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Department of Internal Medicine, Harold C. Simmons Arthritis Research Center, University of Texas Southwestern Medical Center, Dallas, TX 75235
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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5% of B cells expressed both RAG1 and RAG2. These cells were
uniformly postswitch memory B cells as documented by the coexpression
of IgG mRNA. These results indicate that coordinate RAG expression is
not found in normal peripheral naive B cells but is up-regulated in
naive B cells which are activated in the tonsil. With the exception of
centroblasts, RAG1 and RAG2 expression can be found in all components
of the GC, including postswitch memory B cells, some of which may
circulate in the blood of normal subjects. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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) light chain
has been demonstrated in human GC reactions (30), although
TdT does not appear to be expressed in murine GC reactions
(23). It has been proposed that secondary V(D)J
recombination in secondary lymphoid organs might be a mechanism to
rescue B cells whose Ag receptor avidity has been decreased as a result
of somatic hypermutation (25). Recent evidence using
transgenic mice expressing RAG promoter constructs, however, has
questioned whether transcription of RAG genes is up-regulated in
secondary lymphoid organs. In this regard, transcription of RAG genes
was limited to B cell precursor cells and transitional cells that had
left the bone marrow and were completing maturation in the spleen
(33, 34).
We had previously reported evidence that patients with a systemic
autoimmune disease, systemic lupus erythematosus, manifested evidence
of secondary rearrangement of V
genes in the
periphery (35, 36). Therefore, it was important to
determine whether B cells re-expressed RAG enzymes in secondary
lymphoid organs during immune responses.
RAG expression was assessed by using a combination of multiparameter flow cytometric cell sorting and RT-PCR analysis of tonsil and peripheral blood B cell mRNA content at both the subset and individual cell level. RAG1 and RAG2 mRNA expression by activated naive B cells in the tonsil as well as by B cells at subsequent stages of maturation in the GC was documented. Moreover, postswitch IgG expressing activated memory B cells in both blood and tonsil expressed RAG1 and RAG2 mRNA. These results imply that many B cells generated during GC reactions may have the molecular machinery to accomplish secondary V(D)J rearrangement.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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PBMC were separated by Ficoll-Hypaque density gradient centrifugation from heparinized peripheral blood of 14 healthy adult Caucasian donors. B cells were purified from PBMCs by negative selection using magnetic separation according to the manufacturer’s instructions (Stemcell Technologies, Vancouver, British Columbia, Canada). Purity of the B cell enrichment was determined by indirect immunofluorescence staining using a PE-conjugated anti-human CD19 mAb (PharMingen, San Diego, CA) and analysis with a FACScan flow cytometer (Becton Dickinson, San Jose, CA). B cells in the 14 different samples were 93–96% CD19 positive.
Preparation of B cells from tonsil tissue and of mononuclear cells from thymus
Human tonsils from six healthy children were obtained after routine tonsillectomy. Suspensions of tonsilar mononuclear cells were prepared by collagenase digestion (Worthington Biochemical, Lakewood, NJ) of the tissue for 30 min, followed by Ficoll-Hypaque density gradient centrifugation, as described previously (32).
Human thymus tissue from one otherwise healthy child was obtained during heart surgery for congenital heart disease. Suspensions of thymus mononuclear cells were prepared as described above.
Cell sorting
Tonsil B cells subsets from six subjects were defined by the
surface expression of CD19, IgD, and CD38 and were isolated with a
FACStarPlus or FACSVantage flow cytometer (Becton
Dickinson) as described previously (32) (see Fig. 1
).
Indirect immunofluorescence staining was conducted using a biotinylated
anti-human CD19 mAb (Coulter, Miami, FL) followed by
streptavidin-RED670 (Life Technologies, Gaithersburg, MD), a
PE-conjugated anti-human CD38 mAb (Becton Dickinson), and a
FITC-labeled goat anti-human IgD polyclonal Ab (Caltag, San
Francisco, CA). Postsort analysis of isolated populations revealed a
purity of >90%.
