) and B220-allophycocyanin-Cy7. Cells were washed and finally suspended in 5 µg/ml 7-aminoactinomycin-D (Sigma-Aldrich) for analysis with a MoFlo (DakoCytomation). At least 2,000,000 events were collected, and the frequency of NP-binding GC B cells, B220⁺ memory B cells in the viable lymphocyte gate was determined with Summit software version 4 (DakoCytomation).

Cell sorting

To enrich memory B cells, spleen cells were pretreated with Fc-block and incubated for 45 min with a lineage depletion biotinylated Ab mixture (as described in Flow cytometry) including anti-CD138 Ab in RPMI 1640/2% FCS. To enrich only MHC-II^– memory B cells, biotinylated anti-IA^b Ab was also included in the mixture. After washing, cells were incubated with anti-biotin microbeads (Miltenyi Biotec). Biotin⁺ cells were depleted with an AutoMACS (Miltenyi Biotec). For isolation of NP-specific memory B cells, the enriched cells were incubated with a mixture of anti-IA^b-FITC, NP₂₀-PE, streptavidin-PE-Cy5, anti-CD38-allophycocyanin, and B220-allophycocyanin-Cy7. Among the NP-binding/lineage^–/B220⁺ cells, CD38⁺ cells, and CD38^– cells were purified by MoFlo as B220⁺ memory B cells and GC B cells, respectively.

Adoptive transfer

MHC-II⁺ or MHC-II^– B cells were enriched from control mice or IA-B mice after depleting non-B cells from spleen cells by magnetic cell sorting or complement lysis. For magnetic cell sorting, B cells were incubated with a lineage depletion biotinylated Ab mixture (as described in Flow cytometry). To enrich only MHC-II^– memory B cells, biotinylated anti-IA^b Ab was also included in the mixture. For complement lysis, spleen cells were incubated with 10 µg/ml anti-CD90.2 Ab and anti-NK.1 Ab for 30 min on ice. After washing, the cells were incubated with rabbit complement for 30 min at 37°C then washed three times with RPMI 1640/10% FCS. CD4⁺ T cells were enriched from C57BL6/J mice that had been primed with 50 µg of CGG plus alum 30 days before by using magnetic cell sorting with CD4⁺ magnetic beads (Miltenyi Biotec). B cells (2 x 10⁷) and T cells (2 x 10⁶) were injected i.v. into RAG-1^–/– mice and, 24 h later, were challenged i.p. with 50 µg of soluble NP-CGG. Anti-NP response was estimated by ELISA and FACS at day 10 days after the challenge.

Histology

Spleens were snap frozen in OCT embedding compound in a dry ice/methylbutane bath. Frozen spleen tissue sections of 7-µm thickness were prepared with a cryostat microtome (Leica Microsystems), fixed in cold acetone for 20 min, air-dried, and stored at –80°C until staining. Thawed sections were rehydrated in PBS and then preblocked before being incubated with either anti-I-A^b-PE, NP₂₀-PE, anti-CD8-allophycocyanin, or anti-CD19-PE, and biotin-anti-IgD followed by streptavidin Alexa 488 (Molecular Probes). Images were acquired using an LS51confocal microscope system (Zeiss).

ELISA and ELISPOT assays

ELISA and ELISPOT assays were performed as previously described (34). Total and high-affinity NP-specific Abs in sera were measured by using a Clonotyping System-AP with pNPP substrate (Southern Biotechnology Associates) using plates coated with NP₁₅-BSA and NP₂-BSA, respectively. The reciprocal endpoint titer for total (NP₁₅-BSA) and high-affinity (NP₂-BSA) NP-specific Abs in sera was defined as the dilution of serum giving an OD at 405 nm of 0.05. Pooled sera from NP-CGG-immunized control mice between 12 and 20 wk postimmunization were used as a standard control. Similarly, total and high-affinity NP-specific ASCs in spleen and BM were determined by 3-h culture on plates coated with NP₁₅-BSA and NP₂-BSA, respectively, followed by detection with a Clonotyping System-AP as above but with a Liquid BCIP/NBT Substrate Kit (Zymed Laboratories).

Sequence analysis of VDJ DNA segments

VDJ sequences were determined as previously described (34). Total RNA was prepared from sorted TRIzol-solubilized GC B cells and memory B cells from pooled spleen cells of at least three mice at each group. First-strand cDNA was synthesized with an oligo(dT) primer by use of a Superscript Kit (Invitrogen Life Technologies). Two microliters of cDNA solution was used as a template in a reaction volume of 50 µl for two rounds of nested PCR for amplifying the V186.2 gene rearranged to the JH2 region by use of Pfu DNA polymerase (Stratagene). PCR products were size-fractionated by agarose gel electrophoresis and purified with a PCR Purification Kit (Qiagen). The purified fragments were cloned into the PCR-Script Amp cloning vector (Stratagene) according to the manufacturer’s instructions, and the sequence of the VDJ segment was determined. Assignment of gene usage and somatic mutations was performed with the BLAST and CLUSTALW programs provided by the DNA Data Bank of Japan (www.ddbj.nig.ac.jp/).

Statistical analysis

The Student t test (two-tailed) was used. A value of p < 0.05 was considered to indicate a significant difference.

