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*
Division of Geriatrics and Gerontology, Weill Medical College of Cornell University, New York, NY 10021; and
Laboratory of T Cell Development, Immunology Program, Memorial Sloan-Kettering Cancer Center, New York, NY 10021
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The objectives of the present study were to investigate what type of B lineage cells dominate the clonal expansions, their origin, and stability in old C57BL/6 mice. We report here that the clonal B cell populations in old mice develop in the peripheral lymphoid compartments. They are not detectable among bone marrow B cell precursors or in the peritoneal cavity, the sites of development of B2 or B1 lymphocytes, respectively. We report that two types of B cell clonal expansions can be identified based on their size. One type of clonal expansion of B lineage is made up of a small number of cells, which are found largely in CD45R- and B220-sorted cells, do not express the membrane-bound form of the µ Ig heavy chain mRNA, and thus have characteristics of plasma cells. The other type of clonal expansion of B lineage is made of many cells that are found largely in populations that express CD45R and B220. These cells express the mRNA that codes for the membrane-bound form of the µ Ig heavy chain. There is also a difference in the localization of the two types of clonal B cells. The large clonal populations are detected in the lymph nodes, bone marrow, and thymus. The small clonal populations are detected only in the spleen.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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We studied splenic B cell clonal populations detected by Ig mRNA
CDR3 size analysis in five 20-mo-old female C57BL/6 mice purchased from
the NIA colony at Charles River Laboratories (Wilmington, MA). These
mice had been partially splenectomized 2 mo earlier to identify splenic
B cell clones and then maintained for 2 mo in our animal facility. Ten
stable IgM splenic B cell clonal populations expressing the
VHJ558, VHQ52, or VH7183 gene
families were identified in these five mice as previously described
(4). The sequences of the heavy chain CDR3s of these clonal B cell
populations are shown in Table I
. An
exceptional 2-mo-old female C57BL/6 mouse with an IgM splenic clone was
included in the study.
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Cell surface staining and sorting
Single-cell preparations of thymus, bone marrow, mesenteric lymph nodes, or spleen were prepared as previously described (4). B cell subsets were separated after incubation with anti-CD5 or anti-CD45R Abs by flow cytometry. From 50,000 to 500,000 cells were stained with anti-mouse CD5 PE, CD43 PE, CD45R CyChrome, or Ig FITC Abs (PharMingen, San Diego, CA) as previously described (9). Living cells were selected for analysis and sorting based on forward and side scatter gates that excluded dead cells and debris. Data analyses made use of the LYSIS II software (Becton Dickinson, Mountain View, CA).
DNA, RNA, and cDNA samples
Total RNA and genomic DNA were extracted from B cell preparations using the RNA-Isolator Kit (Genosys Biotechnologies, The Woodlands, TX) and quantitated by optical density. For cDNA synthesis, up to 5 µg of total RNA were reverse transcribed using the avian myeloblastosis virus-reverse transcriptase kit (Boehringer Mannheim, Indianapolis, IN) and oligo(dT)15 primers in a 30-µl reaction. After the reaction was completed, 30 µl of double-distilled water were added.
Oligonucleotides
Oligonucleotides specific for murine VHJ558,
VHQ52, and VH7183 families and µ-chain IgM as
well as the four 5-carboxyfluorescein-labeled JH runoff
primers (Table II) have been previously
described (4, 10). The reverse primers specific for mRNA that codes for
the membrane-bound form of the µ Ig heavy chain and for mRNA coding
for the secreted form of the µ Ig heavy chain, as well as the three
pairs of clonotypic primers used in these studies, are described in
Table II.
