Rectification of age-related impairment in Ig gene hypermutation during a memory response

Shuhua Han1, Ekaterina Marinova1 and Biao Zheng1

1 Department of Immunology, Baylor College of Medicine, Houston, TX 77030, USA

Correspondence to: B. Zheng; E-mail: bzheng{at}bcm.tmc.edu
Transmitting editor: K. Takatsu


    Abstract
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
The deficiency in generating high-affinity antibodies due to impaired somatic hypermutation of Ig genes in the germinal center (GC) is considered the major mechanism responsible for the compromised humoral responses in aging. Since the intrinsic capability of aged B lymphocytes to respond to initial antigenic stimuli is largely intact and the expression of activation-induced cytidine deaminase, a key component required for Ig somatic hypermutation, is comparable between B cells from aged and young mice, it is possible to restore the age-related deficiency in the humoral response by circumventing the requirement for signals from other immune components. Here, we show that GC B cells from aged mice during a memory response carried mutated Ig genes with mutational frequencies comparable to that of GC B cells from young mice. Additionally, characterization of mutations in VDJ segments, and analysis of antibody-forming cells and antibodies demonstrated that the processes of antigen-driven clonal selection and affinity maturation are largely intact in aged animals. Thus, we conclude that the diminished antibody responses in aged animals may be significantly improved by repeated immunizations. These findings may have important implications in designing vaccines and immunization protocols for the elderly population and patients with certain immune deficiencies such as AIDS.

Keywords: antibody, germinal center, immunosenescence, memory, somatic hypermutation


    Introduction
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
After infection or immunization, aged individuals often generate significantly less antibodies (1), maintain protective titers of serum antibodies for much shorter periods (2) and produce antibodies with affinity lower than that of young controls (3,4). These deficits may be responsible for increased susceptibility to infection in aged populations. One of the major mechanisms responsible for aged-associated immune dysfunction is the impaired germinal center (GC) pathway of B cell differentiation and maturation. In aged mice, not only is the formation of GC diminished (57), but also somatic hypermutation of Ig genes is absent or reduced (8,9). However, it has been shown that the intrinsic capability of aged B cells to be activated by the initial antigen stimulation is largely intact (7). It is of great importance to identify means to restore the age-associated GC dysfunction by bypassing the B cell requirement for signals from other components of the immune system, such as Th cells.

Earlier work demonstrates that although somatic hypermutation of Ig genes is diminished in GC of aged mice, antigen-driven clonal selection for higher-affinity B cells appears to occur, resulting in accumulation of clones with relatively higher affinity (79). Thus, to a certain degree, memory B cells expressing relatively higher-affinity BCR are enriched in aged animals after primary immunization. On the other hand, in primary antibody responses to protein-based antigens, T cell help for B cell activation and differentiation is a major limiting factor (10). This limited T help becomes even more profound in old animals (11). It has been shown that the follicular B cell response was significantly more robust in mice whose T cells were primed with carrier proteins (12). These findings provide the rationale that the age-related impairment in the B cell response may be significantly corrected during a secondary response, including Ig somatic hypermutation and clonal selection.

In the present study, we have analyzed the anti-(4-hydroxy-3-nitrophenyl) acetyl (NP) response in aged and young control mice. In mice of the IgHb allotype, the antibody response to NP is highly restricted. Most primary NP-specific antibodies bear the {lambda}1 L chain and are encoded by the VH186.2 gene segment (13). Our results demonstrate that diminished Ig somatic hypermutation in aged animals can be largely restored in GC elicited during an anamnestic response. Significantly, the process of antigen-driven clonal selection appears functional and affinity maturation is evident in aged mice. Our findings suggest that an impaired humoral response in aging can be overcome by repeated immunizations, which may have clinical implications in designing vaccines and immunization protocols for the elderly population.