	Results

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

 
Generation of mice lacking MHC-II on B cells

We generated mice that lack MHC-II specifically from B cells by crossing the cd19^cre allele (27) onto an iab^neo/neo mouse background (28) (iab^neo/neocd19^cre/+: IA-B mice). The conditional iab^neo allele was generated such that loxP sites flank the promoter and the first exon of the iab gene encoding IA chain of MHC-II (28). Cre recombinase-mediated deletion of this iab^neo allele is known to cause loss of cell surface MHC-II expression (28), as is observed in the case of conventional IA chain knockout mice (29). Selective loss of cell surface MHC-II on B220⁺ B cells was confirmed by staining MHC-II -chain or -chain, with no apparent loss of MHC-II among CD11c⁺ DCs (Fig. 1A). Both CD8⁺ and CD8^– subpopulations among B220^–CD11c^high DCs (data not shown) as well as CD11c^low120G8⁺ plasmacytoid DCs (32) (Fig. 1B) in IA-B mice showed normal MHC-II expression. Loss of MHC-II was also observed among peritoneal B-2 (B220⁺CD11b^–CD5^–), B-1a (B220^lowCD11b⁺CD5^–), and B-1b (B220⁺CD11b⁺CD5^–) B cells (Fig. 1C).

View larger version (40K): [in this window] [in a new window]   FIGURE 1. Selective loss of MHC-II expression on B cells in IA-B mice. A, Cell surface MHC-II on B220+CD11c– B cells and B220–CD11c+ DCs in spleen was stained with Abs specific to MHC-II -chain (IA) or -chain (IA) (bold open histograms), with anti-CD90.1 as an isotype control (gray histograms). C57BL/6J mice and iabneo/neo mice express MHC-II on B cells and DCs, whereas iab/ mice lack MHC-II from both B cells and DCs. IA-B (iabneo/neocd19cre/+) mice lack MHC-II selectively from B cells. B, MHC-II expression on spleen plasmacytoid DCs (CD11clow120G8+) was compared between wild-type mice (thin line) and IA-B mice (bold line) by staining with anti-IA-PE, using anti-TCR-PE as a negative control (gray histogram). C, MHC-II expression on peritoneal B-2 (B220+CD11b–CD5–), B-1a (B220lowCD11b+CD5+), and B-1b (B220+CD11b+CD5–) cells (56 ) in wild-type mice and IA-B mice is shown by staining with anti-IAb-FITC (filled histograms) using anti-CD90.1-FITC as an isotype control (open histograms). D, CD19 expression was compared between MHC-II+ (thin line) and MHC-II– (bold line) B cells and B220– CD4+ T cells (gray histogram) in the spleen of IA-B mice. Data in A–D are representative of one of at least three mice per group.

neo/

The numbers of B cells, DCs, marginal zone B cells, T-1 and T-2 immature B cells, as well as CD4⁺ T cells in the spleen of IA-B mice were normal, and there was no apparent histological abnormality in follicular structure and no difference in serum Ig titer for all the isotypes between wild-type and IA-B mice at 8–12 wk of age (data not shown).

IA-B mice develop delayed but otherwise normal humoral response in association with GC formation

When immunized with a T cell-independent (TI) Ag, NP-haptenated Ficoll (NP₂₄-Ficoll), IA-B mice developed relatively normal anti-NP IgM, IgG3, and IgG1 response during the first 2 wk postimmunization (Fig. 2A). To investigate TD response, IA-B mice and control mice were immunized with 50 µg of NP-CGG with alum as an adjuvant. Age-matched littermate (iab^neo/neocd19^+/+) mice were used as control mice because heterozygous expression of CD19 due to the presence of cd19^cre in IA-B mice did not affect Ag-specific Ab production and serum Ig affinity maturation (data not shown).

View larger version (25K): [in this window] [in a new window]   FIGURE 2. Early TD but not TI immune response is impaired in IA-B mice. A, IA-B mice and control mice were immunized with 50 µg of NP24-Ficoll and sera were collected at each time point. Endpoint titers of serum anti-NP Ig of each isotype for control mice () and IA-B mice (•) were defined as the dilution of serum giving an OD at 405 nm of 0.05 by ELISA. Data shown are representative of one of three experiments with at least three mice per time point. B, Similar to A, except IA-B mice and control mice were immunized with 50 µg of TD Ag, NP-CGG with alum as an adjuvant. Data shown are representative of one of three experiments with at least three mice per time point.

neo/

neo/

View larger version (15K): [in this window] [in a new window]   FIGURE 3. Mice having >2% MHC-II+ B cells develop normal levels of anti-NP humoral immune response upon immunization with TD Ag. Various iabneo/cd19cre/+ mice having slightly different frequencies of MHC-II+ B cells (as determined by FACS of peripheral blood cells at 6 wk of age) and wild-type control mice were immunized with NP-CGG in alum, and endpoint anti-NP IgG1 titers in sera were defined as the dilution of serum giving an OD at 405 nm of 0.05 by ELISA at 4 () and 8 (•) wk postimmunization. The endpoint titer for each individual mouse was then plotted against the frequency of MHC-II+ B cells in each mouse, determined at 6 wk of age before immunization.

View larger version (53K): [in this window] [in a new window]   FIGURE 4. MHC-II+ GC development in IA-B mice. Frozen sections of spleen from control mice (A) and IA-B mice (B) at 2 wk postimmunization with NP-CGG in alum were stained with anti-IgD-biotin and streptavidin Alexa 488 (green), anti-IA-PE (red), and anti-CD8-allophycocyanin (blue). T cell areas are stained as CD8+. In IA-B mice, a typical IgD– GC (indicated as GC) is clearly stained with anti-IA-PE, whereas IgD+ follicular B cells are not. In a merged image, the GC is stained in red in both control mice and IA-B mice, whereas follicular B cells stained in red in IA-B mice and yellow in control mice. Data represent results from one of at least three mice per group.

