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Repertoire of Antibody Response in Bone Marrow and the Memory Response Are Differentially Affected in Aging Mice¹

Department of Microbiology and Immunology, University of Maryland School of Medicine, Baltimore, MD 21201

	Abstract

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The primary burst of Ab and germinal center (GC) formation in response to T-dependent Ag is compromised in aging mice. Here we examine the effects of aging on the post-GC phase of memory B cell differentiation and the late Ab repertoire maturation in bone marrow (BM) in mice immunized with a hapten nitrophenyl coupled to chicken -globulin. Specific Ab-forming cells (AFC) with mutated V_H genes accumulated preferentially in the BM of aged mice, although the AFC numbers and average number of mutations per V_H were lower, and the D gene usage was less restricted compared with those in the young animals. However, the repertoire of AFC after an Ag boost demonstrated the hallmarks of Ag selection, including the recurrent mutations and canonical VD rearrangements, similar to the late primary response in young animals. It is postulated that the Ab repertoire maturation in aged mice is delayed and may be notably improved by repeated immunizations.

	Introduction

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Aging is marked by functional impairment of the adaptive phase of the Ab response in both humans and laboratory animals (1, 2). The aged immune system tends to generate Abs with lower avidity and/or affinity compared with young controls (3, 4, 5, 6, 7). Thus, even if the response of am aged organism to a specific Ag remains vigorous, the Abs are functionally insufficient and fail to protect against infection (8, 9, 10). One of the mechanisms of age-related immune dysfunction appears to be related to the germinal center pathway of Ab response. Germinal centers (GC)³ are formed in B cell follicles in lymphatic tissues within 1 wk after Ag exposure. The GC B cells, which undergo cycles of somatic hypermutation in their B cell Ag receptor genes, followed by affinity-based selection, are the progenitors of memory B cells as well as the cells that produce the Ab with higher affinity during the progression of the immune response (11). Thus, the paucity of high affinity Ab in aged animals pointed to a defect in GC. Indeed, Tew and his colleagues (12, 13) found a delayed and diminished formation of GC in aged mice. Miller and Kelsoe (14) and Yang et al. (15) subsequently showed a reduced frequency of somatic mutations (14, 15) and altered clonal selection (15) in GC of aged mice immunized with a hapten, (4-hydroxy-3-nitrophenyl)acetyl (NP). These studies further revealed that GC dysfunction was linked in part to intrinsic changes in aging B cells as well as changes in aging Th cells (15), which play a pivotal regulatory role in the GC reaction.

The initial bursts of Ab response and GC formation in the lymphatic tissue are followed by an extended period of Ab repertoire maturation and memory B cell differentiation that involves bone marrow (BM), which is a major source of Ig in both man and mouse (16). Animal experiments have shown that, 2–3 wk after the systemic immunization, specific Ab-forming cells (AFC) begin to accumulate in BM (16, 17, 18, 19, 20) where they persist for a long time (17, 21). The kinetics and magnitude of the BM AFC response is influenced by the immunizing dose of Ag (22) and use of adjuvant (23). Recent studies using NP hapten as a model Ag suggested that BM plays an important role in Ab repertoire maturation that continues for months after the involution of GC (18, 19, 20). Ag-reactive B cells and AFC in BM contain more somatically introduced mutations than their counterparts in the spleen (18, 20) and produce Abs with increasing affinity for NP (18, 19). The maturation of Ab repertoire in BM and the development of B cell memory appear to be differentially regulated. Smith et al. (24) examined affinity maturation of anti-NP response in mice expressing the bcl-2 transgene. They found decreases in somatic mutation as well as in affinity of Ab secreted by splenic memory B cells, whereas the BM AFC remained unaffected. A discriminatory effect of the bcl-x_L transgene on the repertoire of GC B cells and BM AFC was reported also by Takahashi et al. (25). Studies from our laboratory have shown that the late, NP-specific Ab response in BM and the anamnestic response to Ag boost may be dominated by different B cell clones (20).