    Methods
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Antigens, mice and immunization
Young (2–4 months old) and aged (20–24 months old) C57BL/6J (H-2b) mice were purchased from Charles River (Wilmington, MA) from cohorts maintained by the National Institute on Aging, NIH. All animals were maintained in autoclaved microisolator cages, and provided with sterile bedding, food and water. To elicit the primary immune responses, mice were immunized i.p. with 100 µg alum-precipitated NP (Cambridge Research Biochemicals, Cambridge, UK) conjugated to chicken {gamma}-globulin (CGG; Accurate Chemical & Scientific, Westbury, NY) as described (7). For secondary immunization, mice were given an i.p. injection of 20 µg soluble NP-CGG in PBS 70 days after primary immunization.

Immunohistology
Spleens were flash frozen in OCT embedding media; serial, 6-µm thick frozen sections were cut in a cryostat microtome, thaw mounted onto poly-L-lysine-coated slides, air-dried, fixed in ice-cold acetone for 10 min and stored at –80°C (7,14,15). Immunolabeling of tissue sections was performed as described (14,15). Briefly, splenic GC were labeled by peanut agglutinin (PNA) conjugated to horseradish peroxidase (HRP; E-Y Laboratories, San Mateo, CA) or by biotinylated GL-7 antibody followed by streptavidin–HRP (Southern Biotechnology Associates, Birmingham, AL). Bound HRP activity was then visualized using 3-aminoethyl carbazol as previously described (7).

Microdissection of cells from tissue sections
Spleen sections were stained with PNA–HRP to visualize GC (15,16). About 20–100 cells were picked from individual PNA+ GC using a set of micromanipulators as described previously (14,15).

PCR amplification and sequencing of rearranged Ig heavy chain genes from single splenic GC
Microdissected cellular materials from splenic sections were incubated with proteinase K (Boehringer Mannheim, Indianapolis, IN) overnight at 37°C. After heat inactivation of the enzyme at 96°C for 10 min, the cellular lysate was subjected to two rounds of 40 cycles of PCR amplification using the Expand High Fidelity PCR Kit (Boehringer Mannheim). According to the manufacturer, a PCR error rate of 8.5 x 10–6 per cycle is expected from the polymerase. Therefore, 80 cycles of amplification would generate one misincorporation in ~1700 bp (~0.06%). We have confirmed this polymerase’s high fidelity by sequencing eight VDJ clones from two independent amplifications of DNA recovered from pEVHC{gamma}1 transfectoma (germline VH186.2–DFL16.1–JH2/{lambda}1) and found no mutations. All procedures for PCR amplification, DNA cloning and sequencing were performed as described previously (14,15). All sequence data are available from the data base of EMBL/GenBank/DDBJ under the following accession numbers: AY035667AY035700 (VDJ sequences from primary responses in young C57BL/6 mice), AY239754AY239819 (VDJ sequences from secondary responses in young C57BL/6 mice), AY246565AY246625 (VDJ sequences from primary responses in aged C57BL/6 mice) and AY246626AY246694 (VDJ sequences from secondary responses in aged C57BL/6 mice).

B cell purification and stimulation in vitro
Splenic B cells were purified by MACS. Briefly, cell suspensions were incubated with biotinylated mAb specific for CD4, CD8, Thy-1, Mac-1, Gr-1, and Ter-119 (all purchased from PharMingen, La Jolla, CA), and labeled T cells, macrophages, dendritic cells, neutrophils and erythroid cells were removed by incubating with streptavidin-microbeads (Miltenyi Biotec), and passing through a magnetic column. Purified B cells (>95% of purity) were cultured at 3 x 106 cells/ml in 24-well plates with 10 µg/ml anti-CD40 antibody and 10 µg/ml anti-mouse IgM for 48 h.