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For the deposition of individual cells into microtiter wells, tonsilar mononuclear cells were stained as described above. Single cells were sorted into 96-well PCR plates (Robbins Scientific, Sunnyvale, CA) using a FACStarPlus flow cytometer (Becton Dickinson) outfitted with a single-cell deposition unit, as described previously (37). For the sorting of tonsil naive and memory B cells into CD69-positive and -negative cells (38), four-color immunofluorescence staining was performed using a biotinylated anti-human CD19 mAb (Coulter) followed by streptavidin PerCP (Becton Dickinson), a PE-conjugated anti-human CD38 mAb (Becton Dickinson), a FITC-labeled goat anti-human IgD polyclonal Ab (Caltag), and a tricolor-conjugated anti-human CD69 mAb (Caltag). Single cells were sorted with a FACSVantage flow cytometry system (Becton Dickinson). In some experiments, cells were sorted in bulk and then deposited in the wells manually by limiting dilution.
Preparation of RNA and cDNA from sorted bulk populations
Total RNA was extracted using the RNAeasy RNA isolation kit (Qiagen, Chatsworth, CA). The cell numbers subjected to RNA preparation were as follows: 0.5 x 106 FACS-sorted B cells of each tonsil subset and 2.0 x 106 negatively selected B cells from normal peripheral blood of each donor. Contaminating genomic DNA was removed using RQ1 RNase-free DNase according to the manufacturer’s instructions (Promega, Madison, WI). For conversion of mRNA into cDNA, Superscript II RNase H-reverse transcriptase (RT; Life Technologies) was used according to the manufacturer’s instructions. From each RNA, one sample was prepared by omitting the RT and used as control.
Preparation of RNA and cDNA from sorted single cells
A total of 5 µl of lysis solution (1 µl of 5x first-strand buffer B (Life Technologies), 0.01 M DTT (Life Technologies), 1% Nonidet P-40 (Sigma, St. Louis, MO), 5 U of recombinant RNasin ribonuclease inhibitor (Promega), 800 µM each dATP, dCTP, dGTP, and dTTP (Sigma), 0.05 µg of oligo(dT)12–18 (Pharmacia Biotech, Piscataway, NJ)) was added into each well of the PCR plate before sorting individual cells into the wells. The conversion of mRNA from a single cell was conducted with Superscript II RNase H-RT (Life Technologies) as described previously (1). In short, after annealing for 1 min at 65°C, followed by a 2-min incubation at 20°C, 3 µl of RT reaction mix (0.6 µl of 5x first-strand buffer B, 0.01 M DTT, 10 U of RT) was added. Reverse transcription was conducted for 50 min at 42°C, followed by heat inactivation at 65°C for 10 min. For removal of the remaining mRNA from the newly synthesized cDNA, RNase H (0.08 U/reaction; Amersham Life Science, Cleveland, OH) was used.
PCR amplification
The relative amount of RAG1-, RAG2-, TdT-, 8-oxoguanine DNA
glycosylase (OGG)-, VpreB-, 14.1- encoding cDNA in the different
samples was determined by amplification of glucose-6-phosphate
dehydrogenase (G6PD) or 
-actin adjusted samples using the PCR
Southern blot technique, as described below. The primer pairs used are
listed in Table I. In the first round of
PCR, 30 (OGG, VpreB, 14.1, IgD, IgM, IgG) or 38 cycles (RAG1 and RAG2,
TdT) of 30 s at 94°C, 1 min at 60°C, and 2 min at 72°C with
a final 5-min extension at 72°C were conducted. A second nested step
of PCR was performed only in the analysis of cDNA generated from single
cells. Five microliters of the first PCR product was subjected to
nested PCR amplification using primers as indicated in Table I. For
analysis of human RAG2 mRNA, two different 5' primers for the
alternative exon 1a and exon 1b were used. The RAG2 exon 1a primers
were intron spanning, whereas the RAG1 and RAG2 exon 1b forward primers
spanned an intron. These primers were chosen because completely intron
spanning primers for RAG2 exon 1b and RAG1 did not yield reproducible
results with single-cell RT-PCR. Control samples of each preparation
carried out without RT were run in parallel and were negative for the
presence of contaminating DNA (data not shown). In addition, PCR
without adding cDNA were run in parallel and did not yield a
product.