These new findings prompted us to examine how aging may affect the repertoire of the post-GC phase of Ab response in BM and that of anamnestic response upon immunization with NP coupled to the chicken -globulin (NP-CGG). In mice with IgH^b allotype, this response is dominated by cells that produce Ab with H chain encoded by the V_H186.2 segment of the J558 gene family (26, 27, 28). Thus, we analyzed the V/D rearrangements and somatic mutations in V_H186.2⁺ AFC in cohorts of young/adult (2- to 3-mo-old) and aged (20- to 24-mo-old) C57BL/6 mice immunized with NP-CGG. Results from young mice were reported in part previously (20). Here we show that aging BM accumulates more AFC with mutated V_H compared with the spleen, and that the memory response in the old mice has the characteristic features of normal repertoire selection in GC. The implication of these findings for vaccination of elderly are briefly discussed.

	Materials and Methods

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Mice, Ags, and immunization

Strain C57BL/6 mice, young (2–3 mo old) and aged (19–21 mo old), were purchased from the Charles River Laboratory (Wilmington, MA) from cohorts maintained by the National Institute of Aging and maintained in sterile microisolator cages (Lab Products, Maywood, NJ). The aged mice had a healthy appearance, and no gross pathological changes were observed during the autopsy. NP or its analog (4-hydroxy-5-iodo-3-nitrophenyl) acetyl (NIP) (Cambridge Research Biochemical, Cambridge, U.K.) were conjugated to CGG (Sigma-Aldrich, St. Louis, MO) or BSA (Amersham Life Science, Cleveland, OH) as previously described (29). Mice were immunized with a single i.p. injection of 10 µg of an Ag in alum and challenged with 10 µg of a soluble Ag in PBS i.p.

Preparation of NIP-RBC

SRBC in Alsever’s solution (Colorado Serum, Denver, CO) were washed in DPBS and resuspended to 10% (v/v) in 0.1 M bicarbonate buffer, pH 9.4. Succinimide ester of NIP (Cambridge), dissolved in dimethylformamide (Sigma-Aldrich), was added to a final concentration of 25 µg/ml of SRBC, and the mixture was incubated at room temperature for 30–60 min. Cells were then washed and resuspended to 20% (v/v) in DPBS.

Preparation of cells and AFC assay

Splenocyte suspension was prepared by mashing spleens in RPMI 1640 medium supplemented with 25 mM HEPES (Life Technologies, Gaithersburg, MD) and 0.5% BSA (Amersham). Bone marrow cells were recovered by flushing the femurs through a 23-gauge needle with the same medium. Lymphocytes producing the NP-specific IgG Ab were detected with a modified plaque assay (30) using SRBC coupled with hapten as described previously in detail (20). Briefly, a mixture containing 0.3% agarose in Basal Medium Eagle (Life Technologies), lymphocytes, NIP-RBC, goat anti-mouse IgM (Southern Biotechnology Associates, Birmingham, AL), and rabbit anti-mouse IgG (Sigma-Aldrich) was poured on petri dishes and incubated at 37°C in 5% CO₂ for 2 h. The hemolytic plaques were visualized by covering the plates with guinea pig complement (Life Technologies). The hemolytic plaque assay readily detects the IgM-producing cells, whereas the IgG-producing cells become detectable in the presence of an exogenous anti-IgG Ab (30). The Abs added to the assay mixture were previously titrated to inhibit the IgM producers and to visualize the IgG producers, as previously described (20). For the isolation of single AFC from the center of the plaque, the cell suspensions were highly diluted to increase the probability of sampling one cell (20). On the average, 40–70% of samples yielded a PCR product using the specific primers (see below). In contrast, no products were obtained from random sampling of the RBC lawn.

Ab measurement

Serum levels of NP-specific IgG Ab that exhibit heteroclitic binding to the NIP analog were determined by standard ELISA techniques using NIP-BSA conjugate as Ag in solid phase and isotype-specific goat anti-mouse Ig labeled with HRP (Southern Biotechnology Associates) as secondary Abs, followed by a tetramethylbenzidine hydrogen peroxide substrate kit (Bio-Rad, Richmond, CA). The Ab titers were expressed as reciprocal end-point dilutions of the sera.

Recovery and molecular analysis of GC B cells

Spleens were removed from mice on day 12 after primary immunization, frozen sections were prepared for immunohistochemistry, and NP-reactive GC B cells were identified by dual staining with peanut agglutinin (PNA) and NIP-BSA as previously described (15). Cells (100) from individual GC were microdissected (15, 31) and transferred into microcentrifuge tubes for PCR DNA amplification. The initial round of amplification used primers corresponding to the V186.2 genomic DNA 5' transcription start site sequence and the intron J_H2 sequence, respectively. An aliquot of the reaction mixture was reamplified using nested primers complementary to the initial 20 nucleotides of the V186.2 gene and to the J_H2 segment, with additional restriction enzyme sites. The details of the PCR reactions and sequences of the primers were published previously (15, 32). The PCR product was digested with the restriction enzymes, ligated to a plasmid, cloned in competent Escherichia coli, and sequenced as previously described (32).