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PCR and runoff reactions were performed as previously described
(4, 10). Briefly, for PCR using cDNA, classical PCR reactions with a
sense primer specific for a VH family and an antisense
primer specific for the constant IgM region (Table II
) were performed
on 2–5 µl of cDNA in 25 µl, with 0.5 µM concentrations of each
primer, 1.5 mM MgCl2, and 1 unit of Taq DNA
polymerase (Perkin-Elmer, Roche Molecular Systems, Branchburg, NJ). For
PCR using genomic DNA (100 ng/reaction), the same conditions were used,
except that VH-JH PCRs were directly performed
for each sample, using one VH sense primer and one of the
four antisense fluorescent JH primers. The PCR products
were visualized on a 1.5% agarose gel by ethidium bromide staining.
Two microliters of the VH-IgM (cDNA-derived) PCR products
were reamplified by 10 cycles of runoff elongation using the
fluorescent JH primers (Table II). Two microliters of the
VH-JH PCR products (for DNA-derived samples) or
runoff elongation products (for RNA-derived samples), mixed with the
same volume of 95% (v/v) formamide/10 mM EDTA, were then run on a 6%
acrylamide, 8 M urea, 89 mM Tris, 89 mM boric acid, 2 mM EDTA, pH 8.3
(1x TBE) sequencing gel loaded on an automated ABI 373A DNA sequencer
(Applied Biosystems, Foster City, CA).
6-Carboxy-X-rhodamine-labeled size markers, synthesized in
the laboratory, were also loaded. Size determination of the runoff
products and CDR3 size analysis was performed with Immunoscope software
(11).
Clonotypic PCR
Forward and reverse primers specific for CDR3 sequences of three B cell clones from old mice (clonotypic primers) were designed as follows. Clonotypic forward primers encompassed the 5'-end of the CDR3 (3'-end of the VH region, the VH-D junction, and the 5'-end of the D region). These primers were designed to pair with the reverse IgM primer. Similarly, the clonotypic reverse primers encompassed the 3'-end of the CDR3 (3'-end of the JH region, the D-JH junction, and the 3'-end of the D region). These primers were designed to pair with the forward VH primers. Clonotypic PCRs were performed for both the VH-clonotypic reverse and the clonotypic forward-IgM primer combinations. For each study, four controls were included: cDNA samples two other old mice; and two cDNA samples from young mice. Specificity was documented by the appearance of bands for both the VH-clonotypic reverse and the clonotypic forward-IgM PCRs with the appropriate cDNA samples but not with other cDNA samples.
Sequence analysis of runoff products
CDR3 were sequenced using 2–5 µl of the VH-IgM (for cDNA) or VH-JH (for DNA) PCR products which exhibited a dominant peak for a particular VH-JH combination as previously described (4). Nucleotides contributed by the VH, JH or D genes were identified by alignment with germline gene sequences (12).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Most old mice have stable splenic clonal population, detectable as
dominant peaks (peaks that make up 70% or more of the profile) in IgM
by CDR3 size analysis. In some mice, the same clonal populations
detected in unfractionated spleen are also detected in lymph nodes,
thymus, or bone marrow B cell populations, but not in the blood (4). To
determine whether these B cell clonal populations were generated in the
precursor populations or selected and expanded in the peripheral B cell
populations, IgM mRNA CDR3 size analysis was performed on purified bone
marrow pre-B cells (SIg-, CD45R+,
CD43-) and peritoneal cells from the five old mice. In
adult life, bone marrow Pre-B cells are precursors of B2 cells whereas
the peritoneal cavity is the richest source of self-renewing B1a cells
(13, 14). In some mice, the same clonal population was detected in both
spleen and unfractionated bone marrow cells. In these mice, however,
the clonal populations were not detected in the sorted bone marrow
pre-B and unfractionated peritoneal cavity cells (Fig. 1). The three representative B cell
clones detected in both the unfractionated bone marrow and spleen
preparations were not detectable in bone marrow pre-B cells or in
peritoneal B cells. It was possible that the clonal population was not
large enough to be detected as a dominant peak. For this reason, we
designed clonotypic primers specific for the CDR3 region of three
clonal populations from two mice. These primers gave a PCR product only
when mRNA from the spleen or unfractionated bone marrow from the
appropriate mouse was used. In contrast, no PCR product was found when
mRNA from sorted pre-B or unfractionated peritoneal cavity cells were
used (data not shown).