RNA preparation and RT-PCR
Total RNA from purified B cells was prepared and reverse transcribed into cDNA with oligo(dT) using the Superscript kit (Gibco, Grand Island, NY) as described (16,17). One round of PCR reactions was performed with the following primer sets: activation-induced cytidine deaminase (AID) sense 5'-ATG GACAGCCTTCTGATGAAGC-3', antisense, 5'-ATCTCAGAA ACTCAGCCACG-3'; HPRT sense, 5'-GCTGGTGAAAAGGAC CTCT-3', antisense, 5'-CACAGG ACTAGAACACCTGC-3'. PCR conditions were as follows: for AID amplification, 94°C for 2 min, followed by 35 cycles of 94°C for 1 min, 52°C for 1 min and 72°C for 1 min, and 72°C for an additional 7 min; for HPRT amplification, 94°C for 2 min, followed by 35 cycles of 94°C for 1 min, 55°C for 1 min and 72°C for 1 min, and 72°C for an additional 7 min. PCR products were electrophoresed on agarose gels and detected by staining with ethidium bromide (16,17).

Flow cytometry
Single-cell suspensions from mouse spleens were prepared and red blood cells were depleted by incubation in 0.83% NH4Cl; cells were then washed with PBS (pH 7.4) containing 2% FCS and 0.08% sodium azide at 4°C. To estimate the prevalence of GC B cells, splenic cells were stained with FITC-labeled GL-7, phycoerythrin-conjugated anti-Fas and TriColor-conjugated anti-B220 (all from PharMingen) after incubation with anti-Fc{gamma}III/IIR (PharMingen) to block Fc{gamma}R-mediated binding. Cells were washed 3 times between each staining step. Samples were collected on a FACScalibur machine (Becton Dickinson, Mountain View, CA) and analyzed using Flow Jo software (Tree Star, San Carlos, CA).

Measurement of antibody-forming cells (AFC)
The frequencies of NP-specific AFC from both splenocytes and bone marrow (BM) cells were estimated by ELISPOT assay using two different coupling ratios of NP-BSA as described (7). Briefly, nitrocellulose filters were coated with 50 µg/ml NP5-BSA, NP25-BSA or BSA in PBS at 4°C overnight and then blocked with 10% FCS in PBS. Splenocytes (5 x 105 cells/well) or BM cells (106 cells/well) were incubated on the nitrocellulose filters in 96-well plates at 37°C, 5% CO2. After a 2-h incubation, nitrocellulose filters were washed with PBS containing 50 mM EDTA once, followed by PBS containing 0.1% Tween 20 twice and PBS once. Filters were double-stained with alkaline phosphatase-conjugated anti-mouse IgM and HRP-conjugated anti-mouse IgG1 antibodies. Alkaline phosphatase and HRP activities were visualized using 3-aminoethyl carbazol and napthol AS-MX phosphate/Fast Blue BB respectively. The frequencies of high-affinity and total AFC were determined from NP5-BSA- and NP25-BSA-coated filters after background on BSA-coated filters was subtracted.

The threshold of antibody affinity which can be detected by each NP-BSA conjugate was determined using several J558L myeloma lines (H, {lambda}1+) transfected with an Ig{gamma}1 expression vector carrying different VDJ rearrangements derived from NP-binding B cells (18). Transfectomas secreting NP-binding antibodies with an association constant (Ka) = 2.0 x 107 M–1 could be detected by both NP5-BSA and NP25-BSA. However, transfectomas with Ka = 106 M–1 could be detected by NP25-BSA, but not by NP5-BSA. Transfectomas with Ka = 2.3 x 105 M–1 could not be detected by either NP-BSA coat. Thus, AFC secreting antibodies with Ka >= 2.0 x 107 M–1 can be detected with NP5-BSA, while those with Ka >= 106 M–1 can only be detected with NP25-BSA.