Amplification and sequencing of VDJ DNA recovered from individual AFC

The individual NIP⁺, IgG AFC were picked manually using 50-µl disposable micropipettes (Fisher Scientific, Pittsburgh, PA) and transferred into microcentrifuge tubes with PCR buffer, proteinase K, and Tween 20 exactly as previously described (20). DNA amplification of the crude cell lysate was conducted by two rounds of PCR using nested primers corresponding to V186.2/JH2 genomic sequences described above. The PCR product isolated with the QIA quick gel extracting kit (Qiagen, Hilden, Germany) was directly sequenced by the Biopolymer Laboratory of the University of Maryland School of Medicine using an automatic DNA sequencing system (PE Applied Biosynthesis, Foster City, CA). DNA codons are numbered according to the method of Kabat et al. (33).

Assessment of somatic mutations and identification of V_H186.2 gene

An error rate of 8.5 x 10^-6 misincorporations bp PCR cycle is expected from the Expand High Fidelity polymerase according to the manufacturer. Thus, approximately five VDJ fragments (1715 bp) recovered from tissue by 80 cycles of amplification would be expected to contain one mutation attributable to the polymerase error. We found no mutations after sequencing six clones recovered from two independent amplifications of germline-encoded NP-reactive B1-8 hybridoma cells (V186.2, DFL 16-1, and J_H2; not shown). Thus, we assume that mutations in excess of the theoretical average value (0.2 mutation/VDJ) have resulted from a somatic process. Mutation frequencies shown in Results are based on scores of base substitutions in the V_H sequences only. Shared mutations within a set of clonally related sequences (according to the complementarity-determining region (CDR) 3 region) were counted as one mutational event. The sequences representing the V186.2 gene differed from their presumptive germline counterpart by up to 20 nucleotides, indicating that the V segments were somatically mutated or that they were derived from a different germline gene of the V186.2/V3 gene family (J558) (34). Distinction was possible because the nucleotide differences between the recovered sequences and the V186.2 germline sequence typically fell into the positions shared by members of the V186.2/V3 gene family and spared those positions that characterize individual germline genes. Thus, codons 11, 20, 43, 50, 74, and 91 (and others) that distinguish the 186.2 gene were never mutated.

Statistics

The data were analyzed by the Wilcoxon nonparametric test.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
The responses of aged mice to a single immunization with 10 µg NP-CGG in alum were overall diminished compared with young/adult animals. The formation of PNA⁺ GC in the spleen of aged mice was reduced by more than half (Fig. 1a), as expected (14, 15); however, the proportion of GC that contained the NP-specific B cells was comparable in both age groups (Fig. 1b). The IgG AFC response in the spleen was also lower, particularly during the early peak (Fig. 2), yet the NP-specific AFC appeared, characteristically in the bone marrow of aged mice at later stages of the response (Fig. 2b), although in smaller numbers compared with the young animals (Fig. 2a). Serum IgG Ab levels in aged mice were reduced by 10-fold or more (Table I).

View larger version (15K): [in this window] [in a new window]   FIGURE 1. GC reaction in the spleen of young ( and ) and aged (• and ) mice on day 12 after immunization with NP-CGG/alum. a, The number of GC is expressed as the percentage of follicles stained with PNA. b, The number of GC containing NP-specific cells is expressed as the percentage of PNA+ follicles double-stained with biotinylated NIP-BSA.

View larger version (11K): [in this window] [in a new window]   FIGURE 2. Kinetics of NP-specific IgG-producing cells (AFC) as detected by hemolytic plaque assay in the spleen () and BM () of young (a) and aged (b) mice after immunization with NP-CGG. Three or four animals from each cohort were boosted with 10 µg NP-CGG/PBS on day 62 (arrows), and NP-specific IgG AFC were enumerated 5 days later in the spleen () and BM (). Each point represents mean from three or four mice ± SEM (vertical bars).

View larger version (12K): [in this window] [in a new window]   FIGURE 3. Distribution of mutations in the rearranged VH186.2 genes among NP-specific AFC in BM of young () and aged (•) mice in late primary response (days 35–65). Data for young animals were adapted from Ref. 20 .