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For quantification of cDNA in the different samples, PCR
amplification for the housekeeping genes, G6PD or -actin, was done
in triplicate at 20, 25, 30, and 35 PCR cycles each. RT-PCR products
were analyzed on a 1.8% agarose gel and transferred to a nylon
membrane by alkaline vacuum transfer (Bio-Rad, Hercules, CA). PCR
Southern blots were incubated in hybridization fluid containing
-32P-labeled probes for G6PD or -actin PCR
products, as listed in Table II. The
amount of hybridized probe for the housekeeping gene was quantified by
phosphor imager. The linear range of PCR was determined by plotting the
amount of phosphor imager counts (above background) of the hybridized
probe as a function of the number of cycles run. The mean counts of
these triplicates in the linear range of PCR amplification were
calculated and were used to calculate an amount of cDNA (in µl) of
each preparation that contained the same relative content of
housekeeping gene cDNA. This calculated volume of cDNA was subjected to
further PCR amplification for specific cDNAs (39). The
amplified PCR products were again analyzed by agarose gel
electrophoresis and transferred to a nylon membrane and blotted. The
number of PCR cycles in which the PCR product was amplified in a linear
range was again determined for each primer set, as described above for
housekeeping genes. After hybridization using specific probes (Table II), the mean of the counts of the hybridized probe from six different
tonsil donors was calculated for each tonsil B cell subset. Finally,
the total number of counts was calculated by summing up the mean counts
of each tonsil B cell subset and the fraction of total cDNA in each B
cell subset was expressed as a percentage.
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-actin products
from single-cell RT-PCR. This occurred in 1–3% of the wells with a
detectable RAG product and were omitted from the analysis. Indirect immunohistochemistry
For immunohistochemical staining, 5-µm cryostat serial sections of unfixed frozen tonsil tissue were prepared. The following Abs were used: biotinylated anti-human IgD (Sigma), followed by alkaline phosphatase-conjugated streptavidin (Vector Laboratories, Burlingame, CA), and anti-human RAG2 polyclonal goat Ab (Santa Cruz Biotechnology, Santa Cruz, CA), followed by a peroxidase-conjugated rabbit anti-goat polyclonal Ab (Dako, Carpinteria, CA), followed by a peroxidase-conjugated goat anti-rabbit polyclonal Ab (Dako). Control sections were stained with a polyclonal preimmunization goat serum with the omission of the anti-IgD Ab. All Abs were diluted in Tris-hydroxymethylaminomethane buffer (pH 7.4) containing 0.5% BSA. Fast Red TR salt (Sigma) was used for detection of Ab-conjugated alkaline phosphatase. Diaminobenzidine (Sigma) was used for detection of Ab-conjugated HRP as described previously (40).
Statistical analysis
The differences in RAG1, RAG2, IgG, and IgD mRNA expression in
single cells of the various B cell subsets were analyzed using the
2 test or Fisher’s test as indicated.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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B cell subsets were characterized by surface expression of IgD and
CD38 as described previously (32) and identified as
follows: naive tonsil B cells were IgD+ and
CD38-; pre-GC/GC founder cells were
IgD+ CD38+ and
IgD+ and CD38++;
centroblasts were IgD- and
CD38++; centrocytes were IgD-
and CD38+; and memory B cells were
IgD- and CD38- (Fig. 1
). In the six tonsils analyzed, RAG1
mRNA was found in each B cell subset. The greatest quantity of
RAG1 mRNA was found in the naive B cell population, which contained
about 3 times more than pre-GC/GC founder cells, 8 times more than
centroblasts, about 10 times more than centrocytes, and twice as much
as memory B cells (Fig. 2). RAG2 mRNA was
also prominent in tonsil naive B cells. A comparable amount was found
in centrocytes, whereas 20% of the amount was found in
centroblasts. No RAG2 mRNA was detected in GC founder cells or memory B
cells (Fig. 2).
