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Increased VH 11 and VH Q52 gene use by splenic B cells in old mice associated with oligoclonal expansions of CD5+ B cells
Mechanisms of Ageing and Development 103 (1998) 111 – 121
Increased VH 11 and VH Q52 gene use by splenic B cells in old mice associated with oligoclonal expansions of CD5+ B cells Arie Ben-Yehuda a,1, Paul Szabo b, Joel LeMaoult b, J. Sanil Manavalan b, Marc E. Weksler b,* a
Department of Medicine, Geriatric Unit, Hadassah Uni6ersity Hospital, Ein-Karem, Jerusalem, 91010, Israel b Di6ision of Geriatrics and Gerontology, Cornell Uni6ersity Medical College, 1300 York A6enue, New York, NY 10021, USA Received 31 October 1997; accepted 16 December 1997
Abstract A significant increase in the utilization of the VH gene families VH11 and Q52 was observed in LPS-stimulated splenic B lymphocytes from aged mice compared to young mice. VH gene usage was assayed by in situ DNA/RNA hybridization using VH family-specific and kappa chain probes. The observed age-dependent differences appear to reflect the preferential use of VH11 and Q52 VH gene use by the CD5+ B lymphocyte subset whose numbers in the spleen increase with age. The increased use of VH11 by splenic cells from old mice is associated with clonal expansions of splenic CD5 + B lymphocytes. © 1998 Published by Elsevier Science Ireland Ltd. All rights reserved. Keywords: Aging; VH 11 and VH Q52 genes; Splenic B cells; CD5 + B cells
* Corresponding author. Tel.: + 1 212 7466478; fax: + 1 212 7466382; e-mail: weksler@ med.cornell.edu 1 Tel.: + 972 2 6777627/8; fax: +972 2 6413147; e-mail: [email protected] 0047-6374/98/$19.00 © 1998 Published by Elsevier Science Ireland Ltd. All rights reserved. PII S0047-6374(98)00004-9
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1. Introduction The generation of a diverse B cell repertoire requires the rearrangement of immunoglobulin gene segments within the heavy and light chain gene loci. The first step in this process is the joining of D and JH segments followed by recombination with one gene from one of the 14 VH families. Subsequently, VL and JL segments are joined leading to the creation of a functional light chain gene and the expression on the surface of B lymphocytes of a complete immunoglobulin (Ig) molecule. The use of VH gene segments by splenic B cells in fetal and neonatal mice less than 1 week old shows a strong bias for 3%-end, D proximal VH gene families, predominantly 7183 and Q52 (Jeong and Teale, 1984; Yancopoulos et al., 1984). Thereafter, VH gene use by adult spleen cells is proportional to the size of the VH family and shows no 3% preference (Yancopoulos et al., 1988). Aging also affects VH use. Thus, VH use by antigen-specific B cells following immunization with PC (Riley et al., 1989) or NP (Yang et al., 1996) differs in old and young mice although analysis of LPS-induced B cell colonies and B cell hybridomas prepared from young and old mice showed, with one exception, no difference in VH gene family use (Miceli et al., 1990; Schultze et al., 1992). As fewer B-cell colonies and B-cell hybridomas are generated from old compared to young mice (Miceli et al., 1990; Schultze et al., 1992), we used direct DNA/RNA hybridization to assess VH use by individual splenic and peritoneal B cells from old and young mice cultured with LPS. We found that old mice had significantly more CD5 + splenic B cells and that the use of VH11 and Q52 immunoglobulin VH gene families by spleen cells was significantly increased. The VH11 gene family is used almost exclusively by CD5+ B cells (Andrade et al., 1991). Furthermore, oligoclonal populations of CD5+ B cells have been demonstrated within the spleens of old mice (Stall et al., 1988; Hardy and Hayakawa, 1994. In this report we demonstrate monoclonal CD5+ B cell populations using the VH11 VH gene family.
2. Materials and methods
2.1. Animals Young (2 – 3 months old) and old (16–22 months old) female C57BL/6 mice were purchased from the National Institute on Aging Colony at the Charles River Laboratories (Kingston, NY) and maintained in the Animal Resource Facility at Cornell University Medical College under laminar flow hoods. Routine serological assays for viral, bacterial and parasitic pathogens were negative. All experiments were performed in accordance with protocols approved by the Institutional Animal Care and Use Committee of Cornell University Medical College.
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2.2. Cell lines Cloned B cell hybridomas known to use certain VH gene were used as to establish the optimal conditions for in situ hybridization. The cell lines 7023 (J558), NAb-1 (7183), NAb-4 (Q52) and 582C10 (J606) were all provided by Dr D.L. Kastner (Bethesda, MD) The cloned cells were passaged in complete culture medium (RPMI 1640 with 10% FCS, 2 mM L-glutamine, 1 mM Na-Pyruvate, 10 mM HEPES, 5× 10 − 5 2ME, non-essential amino acids and penicillin/streptomycin).
2.3. VH Ig family gene probes Probes for Ig VH gene families that make up more than 5% of the B cell repertoire: VH81X (7183), VHQ52 (Q52), VHB4 (J558) and VHJ606 (J606) were kindly provided by Dr Frederick W. Alt (Boston, MA). The pCP12 (VH11) probe was kindly provided by Dr Reininger (Geneva, Switzerland), and the kappa light chain constant region probe was a gift from Dr J. Stavnezer (Worchester, MA).