During GC reaction, high-affinity GC B cells are selected and differentiate into memory B cells and long-lived PCs (4, 5, 6). To further study whether or not IA-B mice can restore and maintain normal numbers of GC and memory B cells, the kinetics of differentiation of GC and B220⁺ memory B cells in IA-B mice and the expression levels of MHC-II in these populations were followed by FACS. After gating out non-B cells, B-1, and plasma B cells, as well as naive (IgM⁺/IgD⁺) B cells with lineage markers (CD90.2, Gr-1, CD49b, CD11b, CD5, CD43, IgD, and IgM), the B220⁺CD38^– GC and B220⁺CD38⁺ memory B cells (23, 24, 33, 34) were identified among NP-binding B cells (Fig. 5A). IA-B mice proved to have delayed kinetics of NP-binding GC B cell generation (Fig. 5B). This did not appear to be a result of an intrinsic difference in NP-specific precursor frequency between control mice and IA-B mice but a difference in the frequency of MHC-II⁺ cells among the anti-NP repertoire because IA-B mice and control mice had a similar frequency of NP-binding naive IgM⁺IgD⁺B220⁺ cells (0.03% of total spleen cells) before immunization. However, IA-B mice restored a maximum frequency of GC B cells similar to or even higher than that in control mice between 2 and 10 wk postimmunization (Fig. 5B). Also, genetic analysis of V186.2DJ rearrangements, which dominate in anti-NP response in C57BL/6 background (37), showed that clones with somatic mutations as well as high-affinity clones carrying an affinity-enhancing Try to Leu mutation at amino acid position 33 (38, 39) were enriched among GC B cells of IA-B mice at 4 wk postimmunization (Table I). Thus, despite the delayed kinetics, accumulation of somatic mutations and GC selection in IA-B mice seemed to be normal. Also, >93% of the GC cells in IA-B mice expressed MHC-II (Fig. 5C), emphasizing the strict requirement for MHC-II expression on B cells for GC differentiation.

View larger version (35K): [in this window] [in a new window]   FIGURE 5. Memory B cells in IA-B mice lose MHC-II expression without significant loss of frequency. A, Representative flow cytometry profile of spleen cells from a control mouse 2 wk postimmunization with 50 µg of NP-CGG in alum. Among lineage-negative (CD90.2, Gr-1, CD49b, CD11b, CD5, CD43, IgD, and IgM) B220+ B cells (R1), NP-binding cells identified with NP20-PE were separated by differential expression of CD38 as CD38dull/– GC cells (R2) and CD38+ memory B cells (R3). B, The frequency of NP-binding GC B cells of control mice () and IA-B mice (•) was analyzed as in A at the indicated weeks postimmunization. Data represent the average of at least four mice per time point. NP-binding cells were not detectable (<5 x 10–5% of total spleen cells) in the lineage-negative B220+ cell population in naive mice (i.e., wk 0). C, The percentage of MHC-II+ B cells among NP-binding GC B cells was determined at the indicated weeks postimmunization. Data represent the average of at least four mice per time point. D, Similar to B, the frequency of NP-binding memory B cells was analyzed. E, Similar to C, the percentage of MHC-II+ B cells among NP-binding memory B cells was analyzed. F, Representative flow cytometry profiles of spleen cells from wild-type mice (left) and IA-B mice (right) at 4, 12, 20, and 30 wk postimmunization as in A. Histograms show MHC-II IAb expression on memory B cells (gray plot) as well as anti-CD90.1 isotype control staining (empty plot).

Genetic analysis showed that comparable frequencies of high-affinity clones, which are generally recovered from the late memory B cell compartment (33, 39), were recovered at 30 wk postimmunization from both purified MHC-II^– and MHC-II⁺B220⁺ memory B cells of IA-B mice and control mice, respectively (Table I). Thus, these results suggest that once selected to differentiate into memory B cells, MHC-II expression (i.e., cognate T cell-B cell interaction) is no longer required for the maintenance of B220⁺ memory B cell frequency.

Impaired establishment of long-lived PC compartment in IA-B mice

To study whether or not IA-B mice established and maintained a normal long-lived PC compartment, affinity maturation of serum anti-NP IgG1 and BM anti-NP IgG1 ASCs in IA-B mice was followed. When mice were immunized with a low dose (5 µg) of NP-CGG rather than a high dose (50 µg) as above, IA-B mice failed to show normal levels of Ag-specific serum Ig titer (Fig. 6A), and post-GC serum affinity maturation was not evident between 8 and 20 wk postimmunization even in control mice (Fig. 6B). Thus, consistent with other studies (17, 19), Ag supply was critical for post-GC affinity maturation.

View larger version (29K): [in this window] [in a new window]   FIGURE 6. Post GC affinity maturation of Ag-specific serum Ab and generation of high-affinity ASCs in BM is impaired in IA-B mice. A, IA-B mice (•) and control mice () were immunized with 5 µg of NP-CGG with alum as an adjuvant and sera were collected at each time point shown. Endpoint titers of anti-NP IgG1 Ab in sera defined as the dilution of serum giving an OD at 405 nm of 0.05 by ELISA were determined with NP15-BSA-coated plates (detecting high-affinity Abs) and NP2-BSA-coated plates (detecting total Abs). Data represent one of four similar experiments with the average of at least four mice per time point. B, Serum Ig affinity maturation in the same mice shown in A was estimated as the ratio of high-affinity anti-NP IgG1 Ab captured with NP2-BSA-coated plates vs total anti-NP IgG1 Ab captured with NP15-BSA-coated plates. C, Similar to A, except mice were immunized with 50 µg of NP-CGG with alum. D, Similar to B, serum Ig affinity maturation in the same mice shown in C was estimated. E, The numbers of anti-NP ASCs in BM of mice immunized with 50 µg of NP-CGG plus alum were measured by ELISPOT at various time points. Data represent the average of three to eight mice per time point. F, The ratio of high-affinity anti-NP IgG1 BM ASC captured with NP2-BSA-coated plates vs total anti-NP IgG1 BM ASC captured with NP15-BSA-coated plates in the same mouse shown in E was measured by ELISPOT at various time points. *, Statistically significant (p < 0.05).