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Expression of TdT mRNA was predominantly found in centroblasts
with only minimal expression in naive B cells (Fig. 2). No other subset
expressed TdT mRNA.
As has been previously noted (41), OGG mRNA was found in
equal amounts in CD38++ GC founder cells and in
centroblasts. Only a marginal amount of OGG mRNA was found in the
remaining subsets (Fig. 2).
Analysis of VpreB mRNA expression revealed the highest amount in
CD38++ pre-GC/GC founder cells. They contained
about six times more than naive B cells, twice that in
CD38+ pre-GC/GC founder cells, 1.5 times more
than centroblasts, about 2 times more than centrocytes and nearly three
times more than memory B cells (Fig. 2). The content of the surrogate
() light chain constant region encoding human gene 14.1 mRNA was the
highest in centroblasts, which contained 4 times more than naive B
cells, 12 times more than CD38+ pre-GC/GC founder
cells, 3 times more than CD38++ pre-GC/GC founder
cells, and 22 times more than memory B cells. No expression of 14.1
mRNA was found in centrocytes (Fig. 2). No correlation (r)
between the data sets of VpreB and 14.1 was found (r =
0.452, p > 0.1).
RAG2 expression in tonsil tissue
To confirm the expression of RAG protein in tonsil B cells,
indirect immunohistochemistry of four different tonsil tissues was
performed. GC were visualized by the typical pattern of IgD expression
in the mantle zone (Fig. 3A).
RAG2 protein-expressing cells were present in the GC (Fig. 3, B and C). A significant number of RAG2-positive
cells were also identified in the mantle zone and the interfollicular
area. Higher magnification revealed a nuclear distribution of RAG2 with
the greatest expression at the periphery of the nuclei (Fig. 3C). Control sections stained with a preimmunization sera
were negative (Fig. 3D). Representative consecutive sections
of one tonsil are shown.
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No RAG1 mRNA was found in normal blood B cells isolated from 14
healthy individuals, whereas RAG2 was detected in three samples (Fig. 4). No TdT mRNA expression could be
detected in normal peripheral B cells. Eight of 14 peripheral B cell
preparations showed low expression of OGG mRNA compared with tonsil
mononuclear cells or thymus mononuclear cells. VpreB mRNA expression
could be detected in 3 of 14 samples. The 14.1 mRNA was detected at a
low level (lane 2) in one sample which did not
contain VpreB mRNA (Fig. 4).
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To analyze RAG1 and RAG2 mRNA expression in greater detail, a very
sensitive single-cell RT-PCR method was employed that made it possible
to examine the expression of multiple mRNAs by individual
phenotypically defined B cells. Analysis of individual tonsil B cells
from one patient revealed that 15–26% of individual
-actin-positive cells in each subset expressed RAG1 mRNA (Fig. 5A). The differences in RAG1
mRNA expression by the various subsets were not statistically
significant (p > 0.05,
2 test). RAG2 mRNA was frequent in naive, GC
founder/pre-GC cells, and memory B cells, but significantly less so in
centroblasts and centrocytes (p < 0.05,
2 test) (Fig. 5A). The frequency of
RAG2+ cells in centrocytes was significantly
greater than in centroblasts (p < 0.05,
2 test). Exon 1a and exon 1b of the RAG2 gene
were used comparably by individual cells of tonsil B cell subsets.