2.4. Cell culture Peritoneal lymphocytes from 2 or 18 month old mice were obtained by peritoneal lavage, washed three times with Hanks balanced salt solution (HBSS), and resuspended at a concentration of 106 cells/ml in complete medium: RPMI 1640 with 10% FCS. Single spleen cell suspensions were prepared by teasing the spleens apart in HBSS and passing the dissociated spleen through a sieve. The isolated cells were washed three times with HBSS and resuspended in complete medium at a concentration of 106 cells per ml. Peritoneal and splenic cells were cultured in medium containing 10 mg LPS/ml for 3 days at 37°C in a 5% CO2/95% humidified air environment. The cells were then washed and resuspended at a concentration of 107 cells/ml in complete culture medium. This suspension was pipetted onto an ‘8chamber’ slide (100 ml/chamber). The slides were centrifuged for 5 min (500 rpm/min), the supernatant medium was discarded and cells fixed in freshly prepared 4% paraformaldehyde (Sigma, St. Louis, MO) in phosphate buffered saline (PBS). The slides were stored in 70% ethanol at 4°C until they were used for in situ hybridization.
2.5. In situ hybridization The protocol used for in situ hybridization was a modification of that employed by Harper et al. (1986). All plasmid probes were labeled with 35S-dATP using the nick translation method. Slides bearing appropriate cells were removed from 70% ethanol, washed twice in 1×PBS solution containing 5 mM MgCl2, and then treated for 10 min with 0.25% acetic anhydride in 0.1 M triethanolamine buffer (pH 8.0). Slides were then incubated in 0.1 M Tris, 0.1 M glycine (pH 7.0) buffer for 10 min, and then washed in 2 × SSC, and dehydrated by 1 min incubation in 70 and 90% ethanol. Probes were resuspended in 10 mM Tris/1 mM EDTA buffer
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containing 10 mM dithiothreitol: the activity of the probes was adjusted to 106 cpm/ml. Probes were diluted 1/10 in hybridization buffer to give a final concentration of 50% formamide, 2× SSC, 10 mM dithiothreitol, 10% Dextran solution, 2× Denhart’s solution, 100 ml/ml of solution × DNA and 500 ng (107 cpm/ml) of probe DNA. Twenty microliters of diluted probe were added per chamber and covered with parafilm and incubated for 3 h at 42°C. Slides were then washed twice in 2× SSC at room temperature for 30 min, and then in 2× SSC at 55°C for 20 min. The final wash was in 0.1 × SSC for 20 min at 60°C. After the final wash, the slides were dehydrated in 70 and 90% ethanol and air dried. These conditions and stringencies of hybridization and washing resulted in the identification of only the B cell clones expressing the homologous VH gene family (Table 1).
2.6. Autoradiography In situ hybridization was revealed by autoradiography with NTB-2 emulsion (Eastman-Kodak, Rochester, NY). Exposure times ranged from 3 to 4 days at 4°C in light-tight slide boxes containing desiccant powder. Slides were developed in a 1:1 dilution of Dektol in H2O and fixed in Kodafix. After air-drying, slides were stained in Giemsa for 10 min, then washed in distilled water.
2.7. Calculation of VH frequencies Identically prepared slides were probed for both VH and kappa chain message. More than 200 cells were counted for each probe. Cells were considered positive if they were labeled with more than 50 grains per cell. The total number of B cells was approximated by the number of cells with detectable kappa light chain mRNA as 95% of murine B cells express this light chain. The percentage of VH gene family used by each family was calculated by dividing the percentage of cells positive for a specific VH family probe by the percentage of cells positive for kappa light chain probe. Table 1 Specificity of cDNA probes for the indicated VH gene family B cell hybridoma
7023 (J558) 582C103 (J606) NAb-4 (Q52) NAb-1 (7183)
VH gene family probe J558
J606
Q52
7183
94 2 3 5
8 95 6 4
1 4 95 3
2 1 4 80
The specificity of VH probes was determined by in situ DNA/RNA hybridization using the indicated B cell clones. The percentage of cells from each clone that were positive with each probe are shown. Positive cells were labeled with more than 50 grains/cell. In most experiments, negative cells were labeled with less than 5 grains/cell.
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2.8. Northern RNA analysis Total cellular RNA was prepared from four individual young and four individual old spleens, and was denatured in 50% formamide/2.2 M formaldehyde in 1XMOPS buffer for 5 min at 65°C. Five micrograms of each sample were electrophoresed in 2.2 M formaldehyde/1% agarose gel, and blotted by standard procedures (Sambrook et al., 1989). The membranes were probed with the 32P-labeled VH81 (7183), VHQ52 (Q52), VHB4 (J558) and PCP12 (VH11) cDNAs at 42°C for 18 h, and washed with a final wash in 0.1 × SSPE/0.1% SDS at 42°C, and exposed to X-ray film.
2.9. Separation of CD5+ and CD5 − peritoneal B cells by cell sorting Peritoneal cells obtained as described above were washed and stained with phycoerythrin-conjugated anti-CD5 mAb (4 mg/106 cells) (Pharmingen, San Diego, CA) and 4 mg/106 cells of fluorescein-conjugated goat anti-mouse IgM antibodies (Southern Biotechnology Associates, Birmingham, AL) for 15 min. The cells were then washed three times in complete culture medium. The stained cells were sorted into CD5+ and CD5− B cells using a FACStar Plus apparatus (Becton Dickinson, Franklin Lakes, NJ). Sorted B cells were reanalyzed by flow cytometry. Ninety percent of B cells in one fraction expressed CD5 and less than 5% of B cells in the other fraction expressed CD5. B cells from each preparation were then spun onto slides and fixed for in situ hybridization. To assay the frequencies of the various B cell populations in young and old spleens by FACS analysis, lymphocytes from five 18 month and five 6 week old mice were stained with PE conjugated anti-CD5 (LY-1) mAb and FITC conjugated goat anti-mouse IgM mAb.