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, A and
B) at the time of thymic transplantation. These clonal B
cells were identified in the transplanted thymus 2 mo later. In the
third old mouse studied, the clonal B cells in the transplanted thymus
was also present in the thymus but not the spleen of the old recipient
(Fig. 2C). The appearance of the clonal B cells in the
transplanted thymus indicates that the clonal B cells in old mice can
colonize a young thymus. Furthermore, the colonization of the
transplanted thymus suggests that the clones detected in the thymus do
not result from possible contamination by lymph nodes adjacent to the
old thymus.
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A dominant peak in the Ig mRNA CDR3 size profile might result from
a large clonal population of B cells or from a small clonal population
of Ab-producing cells expressing high levels of Ig mRNA, or both (4).
To investigate the size of the clonal population that produces the
stable splenic dominant peaks, three mice that had eight splenic stable
clonal B cell populations, as shown by CDR3 size analysis on RNA
samples from two sequential partial splenectomies, were selected for
study. From these three mice, CDR3 size analysis was performed on both
mRNA and DNA obtained from unfractionated spleen. Three of the eight
clonal B cell populations identified in RNA could also be detected in
DNA as a size-matched alteration in the CDR3 size profiles. These
clonal B cell populations will be referred to as "large." In five
of eight clonal B cell populations originally detected in RNA, no
size-matched alteration of the CDR3 size profiles could be detected in
DNA. These clonal B cell populations will be referred to as
"small." In one exceptional young mouse, originally investigated as
a control mouse, we detected a splenic small clonal population. Two of
the five small clonal populations are shown in Fig. 3, A and B. Two of
the three large clonal populations are shown in Fig. 3, C
and D. These results suggest that those dominant peaks seen
only in mRNA are mainly expressed by a small number of clonal cells
expressing a high level of Ig mRNA. In contrast, those dominant peaks
seen in both mRNA and DNA are expressed by a greater number of clonal
cells. Next, we characterized the large and small clonal populations
with regard to their expression of CD45R, the expression of mRNA coding
for the membrane-bound form or for the soluble form of the µ Ig heavy
chain, and to their tissue distribution.
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We suspected that the smaller clonal populations were dominated by
plasma cells whereas the large clonal populations were dominated by B
lymphocytes. Because plasma cells do not express the lymphocyte surface
marker CD45R (15), we sorted spleen cells from five old mice and one
young mouse into CD45R+ and CD45R-
populations. These five old mice included the three mice described in
the previous paragraph, two other mice with a single stable splenic
clonal B cell expansion, and a young mouse with a small clonal B cell
population described in the previous paragraph. All six small clonal
populations (five from old mice and one from a young mouse) were
recovered predominantly in the CD45R- cell populations
(two examples given in Fig. 4,
A and B). In contrast, all three large clonal
populations were recovered predominantly in the CD45R+ cell
populations (two examples in Fig. 4, C and D).