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B shows the -actin, RAG1, RAG2 exon 1a, and RAG2
exon 1b mRNA expression of
CD38+IgD- B cells
(centrocytes) sorted as individual cells into a 96-well plate.
Fifty-four of 96 attempts to sort an individual cell yielded a positive
product by single-cell RT-PCR for -actin (56%). Seven of 54
-actin-positive wells were also positive for RAG1 and RAG2 mRNA
using either RAG2 exon 1a or RAG2 exon 1b (13%).
Only the coordinate action of RAG1 and RAG2 together can lead to
recombination of the B cell receptor genes (7). Therefore,
the frequency of RAG1 and RAG2 mRNA-positive B cells was analyzed as
shown in Fig. 5B. RAG1 plus RAG2 expression was found in
individual cells in all populations, except centroblasts, which showed
no coordinate expression of RAG1 and RAG2 mRNA (Fig. 5A).
To assess the stage of maturation of the cells expressing RAG1 plus
RAG2 mRNA, IgG mRNA was examined in these individual cells to determine
whether they were pre- or postswitch B cells. The frequency of RAG1
plus RAG2 mRNA-positive cells expressing IgG mRNA increased from
centrocytes to memory B cells, with 75% of RAG1 plus RAG2 expressing
memory B cells also expressing IgG mRNA (Fig. 6). Further analysis of the RAG
double-positive cells in the naive and memory B cell subsets in the
tonsil was conducted to determine the influence of activation. As can
be seen in Fig. 7, the frequency of RAG1
and RAG2 double-positive cells in activated CD69+
memory cells (surface (s) IgD- and
CD38-) was 10 times higher than in resting
CD69- memory B cells. Of note, the RAG1- and
RAG2-positive, CD69+ memory cells uniformly
expressed IgG mRNA (data not shown). As can be seen in Fig. 8, no RAG1 and RAG2 double-positive cells
were found in the CD69- resting naive B cells
(sIgD+ and CD38-). In
contrast, 5% of activated CD69+ naive cells
expressed RAG1 and RAG2 mRNA (Fig. 8). Of note, these RAG 1- and
RAG2-positive, CD69+ naive B cells were all
positive for IgD mRNA (data not shown).
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Analysis of peripheral blood B cells of two donors was conducted
to investigate whether cells entering the tonsil from the blood
expressed RAG1 and RAG2 mRNA. Using single-cell analysis, 3% of CD19-
and -actin-positive B cells expressed both RAG1 and RAG2 mRNA (Fig. 9). All of these RAG double-positive
cells (n = 3) expressed IgG mRNA. None of the RAG
double-positive cells were positive for IgD mRNA (Fig. 9). Sixteen
percent of CD19- and -actin-positive B cells were positive for RAG1
mRNA alone and 26% were positive for RAG2 mRNA alone. Evaluation of
the expression of either RAG1 or RAG2 mRNA and the Ig expressed by a
particular cell revealed that 5% of -actin-positive cells were
positive for only RAG1 and IgG, 9% for RAG2 and IgG, 1% for RAG1 and
IgD, and 12% for RAG2 and IgD mRNA.