View larger version (26K): [in this window] [in a new window]   FIGURE 4. VH repertoire in aged mice after primary immunization. Partial nucleotide sequences of rearranged VH186.2/D genes recovered from individual AFC in the spleen and BM at late primary response (days 35–65) after immunization with NP-CGG/alum. The germline-encoded VH186.2 sequence is around positions 33 in CDR1 and 58 in CDR2, and CDR3 with part of the DFL16.1 segment is shown at the top. The positions of nucleotide changes in the sequences from individual AFC are indicated below.

Analysis of V186.2/D rearrangements from NP-specific AFC that were recovered after the antigenic boost was insightful. All secondary AFC in aged mice were somatically mutated, containing, on the average, half as many mutations (five or six mutations per V_H) than the secondary AFC from the young (11–12 mutations per V_H; p > 0.01; Table V). More surprisingly, the repertoire of secondary AFC in aged mice had the hallmarks of typical GC-derived precursor B cell that are found in young NP-immunized mice (36, 37, 38): shared W33L replacement, Tyr⁹⁵, and canonical rearrangement V186.2/DFL16.1/2 (Fig. 5 and Table IV); note that this clonotype was relatively rare in the late primary AFC in the aged mice (Fig. 4 and Table III). The distinct repertoires of NP-reactive cells in aged mice, the late primary BM AFC, and the AFC triggered by antigenic challenge are summarized in Fig. 6, b and d. It can be seen that the anamnestic AFC in aged mice (Fig. 6d) had a repertoire similar to that of late primary AFC in BM of young mice (Fig. 6a). In contrast, as detailed previously (20), the anamnestic AFC in the young represented a novel clonotype that is seldom found among NP-specific GC B cells (Fig. 6c); most AFC shared a mutation that replaced lysine (K) with arginine (R) in position 58 (instead of the W33L) and an N residue Gly⁹⁵ (instead of Tyr⁹⁵); such cells were virtually absent from the repertoire of anamnestic response in aged mice (Fig. 6d).

View larger version (26K): [in this window] [in a new window]   FIGURE 5. VH repertoire in aged mice after secondary immunization. Partial sequences from anamnestic AFC recovered from the spleen and BM of aged mice are shown. Mice were immunized as shown in Fig. 2, and AFC were obtained on day 5 after the Ag boost. Data are presented as described in Fig. 4.

View larger version (76K): [in this window] [in a new window]   FIGURE 6. Graphic representation of somatic mutation patterns in VH186.2 in AFC from young (a and c) and aged (b and d) mice during late primary (a and b) and anamnestic (c and d) responses to NP-CGG. The size of each pie section is proportional to the following AFC: unmutated (), mutants with W33L replacement and Tyr95 (), mutants with K58R replacement and Gly95 (), and mutants w/o the recurrent replacements (). The diagram shows combined results from splenic and BM AFC; the data from young mice were adapted from Ref. 20 .

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

We found it interesting that the skewed pattern of V/D diversity, which was observed in aged AFC in both BM and spleen, is strikingly similar to the pattern that was found by Takahashi et al. (25) in young mice expressing the bcl-x_L transgene. Overexpression of bcl-x_L (24) or bcl-2 (24) causes a promiscuity in affinity-based clonal selection in GC due to inhibition of apoptosis. The present data suggest that aging may similarly compromise the GC selection process, although the exact mechanism remains to be determined.

The second observation was that the aged BM was enriched for somatically mutated AFC to the extent that is seen in young BM, except that highly mutated cells (10 mutations LV_H) were rare. Moreover, 50% of the mutant AFC contained the V_H replacement W33L that is associated with increased affinity of anti-NP Ab (35). Therefore, it appears that the general process of repertoire maturation in BM is partially operative even in very old mice. Why the Ag-specific B cells/AFC in BM are more somatically mutated than the cells in the spleen is not known. It has been proposed that the mutated B cells/AFC may home selectively to BM (18, 39), that BM provides stimuli for mutation and selection in situ, or both (19, 20, 40). The pattern of mutations in BM AFC observed in aged mice, i.e., a high proportion of mutants with fewer mutations per cell, is consistent with the possibility that both hypothetical mechanisms exist, and that immunosenescence spares the homing mechanism, but compromises the in situ activity.