Measurement of serum antibodies
Antibodies specific for the NP hapten were detected by ELISA using two different coupling ratios of NP-BSA as the coating antigens as described (7). Briefly, 96-well flat-bottom plates (Falcon; Becton Dickinson, Oxnard, CA) were coated with 50 µg/ml NP5-BSA or NP25-BSA in 0.1 M carbonate buffer (pH 9.0) at 4°C overnight. On each plate, mAb specific for NP, H33L{gamma}1/{lambda}1 (18) or B1-8 (19) were included as controls. After washing with PBS containing 0.1% Tween 20, HRP-conjugated goat anti-mouse IgG1 or IgM was added and incubated at room temperature for 1 h. HRP activity was visualized using a TMB peroxidase substrate kit (Bio-Rad, Hercules, CA) and optical densities were determined at 450 nm. The concentrations of anti-NP IgG1 or IgM antibodies were calculated by comparison to standard curves created from the H33L{gamma}1/{lambda}1 or B1-8 control antibodies respectively on each plate. To estimate the affinity of NP-binding antibodies in the sera, the ratios of NP5-binding to NP25-binding antibodies were determined.


    Results
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Somatic hypermutation is not equally diminished among GC B cells carrying different VDJ gene segments in aged mice
The most remarkable difference between the GC reaction of a primary response to NP in aged and young mice is the almost complete absence of mutated VH186.2 rearrangements in old mice (8). However, it should be noted that the NP-specific B cell repertoires in young and aged C57BL/6 mice are quite different. In young mice, the majority of NP-responding GC B cells express BCRs encoded by VH186.2–DFL16.1–JH rearrangement (20); only a small minority of NP-specific GC B cells expresses closely related analogue genes of the JH558 family (21). By contrast, in aged mice, the clonotypic repertoire of NP-responding B cells exhibits a significant shift toward analogue genes (11). To date, it is not known whether somatic hypermutation in analogue genes is equally diminished as VH186.2 segments in aged mice.

To investigate whether the age-associated impairment of somatic hypermutation equally affects all VDJ segments encoding NP-binding BCR/antibodies during a primary response, we analyzed both VH186.2 and analogue gene segments recovered from dissected splenic GC B cells of young and aged mice 12 days after immunization.

All of the six GC from three young mice contained mutated VH segments, whereas only four of the seven GC from two aged mice had mutated VH segments (Table 1). All of the 34 VH segments from GCs of young mice were encoded by the VH186.2 gene and mutated, with a base substitution rate of 1.8% or 5.5 point mutations per rearrangement (Table 1). By contrast, in aged mice, the NP-specific GC B cell repertoire consisted of mostly analogue genes instead of VH186.2 and all the VH186.2 rearrangements recovered from aged GC were not mutated (Table 1). However, 80% of the unique analogue clones (four of five) or 77% (24 of 31) of the analogue sequences from aged GC contained low levels of mutations, reaching a mutational frequency of 0.5% or 1.5 mutations per rearrangement (Table 1). Thus, the suppression in somatic hypermutation is more profound in GC B cells carrying the VH186.2 rearrangements than GC B cells expressing analogue genes. These findings are opposite to the results from young mice showing that analogue genes from primary GC contain mutations below that of VH186.2 segments (20). This discrepancy in susceptibility of different VDJ genes to the mutational process may result from altered Th function in aging. It is known that the antibody repertoire of NP-specific B cells is strongly influenced by CD4+ T cells (11). T cells, but not B cells, from aged mice are responsible for the VH gene repertoire shift to analogue genes (11). Therefore, the altered T cell help in aging may cause the alteration in targeting the mutational machinery.


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Table 1. Recovery of Ig hypermutation during a secondary response in old mice
 