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IgM Ab molecules exist in two forms, a monomeric, membrane-bound
form and a pentameric, secreted form. Two mRNAs, with different
3'-ends, encode the membrane-bound and the secreted forms of the Ig µ
chain (16). The two types of mRNA are produced from a single Ig gene by
alternative RNA processing pathways (17). The relative amount of the
two mRNAs correlate with the stage of B cell differentiation. To
investigate which form of IgM is coded for by the dominant mRNA peaks,
we designed two reverse primers. The first is specific for the
3'-untranslated region of the mRNA coding for the secreted form of
the µ Ig heavy chain. The second is specific for the mature mRNA
coding for the membrane-bound form of the µ Ig heavy chain, since it
encompasses the junction of the µ-1 and µ-2 IgM exons, linked only
when the membrane-bound form of the IgM is expressed (Table II). CDR3
size analysis was performed on mRNA coding for the secreted or
membrane-bound forms of IgM. For this study, we used RNA samples from
the unfractionated splenic cells from the same 6 mice as above (5 old
mice with altogether 10 stable clonal B cell populations, and 1 young
mouse with 1 clonal B cell population) (Fig. 5). The profiles were compared with those
obtained from total IgM mRNAs. In both large and small B cell clonal
populations, the dominant peaks that were detected in the total IgM
mRNA CDR3 size profiles also dominated the CDR3 size profiles derived
from mRNA coding for the secreted form of the µ Ig heavy chain. In
contrast, the same overwhelming dominance of the CDR3 size profiles of
the mRNA coding for the membrane-bound form of the µ Ig heavy chain
was detected only for the 3 large B cell clonal populations (Fig. 5).
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We investigated the presence in different lymphoid compartments of
the previously described five small clonal populations from old mice,
one small clonal population from the young mouse, and three large
clonal populations from old mice. Two examples of the results obtained
for large and two examples for small clonal populations are illustrated
in Fig. 6. All three large clonal
populations were detected in the, spleen, thymus, and bone marrow. In
contrast, none of the 6 small clonal populations originally detected in
the spleen were detected in thymus or bone marrow.
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B cells have been divided into B1a, B1b, and B2 subsets, distinguishable by the expression of cell surface Ags. We examined whether the B cell clonal populations detected in unfractionated spleen cells were detectable in the B1a (CD5+, CD45Rlow cells) and/or the B2 (CD5-, CD45R+ cells) B cell subsets. We sorted unfractionated spleen cells into CD5+, CD45Rlow B1a and CD5-, CD45R+ B2 cell subsets from the same mice as above (5 old mice with altogether 10 stable clonal B cell populations, and 1 young mouse with 1 clonal B cell expansion). The purity of the sorted preparations was >95% based on re-analysis of the sorted populations by FACS (not shown). As previously reported (5, 14, 18), the B1a cell subset represented less than 5% of the CD45R+ splenic cells in the young mouse and up to 15% of the CD45R+ splenic cells in the old mice (data not shown).
Representative CDR3 Ig mRNA size profiles obtained from unfractionated
and the 2 sorted splenic B cell populations from the 5 old mice are
shown in Fig. 7. Six splenic stable
clonal B cell populations were detected in the B1a but not in the B2
cell preparations (Fig. 7A). Only 1 splenic stable clonal B
cell population could be detected in the B2 but not the B1a cell
preparation (Fig. 7B). Finally, 3 splenic stable clonal
populations were detected in both the B1a and the B2 cell preparations
(Fig. 7C). The identity of the B cell populations detected
in the B1a and in the B2 B cell subsets was established by sequencing.
The results for all 11 clonal populations from the 5 old mice and the
young mouse are reported in Table III.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Ten stable splenic B cell clonal populations from five 20-mo-old mice, which expressed a dominant mRNA CDR3 size in the VHJ558, VHQ52, or VH7183 families in association with IgM, were selected for detailed study. mRNA was extracted from unfractionated spleen, bone marrow, thymus, and peritoneal cavity cells from these five mice, as well as from sorted splenic subpopulations and bone marrow pre-B cells. In addition, DNA was extracted from unfractionated spleen cells from three old mice (in which eight stable clonal B cell populations had been identified) and one young mouse with a B cell clonal expansion. CDR3 size analysis was performed on each of these samples as well as on mRNA coding for the membrane-bound or for the secreted forms of the µ Ig heavy chain.