2.10. Ig CDR3 size analysis PCR and run-off reactions were performed as previously described (LeMaoult et al., 1997). Briefly, classical PCR reactions with a sense primer specific for the VH11 (5%-GTGATGGCAGTGCAATAAACTACGCACC-3%) VH family and an antisense primer specific for a constant CH region were performed on 2 ml of conventional oligo dT-primed cDNA or genomic DNA using 0.5 mM of each primer, 1.5 mM MgCl2 and 1 ml of Taq DNA Polymerase (Perkin-Elmer, Roche Molecular Systems, Branchburg, NJ). The PCR products were visualized on a 1.5% agarose gel with ethidium bromide before using 2 ml of the amplified products for a run-off elongation with FAM-labeled JH primers. The elongation products (2 ml), mixed with an equal volume of 95% (vol/vol) formamide/10 mM EDTA were mixed, run on a 6% acrylamide/8 M urea/1 × TBE sequencing gel using an automated ABI 737 DNA sequencer (Applied Biosystems, Foster City, CA). ROX-labeled size markers, synthesized in the laboratory, were also run with the run-off products. Size determination of the run-off products and CDR3 size analysis were performed using the Immunoscope software (Pannetier et al., 1995).
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Table 2 Percentage of splenic B-lymphocytes from old and young mice using different VH gene family usage Age
Young Old PB
VH gene family probe VH11
Q52
7183
J558
J606
6.09 1.4 16.79 2.5 0.008
7.291.5 13.89 2.5 0.03
8.9 91.0 10.1 90.6 0.18
31.0 9 3.7 27.0 9 2.2 0.37
14.6 92.5 11.4 91.4 0.74
Total spleen cells from five young and nine old mice were hybridized with VH and kappa light chain probes. The percentage 9 S.D., of cells expressing kappa light chain message using VH11, Q52, 7183, J558 and J606 is shown.
2.11. Direct sequencing of run-off products Direct sequencing of run-off products was performed using the Perkin-Elmer AmpliCycle Kit on 2 ml of PCR product with the appropriate FAM-labeled JH primer and loaded on an automated sequencer. Nucleotides contributed by the VH, JH or D genes were identified by alignment with germline gene sequences (Kabat et al., 1987).
2.12. Statistical analysis The Student’s t-test was used to compare differences between means of replicate observations. 3. Results
3.1. VH gene gamily usage by splenic B cells differs in old and young mice In situ hybridization using the panel of VH gene family probes described above was used to assay VH gene family use by LPS-activated lymphocytes from the spleen of five young and nine old mice. Two VH gene families, VH11 and Q52, were expressed significantly more frequently by LPS-activated spleen cells from old rather than young mice (Table 2). These results were confirmed by Northern analysis which revealed an approximately 6-fold increase in VH11 and a 2-fold increase in Q52 gene usage by splenic B cells from four old compared to four young C57B1/6 mice. The age-associated difference was especially clear in the case of VH11 where the level of mRNA was barely detectable on Northern blots of splenic B cells from young mice. In one exceptional old mouse, the majority of splenic immunoglobulin mRNA appeared to be derived from the Q52 VH gene family suggesting a monoclonal expansion of B cells expressing a member of this VH gene family. However, no other old mouse studied exhibited such an increase in the use of a single VH gene family although monoclonal splenic B cell expansions using VH11 VH gene family were documented by Ig CDR3 size analysis (see below).
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Table 3 Percent VH gene family usage by peritoneal B-lymphocytes from old and young mice Age
Young Old PB
VH gene family probe VH11
Q52
7183
J558
J606
17.39 4.9 17.69 2.2 0.89
12.592.2 13.09 2.3 0.83
7.6 92.5 10.5 91.6 0.31
24.7 9 1.0 26.8 9 3.7 0.51
11.7 9 0.7 11.9 92.6 0.85
Peritoneal B-lymphocytes from old and young mice were analyzed by in situ hybridization with VH family-specific probes as described in Table 2.
3.2. VH gene family use by peritoneal cells from old and young mice does not differ The number of peritoneal cells increases 5–10-fold during aging and a portion of this increase has been attributed to oligoclonal expansions of CD5+ cells (Stall et al., 1988; Weksler et al., 1990). For this reason, we were interested in knowing whether VH gene family usage was different in peritoneal B cells from old and young mice. As seen in Table 3, there was no significant difference in VH gene usage by peritoneal B cells from old and young mice. As a significant percentage of peritoneal B cells express CD5, we next examined VH gene usage by CD5 + and CD5− peritoneal B cells.
3.3. Different VH usage by CD5+ and CD5 − B cells It has been reported that CD5 + and CD5 − B cells differ in VH gene family usage (Pennel et al., 1989; Andrade et al., 1991). In order to examine the possibility that the increased use of the VH11 and Q52 gene families by splenic B cells from old mice might be due to an expanded population of CD5+ B cells, we examined VH gene family use by CD5 + and CD5 − peritoneal B cells (Table 4). CD5 + B cells use the VH11 (P B 0.002) and the Q52 (PB 0.07) VH more frequently than CD5− B cells. This raised the possibility that there were more CD5+ B cells in Table 4 Percent of CD5+ and CD5− peritoneal B-lymphocytes using different VH gene families Cell type
CD5+ CD5− PB
VH gene family probe VH11
Q52
S107
J558
J606
18.69 0.8 6.89 0.2 0.002
13.39 1.8 8.091.1 0.07
8.2 92.3 4.8 9 0.2 0.14
31.0 96.0 30.0 9 5.1 0.88
8.4 92.2 7.1 9 1.6 0.84
Purified peritoneal CD5+ and CD5− B-lymphocytes were analyzed by in situ hybridization with VH family-specific probes as described in Table 2.
