A cholesterol-dependent CD20 epitope detected by the FMC7 antibody


Accurate diagnosis of lymphoid malignancies is essential for appropriate therapeutic intervention. In conjunction with other diagnostic determinants, immunophenotypic analysis of differentially expressed cell surface markers, such as CD5, CD20, CD23 and FMC7, is useful in the subclassification of lymphomas and leukemias arising from the B-cell lineage. Recent evidence suggesting that CD20 predicts FMC7 expression has prompted reappraisal of the utility of monitoring both markers. Here, we report that the FMC7 monoclonal antibody (mAb) specifically and strongly recognized CD20 ectopically expressed in hematopoietic and nonhematopoietic cell lines. The reactivity of FMC7 was abolished by mutations in the extracellular domain of CD20. These data confirm the CD20 specificity of FMC7. Like other CD20 mAbs, FMC7 binding was temperature dependent and induced detergent insolubility of CD20. Of significant interest, the CD20 epitope recognized by FMC7 was unusual in that it was exceptionally sensitive to membrane cholesterol. Cholesterol depletion profoundly reduced expression of the FMC7 epitope, whereas cholesterol enrichment enhanced its expression. FMC7 mAb binding thus appears to be a sensitive indicator of the level of plasma membrane cholesterol and reveals a conformational state of CD20 that is regulated by cholesterol.


B-cell malignancies are a broad spectrum of neoplasms subclassified based on the stage of cellular differentiation and pathology. Those malignancies derived from cells of a mature phenotype comprise several lymphoproliferative disorders (LPDs) varying in clinical presentation and outcome. Diagnosis has been based on clinical features, cellular morphology, cytogenetics and the expression of antigenic determinants. One of the most reliable and widely used distinguishing features of B-cell malignancies is the differential expression of the cell surface marker FMC7. Monitoring FMC7 in conjunction with other cell surface determinants, including CD5, CD20 and CD23, has been useful in evaluating the subclassification of B-cell LPDs.1,2,3,4

There has been some controversy surrounding the identity of the cell surface marker recognized by the FMC7 antibody and, as a result, the utility of its broad use in diagnosis. Recent studies have suggested that FMC7 may recognize an epitope on CD20,5 raising uncertainty about its added benefit. CD20 is highly expressed on all normal B lymphocytes from the late pre-B-cell stage of development, until, like most surface antigens, it is downregulated upon terminal differentiation.6 FMC7 is reported to recognize a subset of CD20-positive cells, specifically during the late stages of B-cell development.7,8,9,10 LPDs with low or absent CD20, such as B-cell chronic lymphocytic leukemia, are FMC7 negative, while those in which CD20 has high expression, such as hairy-cell leukemia, are FMC7 positive.2,3,11,12,13,14,15,16 Binding of FMC7 to patient samples and to B-cell lines is blocked by prior incubation with anti-CD20 antibody,5,17 indicating apposition of FMC7 and CD20 epitopes. The detection of FMC7 after transfection of CD20 cDNA into a myeloid cell line provided the first direct evidence that FMC7 recognizes CD20.5 However, in this system CD20 was expressed on only 18% of cells and FMC7 reactivity was detected at a low level on a minor fraction of these. It has been argued that ectopic expression of CD20 in myeloid cells may have induced expression of additional cell surface markers.

Although the expression of CD20 generally predicts that of FMC7 in both normal and malignant B-cells, the level of FMC7 expression does not strictly correlate with that of CD20.18 In this report, we confirm the identity of FMC7 as CD20 and demonstrate that the level of expression of the FMC7 epitope is dependent on membrane cholesterol. These observations resolve the controversy regarding the identity of the FMC7 antigen, and explain why FMC7 monoclonal antibody (mAb) binding characteristics differ from those of other CD20 mAbs.

Materials and methods


Ramos Burkitt's lymphoma B-cells were grown in RPMI/7.5% fetal bovine serum (FBS). Molt 4 T cells expressing stably transfected human wild-type (WT) CD20 (Molt 4.CD20) or empty vector (Molt 4.V), previously established in this laboratory,19 were grown in RPMI/10% FBS, with 0.4 mg/ml geneticin (Life Technologies, Gaithersburg, MD, USA). Chinese hamster ovary (CHO) cells were grown in αMEM/10% FBS, and HEK 293 cells in DMEM/10% FBS.