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, no double
RAG1- and RAG2-positive cells were found in the
sIgD+ subset. However, in the
sIgD- subset, 5% of the cells expressed both
RAG1 and RAG2 (n = 4) (Fig. 9). In the
IgD- subset, 3 of 4 (75%) of the RAG
double-positive cells were positive for IgG mRNA; none was positive for
IgD mRNA (Fig. 9). Evaluation of either one of the RAG mRNAs and Ig
mRNA expressed by individual cells in the sIgD+
subset revealed that 1% of -actin-positive cells were positive for
RAG1 and IgG, 1% for RAG2 and IgG, 9% for RAG1 and IgD, and 9% for
RAG2 and IgD mRNA. Evaluation of the expression of either one of the
RAG mRNAs and the Ig expressed in the sIgD-
subset revealed that 12% of -actin-positive cells were positive for
RAG1 and IgG, 6% for RAG2 and IgG, 4% for RAG1 and IgD, and 1% for
RAG2 and IgD mRNA. In summary, these results have demonstrated that each B cell expressing both RAG1 and RAG2 mRNA in the peripheral blood was positive for IgG mRNA and not IgD mRNA. RAG double-positive cells in the peripheral blood, therefore, are found in the postswitch memory B cell compartment.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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One of the unique findings of the current study was the marked discordance in expression of RAG1 and RAG2 mRNA in B cell subsets and, importantly, in individual B cells. The frequency of RAG2 mRNA expressing individual tonsil B cells was 2-fold higher than RAG1 mRNA-expressing cells, indicating that approximately half of the RAG2 mRNA-positive cells were RAG1 mRNA negative. It is unlikely that this discrepancy reflected differential effectiveness of the RAG primers, as careful titrations suggested that each was comparably effective. Moreover, it is unlikely that DNA contamination contributed to the results, as all samples were treated with DNase before analysis and intron spanning primers were used. Rather, the data suggest that RAG1 and RAG2 are differentially expressed. Although both RAG1 and RAG2 were down-regulated in individual centroblasts, in other B cell subsets, regulation appeared to be stochastic. RAG mRNA levels are known to be regulated transcriptionally and posttranscriptionally (42, 43, 44, 45). The current data do not permit a conclusion about the mechanisms underlying the discordant expression of RAG1 and RAG2 mRNA in tonsil B cell subsets, but the available data suggest that transcriptional and posttranscriptional effects may play roles. In this regard, coordinate transcription of RAG1 and RAG2 genes has been demonstrated in a murine system using transgenic reporter constructs (46). Even though the regulation was in general coordinate, a considerable number of bone marrow B cells expressed RAG2 promoter activity in the absence of RAG1 expression (46). This differential transcription of RAG2 may contribute to the increased frequency of RAG2 mRNA-expressing cells in the current study. RAG2, and to a lesser extent RAG1, can also be regulated posttranslationally by proliferation-dependent phosphorylation and subsequent degradation of the RAG proteins (43, 44). Currently, there are no available data about whether posttranscriptional regulation of RAG1 and RAG2 mRNA is different. However, in view of the generally coordinate regulation of transcription, it is likely that the discordant expression of RAG1 and RAG2 mRNA levels may reflect differential posttranscriptional regulation. It is notable that the frequency of single RAG-positive cells increased from centrocytes to memory B cells, whereas the frequency of double RAG1- and RAG2-positive individual cells decreased. These results suggest that the capacity for receptor editing may decrease as B cells mature from centrocytes to memory B cells. Since both the RAG1 and RAG2 enzymes are needed for recombination of the Ig genes, the physiological function of isolated expression of RAG1 or RAG2 mRNA is unknown.
To analyze potential influences that might induce coordinate expression of RAG1 and RAG2 mRNA in human B cells, we further analyzed the naive B cell subset in the peripheral blood and the tonsil. Peripheral naive B cells did not show coordinate expression of both RAGs, as was also found for resting CD69- naive B cells in the tonsil. However, a significant number of activated, CD69+, naive B cells in the tonsil exhibited coordinate expression of RAG1 and RAG2 mRNA. The absence of RAG double-positive B cells in the CD69- naive tonsil B cell compartment, along with the absence of RAG1 and RAG2 double-positive naive B cells in the peripheral blood, suggest that RAG expression is induced coordinately after naive cells enter the tonsil and become activated, as activation of naive tonsil B cells by Ag is associated with up-regulation of CD69 (47). There are only limited and conflicting data on the induction (48, 49) or suppression (30, 49) of RAG expression after cross-linking the B cell receptor to simulate Ag contact by B cells. Induction of RAG in B cells was observed to be induced by sIg engagement (50, 51) and maintained by CD40 engagement (22, 30), possibly by a B cell lineage-specific activator protein-dependent mechanism (52). In contrast, engaging sIg on B cells that are already positive for RAG mRNA down-regulated RAG expression (30). The current results support the hypothesis (23) that activation of naive B cells in secondary lymphoid organs might induce up-regulation of both RAG1 and RAG2 expression following Ag stimulation.
