A B220, CD19 population of B cells in the peripheral blood of quasimonoclonal mice
Marilia Cascalho,
Jamie Wong,
Jeffrey Brown1,
Hans-Martin Jäck1,
Charles Steinberg and
Matthias Wabl
Department of Microbiology and Immunology, University of California, San Francisco, CA 94143-0670, USA
1 Department of Microbiology and Immunology, Stritch School of Medicine, Loyola University Chicago, Maywood, IL 60153, USA
Correspondence to:
M. Wabl
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Abstract
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We describe a new population of non-naive B cells in the peripheral blood of quasimonoclonal (QM) mice. Surface Ig of switched isotypes is expressed, but not B220 nor CD19. These cells are larger and denser than naive B cells but smaller than blasts or plasma cells; they do not stain with syndecan, a marker for plasma cells. Telomerase, which is usually expressed in B cell blasts, was not present in this population. We sorted the switched, idiotype-positive, B220 B cells from the peripheral blood of QM mice and sequenced Ig H chain and
L chain cDNA. There were many point mutations but no V gene replacements, gene conversions or other type of diversifications. As they express switched isotypes and have mutated their Ig genes, cells in the B220, CD19 population must have been in an immune response and we suggest that it includes the memory B cell subset.
Keywords: B lymphocytes, cellular differentiation, FACS, generation of diversity, memory
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Introduction
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CD19 and B220 (CD45R), an isoform of CD45, are surface markers of mature, resting B cells. CD45 is a tyrosine phosphatase that participates in the modulation of the immune response in both B and T cells. It is present on immature and mature B cells, but it is lost upon differentiation into plasma cells (13). CD19 is a component of a multimeric signal transduction complex that includes CD21 (complement receptor type 2), which is thought to mediate a bridging between CD19 and surface Ig by antigen and C3d (46). T cells express different splice variants of CD45 upon activation (1,2,7,8). Such a `switch' is thought to be crucial in the later stages of T cell development, but no equivalent phenomenon has been described for B cells in vivo. Nevertheless, several forms of CD45 have been identified in B cells differentiating in vitro (9,10) and CD45 has been firmly established as a positive regulator of the B cell response (11,12). Moreover, CD19 is crucial for the B cell response to T cell dependent antigens and both markers are thought to set the threshold for activation (1316).
The quasimonoclonal (QM) mouse has been engineered to contain a single functional Ig H chain allele with a rearranged VHDJH exon encoding an H chain V region. Due to targetted deletions at both
L chain alleles, it only produces
light L chain. An available anti-idiotypic (Id) antibody recognizes H chains with an unmodified V exon combined with any
chain. The primary antibody repertoire of QM mice is exceedingly limited (17, 18), a state of affairs that results in an extraordinary selection pressure for B cells bearing receptors altered by V gene replacement, which invariably results in loss of Id, or by somatic hypermutation, which only rarely causes Id loss.
In this paper we describe a novel population of B cells in the peripheral blood of QM mice. This population is composed of cells that express neither B220 nor CD19, that are highly mutated in both the VHDJH and V
J
exons, that have undergone the H chain class switch, and that lack telomerase. This population may include the memory B subset.
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Methods
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Antibodies
The mAb phycoerythrin (PE)-labeled anti-B220/RA3-6B2, anti-CD19/1D3, anti-IgMa/Igh-6a, anti-CD45RB/C363-16A and FITC-labeled anti-
1 and
2 light chain/R2646 were purchased from PharMingen (San Diego, CA). FITC-labeled anti-mouse IgG was purchased from Sigma (St Louis, MO). Biotinylated anti-Id was kindly supplied by Dr Abul Abbas.
Cell preparation
Cell preparation, cell sorting and analysis, cDNA preparation, PCR amplification, sequencing, and data analysis were previously described (17). The order of triple staining was first anti-B220 plus biotinylated anti-Id, followed by anti-CD19, CD45RB or anti-µa plus streptavidinCyChrome (PharMingen); alternatively the cells were first stained with anti-IgG, followed by non-labeled mouse IgG1, anti-µa plus biotinylated anti-Id and finally by streptavidinCyChrome. Cell sorting was done on a FACStar and analysis on a FACScan with CellQuest software (Becton Dickinson, Mountain View, CA).
Telomerase assay
The telomeric repeat amplification protocol (TRAP) was used as described (14), except that PBS was used instead of wash buffer, reaction buffer contained no T4g32, cells were lysed at 2.510x103 live cells/µl, and hot-start PCR.
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Results and discussion
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A B220 B cell population in the peripheral blood of QM mice
Peripheral blood lymphocytes (PBL) from QM mice were stained with antibodies specific for either µa or B220 and for either Id or
L chain; cells stained with three combinations were analyzed by two-color flow cytometry (Fig. 1
, left panels). Two populations of B cells are defined by rectangular gates for each of the stainings. In R1 are naive QM B cells. In R2 are the novel population of B220 B cells that forms the subject of this paper. As the R2 cells are either Id+ or
+, they must be B cells, which means that failure to stain with anti-µa implies that the cells have switched their H chain isotype. When these cells were back-gated on R1 or R2, each of the three staining combinations yielded a cluster with similar forward (FSC) and side (SSC) scatter. R2-gated cells are somewhat larger (higher FSC) and denser (higher SSC) than R1-gated cells. As they occupy a very narrow range of FSC and SSC, they are not likely to be blasts. For the same reason, they are even less likely to be plasma cells; moreover, unlike plasma cells, they do not stain with anti-syndecan (19,20).