B1 mAb (IgG2a) was purchased from Coulter (Miami, FL, USA), FMC7 (IgM) from Serotec (Raleigh, NC, USA), and actin antibody from Boehringer Mannheim (Mannheim, Germany). Isotype control IgG2a mAb was purchased from Southern Biotechnology Associates (Birmingham, AL, USA), while IgG2b and IgM mAbs were purchased from Sigma (St Louis, MO, USA). 2H7 mAb (IgG2b) was provided by Dr J Ledbetter (Bristol-Myers Squibb, Seattle, WA, USA). FITC-conjugated goat anti-mouse IgG and goat anti-mouse IgM were from Southern Biotechnology Associates, and Caltag (Burlingame, AL, USA), respectively. Rabbit antiserum to CD20 (anti-CD20C2) used for immunoblotting was generated using a peptide corresponding to the intracellular C-terminal (amino acids 280–297) of human CD20 conjugated to GST.

Mutagenesis and transfections

Transfection of HEK 293 cells was performed using constructs and methodology described previously.20 Briefly, the cells were grown to 50% confluence and transfected by the calcium phosphate method with human WT CD20 cDNA or extracellular domain mutant constructs. At 2 days post-transfection, cells were washed with phosphate-buffered saline (PBS; 140 mM NaCl, 2.5 mM KCl, 10 mM Na2HPO4, 2 mM KH2PO4), lifted off the culture plates by scraping, and then divided for analysis by flow cytometry. Stable transfectants of CHO cells expressing human WT CD20 were generated by electroporation at 250 V, 960 μF, with 20 μg CD20 cDNA in the BCMGSneo plasmid.21 CD20-positive cells were sorted by flow cytometry and maintained with geneticin at 0.5 mg/ml.


Cells (1 × 106 per sample) were incubated in PBS/2% FBS with FMC7, anti-CD20, or control mAbs, either at room temperature for 15 min or on ice for 30 min. Bound antibody was detected with FITC-conjugated secondary antibody and a FACScan cytometer (Becton Dickinson, San Jose, CA, USA). Mean fluorescence values were obtained using a log scale. Isotype controls were gated so that the fluorescence was within a consistent range. Calibration beads (CaliBRITE Beads; Becton Dickinson) and FACSComp software (Becton Dickinson) were used daily to monitor instrument performance, set fluorescence compensation and maintain quality control. CellQuest Pro software (Becton Dickinson) was used for data analysis.

Cholesterol modulation

Ramos cells were treated with 10 mM methyl-beta-cyclodextrin (MβCD; Sigma, Oakville, ON, Canada) or with cholesterol: MβCD complex (1.4 mM cholesterol in 10 mM MβCD) (Sigma) for 15 min at room temperature, and washed prior to labeling. Where indicated, cells were treated with cholesterol:MβCD complex at 15 min intervals for 2 h. Subsequent labeling steps were performed in PBS to avoid cholesterol repletion by FBS.

Estimation of CD20 detergent solubility

Experiments examining CD20 solubility in the nonionic detergent, Triton X-100, were performed essentially as previously described.22 Briefly, 106 cells were incubated with 1 mg FMC7, 2H7 or isotype control antibody for 15 min at 37°C, washed and then lysed in 1% Triton X-100 lysis buffer containing protease inhibitors (1 μg/ml aprotinin, 1 μg/ml leupeptin, 1 mM NaVO4, 1 mM NaMoO4, 1 mM PMSF, 1 mM EDTA). The detergent-insoluble material was pelleted at 13 000 rpm for 15 min at 4°C. The supernatants were transferred to clean tubes containing an equal volume of 2 × SDS sample buffer. The insoluble pellets were washed three times in lysis buffer and then solubilized in 2 × SDS sample buffer. Samples were separated by SDS-PAGE under reducing conditions and transferred to Immobilin P (Millipore, Bedford, MA, USA). Prestained MW markers (New England Biolabs, Beverly, MA, USA) were run on each gel. Membranes were blocked in 5% bovine serum albumin, and incubated with anti-CD20C2 or antiactin Abs detected with protein A–horseradish peroxidase (Bio-Rad, Richmond, CA, USA). Proteins were visualized using enhanced chemiluminescence (Pierce, Rockford, IL, USA) and recorded using a Fluor-S MAX imager (Biorad, Mississauga, ON, Canada).


