Cell Characterization
Characterization of
specific subpopulations, developmental stage, and activation status of isolated
splenic B cells was performed by multiparameter FACS analysis 
. The analysis
described here uses specific antibodies coupled to one of four fluorochromes:
fluorescein isothiocyanate (FITC), R-phycoerythrin (PE), peridinin chlorophyll-a
(PcP), or allophycocyanin (APC). These fluorochromes were used concurrently to
assess the cell-surface expression of four different markers within the same
sample. Isotype-matched antibodies against keyhole limpet hemocyanin (KLH) were
used as controls.
Purity. The purity of the B cells obtained from mixed splenocyte populations was assessed in part by expression of the B220 isoform of CD45 (leukocyte common antigen, Ly-5, or T200) (13, 14, 15). Although B cells are the predominant leukocyte expressing B220, the marker is also displayed by subsets of NK cells, activated T cells, and a subpopulation of dendritic cells (16). Hence, the purity of our preparations cannot be assessed by expression of B220 alone. The absence of T-cell and myeloid-cell markers, in addition to the concomitant expression of CD23 and membrane-bound IgD and IgM, were also used to evaluate heterogeneity within the cell population.
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Fig. 4. Size and uniformity of splenic B cells. B cells profiled using forward scatter (FSC) and side scatter (SSC), viewed on dot plot (A) or contour plot (B). Dead cells falling in the bottom left corner of region 1 (R1) are colored red. R2 and R3 are drawn within R1, marking smaller less granular cells (R2, green) and larger more granular cells (R3, blue). |
Although the vast majority of negatively selected cells are characterized as B220pos, CD3neg, Mac-1neg, and IgDpos (consistent with mature resting B cells), the enriched B cells are not a homogeneous population (Figs. 4 and 5). Forward scatter (FSC) and side scatter (SSC) analysis of the selected B cells consistently showed a subpopulation of relatively larger cells, marked as region 3 (R3) (Fig. 4), that carry the same markers as the smaller cells in region 2 (R2). These larger cells may represent cell pairs. If so, pairs are maintained in the presence of 0.5 mM EDTA, and these cellular interactions may be particularly tight. These cells typically constitute less than 15% of the negative fraction. Cells in the bottom left corner of region (R1) (Fig. 4) are dead.
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Fig. 5. Purity of B-cell preparations. Live cells (R2 and R3, Fig. 4) were examined. Cells stained positively for B220 but not (A) CD3 (T-cell marker) or (C) Mac-1 (myeloid cell marker). (E) The majority (~81%) of B220 positive cells also stained positively for membrane IgD, which is present on mature B cells. (G) Most cells (64.8%) stained for both IgD and IgM, although some were positive only for IgD (13.2%) or IgM (7.7%). Plots of cells stained with isotype-matched control antibodies against keyhole limpet hemocyanin (KLH) coupled to FITC, PE, PcP, or APC (B,D,F,H) are shown to the right of their corresponding test pair (A,C,E,G).
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B cells are classified into two major groups based on their ontogeny (17). B2 cells are bone marrow-derived and make up the bulk of splenic B cells. B2 cells characteristically have high levels of B220, membrane-bound IgD, and CD23 (IgE Fc Receptor, Fce). Conversely, B1 cells arise from precursors present during fetal development and reside predominantly in the pleural and peritoneal cavities of the adult mouse. B1 cells usually display low-to-intermediate amounts of B220, no or intermediate amounts of CD23, and no or low levels of IgD. B1 cells share some of these characteristics with marginal zone (MZ) B cells. Hence, examination of the levels of B220 and CD23 allow the B2 cells to be distinguished from B1, MZ, and transitional immature B cells (T1 or T2, newly arrived in the spleen). Analysis of the B220pos fraction suggests that roughly 80% to 85% of the selected B cells are of the B2 type, as defined by expression of IgD (Fig. 5E) and CD23 (Fig. 6B and D).