Comparable expression of AID between B cells from young and aged mice
The fact that analogue gene segments recovered from primary GCs of aged mice contain low levels of point mutations suggests that, at least to a certain extent, the mutational machinery can be activated in aged mice. Additionally, our previous work has found that the intrinsic capability of B cells to be activated by the initial antigen stimulation is largely intact in aged mice (7). Since it has been shown that AID is a key component required for Ig hypermutation and isotype switching (22,23), we examined whether the Ig hypermutational mechanism in B cells of aged mice can be activated in vitro by measuring the expression of AID in B cells that had been fully activated in cultures. Although AID alone is not sufficient in inducing Ig hypermutation and other components are necessary for a fully active mutational machinery, the expression level of AID may be a good indicator of the intrinsic potential of B cells to undergo Ig hypermutation. Before in vitro activation, naive B cells from young and aged mice expressed little or no AID mRNA (data not shown). Interestingly, when B cells were activated by cross-linking BCR and CD40 with antibodies to IgM and CD40, the expression levels of AID mRNA in B cells from aged mice were comparable to that of B cells from young control animals (Fig. 1). These findings suggest that the Ig somatic hypermutation machinery in B cells may be preserved in aged mice and provides the basis for intervention to correct the age-related deficiency in Ig somatic hypermutation.



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Fig. 1. Comparable levels of AID expression between B cells from young and aged mice. Purified splenic B cells from young and aged mice were activated by anti-mouse IgM (10 µg/ml) and anti-mouse CD40 (10 µg/ml) for 48 h. (A) Representative RT-PCR assays are shown as gels stained with ethidium bromide. Titrations of 10-fold serial dilution of cDNA isolated from B cells from young or aged mice are shown. (B) Relative intensity of AID expression in young or aged B cells is expressed as ratios of AID/HPRT. Data (mean ± SE) for relative intensity of AID expression were calculated based on independent experiments on B cells from four individual animals of each group.

 
Somatic hypermutation is restored in aged mice during a memory response
To determine whether the diminished mutational machinery in aging can be reactivated by repeated immunization, we analyzed GC B cells during a memory response. Seventy days after primary immunization, mice were given an injection of 20 µg soluble NP-CGG and sacrificed 12 days later. Remarkably, somatic hypermutation in both analogue and VH186.2 rearrangements from GCs of aged animals was significantly increased compared to that of the primary response, reaching a level close to that of young mice (Table 1); 73% of individual GCs and 70% of unique clones from three old animals carried somatic mutants (Table 1). Importantly, in most cases, we were able to recover unmutated sequences containing the same complementarity-determining region (CDR) 3 as in mutated Ig sequences from the same GC, excluding the possibility that unknown germline sequences were misidentified as mutants.

Point mutations in analogue genes from primary GC of aged mice were not concentrated in the CDR (Table 1). In contrast, analogue genes from secondary GC of aged mice showed a significant enrichment of mutations in CDR over the framework regions (FR). The mutational frequency in CDR is >3-fold higher than that in FR (Table 1). Additionally, ratios of replacement versus silent mutations in CDR were significantly higher than those in FR (Table 2), further indicating that point mutations in the antigen-binding sites are selected and preserved during the secondary response. These findings demonstrate that antigen-driven selection of B cell mutants in aged mice was more evident in memory response than in primary response.


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Table 2. Ratios of replacement/silent point mutations in VH gene segments from GC of secondary responses
 
Clonotypic diversification during secondary responses
We have also determined the repertoire composition of GC B cells in young and aged mice during the secondary response. In young animals, there was a significant shift of clonotypic repertoire toward analogue genes during the secondary response (Fig. 2), consistent with findings that the AFC during an anamnestic response were more diversified than those in primary responses (24). However, the repertoire shift toward analogue genes was more profound in GC B cells from aged mice than young controls (Fig. 2). This may be due to the earlier shift of repertoire during the primary response in aged animals.



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Fig. 2. Repertoire composition of GC B cells during the secondary antibody response. At 12 days after secondary challenge, spleens of young and aged mice were taken, and the VH genes recovered from GC were sequenced and analyzed. (A and B) Percentages of individual GC containing VH186.2 (open segments), analogue (gray segments) or mixed rearrangements (black segments) are indicated around the periphery of each chart. (C and D) Percentages of unique clones carrying VH186.2 (open segments) or analogue (gray segments) genes are shown around the periphery of each chart. (E and F) Percentages of all VDJ sequences containing VH186.2 (open segments) or analogue (gray segments) genes are shown around the periphery of each chart. The total number of GC, clones or VDJ sequences analyzed in each group is indicated in the center of each chart.