FMC7 recognizes CD20 ectopically expressed in hematopoietic and nonhematopoietic cells

FMC7 was previously shown to recognize an epitope expressed in a myeloid cell line after transient transfection of CD20 cDNA.5 However, in that study FMC7 bound to only 7% of transfected cells, 18% of which were CD20 positive, and the level of binding was low. We investigated FMC7 reactivity using a human T-cell line stably expressing a high level of CD20 on all cells (Molt4.CD20).19 Figure 1 shows that FMC7 bound strongly to Molt4.CD20, but not to cells transfected with the vector alone. In order to address the unlikely possibility that ectopic expression of CD20 in a human lymphoid cell line may have upregulated an unrelated protein recognized by FMC7, we expressed CD20 in rodent fibroblasts (CHO.CD20). Again, FMC7 bound strongly to CHO.CD20, but not to CHO cells transfected with the vector alone (Figure 1).

Figure 1

FMC7 binds to CD20 ectopically expressed in hematopoietic and nonhematopoietic cells. Molt 4 cells and CHO cells stably transfected with either CD20 (Molt 4.CD20; CHO.CD20) or vector alone (Molt 4.V; CHO.V) were incubated at room temperature with isotype control (dashed line), B1 or FMC7 (solid lines). Bound antibody was detected with appropriate FITC-labeled secondary antibody. Mean fluorescence intensities are indicated in the top right corner of each panel. Data shown are representative of seven (Molt 4) and two (CHO) independent experiments.

FMC7 reactivity is abrogated by mutagenesis of CD20

Recently, we demonstrated that CD20 extracellular epitopes depend on two residues, alanine and proline at positions 170 and 172, respectively (AxP; single-letter amino-acid codes, x indicates the identical amino acid at the same position in both murine and human CD20 sequences). Mutation of these residues to serines (AxP/SxS) abolished binding of CD20 mAbs.20 FMC7 reactivity against the AxP/SxS construct was tested to provide additional confirmation that FMC7 recognizes CD20 and not a molecule coordinately upregulated in transfected cells with the CD20 cDNA construct. FMC7 binding to HEK 293 cells that transiently expressed WT CD20 was similar to that of CD20 mAbs 2H7 and B1 (Figure 2). Replacement of the extracellular domain of human CD20 with the murine sequence (h/m CD20) eliminated binding of FMC7, as well as that of mAbs 2H7 and B1. As previously described, the B1 epitope, but not the 2H7 epitope, was completely reconstituted in the h/m CD20 chimera by replacing serine residues in the murine sequence with alanine and proline from the equivalent positions in the human sequence (SxS/AxP).20 The FMC7 epitope was reproducibly recovered to a low level in independent experiments. Taken together with the data shown in Figure 1, these results confirm that the FMC7 epitope is defined by CD20 amino-acid residues.

Figure 2

FMC7 reactivity is abolished by CD20 mutagenesis. HEK 293 cells transiently expressing either human WT CD20 or extracellular domain mutant constructs were incubated at room temperature with 2H7, B1, FMC7, or a mixture of appropriate isotype controls (CT). AxP/SxS represents mutation of alanine and proline at positions 170 and 172 in the human sequence to serine residues. Human CD20 containing the murine extracellular domain sequence is represented as h/m CD20. Mutation of serines at positions 170 and 172 in the h/m CD20 construct to alanine and proline is represented by SxS/AxP. Mean fluorescence intensities (MFI) from the experiment shown in the upper panel are expressed in the bar graph below. Data are representative of two independent experiments.

FMC7 induces CD20 insolubility in the nonionic detergent 1% Triton X-100

Previously, we have shown that mAbs directed against extracellular CD20 epitopes induce translocation of CD20 from the soluble to the insoluble fractions of 1% Triton X-100 cell lysates, and that this reflects association with cholesterol and glycosphingolipid-enriched plasma membrane microdomains known as lipid rafts.22 To determine whether FMC7 also exhibited this property, CD20 solubility was examined after FMC7 ligation and compared to that induced by 2H7. In Molt 4.CD20 cells, FMC7 ligation increased the amount of CD20 in the detergent-insoluble fraction, to an extent similar to that of 2H7, with a corresponding reduction in the soluble fraction (Figure 3). Similar results were obtained using Ramos cells, except that the degree of translocation induced by FMC7 was less than that of 2H7. This could be attributed to the reduced binding of FMC7 to Ramos cells (see below).