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Table 5 Percentage of B-lymphocytes expressing CD5 in spleens from young and old mice Surface phenotype
Young (individual mice; %)
Mean 9 S.D. (%)
IgM+, CD5+
4.4, 5.2, 2.6, 3.5, 5.2
4.2 9 0.5
Old (individual mice; %) IgM+, CD5+
8.6, 17.5, 12.3, 12.8, 7.1
11.6 91.8
Percent of total splenic B-lymphocytes expressing IgM and CD5 cell surface antigens are shown. Our previous studies had shown that there was no change in the total number of spleen cells or splenic B cells during aging.
the spleen of old compared to young female C57B1/6 mice. The frequency of CD5 + B cells was 3-fold higher in old compared to young mice (Table 5). Although VH11 is used almost exclusively by CD5+ B cells, less than 10% of the CD5 + B cells use this VH gene family (Andrade et al., 1991; Hardy et al., 1994). For this reason the increase in the number of splenic B cells expressing the VH11 gene family cannot be explained by the random colonization of the spleen by peritoneal CD5+ B cells.
3.4. Monoclonal splenic B cell expansions express VH11 gene family members Monoclonal expansions of splenic B cells have been detected in more than 85% of mice 18 months of age and older (LeMaoult et al., 1997). In preliminary studies we have found that more than 90% of the expanded B cell clones express the CD5 cell surface marker. As VH11 gene family use and the number of splenic CD5+ B cells were increased in the old mice, we used CDR3 DNA size analysis to investigate whether clonal expansions of CD5+ B cells explain this preferentially use the VH11 gene family. The B cell repertoire is characterized by size heterogeneity of Ig CDR3 sizes (LeMaoult et al., 1997). A clonal B cell expansion is suggested by the presence of a peak which skews the usual Gaussian distribution of CDR3 sizes. Old mice exhibit clonal B cell expansions using the VH11 gene family. Fig. 1 shows a representative example of a unique CDR3 size peak, coding for eight amino acids, in splenic RNA from an old mouse. This peak corresponds to an expanded CDR3 size peak in splenic DNA; such expansions were not seen in DNA from young mice although a low level of VH11 mRNA was found (data not shown). The clonal nature of this peak was confirmed by obtaining a readable sequence from the PCR products (data not shown). This sequence was identical to that previously reported for an anti-phosphatidylcholine antibody produced by CD5 + B cells (Hardy et al., 1994). In order to determine the cellular basis for the clonal expansions, splenic B cells were sorted into CD5 + and CD5 − B cell subpopulations and subjected to CDR3 size analysis. As can be seen, the clonal expansion appears only in the CD5+ B cell population. The identical clonal expansion was also detected in peritoneal B cells.
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4. Discussion The expression of the antibody repertoire shifts with age with respect to antibody specificities, affinity, isotype and heterogeneity (LeMaoult et al., 1997). Several factors contribute to these changes including thymic involution and compromised T cell function which are necessary for B cell development, somatic mutation and isotype switching. Our previous studies of VH usage had provided evidence for individual and unique VH gene family over-utilization in old mice (Viale et al., 1994). These results may be explained by B cell clonal expansions. Other published studies had found no difference in VH usage by LPS-stimulated B cell colonies or by B cell hybridomas from old compared to young mice (Miceli et al., 1990; Schultze et al., 1992). However, the decreased number of B cell colonies and B cell hybridomas generated from old mice raised the possibility that neither technique assayed the entire B cell repertoire in old mice. However, two published studies did demonstrate age-related differences in VH use by antigen-specific B cells (Riley et al., 1989; Yang et al., 1996). In both cases, the VH families used by antigen-specific B cells differed in young and old animals. An increase in the frequency of B cells producing anti-autologous erythrocytes antibodies coded for by the VH11 gene segment have been reported (Weksler et al., 1990; Ravichandran et al., 1994). This is compatible with our finding of an increased percentage of splenic B cells using this VH gene family. However, the increased VH11 gene family use cannot be explained by a random selection of CD5+ B cells within the spleen as only 10% of CD5+ B cells use VH11 (Hardy et al., 1994). This suggests that the increased VH11 usage by splenic CD5 + B cells can be explained by a clonal expansion of CD5 + B cells using the VH11 gene family.
Fig. 1. Immunoglobulin CDR3 size diversity in spleen and peritoneal cells from a representative old mouse. DNA or cDNA derived from the indicated cells, was amplified with the appropriate primers as described in Materials and methods. Each pattern represents the CDR3 size diversity of the VH11-JH1Cm configuration.
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The origin of the clonal CD5+ B cells in the spleen is not certain. Clonal B cells expressing the VH11 gene family is also detected in the peritoneal cavity of old mice. Thus, it is likely that B cells expressing VH11 originate within the peritoneal cavity as has been previously suggested (Stall et al., 1988; Hardy and Hayakawa, 1994). In contrast, in preliminary studies, we have not detected any clonal B cell populations in bone marrow B cell precursors suggesting that such clones expand in the peripheral lymphoid tissues. In summary, the age-associated changes in VH gene utilization by splenic B cells in old mice appear to reflect clonal expansions of CD5+ B cells within the spleen. Whether clonal CD5 + peritoneal B cells can disseminate to the spleen or whether these clonal CD5+ splenic B cells develop in situ is not known.