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Fig. 1. Flow cytometric analyses of PBL from QM mice. Left and rightmost panels are fluorescence plots; y-axis, PE-labeled mAb, x-axis, FITC-labeled mAb. Middle panels are light scatter analyses of R1- and R2-gated cells from the left panels: y-axis, side scatter (SSC); x-axis, forward scatter (FSC). Left panels were pre-gated with a standard lymphocyte gate and with exclusion of dead cells by propidium iodide staining; right panels were gated only with R3. Gate R3 is defined on the FSC/SSC plot. It includes the area occupied by the main cell cluster in the R2-gated light scatter plots shown. Its coordinates are: FSC axis, between 400 and 600; SSC axis, between 100 and 300.
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The cells shown in the left panels of Fig. 1
had already been gated by a standard `lymphocyte gate'. Starting with total PBL, we used a triangular gate R3 covering the R2-gated clusters in the SSC versus FSC plots (Fig. 1
) instead of the lymphocyte gate. When stained with the same antibodies as the left panels, few B220+ or µa+ cells are seen (Fig. 1
, right panels).
The phenotype of R2 cells in the blood of QM mice was confirmed and extended with three-color flow cytometry. PBL were stained with antibodies specific for Id, B220 and either µa, CD19 or CD45RB; or, alternatively, with antibodies specific to Id, µa or mouse IgG. The left panels of Fig. 2
display fluorescence plots for Id and one of the other markers with R2 marked as in Fig. 1
. These cells are then back-gated on R2, and fluorescence plots for Id and the other marker are displayed on the right panels. This establishes the surface phenotype of the novel B cell population as B220, CD19, Id+ and mostly µa. The B220 population expresses intermediate amounts of CD45RB (Fig. 2B
) is mostly IgG+ (Fig. 2C
) and is negative for the myeloid cell marker MAC-1, i.e. the novel B cell population consists of switched B cells lacking the two principal markers of resting B cells, B220 and CD19.

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Fig. 2. Flow cytometric analyses of PBL from QM mice. All diagrams are fluorescence plots: anti-B220 (upper left, middle right in A), anti-CD19, anti-CD45RB and anti-µa were PE-labeled mAb; the remaining anti-B220 (lower left in A and in B) and the anti-IgG were FITC-labeled mAb; biotinylated anti-Id mAb was followed by staining with TriColor-conjugated streptavidin. The left panels were pre-gated as in Fig. 1 . The right panels have been back-gated with R2.
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Could they be non-B cells that bind Ig via Fc
receptors? As both antibodies, to B220 and to
, are monoclonal rat antibodies of the same class (IgG2a), the B220 antibody actually represents an internal control for excluding Fc receptor binding during staining. While the B220 cells still could have picked up Ig from the serum, they make cytoplasmic
chain (not shown), and produce (mutated) mRNA encoding both H and L chain; therefore they are, by definition, B cells.
B220 cells in the peritoneum
Previously activated B cells, which are B220 or B220lo, are known to be prevalent in the peritoneum (18). Indeed,
+, B220 B cells can be seen in the peritoneum of 20-week-old QM and normal C57BL/6 mice (Fig. 3
). They are more frequent in the QM mouse because the
locus has been knocked out. In contrast to what was seen in PBL, the R1 and R2 populations are not clearly separated in the peritoneum; there is a continuum of B220 expression from highly positive to really negative. This continuum would be expected for cells that are in various stages of activation and the separation of R2 from R1 in PBL is another argument that these are not blasts. In QM mice the number of B220 and B220lo cells varies considerably between individuals.