Figure 3

FMC7 induces CD20 translocation to detergent-insoluble fractions. Ramos B-cells and Molt 4.CD20 cells were incubated with either isotype control (CT), FMC7 or 2H7 at 37°C for 15 min and lysed in 1% Triton X-100. Detergent-insoluble pellets (1.2 × 106 cell equivalents/sample) and soluble lysates (3 × 105 cell equivalents/sample) were analyzed by SDS-PAGE and anti-CD20 immunoblot. Actin blots were performed on the same membranes. Data are representative of four independent experiments.

FMC7 binding to CD20 is temperature dependent

Analysis of cell surface antigen expression is often performed with antibody incubations on ice. In the case of CD20, we have observed that antibody binding is enhanced at room temperature. Like B1, FMC7 binding to Molt 4.CD20 was significantly increased at room temperature, as compared to cells stained on ice (Figure 4). In Ramos cells, FMC7 binding was very low when incubations were performed on ice, and a dramatic increase was observed with incubation at room temperature (Figure 4). Interestingly, however, FMC7 binding to Ramos cells never reached the high level of binding observed with transfected Molt 4 or CHO cells.

Figure 4

FMC7 binding to CD20 is temperature dependent. Molt 4.V, Molt 4.CD20 and Ramos B-cells were incubated with either isotype control (dashed line), FMC7 or B1 (solid lines) followed by appropriate FITC-labeled secondary antibodies, either on ice or at room temperature, as indicated. Dotted vertical lines mark peak binding of antibody to each cell line at 22°C. Numbers in each panel are mean fluorescence intensity. Data are representative of four independent experiments.

The FMC7 epitope is cholesterol dependent

The temperature dependence of FMC7 reactivity suggested that membrane fluidity – and, in turn, CD20 mobility – might influence antibody binding. Since membrane fluidity is also related to cholesterol content,23 the effect of cholesterol depletion on FMC7 binding was examined. MβCD is a cyclic oligosaccharide with a hydrophobic core that extracts cholesterol from the plasma membrane without binding to or crossing the membrane.24,25 Remarkably, FMC7 binding to MβCD-treated Ramos cells was greatly diminished as compared to untreated cells (Figure 5a). This effect was specific for cholesterol since treatment with MβCD that was pre-loaded with cholesterol enhanced FMC7 binding (Figure 5a). To a much lesser extent, cholesterol depletion also reduced binding of B1 (Figure 5a) and other CD20 mAbs, but not a control (anti-CD45) mAb (not shown). When cholesterol was replenished after depletion, FMC7 binding was completely recovered (Figure 5b).

Figure 5

FMC7 recognizes a cholesterol-dependent CD20 epitope. (a) Ramos cells were either untreated (top panel), treated with 10 mM MβCD (middle panel) or with cholesterol: MβCD complex (MβCD/cholesterol; bottom panel) for 10 min at room temperature before incubating with either isotype control (dashed line), B1 or FMC7 (solid lines). Data are representative of six independent experiments. (b) Ramos cells were either untreated or treated with 10 mM MβCD for 15 min at room temperature. Cells were then either untreated or treated with cholesterol:MβCD complex (MβCD/cholesterol) added at 15 min intervals for 2 h at room temperature. Cells were labeled with either isotype control (dashed line) or FMC7 (solid line). Data are representative of three independent experiments.


The specificity of the FMC7 mAb has been the subject of long-standing interest because of its importance as a diagnostic reagent, yet it has been difficult to elucidate. The results reported here confirm the identity of the FMC7 antigen as CD20 by several criteria including: (1) strong recognition of CD20 ectopically expressed in hematopoietic and nonhematopoietic cell lines, (2) elimination of FMC7 reactivity by CD20 extracellular domain mutations, (3) partial recovery of FMC7 reactivity in the SxS/AxP h/m CD20 chimera; and (4) FMC7 induction of CD20 detergent insolubility. However, although the FMC7 mAb is clearly directed against CD20, the epitope is cholesterol dependent and its reactivity is low in cells with reduced membrane cholesterol even when CD20 expression is high. Interestingly, FMC7 binds much more strongly to CD20 expressed ectopically in Molt 4 T cells and CHO cells than to endogenously expressed CD20. This suggests that B lymphocytes have low membrane cholesterol relative to other cell types, or that there is an additional factor in B-cells suppressing expression of the FMC7 epitope. For example, the association of CD20 with another cell surface protein expressed in B-cells, but not in T cells or CHO cells, may mask the FMC7 epitope.