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Fig. 6. Heterogeneity of B cells. Live cells (R2 and R3, Fig. 4) were examined. (A,C) B-cell subpopulations were identified by anti-CD21-FITC versus anti-CD23-PE staining. Follicular B cells express high levels of CD23 and CD21 and are marked in the FO region. Marginal zone B cells express high levels of CD21 but do not express CD23, and are marked in the MZ region. (B,D) B-cell subpopulations were identified by anti-B220-PcP and anti-CD23-PE staining. B2 cells express B220 and high levels of CD23. B1 cells express B220 but have little or no CD23. (E,F) Cells stained with isotype-matched antibodies against keyhole limpet hemocyanin (KLH) coupled to FITC, PE, or PcP. |
Splenic MZ B cells, like B1 cells, are a noncirculating population of B cells distinct from the predominant B2 follicular (FO) population (18). MZ B cells express high levels of membrane-bound IgM and the complement receptors, CR2 and CR1 (CD21 and CD35), while having little or no CD23. MZ B cells normally compose only a small fraction of the splenic B-cell population, as shown in Fig. 6A and C, and B1 cells constitute even a smaller fraction. (Note that the monoclonal antibody used to stain CD21 recognizes an epitope shared by CR2 and CR1).
Analysis of 23 distinct B-cell preparations is shown in Supplemental Table A. Purity was determined as described above. The data show that we consistently isolate splenic B cells that are primarily (>80%) composed of mature B2 cells.
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Fig. 7. Expression of activation and adhesion markers on isolated B cells. Expression of CD25, CD69, CD80, CD86, CD30, Ly-77, CD29, CD54, and CD62L was evaluated on B cells by four-color flow cytometry using fluorochrome-conjugated antibodies. All samples were gated on B220. Each histogram is representative of four experiments. The filled histograms represent cells stained with an isotype control antibody. The open histograms represent staining for the specific surface marker.
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Activation State. The expression of common lymphocyte activation markers was also examined by flow cytometry. Freshly isolated cells were stained for B220 and CD69, CD25, CD80, CD86, CD30, or Ly-77. Fig. 7 shows histograms of cells stained with each marker compared to a corresponding isotype-matched antibody control. B220pos-gated cells show low levels of CD69 (very early antigen) and CD25 (a chain of the IL-2 receptor), both of which can be up-regulated on T and B cells after activation (19, 20). Similarly, CD80, a coreceptor induced on B cells and necessary for T-cell activation (21), is expressed at low levels in our B-cell population. CD86 (B7-2) and CD30 were not detected. Interestingly, Ly-77 is expressed heterogeneously within this cell population. Taken together with the low percentage of cells observed within the S, G2, and M phases of the cell cycle (Fig. 3), these data indicate that the cells are in a resting state.
We also evaluated expression of adhesion markers commonly found in B cells (Fig. 7). Within the isolated B220pos B-cell fraction, CD29 (integrin b1 chain) is expressed at a low level by some cells. CD62L (L-selectin) is highly expressed with a broad distribution, and CD54 (LFA-1) is expressed rather homogeneously. This again demonstrates some heterogeneity within the isolated B-cell population.
Gene
Expression. A profile of gene expression in isolated splenic B cells was
obtained in a collaborative effort with the Genomics Institute of the Novartis
Research Foundation (GNF)(22). Two independent experiments were performed using total RNA purified from
separate B-cell preparations . Expression was examined with mouse Affymetrix GeneChip arrays (MGU74Av2)
using a standard protocol described by Affymetrix. Average difference values that estimate transcript expression levels were
calculated, and judgments were made as to the presence or absence of each transcript. Equivocal results were termed marginal.
Data can be viewed in the Supplemental Data file.
Of 12,488 probe sets screened, 3,664 (29%) yielded positive results in both hybridization experiments. As expected, many probe sets with high expression values represent genes normally expressed in B cells. For instance, transcripts for CD45, CD22, CD23, CD37, CXCR4, CXCR5, and B-cell receptor components (e.g., heavy chain of IgM) were present, whereas transcripts for proteins typically expressed in T cells such as CD3 and CD8, were absent. Many transcripts that are restricted to the myeloid lineage were also absent, although expression of CD14 and CD68 was suggested. Several transcripts typically restricted to erythroid cells (e.g., kruppel-like factor 1, hemoglobin alpha and beta chains) were also present. The data are consistent with the characterization performed by FACS and suggest the presence of few myeloid and erythroid cells in our preparations.