These analyses allowed us to define two types of stable B cell clonal
populations in old mice. For one type, the characteristic dominant peak
in the CDR3 size analysis profiles from total spleen cells mRNA was
expressed by a relatively small number of B lineage cells. These cells
were detected only in the spleen, were predominantly recovered from
CD45R-sorted cell preparations not expressing mRNA coding for the
membrane-bound form of the µ Ig heavy chain. (Fig. 8A). This phenotype is
expressed by plasma cells. For the other type of clonal B cell
population, the characteristic dominant peak in the CDR3 size analysis
profiles from total spleen cells mRNA was expressed mainly by a larger
number of clonal B cells, which were detected not only in the spleen
but also in the thymus and bone marrow. These cells were predominantly
recovered from CD45R+-sorted cell populations and expressed
mRNA coding for the membrane-bound form of the µ Ig heavy chain (Fig. 8B). This phenotype is characteristic of mature B
lymphocytes. These two populations are not mutually exclusive, since
they seem to represent two stages of B cell differentiation, one more
advanced than the other. It is therefore not surprising to find
indications of the presence of each of them in almost every sample
studied, as illustrated by CDR3 size expansions of small amplitude in
non characteristic types of CDR3 size profiles. For example, for large
clonal B cell populations, it is possible to see CDR3 size expansions
at the size of the original clonal peak in the profiles of
CD45R--sorted cells. Similarly, for small clonal B cell
populations, one can see CDR3 size expansions for mRNA coding for the
membrane-bound form of the IgM, at the size of the originally detected
dominant peak. However, none of those expansions can compare with the
dramatic dominant peaks, which represent 70–100% of the total area of
a given CDR3 size pattern.
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B cells have been divided into 2 subsets (13). The B1a subset consists of CD5+ and CD45Rlow cells. These cells are associated with lymphoproliferative and autoimmune disorders as well as clonal expansions that appear with age (22, 23, 24, 25, 26). Of the 10 B cell clones, 9 were sorted and could be detected in the B1a subset. One B cell clonal population was not be detected in the B1a subset but rather in the B2 subset (CD5- and CD45R+ cells). Three B cell clonal populations were detected in both B1a- and B2-sorted populations. The presence of the clonal populations in both CD5+- and CD5--sorted samples does not appear to result from cross-contamination as each sorted population was >95% pure (not shown).
Although it is possible that the clonal populations present in both subsets are derived from a common precursor, this appears unlikely because they could not be detected in bone marrow precursors. It is more likely that CD5 expression is either lost by clonal B1a cells or gained by clonal B2 cells. Indeed, it has been reported that murine B1a cells lose CD5 expression in vitro upon cross-linking of their surface Ig with anti-Ig Abs, a model for type 2 thymus-independent stimulation (27). It also has been reported that murine B2 cells acquire CD5 expression in vitro upon cross-linking of their surface Ig with anti-Ig Abs. Furthermore, in these CD5+ B2 cells, IL-6 can down-regulate CD45R expression to the level found in peritoneal cavity B1a cells. Such CD45Rlow, CD5+ B2 cells have an increased viability associated with decreased apoptosis and increased proliferation (28, 29, 30, 31, 32). It is possible that autoantiidiotypic Abs, increased in serum from old mice, that bind to surface Ig, may induce such changes in old mice.
The presence of B cell clonal populations in old mice may have several consequences. One, based on the apparent B1a origin of the majority of B cell clonal populations, is that these cells contribute to the age-associated increase in autoantibody production. Clonal B cells may also interfere with the generation of adaptive Ab responses (33). For example, increased susceptibility to infection is a significant problem in patients with chronic lymphocytic leukemia and multiple myeloma (34, 35). Finally, it is intriguing to consider whether the small or large B cell clones are precursors of the B cell neoplasms chronic lymphocytic leukemia or multiple myeloma, respectively, found in old experimental animals and humans.
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Marc E. Weksler, Department of Medicine, Cornell University Medical Center, 1300 York Avenue, New York, NY, 10021.
3 Abbreviation used in this paper: CDR3, complementarity-determining region 3.
Received for publication October 29, 1998. Accepted for publication March 12, 1999.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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