We have previously reported evidence that CD20 exists in an approximate 200 kDa multimeric molecular complex.20 We considered the possibility that FMC7 reactivity might depend on an intact complex that was disrupted in low cholesterol conditions; however, the size of the complex was unaltered by MβCD treatment, indicating that the integrity of the complex was unaffected (data not shown). Expression of the FMC7 epitope is also highly temperature dependent, with much lower reactivity observed when incubations were performed on ice. Presumably, this effect is because of reduced membrane fluidity and lateral mobility of CD20 at low temperature. However, the cholesterol dependence of the FMC7 epitope cannot be explained by effects on membrane fluidity, since reduced cholesterol is expected to increase fluidity23 and also does not affect the binding of other CD20 antibodies to such a degree.

The FMC7 requirement for cholesterol is likely to be related to alterations in the plasma membrane influencing the conformation of CD20 and/or accessibility of the epitope. Cholesterol is unevenly distributed in the membrane and forms liquid-ordered microdomains, that is, lipid rafts, in the presence of saturated fatty acid membrane components (sphingomyelin and sphingolipids).26,27 The integrity of lipid rafts depends on cholesterol, depletion of which disrupts and dissolves them. We have reported previously that CD20 inducibly associates with rafts after antibody ligation.22 Recent evidence indicates that the association is constitutive and only the affinity of the association is increased with antibody binding (L Ayer et al, manuscript in preparation). FMC7 may therefore detect CD20 that is preferentially associated with lipid rafts.

While the cholesterol dependence of the FMC7 epitope is most likely because of an influence of the plasma membrane microenvironment on the conformation of CD20, it is also possible that there is a direct association between CD20 and cholesterol. The ability of MβCD to reduce FMC7 binding indicates that any direct association between CD20 and cholesterol is noncovalent, as reported for several other integral membrane proteins – for example, the oxytocin receptor and nicotinic acetylcholine receptor.28,29 Cholesterol association modulates the affinity of the oxytocin receptor for its ligand30,31 and is essential for proper ion channel function of the nicotinic acetylcholine receptor.32 Similarly, fluctuating levels of membrane cholesterol during B-cell development or activation may alter the conformation of CD20 and modulate its putative calcium channel function.33,34

Previously, we demonstrated an indirect association between CD20 and src family kinases,35,36 and recently discussed evidence that this is a result of mutual association with lipid rafts.37 Src family kinase-dependent apoptotic signals can ensue from CD20 crosslinking38,39 and this has been proposed to account for some of the efficacy of rituximab, a CD20-specific antibody used in treating B-cell cancers and some autoimmune diseases.40 It is likely that CD20 crosslinking activates src kinases as a consequence of lipid raft aggregation.37 In addition, recent evidence indicates that complement-mediated lysis is mediated efficiently by CD20 antibodies as a consequence of CD20's residency in lipid rafts.41 The efficiency with which CD20 antibodies can induce cell death, either by apoptosis or by complement-mediated lysis, is therefore predicted to correlate with the level of membrane cholesterol. It will be important to assess whether FMC7 reactivity might be a useful predictor of clinical response to rituximab and other CD20-directed therapeutics.

In conclusion, this study reveals an influence of membrane cholesterol on the conformation of CD20. FMC7 reactivity varies with the level of membrane cholesterol, indicating that in those CD20-positive B-cell malignancies where FMC7 is low or negative, membrane cholesterol is likely to be correspondingly low. Plasma membrane cholesterol may be an important diagnostic characteristic, since lymphoma cell lines having an overall greater membrane fluidity have higher metastatic potential.42 The utility of FMC7 as a diagnostic reagent may lie in its ability to report differences in the lipid composition of malignant B-cells.


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We thank L Robertson for assistance with flow cytometry and Dr A Kossakowska for helpful comments on the manuscript. JPD is an Alberta Heritage Foundation for Medical Research Senior Scholar. This research was supported by the Canadian Institutes of Health Research.

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Correspondence to J P Deans.

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This paper is dedicated to the memory of Dr Stefan Serke.

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Polyak, M., Ayer, L., Szczepek, A. et al. A cholesterol-dependent CD20 epitope detected by the FMC7 antibody. Leukemia 17, 1384–1389 (2003).

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  • CD20
  • FMC7
  • cholesterol
  • epitope

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