Acknowledgements Supported by grants AG00541 and AG08707. A.B-Y. was supported in part by Fogarty International Fellowship F05 TWO 04554 ICP.
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Increased VH 11 and VH Q52 gene use by splenic B cells in old mice associated with oligoclonal expansions of CD5+ B cells Arie Ben-Yehuda a,1, Paul Szabo b, Joel LeMaoult b, J. Sanil Manavalan b, Marc E. Weksler b,* a
Department of Medicine, Geriatric Unit, Hadassah Uni6ersity Hospital, Ein-Karem, Jerusalem, 91010, Israel b Di6ision of Geriatrics and Gerontology, Cornell Uni6ersity Medical College, 1300 York A6enue, New York, NY 10021, USA Received 31 October 1997; accepted 16 December 1997
Abstract A significant increase in the utilization of the VH gene families VH11 and Q52 was observed in LPS-stimulated splenic B lymphocytes from aged mice compared to young mice. VH gene usage was assayed by in situ DNA/RNA hybridization using VH family-specific and kappa chain probes. The observed age-dependent differences appear to reflect the preferential use of VH11 and Q52 VH gene use by the CD5+ B lymphocyte subset whose numbers in the spleen increase with age. The increased use of VH11 by splenic cells from old mice is associated with clonal expansions of splenic CD5 + B lymphocytes. © 1998 Published by Elsevier Science Ireland Ltd. All rights reserved. Keywords: Aging; VH 11 and VH Q52 genes; Splenic B cells; CD5 + B cells
* Corresponding author. Tel.: + 1 212 7466478; fax: + 1 212 7466382; e-mail: weksler@ med.cornell.edu 1 Tel.: + 972 2 6777627/8; fax: +972 2 6413147; e-mail: [email protected] 0047-6374/98/$19.00 © 1998 Published by Elsevier Science Ireland Ltd. All rights reserved. PII S0047-6374(98)00004-9
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1. Introduction The generation of a diverse B cell repertoire requires the rearrangement of immunoglobulin gene segments within the heavy and light chain gene loci. The first step in this process is the joining of D and JH segments followed by recombination with one gene from one of the 14 VH families. Subsequently, VL and JL segments are joined leading to the creation of a functional light chain gene and the expression on the surface of B lymphocytes of a complete immunoglobulin (Ig) molecule. The use of VH gene segments by splenic B cells in fetal and neonatal mice less than 1 week old shows a strong bias for 3%-end, D proximal VH gene families, predominantly 7183 and Q52 (Jeong and Teale, 1984; Yancopoulos et al., 1984). Thereafter, VH gene use by adult spleen cells is proportional to the size of the VH family and shows no 3% preference (Yancopoulos et al., 1988). Aging also affects VH use. Thus, VH use by antigen-specific B cells following immunization with PC (Riley et al., 1989) or NP (Yang et al., 1996) differs in old and young mice although analysis of LPS-induced B cell colonies and B cell hybridomas prepared from young and old mice showed, with one exception, no difference in VH gene family use (Miceli et al., 1990; Schultze et al., 1992). As fewer B-cell colonies and B-cell hybridomas are generated from old compared to young mice (Miceli et al., 1990; Schultze et al., 1992), we used direct DNA/RNA hybridization to assess VH use by individual splenic and peritoneal B cells from old and young mice cultured with LPS. We found that old mice had significantly more CD5 + splenic B cells and that the use of VH11 and Q52 immunoglobulin VH gene families by spleen cells was significantly increased. The VH11 gene family is used almost exclusively by CD5+ B cells (Andrade et al., 1991). Furthermore, oligoclonal populations of CD5+ B cells have been demonstrated within the spleens of old mice (Stall et al., 1988; Hardy and Hayakawa, 1994. In this report we demonstrate monoclonal CD5+ B cell populations using the VH11 VH gene family.
2. Materials and methods
2.1. Animals Young (2 – 3 months old) and old (16–22 months old) female C57BL/6 mice were purchased from the National Institute on Aging Colony at the Charles River Laboratories (Kingston, NY) and maintained in the Animal Resource Facility at Cornell University Medical College under laminar flow hoods. Routine serological assays for viral, bacterial and parasitic pathogens were negative. All experiments were performed in accordance with protocols approved by the Institutional Animal Care and Use Committee of Cornell University Medical College.
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2.2. Cell lines Cloned B cell hybridomas known to use certain VH gene were used as to establish the optimal conditions for in situ hybridization. The cell lines 7023 (J558), NAb-1 (7183), NAb-4 (Q52) and 582C10 (J606) were all provided by Dr D.L. Kastner (Bethesda, MD) The cloned cells were passaged in complete culture medium (RPMI 1640 with 10% FCS, 2 mM L-glutamine, 1 mM Na-Pyruvate, 10 mM HEPES, 5× 10 − 5 2ME, non-essential amino acids and penicillin/streptomycin).
2.3. VH Ig family gene probes Probes for Ig VH gene families that make up more than 5% of the B cell repertoire: VH81X (7183), VHQ52 (Q52), VHB4 (J558) and VHJ606 (J606) were kindly provided by Dr Frederick W. Alt (Boston, MA). The pCP12 (VH11) probe was kindly provided by Dr Reininger (Geneva, Switzerland), and the kappa light chain constant region probe was a gift from Dr J. Stavnezer (Worchester, MA).