The apparent retardation of somatic mutation in aged mice is reminiscent of the pattern that has been observed by Diaz et al. (41) in mice with disrupted expression of DNA polymerase . The NP-activated B cells in these mice contain fewer somatic mutations; however, the frequency of mutations increases slowly with prolonged Ag exposure. The authors postulated that a slow kinetics of repertoire maturation may be proportional to the low rate of mutation.

It is thought that BM AFC, like the memory B cells, originate in splenic GC (39). However, the repertoire maturation of these two cell populations appears to be independently regulated (18, 20, 24, 25). This view is further supported by the present finding that the repertoire of anti-NP anamnestic response in the spleen and BM in aged mice is markedly different from the repertoire of AFC present in BM at the time of Ag boost. Although the AFC were sampled randomly, the sharp increase in cell numbers after the boost and the similarity of results in the spleen and BM warrant our inference that the sample was representative of memory AFC. These cells expressed the canonical V186.2/DFL16.2 joints and were uniformly mutated, including the W33L replacement. Thus, the anamnestic anti-NP response in aged mice had all the hallmarks of NP-specific precursor memory B cells that originate from functionally competent GC (36, 37, 38) and, upon stimulation, produce Abs with affinities from NP 10-fold higher compared with the primary, germline-encoded Ab (35, 36, 42). In this model, then, the aged immune system has demonstrated a potential for significant maturation of memory repertoire. However, the aged mice failed to reach a higher order of memory, i.e., cells that produce anti-NP Ab encoded by V186.2/DFL16.1 genes with a distinct N residue, Gly⁹⁵, germline-encoded Trp³³, and the K58R replacement, which dominated the anamnestic response to NP in young mice (19) (see Fig. 6). Affinities of such Ab for NP can increase by another 1–2 logs according to Furokawa et al. (42).

It is apparent that aged mice develop immunological memory after the primary immunization, but the repertoire is less well developed compared with that in young animals. Whether the process is merely slower in the aged or whether there is a limit to the repertoire maturation is an important question that should be addressed in future experiments. If the first alternative is correct, it might be possible to restore the Ab affinity in aged animals by repeated immunizations. Such animal studies could eventually help to design proper vaccination schedules for the elderly population.

	Acknowledgments

 
We thank Dr. Martin Flajnik for his constructive comments on this manuscript.

	Footnotes

 
¹ This work was supported in part by U.S. Public Health Service Grant AG08193.

² Address correspondence and reprint requests to Dr. Jan Cerny, Department of Microbiology and Immunology, University of Maryland School of Medicine, 655 West Baltimore Street, Bressler Research Building 13-15, Baltimore, MD 21201. E-mail address: ojone002{at}maryland.edu

³ Abbreviations used in this paper: GC, germinal center; AFC, Ab-forming cells; BM, bone marrow; CDR, complementarity-determining region; CGG, chicken -globulin; NIP, (4-hydroxy-5-iodo-3-nitrophenyl)acetyl; NP, (4-hydroxy-3-nitrophenyl)acetyl; PNA, peanut agglutinin.

Received for publication May 24, 2002. Accepted for publication August 26, 2002.

	References

Top Abstract Introduction Materials and Methods Results Discussion References

 