 
Clonal selection and affinity maturation are functional during the secondary response in aged mice
A protective response is dependent on the process of antibody affinity maturation, which is the result of Ig hypermutation coupled with selection of B cell mutants with high-affinity BCR. The analysis of point mutations in VDJ rearrangements recovered from secondary GC suggests that antigen-driven clonal competition and selection in the GC are largely intact in aged animals (Tables 1 and 2). To examine antibody-affinity maturation coupled with the selection process in old mice during the memory response, we measured the levels and affinity of AFC and antibodies from mice before and after secondary immunization.

The ratio of high-affinity (NP5-binding)/total (NP25-binding) AFC or antibodies was used as an index for affinity maturation. Our results show that the size of the splenic IgG1 AFC pool in aged mice was comparable to that of young controls during the secondary response (Fig. 3A). Before challenge (70 days after primary response or day 0 of secondary of secondary response), the proportion of the high-affinity splenic AFC was small in both young and aged mice (Fig. 4A). By day 12 after secondary challenge, all of the splenic AFC from young and aged mice secreted high-affinity antibodies. These results demonstrate effective selection and enrichment of high-affinity AFC in the spleen during the memory response (Fig. 4A).



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Fig. 3. Secondary antibody responses to NP in young and aged mice. Seventy days after priming, mice were given an injection of 20 µg soluble NP-CGG. BM, spleen and serum samples from aged (black bars) or young control (open bars) mice were collected before (day 0) and after (day 12) secondary challenge. The number of splenic (A) or BM (B) NP-specific IgG1 AFC was measured by ELISPOT assays. The levels of NP-specific serum antibodies (C) were determined by ELISA. The data for BM AFC from aged mice at day 0 are not shown because the number of these AFC is minimal. The data are representatives (mean ± SE) of two independent experiments with three to five individual mice in each group.

 


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Fig. 4. Affinity maturation during the secondary response in aged mice. BM, spleen and serum samples were collected before (day 0) and after (day 12) secondary challenge from aged (black bars) or young control (open bars) mice. The ratios of NP5- to NP25-binding IgG1 AFC were used as indexes of affinity maturation in the splenic (A) or BM (B) AFC pool from day 0 to 12 of the secondary response. The ratios of NP5 to NP25 binding IgG1 serum antibodies were shown as indexes of affinity maturation of serum antibodies from day 0 to 12 of the secondary response (C). The data for BM AFC from aged mice at day 0 are not shown because the number of the AFC is minimal.

 
A large body of evidence suggests that long-term AFC in the BM are generated in the GC and are responsible for maintenance of long-term antibody levels (2530). In the present study, we found that BM AFC in aged mice were barely detectable before secondary challenge (Fig. 3B), consistent with impaired GC reaction in aged mice (59) and our earlier work showing that aged BM is less supportive for long-lived AFC (7). Nevertheless, at day 12 of the secondary response, a significant number of IgG1 AFC was detected in the BM of aged mice. Although the level of BM AFC in aged animals was ~20% of that in young controls (Fig. 3B), the majority (93%) of BM AFC in aged mice were high-affinity antibody producers (Fig. 4B). These results further support the contention that the process of affinity maturation coupled with clonal selection for higher-affinity mutants was largely functional in aged mice. However, it remains possible that some AFC may have differentiated from memory B cells. The relative contribution of memory B cells and secondary GC B cells to the BM AFC pool is not known.