Ag stimulation alone cannot induce differentiation of memory B cells

The absolute requirement of cognate T cell help in PC differentiation of memory B cells is still questionable (15). If cognate T cell help is required, Ag-specific memory response in IA-B mice, which lost MHC-II from memory B cells, would be defective. When IA-B mice received a secondary challenge with 50 µg of soluble NP-CGG at 8 wk postimmunization, when the majority of memory B cells still expressed MHC-II (Fig. 5E), IA-B mice generated a relatively normal memory response with increased high-affinity anti NP-IgG1 titers (data not shown). Furthermore, IA-B mice generated robust memory response at 2030 wk postimmunization even after the majority (96%, Fig. 5E) of memory B cells lost MHC-II expression. The anti-NP IgG1 titer in both control mice and IA-B mice dramatically increased by day 7 after secondary challenge, and was even more remarkable in IA-B mice (Fig. 7A). The ratio of high-affinity anti-NP Ab (0.75 ± 0.15, n = 6) was similar to that of control mice (0.88 ± 0.18, n = 6) (data not shown). Thus, despite the loss of MHC-II expression from the majority (96%) of the established memory B cells, IA-B mice mounted normal memory response. However, when mice received a secondary challenge with 20 µg of TI Ag NP-Ficoll, neither control mice (Fig. 7A) nor IA-B mice (data not shown) generated detectable memory response.

View larger version (46K): [in this window] [in a new window]   FIGURE 7. Robust Ag-specific memory response in IA-B mice that lack MHC-II from the majority of memory B cells. A, Wild-type mice and/or IA-B mice were given an i.v. secondary challenge with 50 µg of soluble NP-CGG or 20 µg of soluble NP-Ficoll at 30 wk postprimary immunization. Anti-NP IgG1 serum titer defined as the dilution of serum giving an OD at 405 nm of 0.05 by ELISA was measured immediately before () and 7 days after () the secondary challenge. Representative data of four similar experiments with at least four mice per group are shown. B, Spleen cells were recovered from the same mice shown in A 7 days after the secondary challenge with NP-CGG or NP-Ficoll. CD138+ NP-PE+ Ag-specific PCs (boxed) are shown after pregating on B220–CD38– cells. C, GC B cells (R2 gate) and memory B cells (R3 gate) were gated among lineage-negative (CD90.2, Gr-1, CD49b, CD11b, CD5, CD43, IgD, and IgM) B220+ spleen cells (R1 gate, FACS profiles not shown) from the same wild-type mice and IA-B mice shown in A. Histograms show MHC-II IAb expression on GC B cells (R2 gate) and memory B cells (R3 gate). Anti-CD90.1 isotype control staining is shown as the empty plots. Representative flow cytometry profiles of four similar experiments with at least three mice per group were shown. D, Ag capturing B220+ cells with memory phenotype (R4 gate) in the same wild-type mice shown in C were analyzed with or without pretreatment to strip cell surface Ig by incubation at 37°C for 30 min.

Because NP-Ficoll challenge failed to generate memory response in both control and IA-B mice, these results suggest that Ag stimulation itself is not sufficient and cognate T cell help is required for PC differentiation of memory B cells. Thus, the robust memory response in IA-B mice was mounted by the 4% of memory B cells that were MHC-II⁺.

MHC-II^–B220⁺ memory B cells do not differentiate into PCs even in the presence of T cells

To further test whether cognate T cell help is required for PC differentiation of memory B cells, MHC-II^– or MHC-II⁺ B cells from IA-B mice or control mice, respectively, at 30 wk postimmunization (B220⁺ cells, >86%) were transferred into RAG-1^–/– mice with CD4⁺ T cells purified from CGG-primed B6 mice. These RAG-1^–/– recipients were then challenged with soluble NP-CGG. MHC-II⁺ cells (including MHC-II⁺ memory B cells) in IA-B mice were either depleted by complement lysis or magnetic cell sorting before transfer. The remaining MHC-II⁺B220⁺ B cells estimated by FACS were <0.8%. The frequency of NP-binding cells among B220⁺ B cells was similar between control and IA-B mice before transfer (data not shown).

The RAG-1^–/– mice that received MHC-II⁺ memory B cells with T cells and challenged with NP-CGG (recipient 1) generated robust anti-NP IgG1 response in association with high-affinity anti-NP Ab production (Fig. 8A) at day 10 postchallenge, which was also confirmed by ELISPOT (data not shown). In contrast, anti-NP IgG1 response was greatly reduced (>1000-fold) in the RAG-1^–/– mice that received MHC-II^– memory B cells with T cells and challenged with NP-CGG (recipient 2) (Fig. 8A). Anti-NP IgG1 response was undetectable in recipient mice challenged with PBS (recipient 3), those that received naive MHC-II⁺ B cells (recipient 4), and those that received only MHC-II⁺ memory B cells without T cells and challenged with NP-CGG (recipient 5) (Fig. 8A). Thus, consistent with the results of Fig. 7, these results suggest that Ag stimulation is not sufficient for PC differentiation of memory B cells and cognate T cell help through MHC-II-restricted Ag presentation by memory B cells is required.