1. Makinodan, T., M. M. B. Kay. 1980. Age influence on the immune system. Adv. Immunol. 29:287.[Medline]
2. Miller, R. A.. 1996. The aging immune system: primer and prospectus. Science 273:70.[Abstract]
3. Kishimoto, S., T. Takahama, H. Mizumachi. 1976. In vitro immune response to the 2,4,6-trintrophenyl determinant in aged C57BL/6 mice: changes in the humoral response to avidity for the TNP determinant and responsiveness to LPS effect with aging. J. Immunol. 116:294.[Abstract/Free Full Text]
4. Goidl, E. A., J. B. Innes, M. E. Weksler. 1976. Immunological studies of aging. II. Loss of IgG and high avidity plaque forming cells and increased suppressor cell activity in aging mice. J. Exp. Med. 144:1037.[Abstract/Free Full Text]
5. Zharhary, D., Y. Segev, H. Gershon. 1977. The affinity and spectrum of crossreactivity of antibody production in senescent mice: the IgM response. Mech. Ageing Dev. 6:385.[Medline]
6. Doria, G, G. D’Agostaro, A. Poretti. 1978. Age dependent variations of antibody avidity. Immunology 35:601.[Medline]
7. Weksler, M. E., J. B. Innes, G. Goldstein. 1978. Immunological studies of aging. IV. The contribution of thymic involution to the immune deficiencies of aging mice. J. Exp. Med. 148:966.
8. Ammann, A. J., G. Schiffman, R. Austrian. 1980. The antibody responses to pneumococcal capsular polysaccharides in aged individuals. Soc. Exp. Biol. Med. 164:312.[Medline]
9. Romero-Steiner, S., D. M. Musher, M. S. Cetron, L. B. Pais, J. E. Groover, A. E. Fiore, B. D. Plikaytis, G. M. Carlone. 1999. Reduction in functional activity against Streptococcus pneumoniae in vaccinated elderly individuals highly correlates with decreased IgG antibody avidity. Clin. Infect. Dis. 29:281.[Medline]
10. deBruijn, I. A., E. J. Remarque, C. M. Jol-van derZijde, M. J. D. vanTol, R. G. J. Westendorp, D. L. Knook. 1999. Quality and quantity of the humoral immune response in healthy elderly and young subjects after annually repeated influence vaccination. J. Inf. Dis. 179:31.[Medline]
11. Gearhart, P. J.. 1993. Somatic mutation and affinity maturation. W. E. Paul, ed. Fundamental Immunology 865. Raven Press, New York.
12. Kosco, M. H., G. F. Burton, Z. F. Kapasi, A. K. Szakal, J. G. Tew. 1989. Antibody-forming cell induction during an early phase of germinal center development and its delay with aging. Immunology 68:312.[Medline]
13. Szakal, A. K., J. K. Taylor, J. P. Smith, M. H. Kosco, G. F. Burton, J. G. Tew. 1990. Kinetics of germinal center development in lymph nodes of young and aging immune mice. Anat. Rec. 222:475.
14. Miller, C., G. Kelsoe. 1995. Ig V_H hypermutation is absent in the germinal centers of aged mice. J. Immunol. 155:3377.[Abstract]
15. Yang, X., J. Stedra, J. Cerny. 1996. Relative contribution of T and B cells to hypermutation and selection of the antibody repertoire in germinal centers of aged mice. J. Exp. Med. 183:959.[Abstract/Free Full Text]
16. Benner, R., J. J. Haaijman. 1980. Aging of lymphoid system at the organ level with special reference to the bone marrow as site of antibody production. Dev. Comp. Immunol. 4:591.[Medline]
17. Slifka, M. K., J. K. Whitmire, R. Ahmed. 1997. Bone marrow contains virus-specific cytotoxic T lymphocytes. Blood 90:2103.[Abstract/Free Full Text]
18. Smith, K. G. C., A. Light, G. J. V. Nossal, D. Tarlinton. 1997. The extent of affinity maturation differs between the memory and antibody-forming cell compartments in the primary immune response. EMBO J. 16:2996.[Medline]
19. 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 develop in two stages of clonal selection. J. Exp. Med. 187:885.[Abstract/Free Full Text]
20. Lu, Y. F., M. Singh, J. Cerny. 2001. Canonical germinal center B cells may not dominate the memory response to antigenic challenge. Int. Immunol. 13:643.[Abstract/Free Full Text]
21. Manz, R. A., M. Löhning, G. Cassese, A. Thiel, A. Radbruch. 1998. Survival of long-lived plasma cells is independent of antigen. Int. Immunol. 10:1703.[Abstract/Free Full Text]
22. Benner, R., A. Van Ovdenaren. 1976. Antibody formation in mouse bone marrow: the response to thymus-independent antigen Escherichia coli lipopolysaccharide. Immunology 30:49.[Medline]
23. Koch, G., D. G. Osmond, M. H. Julius, R. Benner. 1981. The mchanism of thymus-dependent antibody formation in bone marrow. J. Immunol. 126:1447.[Abstract]