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Fig. 3. Flow cytometric analysis of peritoneal cells from three different QM (left panels) and C57BL/6 (right panels) mice. y-axis, PE-labeled mAb; x-axis, FITC-labeled mAb.
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B220 B cells do not express telomerase
Telomerase has been shown to be expressed in B cell blasts (J. Brown and H.-M.Jäck, unpublished), which are also B220 or B220lo. Id+, B220 (R2) and Id+, B220+ (R1) B cells were sorted from pooled PBL of six QM mice, lysed, and tested for telomerase expression. Figure 4
depicts the electrophoretic analysis of the products from the telomerase assay in vitro. The presence of telomerase activity results in a ladder of DNA fragments as in the positive controls (lipopolysaccharide-activated blasts and tumor cell lines) in Fig. 4
(lanes 36). There are no ladders in the lanes corresponding to the Id+, B220 or B220+ B cells. This is additional evidence that the B220- cells are not likely to be blasts.

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Fig. 4. Telomerase assay. `B220-pos' and `B220-neg' are the R1 and R2 populations from QM PBL. Lipopolysaccharide cultures are from C57BL/6 spleen cells. NYC and WEHI279 are B cell lines.
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H and L chain cDNA sequences of the B220 cells are hypermutated
We sorted out switched, Id+ B cells, which were shown to be mostly B220 by the above analysis, from QM peripheral blood, prepared mRNA, reverse transcribed it into cDNA, PCR amplified a stretch of DNA covering most of the VHDJH exon, cloned it into a vector and sequenced it. The 5' primer was sufficiently degenerate to bind to most VH gene segments; the 3' primer bound to a region in JH4, which is contained in the VHDJH 17.2.25 exon inserted into QM mice. In eight sequences with a total of 2435 nucleotides, there were 79 point mutations, yielding a frequency of 3.2% (Fig. 4
). No V gene replacements, gene conversions, or genetic events other than hypermutation were apparent. Only 15 (19%) of the 79 were silent mutations. Many mutations are recurrent, a feature suggestive of antigenic selection of the mutants. In a published experiment with sorted
+, B220 PBL B cells pooled from six QM mice, there were 49 mutations in 3056 nucleotides of sequenced
1 cDNA, yielding a frequency of 1.6% in the
1 sequences (21).
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Concluding remarks
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In this paper we have described a novel population of peripheral blood B cells that lack B220 and CD19 expression. As their Ig genes are hypermutated, and as they express mostly Ig of switched isotypes, these cells are likely to have been activated by antigen. Yet they do not seem to have the size or diversity of blasts. What are these cells and why are they in the bloodstream?
Although the majority of germinal center B cells express normal levels of B220, after immunization there appears a population that expresses lower amounts (22). This population expands and eventually constitutes half of the B cells expressing GL7, a marker of germinal center B cells (22). In mice incapable of expressing the B220, B cells develop, but they cannot be induced to proliferate by cross-linking surface IgM. As the B cell activation threshold seems to be set higher in such mice, it has been suggested that B220 is a positive regulator of the B cell response (12). Similarly, in CD19 mice, there are significantly fewer B cells in the periphery and they are hyporesponsive to T-dependent antigens (1416).
We propose that the R2 population includes long-lived memory B cells. If so, it might be tempting to correlate the lack of expression of B220 or CD19 with an alteration of the threshold for activation, but this would be premature, particularly in view of the likelihood of the presence of another CD45 isoform. We also propose that these cells are in the bloodstream because they are in transit to their final destination (23). Consistent with this proposal is the fact that these cells are less prevalent or absent in mice at weaning age. Throughout the adult life, however, there is no consistent increase in the frequency of the B220/Id+ cells in the peripheral blood. We also have not seen a consistent increase in the frequency of the B220/Id+ population following immunization, which may be due to their relatively short life-span or may point to functions other than memory. In any event, because of their accessibility and likely presence in non-transgenic animals, studies to define their function ought to be facilitated.

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Fig. 5. Comparison of the 17.2.25 sequence with VHDJH sequences of clones, derived from µ, Id+ B cells from a QM mouse. All sequences are from one PCR reaction. FR, framework region; CDR, complementarity determining region. Unmutated nucleotides are labelled as `'; mutations by the incoming nucleotide; unreadable nucleotides by blanks.
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Acknowledgments
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This work was supported by NIH grant R01 AI41570, by the Engalitcheff Award of the Arthritis Foundation, by funds from the Markey Trust, and by a grant from the Junta Nacional de Investigacião Científica e TecnológicaPraxis XXI-BPD/98 to M. C. We thank Thereza Imanishi-Kari for providing anti-idiotypic antibodies to VH17.2.25.
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Abbreviations
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Id idiotype |
PBL peripheral blood lymphocyte |
PE phycoerythrin |
QM quasimonoclonal mouse |
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Notes
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Transmitting editor: L. Du Pasquier
Received 5 August 1999,
accepted 17 September 1999.
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