View larger version (33K): [in this window] [in a new window]   FIGURE 8. MHC-II-deficient memory B cells transferred into RAG-1–/– mice fail to differentiate into PCs upon Ag restimulation even in the presence of Ag-specific CD4+ T cells. A, MHC-II+ B cells and MHC-II– B cells were purified from wild-type mice and IA-B mice, respectively, at 30 wk postprimary immunization and transferred i.v. into RAG-1–/– mice with (no. 1–4) or without (no. 5) CD4+ T cells from CGG-primed mice. As a control, some recipients received MHC-II+ B cells from naive control mice (no. 4). Twenty-four hours after transfer, recipients were given an i.p. challenge of 50 µg of soluble NP-CGG (no. 1, 2, 4, and 5) or PBS (no. 3). Anti-NP IgG1 serum titers defined as the dilution of serum giving an OD at 405 nm of 0.05 were measured by ELISA using NP15-BSA-coated plates (total Abs: ) and NP2-BSA-coated plates (high-affinity Abs: ) 10 days after the challenge. Data shown are representative of four similar experiments with at least four mice per group. B, Spleen cells were recovered from the same RAG-1–/– recipient mice shown in A 10 days after the challenge. Among the viable lymphocyte gate, CD4– NP-PE+ Ag-specific B cells (R1) were identified and their MHC-II and B220 expression levels were analyzed in the second column. Among the NP-binding cells (R1), B220+ memory B cells (R2), and B220low PC precursors (R3) are identified according to their B220 expression levels. For recipient mice nos. 1–3, histograms of MHC-II IAb (bold empty histograms) expression on NP-binding B220+ memory B cells (R2) and NP-binding B220low PC precursors (R3) are also shown with anti-CD90.1 isotype control staining (gray histograms). Representative flow cytometry profiles and histograms of six similar experiments with at least three mice per group are shown.

Consistent with anti-NP serum IgG1 response, a large number of both B220⁺ memory B cells (R2) and B220^low PC precursors (R3) were observed in no. 1 recipient mice that received MHC-II⁺ memory B cells with T cells and challenged with NP-CGG. This B220^+/low population was not observed in no. 4 recipient mice that received MHC-II⁺ naive B cells. As shown in the MHC-II histograms of no. 1 recipient mice, B220⁺ memory B cells expressed higher levels of MHC-II, whereas B220^low PC precursors down-regulated MHC-II expression. Also, significant numbers of B220⁺ memory B cells (R2) and B220^low PC precursors (R3) were identified in no. 2 recipient mice that received MHC-II^– memory B cells with T cells and challenged with NP-CGG. The numbers of B cell populations in R2 and R3 gate of no. 2 recipient mice were less than those in no. 1 recipient mice but much more than those in no. 3 recipient mice that received MHC-II^– memory B cells with T cells and challenged with PBS. Thus, NP-CGG challenge increased the number of MHC-II^– B220⁺ memory B cells. In addition, as evident from the MHC-II histograms of no. 2 recipient mice, NP-CGG challenge also induced expansion of MHC-II⁺ cells among B220⁺ memory B cells (R2) and B220^low PC precursors (R3). In contrast, MHC-II⁺ memory cells hardly responded by NP-CGG challenge in the absence of memory T cells (no. 5). These results suggest that even MHC-II^– memory B cells can proliferate to a certain extent if Ag and bystander T cell help are provided (note that even though there is no cognate T-B interaction in no. 2 recipient mice due to lack of MHC-II on memory B cells, memory T cells can provide bystander help such as cytokines). However, MHC-II^– memory B cells cannot further differentiate into PCs because they cannot receive optimum help from Ag-specific T cells through MHC-II-restricted Ag presentation.

Collectively, these results led to the conclusion that memory response in IA-B mice at 30 wk postimmunization was by selective reactivation of MHC-II⁺ memory B cells and that only 4% of the wild-type level of MHC-II⁺ memory B cells can restore normal memory response within 7 days after secondary challenge. Furthermore, the current results demonstrate that Ag stimulation is not sufficient for the differentiation of memory B cells and cognate T cell help through MHC-II-restricted Ag presentation is required.

	Discussion

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MHC-II is a critical player for TD humoral immune response. However, the role of MHC-II-restricted B cell Ag presentation in humoral immune response and B cell differentiation has never been directly studied in an intact whole animal model such as the one we now present. In this context, the current study provides several findings. First, the current study demonstrated the great impact of clonal expansion of MHC-II⁺ B cells during the TD primary response as well as memory response. In the primary response, as little as 2% of the wild-type level of NP Ag-specific MHC-II⁺ B cells was enough to establish normal levels of GC B cells and memory B cells albeit only with a large Ag dose and with delayed kinetics. In contrast, differentiation of extrafollicular PCs in the primary response was greatly impaired. In fact, Ag-specific ASCs in the spleen of IA-B mice were barely detectable during the primary response until GC-derived PCs developed (data not shown). Presumably, this is because naive MHC-II⁺ B cells activated by Ag and T cells show only very limited proliferation before they differentiate into either extrafollicular PCs or GC B cells. However, once they differentiated into GC B cells, they proliferated dramatically and clearly were able to reach wild-type maximal levels and restore the humoral response. In this regard, it is known at least that IgG-BCR-mediated signaling on IgG⁺ B cells ensures efficient proliferation and IgG production upon Ag restimulation compared with those expressing IgM and/or IgD signaling (40, 41).