2.4. Cell culture Peritoneal lymphocytes from 2 or 18 month old mice were obtained by peritoneal lavage, washed three times with Hanks balanced salt solution (HBSS), and resuspended at a concentration of 106 cells/ml in complete medium: RPMI 1640 with 10% FCS. Single spleen cell suspensions were prepared by teasing the spleens apart in HBSS and passing the dissociated spleen through a sieve. The isolated cells were washed three times with HBSS and resuspended in complete medium at a concentration of 106 cells per ml. Peritoneal and splenic cells were cultured in medium containing 10 mg LPS/ml for 3 days at 37°C in a 5% CO2/95% humidified air environment. The cells were then washed and resuspended at a concentration of 107 cells/ml in complete culture medium. This suspension was pipetted onto an ‘8chamber’ slide (100 ml/chamber). The slides were centrifuged for 5 min (500 rpm/min), the supernatant medium was discarded and cells fixed in freshly prepared 4% paraformaldehyde (Sigma, St. Louis, MO) in phosphate buffered saline (PBS). The slides were stored in 70% ethanol at 4°C until they were used for in situ hybridization.
2.5. In situ hybridization The protocol used for in situ hybridization was a modification of that employed by Harper et al. (1986). All plasmid probes were labeled with 35S-dATP using the nick translation method. Slides bearing appropriate cells were removed from 70% ethanol, washed twice in 1×PBS solution containing 5 mM MgCl2, and then treated for 10 min with 0.25% acetic anhydride in 0.1 M triethanolamine buffer (pH 8.0). Slides were then incubated in 0.1 M Tris, 0.1 M glycine (pH 7.0) buffer for 10 min, and then washed in 2 × SSC, and dehydrated by 1 min incubation in 70 and 90% ethanol. Probes were resuspended in 10 mM Tris/1 mM EDTA buffer
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containing 10 mM dithiothreitol: the activity of the probes was adjusted to 106 cpm/ml. Probes were diluted 1/10 in hybridization buffer to give a final concentration of 50% formamide, 2× SSC, 10 mM dithiothreitol, 10% Dextran solution, 2× Denhart’s solution, 100 ml/ml of solution × DNA and 500 ng (107 cpm/ml) of probe DNA. Twenty microliters of diluted probe were added per chamber and covered with parafilm and incubated for 3 h at 42°C. Slides were then washed twice in 2× SSC at room temperature for 30 min, and then in 2× SSC at 55°C for 20 min. The final wash was in 0.1 × SSC for 20 min at 60°C. After the final wash, the slides were dehydrated in 70 and 90% ethanol and air dried. These conditions and stringencies of hybridization and washing resulted in the identification of only the B cell clones expressing the homologous VH gene family (Table 1).
2.6. Autoradiography In situ hybridization was revealed by autoradiography with NTB-2 emulsion (Eastman-Kodak, Rochester, NY). Exposure times ranged from 3 to 4 days at 4°C in light-tight slide boxes containing desiccant powder. Slides were developed in a 1:1 dilution of Dektol in H2O and fixed in Kodafix. After air-drying, slides were stained in Giemsa for 10 min, then washed in distilled water.
2.7. Calculation of VH frequencies Identically prepared slides were probed for both VH and kappa chain message. More than 200 cells were counted for each probe. Cells were considered positive if they were labeled with more than 50 grains per cell. The total number of B cells was approximated by the number of cells with detectable kappa light chain mRNA as 95% of murine B cells express this light chain. The percentage of VH gene family used by each family was calculated by dividing the percentage of cells positive for a specific VH family probe by the percentage of cells positive for kappa light chain probe. Table 1 Specificity of cDNA probes for the indicated VH gene family B cell hybridoma
7023 (J558) 582C103 (J606) NAb-4 (Q52) NAb-1 (7183)
VH gene family probe J558
J606
Q52
7183
94 2 3 5
8 95 6 4
1 4 95 3
2 1 4 80
The specificity of VH probes was determined by in situ DNA/RNA hybridization using the indicated B cell clones. The percentage of cells from each clone that were positive with each probe are shown. Positive cells were labeled with more than 50 grains/cell. In most experiments, negative cells were labeled with less than 5 grains/cell.
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2.8. Northern RNA analysis Total cellular RNA was prepared from four individual young and four individual old spleens, and was denatured in 50% formamide/2.2 M formaldehyde in 1XMOPS buffer for 5 min at 65°C. Five micrograms of each sample were electrophoresed in 2.2 M formaldehyde/1% agarose gel, and blotted by standard procedures (Sambrook et al., 1989). The membranes were probed with the 32P-labeled VH81 (7183), VHQ52 (Q52), VHB4 (J558) and PCP12 (VH11) cDNAs at 42°C for 18 h, and washed with a final wash in 0.1 × SSPE/0.1% SDS at 42°C, and exposed to X-ray film.
2.9. Separation of CD5+ and CD5 − peritoneal B cells by cell sorting Peritoneal cells obtained as described above were washed and stained with phycoerythrin-conjugated anti-CD5 mAb (4 mg/106 cells) (Pharmingen, San Diego, CA) and 4 mg/106 cells of fluorescein-conjugated goat anti-mouse IgM antibodies (Southern Biotechnology Associates, Birmingham, AL) for 15 min. The cells were then washed three times in complete culture medium. The stained cells were sorted into CD5+ and CD5− B cells using a FACStar Plus apparatus (Becton Dickinson, Franklin Lakes, NJ). Sorted B cells were reanalyzed by flow cytometry. Ninety percent of B cells in one fraction expressed CD5 and less than 5% of B cells in the other fraction expressed CD5. B cells from each preparation were then spun onto slides and fixed for in situ hybridization. To assay the frequencies of the various B cell populations in young and old spleens by FACS analysis, lymphocytes from five 18 month and five 6 week old mice were stained with PE conjugated anti-CD5 (LY-1) mAb and FITC conjugated goat anti-mouse IgM mAb.