Consistently, the antibody levels in aged mice were significantly lower than in young mice before secondary challenge. The concentration of serum NP-specific IgG1 antibodies in aged mice was ~20-fold lower than that in young control mice. The levels of serum NP-specific IgG1 antibodies in aged and young mice were 39 and 776 µg/ml respectively (Fig. 3C). At 12 days after challenge, although the overall concentration of serum NP-specific antibodies was still ~3-fold lower than that in young controls, the level of NP-specific IgG1 antibodies in aged animals had increased >30-fold to 1230 µg/ml (Fig. 3C). Parallel with improved affinity of BM AFC, 96% of the NP-specific serum antibodies in aged mice were high-affinity, NP5 binders (Fig. 4C). Therefore, during the memory response, the mechanism of selection for high-affinity mutants appears largely functional, leading to a significant improvement of affinity in serum antibodies as well as in AFC.


    Discussion
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Our current study demonstrates that age-associated impairment in Ig hypermutation in the GC can be restored during a secondary response. This reactivation of Ig somatic hypermutation, coupled with preserved clonal competition and selection, results in an overall improved pool of secondary specific antibodies in aged animals.

It is generally believed that the humoral responses in the aged are overall diminished. However, the mechanisms responsible for this age-related immune deficiency are not well understood. The evolution of an antibody response can be roughly divided into three phases: the initial pre-GC extrafollicular antibody response, GC response and post-GC differentiation pathway. Currently available evidence suggests that a diminished GC response is the main cause for the impaired antibody responses in aging (59), whereas the pre- and post GC pathways are largely functional except that aged BM is less supportive for long-term AFC generated during a primary antibody response (7,31). It has been suggested that age-related dysfunctions in Th cells and follicular dendritic cells (FDC) play critical roles in GC deficiency in aging (3235). Thus, the GC response in aging may be improved by either circumventing the requirements for Th and/or FDC, or overcoming deficiencies in Th and/or FDC.

Several possibilities may account for the significantly improved Ig hypermutation during a secondary GC response. First, during a memory response, the limited T help in old animals may be significantly overcome by activating memory T cells. It has been demonstrated that the B cell response could be significantly enhanced in mice primed with protein carriers (12). Thus, pre-existing memory T cells may be partly responsible for the improved Ig hypermutation and GC function such as clonal selection and affinity maturation. Second, the immune complexes (IC) formed during a secondary immunization may play an important role in modulating the antibody responses by regulating FDC functions (36,37). It has been shown that the impaired GC formation and hypermutation in athymic mice with limited numbers of T cells were restored by administrating antibodies specific for the immunizing antigen (38). Similarly, immunization with preformed IC enhanced hypermutation and alters the process of clonal competition (39). The BCR affinity threshold for antigen uptake and presentation is significantly lowered by oligomerization of antigens with antibodies (40). The IC may increase the avidity of antigen–BCR interaction and enhance the BCR-mediated signals. Additionally, by fixing complement and bridging BCR with complement, the IC elicit co-stimulatory signals through co-receptors such as CD19 (4143). Currently, we are investigating whether the diminished hypermutation in aging can be restored by immunization with pre-formed IC or sequential injections of antigens and specific antibodies. Finally, other factors may also contribute to the improved Ig hypermutation in aged mice during a secondary response, such as increased precursor frequency of antigen-specific lymphocytes, clonal burst size or improved function of Th cells. All these factors may exert their effects independently or synergistically, resulting an overall enhanced response.

In summary, our findings indicate that impaired humoral responses in aging can be overcome by repeated immunization, which may have important implications in designing vaccine compositions and immunization protocols for the elderly population and certain T-cell deficient patients, such as AIDS.


    Acknowledgements
 
This work was supported by National Institutes of Health Grant R01 AG17149.


    Abbreviations
 
AID—activation-induced cytidine deaminase

AFC—antibody-forming cell

BM—bone marrow

CDR—complementarity-determining region

CGG—chicken {gamma}-globulin

FDC—follicular dendritic cell

FR—framework region

GC—germinal center

HRP—horseradish peroxidase

IC—immune complex

NP—(4-hydroxy-3-nitrophenyl)acetyl

PNA—peanut agglutinin


    References
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 

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