Even more significant was the dramatic memory response by MHC-II⁺ memory B cells in IA-B mice after Ag restimulation. Given the fact that MHC-II^– memory B cells failed to differentiate into PCs in vivo even in the presence of Ag-specific CD4⁺ T cells (Fig. 8), the secondary response in IA-B mice appears to be generated by as little as 4% of the wild-type level of NP Ag-specific MHC-II⁺ memory B cells in IA-B mice, which mounted even greater anti-NP IgG1 response compared with that in control mice within just 7 days after Ag restimulation (Fig. 7). It is not clear why IA-B mice could generate such a robust memory response even though the frequency of MHC-II⁺ memory B cells is small compared with that in control mice. It has been reported that deliberate removal of T cell help promotes specific Ab production with virus-neutralizing ability (42). In addition, NP-binding memory B cells at 2030 wk postimmunization are more enriched in the total memory B cells of IA-B mice compared with that of control mice, as a consequence of the fact that IA-B mice have fewer naturally activated IgD^– B cells (data not shown). Therefore, it is possible that less competition between other MHC-II⁺B220⁺ memory B cells may help anti-NP MHC-II⁺B220⁺ memory B cells of IA-B mice to gain efficient access to memory T cells upon secondary Ag stimulation.

Memory B cells recovered from mice 6 wk after CD4⁺ T cell depletion could still generate memory response when transferred into mice with CD4⁺ T cells and Ag (13), suggesting that memory B cells can be maintained with little CD4⁺ T cell help. In this context, the current study demonstrates that B220⁺ memory B cells do not absolutely require cognate interaction with Ag-specific CD4⁺ T cells through MHC-II restricted Ag presentation for the maintenance of their number because neither a loss of overall B220⁺ memory B cell frequency nor predominance of MHC-II⁺B220⁺ B cells in the memory compartment was seen in IA-B mice. However, it should be noted that a small fraction (4%) of B220⁺ memory B cells still expressed detectable levels of MHC-II even at 30 wk postimmunization. Thus, it remains possible that MHC-II expressing B220⁺ memory B cells in IA-B mice acquired some advantageous survival signals provided by Ag-specific CD4⁺ T cells. Alternatively, it is conceivable that the remaining MHC-II⁺ memory B cells in IA-B mice represent a distinct subset of memory B cells, but we have no evidence at present to support such a possibility. Nonetheless, the survival of B220⁺ memory B cells seems to rely mainly on factors other than cognate T cell help. Certainly, memory B cells regain expression of anti-apoptotic genes such as bcl-2 (33, 43) and TOSO (43) after GC selection. Cytokine receptors such as IL-2R (43) and IL-4R as well as CD130, the signal transducer for IL-6 (44), are significantly up-regulated in human memory B cells compared with naive B cells. Because memory B cells seem to be preferentially localized in particular sites such as marginal zone areas of the spleen (1, 45) and mucosal epithelia (46), the particular localization in addition to the intrinsic survival ability of memory B cells may ensure that memory B cells have access to soluble survival factors and/or to cells providing survival signals.

It has been generally considered that differentiation of memory B cells into PCs upon secondary Ag stimulation requires T cell help (6, 47). Memory B cells differentiate into short-lived PCs in vivo upon Ag stimulation only in the presence of Ag-specific CD4⁺ T cells (20). However, activation of virus-specific memory B cells was intact even in the absence of CD4⁺ T cells (15). In addition, polyclonal stimulation such as CpG ODN or bystander T cells can induce proliferation and differentiation of human memory B cells in vitro (11, 12). Thus, the absolute requirement of Ag-specific CD4⁺ T cell help during memory B cell differentiation is still questionable. In this context, the current study demonstrated that MHC-II^– B cells fail to differentiate into PCs upon Ag restimulation even in the presence of Ag-specific CD4⁺ T cells. In addition, RAG-1^–/– mice receiving only either MHC-II⁺ or MHC-II^– B cells without T cells did not generate memory response upon Ag restimulation. Therefore, our results support a notion that PC differentiation of memory B cells requires both MHC-II expression on memory B cells and Ag-specific CD4⁺ T cells. The discrepancies between the current study and a previous study (15) in regard to the requirement of CD4⁺ T cell help in memory B cell activation may depend on the Ag used for experiments. It is possible that virus Ag may be able to activate memory B cells through innate signaling pathways such as TLRs even in the absence of cognate T-B interaction. In relation to this, early CD4⁺ TD antiviral IgA response can be generated in the absence of MHC-II or CD40 on B cells (48).

Certainly, the role of Ag-specific CD4⁺ T cells is to intimately provide memory B cells with cytokines such as IL-4 and IL-5 that promote PC differentiation through MHC-II-restricted interaction (5). In addition, several studies have reported that MHC-II transduces signals to B cells upon Ag presentation (49, 50). Therefore, it is possible that MHC-II on memory B cells may be required for delivering specific signals for their efficient proliferation and differentiation into PCs. In this context, MHC-II^– memory B cells proliferated to a certain extent in vivo when Ag and bystander T cell help were provided (Fig. 8B). In addition, in vitro culture of purified MHC-II^– memory B cells of IA-B mice at 30 wk postimmunization in the presence of LPS and cytokines induced a similar level of anti-NP IgG1 production compared with MHC-II⁺ memory B cells of control mice (data not shown). However, it remains possible that MHC-II expression on memory B cells (i.e., cognate CD4⁺ T cell help) may be required to maintain their full competence for subsequent PC differentiation and function upon Ag stimulation. Furthermore, we have to concede the possibility that ongoing deletion of MHC-II in IA-B mice might select a certain subset of memory B cells that can survive in the absence of MHC-II but has lost PC differentiation capability. These possibilities are under investigation with V_H Tg memory B cells in an IA-B mouse background.