Activated CD69+ memory B cells contained a 10-fold greater number of coordinate RAG1- and RAG2-expressing cells than resting memory B cells. This suggests that memory B cells that were generated during a GC reaction decrease their coordinate expression of RAG mRNA once they are removed from activation signals. Alternatively, resting memory B cells that enter the tonsil may up-regulate coordinate RAG1 and RAG2 expression upon Ag-mediated activation. Whichever hypothesis is correct, the regulation of RAG expression in memory B cells appears to be tightly linked to activation status, as it is in naive B cells.
The expression of RAG1 and RAG2 mRNA during B cell development in the bone marrow is thought to be tightly controlled and terminated once immature cells express sIgM (2, 42, 44, 45, 53). Two recent reports examining expression of reporter constructs regulated by RAG2 regulatory elements (33, 34) have found that the majority of RAG2 transcription is limited to developing B cells in the bone marrow, but is also found in transitional B cells in the spleen (33). In these studies, no mature murine naive B cells were found to be transcribing RAG genes in vivo, even after immunization (33, 34). In contrast, sIgM+sIgD+ B cells generated from bone marrow cells in vitro expressed RAG2 after B cell receptor cross-linking with anti-IgM (34). Previously, up-regulation of RAG gene expression was found after Ig receptor cross-linking in both Ig-transgenic and wild-type bone marrow B cells (48). Comparing these studies with the current results suggests that RAG mRNA levels may be extensively regulated in tonsil B cell subsets following engagement of sIg. Part of this regulation may be at the level of transcription. In this regard, marked down-regulation of RAG mRNA in centroblasts followed by up-regulation in centrocytes and memory B cells may be compatible with transcriptional regulation of RAG expression in the tonsil.
It has been suggested that the RAG-expressing cells, previously found in GC, represent immature transitional B cells which have been attracted to the secondary lymphoid organ (19, 22, 23, 25, 33), potentially including the tonsil (29, 30). In contrast, the current results show coordinate RAG1 and RAG2 mRNA expression combined with the expression of IgG in centrocytes and in memory B cells of the tonsil. The IgG primers employed could potentially detect sterile transcripts and, thus, not identify actual postswitch IgG-expressing B cells. However, the complete discordance between the ability to detect IgD mRNA and IgG mRNA suggests that the predominant IgG mRNA species detected represents that obtained from postswitch IgG-expressing B cells, and, therefore, is consistent with the conclusion that RAG1 and RAG2 mRNAs are up-regulated in postswitch IgG-expressing B cells. This conclusion is supported by the analysis of blood B cells, in which RAG1 and RAG2 mRNAs were uniquely found in IgG-expressing cells, even though previous data indicated that resting circulating B cells do not express sterile IgG transcripts (54). In summary, these data, therefore, suggest, that many postswitch B cells generated by GC reactions may express RAG and are not consistent with the contention that RAG is uniquely expressed by transitional cells in secondary lymphoid organs.
Surrogate light chain was found to be broadly expressed in all subsets with a maximum in pre-GC/GC founder cells and centroblasts. Therefore, the expression of surrogate light chain was not correlated with the expression of RAG1 or RAG2 mRNA in the various tonsil B cell subsets, in contrast to previous reports showing coordinate expression (25, 30). These findings would appear to conflict with the conclusion that B cell-specific transcription factors regulate not only the human RAG1 and the murine RAG2 promoter (52, 55, 56), but also surrogate light chain gene expression (52, 55). However, the differences in the RAG1 and RAG2 mRNA expression in comparison to VpreB and 14.1 mRNA expression might again be related to posttranscriptional regulation of the various mRNAs (43, 44).