2.10. Ig CDR3 size analysis PCR and run-off reactions were performed as previously described (LeMaoult et al., 1997). Briefly, classical PCR reactions with a sense primer specific for the VH11 (5%-GTGATGGCAGTGCAATAAACTACGCACC-3%) VH family and an antisense primer specific for a constant CH region were performed on 2 ml of conventional oligo dT-primed cDNA or genomic DNA using 0.5 mM of each primer, 1.5 mM MgCl2 and 1 ml of Taq DNA Polymerase (Perkin-Elmer, Roche Molecular Systems, Branchburg, NJ). The PCR products were visualized on a 1.5% agarose gel with ethidium bromide before using 2 ml of the amplified products for a run-off elongation with FAM-labeled JH primers. The elongation products (2 ml), mixed with an equal volume of 95% (vol/vol) formamide/10 mM EDTA were mixed, run on a 6% acrylamide/8 M urea/1 × TBE sequencing gel using an automated ABI 737 DNA sequencer (Applied Biosystems, Foster City, CA). ROX-labeled size markers, synthesized in the laboratory, were also run with the run-off products. Size determination of the run-off products and CDR3 size analysis were performed using the Immunoscope software (Pannetier et al., 1995).
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Table 2 Percentage of splenic B-lymphocytes from old and young mice using different VH gene family usage Age
Young Old PB
VH gene family probe VH11
Q52
7183
J558
J606
6.09 1.4 16.79 2.5 0.008
7.291.5 13.89 2.5 0.03
8.9 91.0 10.1 90.6 0.18
31.0 9 3.7 27.0 9 2.2 0.37
14.6 92.5 11.4 91.4 0.74
Total spleen cells from five young and nine old mice were hybridized with VH and kappa light chain probes. The percentage 9 S.D., of cells expressing kappa light chain message using VH11, Q52, 7183, J558 and J606 is shown.
2.11. Direct sequencing of run-off products Direct sequencing of run-off products was performed using the Perkin-Elmer AmpliCycle Kit on 2 ml of PCR product with the appropriate FAM-labeled JH primer and loaded on an automated sequencer. Nucleotides contributed by the VH, JH or D genes were identified by alignment with germline gene sequences (Kabat et al., 1987).
2.12. Statistical analysis The Student’s t-test was used to compare differences between means of replicate observations. 3. Results
3.1. VH gene gamily usage by splenic B cells differs in old and young mice In situ hybridization using the panel of VH gene family probes described above was used to assay VH gene family use by LPS-activated lymphocytes from the spleen of five young and nine old mice. Two VH gene families, VH11 and Q52, were expressed significantly more frequently by LPS-activated spleen cells from old rather than young mice (Table 2). These results were confirmed by Northern analysis which revealed an approximately 6-fold increase in VH11 and a 2-fold increase in Q52 gene usage by splenic B cells from four old compared to four young C57B1/6 mice. The age-associated difference was especially clear in the case of VH11 where the level of mRNA was barely detectable on Northern blots of splenic B cells from young mice. In one exceptional old mouse, the majority of splenic immunoglobulin mRNA appeared to be derived from the Q52 VH gene family suggesting a monoclonal expansion of B cells expressing a member of this VH gene family. However, no other old mouse studied exhibited such an increase in the use of a single VH gene family although monoclonal splenic B cell expansions using VH11 VH gene family were documented by Ig CDR3 size analysis (see below).
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Table 3 Percent VH gene family usage by peritoneal B-lymphocytes from old and young mice Age
Young Old PB
VH gene family probe VH11
Q52
7183
J558
J606
17.39 4.9 17.69 2.2 0.89
12.592.2 13.09 2.3 0.83
7.6 92.5 10.5 91.6 0.31
24.7 9 1.0 26.8 9 3.7 0.51
11.7 9 0.7 11.9 92.6 0.85
Peritoneal B-lymphocytes from old and young mice were analyzed by in situ hybridization with VH family-specific probes as described in Table 2.
3.2. VH gene family use by peritoneal cells from old and young mice does not differ The number of peritoneal cells increases 5–10-fold during aging and a portion of this increase has been attributed to oligoclonal expansions of CD5+ cells (Stall et al., 1988; Weksler et al., 1990). For this reason, we were interested in knowing whether VH gene family usage was different in peritoneal B cells from old and young mice. As seen in Table 3, there was no significant difference in VH gene usage by peritoneal B cells from old and young mice. As a significant percentage of peritoneal B cells express CD5, we next examined VH gene usage by CD5 + and CD5− peritoneal B cells.
3.3. Different VH usage by CD5+ and CD5 − B cells It has been reported that CD5 + and CD5 − B cells differ in VH gene family usage (Pennel et al., 1989; Andrade et al., 1991). In order to examine the possibility that the increased use of the VH11 and Q52 gene families by splenic B cells from old mice might be due to an expanded population of CD5+ B cells, we examined VH gene family use by CD5 + and CD5 − peritoneal B cells (Table 4). CD5 + B cells use the VH11 (P B 0.002) and the Q52 (PB 0.07) VH more frequently than CD5− B cells. This raised the possibility that there were more CD5+ B cells in Table 4 Percent of CD5+ and CD5− peritoneal B-lymphocytes using different VH gene families Cell type
CD5+ CD5− PB
VH gene family probe VH11
Q52
S107
J558
J606
18.69 0.8 6.89 0.2 0.002
13.39 1.8 8.091.1 0.07
8.2 92.3 4.8 9 0.2 0.14
31.0 96.0 30.0 9 5.1 0.88
8.4 92.2 7.1 9 1.6 0.84
Purified peritoneal CD5+ and CD5− B-lymphocytes were analyzed by in situ hybridization with VH family-specific probes as described in Table 2.