Even though the selection of GC B cells and the generation and maintenance of B220⁺ memory B cells seemed to be fairly normal, the generation of high-affinity long-lived PCs in IA-B mice was reduced in association with impaired post-GC serum affinity maturation (Fig. 6). That is, GC B cell frequency in IA-B mice recovered to a similar or even higher level than that of control mice by 2 wk postimmunization (Fig. 5B). The selection of high-affinity variants in GC of IA-B mice was also normal despite the delayed kinetics (Table I). In addition, the frequency of memory B cells in IA-B mice was normal throughout the analysis (Fig. 5D). In contrast, numbers of high-affinity as well as total numbers of ASCs in BM were significantly reduced in IA-B mice during the post-GC phase (Fig. 6, E and F) in association with impaired post-GC serum affinity maturation in IA-B mice (Fig. 6D). Long-lived PCs are generated by preferential differentiation of high-affinity variants from GC (51). It is possible that generation of high-affinity PCs from GC is impaired in IA-B mice because of delayed GC development and/or a limited MHC-II⁺ Ag-specific repertoire in IA-B mice. However, this alone cannot explain the reduced anti-NP IgG1 long-lived PC numbers and serum titers particularly in the late post-GC phase (Fig. 6, C and D). Alternatively, these results may suggest that accumulation of long-lived PCs during the post-GC phase rather than their generation from GC itself is impaired in IA-B mice. Whether the long-lived PC compartment is established only from the early GC emigrants (4) or by continuous differentiation of precursor cells during the post-GC phase (18) is not yet clear. Considering the fact that MHC-II^– memory B cells were not able to differentiate into PCs in vivo even in the presence of CD4⁺ T cells, it is interesting to speculate that the number and affinity of long-lived PCs are maintained by continuous differentiation of memory B cells and that this process is impaired in IA-B mice because of loss of MHC-II from memory B cells.

Previous studies showed that polyclonal activation such as CpG DNA and bystander T cell help are able to drive PC differentiation of memory B cells in vitro (11, 12). This type of stochastic mechanism may play a role in maintaining a basal level of humoral immunity when Ag is diminished. However, an "instructed" mechanism by Ag-specific T cells is anticipated to have a great advantage in controlling the "quality" of the long-lived PC population when pathogen (Ag) is still present. First of all, high-affinity B cells preferentially capture Ag and present it to Th cells (52). Through MHC-II-restricted Ag presentation to T cells, high-affinity memory B cells, and/or PC precursors differentiate into PCs and provide higher affinity Abs that can clear residual pathogen more efficiently. Second, cognate T cell-B cell interaction would allow differentiation of useful Ag-specific PCs but presumably not help the differentiation of self-reactive PCs. Third, it remains possible that cognate T cell-B cell interaction provides Ag-specific memory B cells with signals that maintain their capability to differentiate into PCs. Finally, in fact, cognate T cell-B cell interaction may be important for the maintenance of memory T cell function. In support of this, memory T cells that had been maintained in the absence of MHC-II showed reduced functional activity upon Ag re-encounter (53).

Our study showed that cognate interaction between memory T cells is important for PC differentiation by memory B cells. Several studies have shown that both memory B cells and T cells home to BM (20, 54, 55). In this context, we are interested in the possibility that memory B cells make cognate interaction with memory T cells and differentiate into long-lived PCs in BM during immunological surveillance. In this regard, IA-B mice should provide an excellent tool to further investigate the mechanisms of TD and TI memory B cell maintenance and differentiation.

	Acknowledgments

 
We thank T. Takemori, T. Tsubata, Y. Takahashi, M. Iwashima, and A. Mellor for critical discussions; Y. Takahashi for technical suggestions on the memory B cell sorting and culture; L. Ignatowicz for Y3P Ab; G. Trinchieri for 120G8 Ab; and K. Miyake for confocal image analysis.

	Disclosures

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The authors have no financial conflict of interest.

	Footnotes

 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ This work was supported in part by Medical College of Georgia Combined Intramural Grants Program grants-in-aid (to M.S. and P.A.K.).

² Address correspondence and reprint requests to Dr. Michiko Shimoda or Dr. Pandelakis A. Koni, Program in Molecular Immunology, Immunotherapy Center, Department of Medicine, Medical College of Georgia, 1120, 15th Street, Augusta, GA 30912. E-mail address: mshimoda@mcg.edu or pkoni{at}mcg.edu

³ Abbreviations used in this paper: MHC-II, MHC class II; PC, plasma cell; GC, germinal center; CGG, chicken gammaglobulin; NP, (4-hydroxy-3-nitrophenyl)acetyl; ASC, Ab-secreting cell; TD, T cell dependent; TI, T cell independent; BM, bone marrow.

Received for publication August 17, 2005. Accepted for publication November 25, 2005.