24. Smith, K. G. C., A. Light, L. A. O’Reilly, S. M. Ang, A. Strasser, D. Tarlinton. 2000. bc1–2 transgene expression inhibits apoptosis in the germinal center and reveals differences in the selection of memory B cells and bone marrow antibody-forming cells. J. Exp. Med. 191:475.[Abstract/Free Full Text]
25. Takahashi, Y., D. M. Cerasoli, J. M. DalPorto, M. Shimida, R. Freund, W. Fang, D. Telander, E. N. Malvey, D. L. Mueller, T. W. Behrens, et al 1999. Relaxed negative selection in germinal centers and impaired affinity maturation in bcl-x_L transgenic mice. J. Exp. Med. 190:399.[Abstract/Free Full Text]
26. Cumano, A., K. Rajewsky. 1986. Clonal recruitment and somatic mutation in the generation of immunologic memory to the hapten NP. EMBO J. 5:2459.[Medline]
27. Bothwell, A. L. M., M. Paskind, M. Reth, T. Imanishi-Kari, K. Rajewsky, D. Baltimore. 1981. Heavy chain variable region contribution to the NP^b family of antibodies: somatic mutation evident in a 2a variable region. Cell 24:625.[Medline]
28. Bothwell, A. L. M., M. Paskind, M. Reth, T. Imanishi-Kari, K. Rajewsky, D. Baltimore. 1982. Somatic variants of murine immunoglobulin light chains. Nature 298:380.[Medline]
29. Weinberger, J. Z., M. I. Green, B. Benacerraf, M. E. Dorf. 1979. Hapten-specific T cell responses to 4-hydroxy-3-nitropheny acetyl. I. Genetic control of delayed-type hypersensitivity by V and I-A region genes. J. Exp. Med. 149:1136.
30. Dresser, D. W., H. H. Wortis. 1965. A localized homolysis in gel method for detection of cells producing 7S antibody: use of antiglobulin serum to detect cells producing antibody with low hemolytic efficiency. Nature 208:858.[Medline]
31. Jacob, J., J. Przylepa, C. Miller, G. Kelsoe. 1993. In situ studies of the primary immune response to (4-hydroxy-3-nitrophenyl) acetyl. III. The kinetics of V-region mutation and selection in germinal centers B cells. J. Exp. Med. 178:1293.[Abstract/Free Full Text]
32. Nie, X., S. Basu, J. Cerny. 1997. Immunization with immune complex alters the repertoire of antigen-reactive B cells in the germinal centers. Eur. J. Immunol. 27:3517.[Medline]
33. Kabat, E. A., T. T. Wu, H. M. Perry, K. S. Gottesman, and C. Foeller. 1991. Sequences of proteins of immunological interest. U.S. Dept. Health and Human Services, Washington, D.C., National Institutes of Health Publication 91-7242.
34. Gu, H., D. Tarlington, W. Muller, K. Rajewsky, I. Forster. 1991. Most peripheral B cells in mice are ligand selected. J. Exp. Med. 173:1357.[Abstract/Free Full Text]
35. Allen, D., T. Simon, F. Sablitsky, K. Rajewsky, A. Cumano. 1988. Antibody engineering for the analysis of affinity maturation of an anti-hapten response. EMBO J. 7:1995.[Medline]
36. Weiss, U., K. Rajewsky. 1990. The repertoire of somatic antibody mutants accumulating in the memory compartment after primary immunization is restricted through affinity maturation and mirrors that expressed in the secondary response. J. Exp. Med. 172:1681.[Abstract/Free Full Text]
37. McHeyzer-Williams, M. G., M. J. McLean, P. A. Lalor, G. J. V. Nossal. 1993. Antigen driven B cell differentiation in vivo. J. Exp. Med. 178:295.[Abstract/Free Full Text]
38. Weiss, U., R. Zoebelein, K. Rajewsky. 1992. Accumulation of somatic mutants in the B cell compartment after primary immunization with a T cell-dependent antigen. Eur. J. Immunol. 22:511.[Medline]
39. Tarlinton, D. M., K. G. C. Smith. 2000. Dissecting affinity maturation: a model explaining selection of antibody-forming cells and memory B cells in the germinal centre. Immunol. Today 21:436.[Medline]
40. Paramithiotis, E., M. D. Cooper. 1997. Memory B lymphocytes migrate to bone marrow in humans. Proc. Natl. Acad. Sci. USA 94:208.[Abstract/Free Full Text]
41. Diaz, M., L. K. Verkoczy, M. F. Flajnik, N. R. Klinman. 2001. Decreased frequency of somatic hypermutation and impaired affinity maturation but intact germinal center formation in mice expressing antisense RNA to DNA polymerase . J. Immunol. 167:327.[Abstract/Free Full Text]
42. Furukawa, K., A. Akasako-Furutawa, H. Shirai, H. Nakamura, T. Azuma. 1999. Junctional amino acids determine the maturation pathway of an antibody. Immunity 11:329.[Medline]

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