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Table 5 Percentage of B-lymphocytes expressing CD5 in spleens from young and old mice Surface phenotype
Young (individual mice; %)
Mean 9 S.D. (%)
IgM+, CD5+
4.4, 5.2, 2.6, 3.5, 5.2
4.2 9 0.5
Old (individual mice; %) IgM+, CD5+
8.6, 17.5, 12.3, 12.8, 7.1
11.6 91.8
Percent of total splenic B-lymphocytes expressing IgM and CD5 cell surface antigens are shown. Our previous studies had shown that there was no change in the total number of spleen cells or splenic B cells during aging.
the spleen of old compared to young female C57B1/6 mice. The frequency of CD5 + B cells was 3-fold higher in old compared to young mice (Table 5). Although VH11 is used almost exclusively by CD5+ B cells, less than 10% of the CD5 + B cells use this VH gene family (Andrade et al., 1991; Hardy et al., 1994). For this reason the increase in the number of splenic B cells expressing the VH11 gene family cannot be explained by the random colonization of the spleen by peritoneal CD5+ B cells.
3.4. Monoclonal splenic B cell expansions express VH11 gene family members Monoclonal expansions of splenic B cells have been detected in more than 85% of mice 18 months of age and older (LeMaoult et al., 1997). In preliminary studies we have found that more than 90% of the expanded B cell clones express the CD5 cell surface marker. As VH11 gene family use and the number of splenic CD5+ B cells were increased in the old mice, we used CDR3 DNA size analysis to investigate whether clonal expansions of CD5+ B cells explain this preferentially use the VH11 gene family. The B cell repertoire is characterized by size heterogeneity of Ig CDR3 sizes (LeMaoult et al., 1997). A clonal B cell expansion is suggested by the presence of a peak which skews the usual Gaussian distribution of CDR3 sizes. Old mice exhibit clonal B cell expansions using the VH11 gene family. Fig. 1 shows a representative example of a unique CDR3 size peak, coding for eight amino acids, in splenic RNA from an old mouse. This peak corresponds to an expanded CDR3 size peak in splenic DNA; such expansions were not seen in DNA from young mice although a low level of VH11 mRNA was found (data not shown). The clonal nature of this peak was confirmed by obtaining a readable sequence from the PCR products (data not shown). This sequence was identical to that previously reported for an anti-phosphatidylcholine antibody produced by CD5 + B cells (Hardy et al., 1994). In order to determine the cellular basis for the clonal expansions, splenic B cells were sorted into CD5 + and CD5 − B cell subpopulations and subjected to CDR3 size analysis. As can be seen, the clonal expansion appears only in the CD5+ B cell population. The identical clonal expansion was also detected in peritoneal B cells.
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4. Discussion The expression of the antibody repertoire shifts with age with respect to antibody specificities, affinity, isotype and heterogeneity (LeMaoult et al., 1997). Several factors contribute to these changes including thymic involution and compromised T cell function which are necessary for B cell development, somatic mutation and isotype switching. Our previous studies of VH usage had provided evidence for individual and unique VH gene family over-utilization in old mice (Viale et al., 1994). These results may be explained by B cell clonal expansions. Other published studies had found no difference in VH usage by LPS-stimulated B cell colonies or by B cell hybridomas from old compared to young mice (Miceli et al., 1990; Schultze et al., 1992). However, the decreased number of B cell colonies and B cell hybridomas generated from old mice raised the possibility that neither technique assayed the entire B cell repertoire in old mice. However, two published studies did demonstrate age-related differences in VH use by antigen-specific B cells (Riley et al., 1989; Yang et al., 1996). In both cases, the VH families used by antigen-specific B cells differed in young and old animals. An increase in the frequency of B cells producing anti-autologous erythrocytes antibodies coded for by the VH11 gene segment have been reported (Weksler et al., 1990; Ravichandran et al., 1994). This is compatible with our finding of an increased percentage of splenic B cells using this VH gene family. However, the increased VH11 gene family use cannot be explained by a random selection of CD5+ B cells within the spleen as only 10% of CD5+ B cells use VH11 (Hardy et al., 1994). This suggests that the increased VH11 usage by splenic CD5 + B cells can be explained by a clonal expansion of CD5 + B cells using the VH11 gene family.
Fig. 1. Immunoglobulin CDR3 size diversity in spleen and peritoneal cells from a representative old mouse. DNA or cDNA derived from the indicated cells, was amplified with the appropriate primers as described in Materials and methods. Each pattern represents the CDR3 size diversity of the VH11-JH1Cm configuration.
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The origin of the clonal CD5+ B cells in the spleen is not certain. Clonal B cells expressing the VH11 gene family is also detected in the peritoneal cavity of old mice. Thus, it is likely that B cells expressing VH11 originate within the peritoneal cavity as has been previously suggested (Stall et al., 1988; Hardy and Hayakawa, 1994). In contrast, in preliminary studies, we have not detected any clonal B cell populations in bone marrow B cell precursors suggesting that such clones expand in the peripheral lymphoid tissues. In summary, the age-associated changes in VH gene utilization by splenic B cells in old mice appear to reflect clonal expansions of CD5+ B cells within the spleen. Whether clonal CD5 + peritoneal B cells can disseminate to the spleen or whether these clonal CD5+ splenic B cells develop in situ is not known.
Acknowledgements Supported by grants AG00541 and AG08707. A.B-Y. was supported in part by Fogarty International Fellowship F05 TWO 04554 ICP.
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