	References

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1. Liu, Y. J., S. Oldfield, I. C. MacLennan. 1988. Memory B cells in T cell-dependent antibody responses colonize the splenic marginal zones. Eur. J. Immunol. 18: 355-362. [Medline]
2. Jacob, J., R. Kassir, G. Kelsoe. 1991. In situ studies of the primary immune response to (4-hydroxy-3-nitrophenyl)acetyl. I. The architecture and dynamics of responding cell populations. J. Exp. Med. 173: 1165-1175. [Abstract/Free Full Text]
3. Kelsoe, G.. 1996. Life and death in germinal centers (redux). Immunity 4: 107-111. [Medline]
4. Blink, E. J., A. Light, A. Kallies, S. L. Nutt, P. D. Hodgkin, D. M. Tarlinton. 2005. Early appearance of germinal center-derived memory B cells and plasma cells in blood after primary immunization. J. Exp. Med. 201: 545-554. [Abstract/Free Full Text]
5. Calame, K. L., K. I. Lin, C. Tunyaplin. 2003. Regulatory mechanisms that determine the development and function of plasma cells. Annu. Rev. Immunol. 21: 205-230. [Medline]
6. McHeyzer-Williams, L. J., M. G. McHeyzer-Williams. 2005. Antigen-specific memory B cell development. Annu. Rev. Immunol. 23: 487-513. [Medline]
7. Schittek, B., K. Rajewsky. 1990. Maintenance of B-cell memory by long-lived cells generated from proliferating precursors. Nature 346: 749-751. [Medline]
8. Slifka, M. K., R. Antia, J. K. Whitmire, R. Ahmed. 1998. Humoral immunity due to long-lived plasma cells. Immunity 8: 363-372. [Medline]
9. Manz, R. A., A. Thiel, A. Radbruch. 1997. Lifetime of plasma cells in the bone marrow. Nature 388: 133-134. [Medline]
10. Maruyama, M., K. P. Lam, K. Rajewsky. 2000. Memory B-cell persistence is independent of persisting immunizing antigen. Nature 407: 636-642. [Medline]
11. Bernasconi, N. L., E. Traggiai, A. Lanzavecchia. 2002. Maintenance of serological memory by polyclonal activation of human memory B cells. Science 298: 2199-2202. [Abstract/Free Full Text]
12. Poeck, H., M. Wagner, J. Battiany, S. Rothenfusser, D. Wellisch, V. Hornung, B. Jahrsdorfer, T. Giese, S. Endres, G. Hartmann. 2004. Plasmacytoid dendritic cells, antigen, and CpG-C license human B cells for plasma cell differentiation and immunoglobulin production in the absence of T-cell help. Blood 103: 3058-3064. [Abstract/Free Full Text]
13. Vieira, P., K. Rajewsky. 1990. Persistence of memory B cells in mice deprived of T cell help. Int. Immunol. 2: 487-494. [Abstract/Free Full Text]
14. Gatto, D., T. Pfister, A. Jegerlehner, S. W. Martin, M. Kopf, M. F. Bachmann. 2005. Complement receptors regulate differentiation of bone marrow plasma cell precursors expressing transcription factors Blimp-1 and XBP-1. J. Exp. Med. 201: 993-1005. [Abstract/Free Full Text]
15. Hebeis, B. J., K. Klenovsek, P. Rohwer, U. Ritter, A. Schneider, M. Mach, T. H. Winkler. 2004. Activation of virus-specific memory B cells in the absence of T cell help. J. Exp. Med. 199: 593-602. [Abstract/Free Full Text]
16. Takahashi, Y., P. R. Dutta, D. M. Cerasoli, G. Kelsoe. 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: 885-895. [Abstract/Free Full Text]
17. Wang, Y., G. Huang, J. Wang, H. Molina, D. D. Chaplin, Y. X. Fu. 2000. Antigen persistence is required for somatic mutation and affinity maturation of immunoglobulin. Eur. J. Immunol. 30: 2226-2234. [Medline]
18. O’Connor, B. P., M. Cascalho, R. J. Noelle. 2002. Short-lived and long-lived bone marrow plasma cells are derived from a novel precursor population. J. Exp. Med. 195: 737-745. [Abstract/Free Full Text]
19. Barrington, R. A., O. Pozdnyakova, M. R. Zafari, C. D. Benjamin, M. C. Carroll. 2002. B lymphocyte memory: role of stromal cell complement and FcRIIB receptors. J. Exp. Med. 196: 1189-1199. [Abstract/Free Full Text]
20. Ochsenbein, A. F., D. D. Pinschewer, S. Sierro, E. Horvath, H. Hengartner, R. M. Zinkernagel. 2000. Protective long-term antibody memory by antigen-driven and T help-dependent differentiation of long-lived memory B cells to short-lived plasma cells independent of secondary lymphoid organs. Proc. Natl. Acad. Sci. USA 97: 13263-13268. [Abstract/Free Full Text]
21. Hayakawa, K., R. Ishii, K. Yamasaki, T. Kishimoto, R. R. Hardy. 1987. Isolation of high-affinity memory B cells: phycoerythrin as a probe for antigen-binding cells. Proc. Natl. Acad. Sci. USA 84: 1379-1383. [Abstract/Free Full Text]
22. Gray, D., H. Skarvall. 1988. B-cell memory is short-lived in the absence of antigen. Nature 336: 70-73. [Medline]
23. Oliver, A. M., F. Martin, J. F. Kearney. 1997. Mouse CD38 is down-regulated on germinal center B cells and mature plasma cells. J. Immunol. 158: 1108-1115. [Abstract]
24. Ridderstad, A., D. M. Tarlinton. 1998. Kinetics of establishing the memory B cell population as revealed by CD38 expression. J. Immunol. 160: 4688-4695. [Abstract/Free Full Text]
25. McHeyzer-Williams, L. J., M. Cool, M. G. McHeyzer-Williams. 2000. Antigen-specific B cell memory: expression and replenishment of a novel B220^– memory B cell compartment. J. Exp. Med. 191: 1149-1166. [Abstract/