Since primary V(D)J recombination is accompanied by TdT-induced n-nucleotide addition, we investigated whether TdT activity is up-regulated in association with RAG expression. There are disparate data concerning this issue in man and mouse (23, 30). In the current study, expression of TdT mRNA was limited to centroblasts. Previously, TdT mRNA expression was also found in centroblasts, but the predominant expression was reported in centrocytes (30). The different definition of centrocytes in the current (CD38+, IgD-) and previous (CD38+, CD77-) studies (30) is unlikely to account for this discrepancy, since CD38+, IgD- tonsil B cells were also found to be predominantly CD77low (32). In light of the far broader expression of RAG1 and RAG2 mRNA compared with TdT mRNA levels in tonsil B cell subsets, the current results suggest that if receptor replacement occurs in peripheral lymphoid organs, it is likely to utilize TdT to a limited extent.
In summary, the use of single-cell PCR analysis has defined the subsets of tonsil B cells that express RAG mRNA and has clearly shown broad expression after initial activation of naive B cells including centrocytes and postswitch memory B cells that simultaneously express IgG mRNA.
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Acknowledgments |
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Footnotes |
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2 Current address: National Institute of Arthritis and Musculoskeletal and Skin Diseases, National Institutes of Health, 9000 Rockville Pike, Bethesda, MD 20892-1820.
3 Address correspondence and reprint requests to Dr. Peter E. Lipsky, National Institute of Arthritis and Musculoskeletal and Skin Diseases, National Institutes of Health, 9000 Rockville Pike, Bethesda, MD 20892-1820.
4 Abbreviations used in this paper: RAG, recombination-activating gene; GC, germinal center; OGG, 8-oxoguanine DNA glycosylase; RT, reverse transcriptase; s, surface; G6PD, glucose-6-phosphate dehydrogenase.
Received for publication February 2, 2000. Accepted for publication October 9, 2000.
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chain receptor editing in systemic lupus erythematosus. J. Clin. Invest. 102:688.[Medline]
gene rearrangements in systemic lupus erythematosus. Clin. Immunol. 92:188.[Medline]
gene utilization in primary biliary cirrhosis. Hepatology 24:1148.[Medline]
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 J. S. Rice, J. Newman, C. Wang, D. J. Michael, and B. Diamond Receptor editing in peripheral B cell tolerance PNAS, February 1, 2005; 102(5): 1608 - 1613. [Abstract] [Full Text] [PDF]
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T.-H. Thai and J. F. Kearney Distinct and Opposite Activities of Human Terminal Deoxynucleotidyltransferase Splice Variants J. Immunol., September 15, 2004; 173(6): 4009 - 4019. [Abstract] [Full Text] [PDF]
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E. H. Sasso, M. Martinez, S. L. Yarfitz, P. Ghillani, L. Musset, J.-C. Piette, and P. Cacoub Frequent Joining of Bcl-2 to a JH6 Gene in Hepatitis C Virus-Associated t(14;18) J. Immunol., September 1, 2004; 173(5): 3549 - 3556. [Abstract] [Full Text] [PDF]
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W. Su, L. Boursier, A. Padala, J. D. Sanderson, and J. Spencer Biases in Ig {lambda} Light Chain Rearrangements in Human Intestinal Plasma Cells J. Immunol., February 15, 2004; 172(4): 2360 - 2366. [Abstract] [Full Text] [PDF]
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 N. Meru, A. Jung, I. Baumann, and G. Niedobitek Expression of the recombination-activating genes in extrafollicular lymphocytes but no apparent reinduction in germinal center reactions in human tonsils Blood, January 15, 2002; 99(2): 531 - 537. [Abstract] [Full Text] [PDF]
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