A flow cytometric rosetting assay for the analysis of IgG-Fc receptor interactions

Journal of Immunological Methods, 127 (1990) 207-214

207

Elsevier JIM 05479

A flow cytometric rosetting assay for the analysis of IgG-Fc receptor interactions W o u t e r B. T u i j n m a n *, J a n G.J. V a n de Winkel a n d P e t e r J.A. C a p e l Department of Experimental Immunology, University of Utrecht, Utrecht, The Netherlands

(Received 3 August 1989, revised received 2 October 1989, accepted 30 October 1989)

We have developed a sensitive and flexible method for the qualitative evaluation of IgG-Fc receptor interactions in cell suspensions. The assay is based on the flow cytometric quantitation of antibody-coated erythrocyte (EA) rosetting using fluorescein-labelled indicator erythrocytes (E). The number of IgG molecules on indicator E, an important parameter in EA rosetting, was estimated by calibrated flow cytometry. EA binding quantitated by this method was correlated with microscopically evaluated rosette formation. Besides automated quantitation of EA binding, this method offers the additional advantage of simultaneously using a second fluorescence parameter, permitting analysis of FcR activity in subpopulations of cells. As an example of the applicability of this approach the binding characteristics of E sensitized with a series of murine heavy chain isotype switch variant monoclonal antibodies against glycophorin A, to the low affinity receptor on K562 ceils were determined. Remarkably, the results suggest a comparable affinity of F c y R I I on these cells for immunoglobulins of the murine IgG1, IgG2a and IgG2b isotypes. Key words: Antibody-coated erythrocyte rosetting; Fc receptor, human; Flow cytometric assay

Introduction Receptors for the Fc portion of IgG play an important role in both the regulatory and effector arms of the immune response. Three different types of F c ' r R are currently recognized on human ieukocytes. These differ in molecular size, affinity for iigands, ligand specificity, cellular distribution

Correspondence to: W. Tuijnman, Department of Experimental Immunology, University Hospital Utrecht, G.04.614, P.O. Box 85500, 3508 GA Utrecht, The Netherlands. Abbreoiations: E, human erythrocytes; EA, erythroeyte-antibody complex; FcyR, receptor for the Fc part of lgG; FCS, foetal calf serum; FITC, fluorescein isothiocyanate; h, human; m, mouse; mAb, monoclonal antibody; PBS, phosphatebuffered saline; BSA, bovine serum albumin; PE, phycoerythrin; PI, propidium iodide; SD, standard deviation.

and in reactivity with monoclonal anti-FcTR antibodies. F c y R I (CD64) has a molecular weight of 72,000 and binds monomeric IgG with high affinity. Fc~,RII (CDw32) is a low affinity 40 kDa receptor that interacts weakly with hlgG, but cross-reacts with mlgG1 in immunecomplex form. The third type, F c ' t R I I I (CD16) is a 45-70 kDa glycoprotein which can bind only multimeric lgG (for reviews see Anderson and Looney, 1986; Unkeless et al., 1988; Fanger et al., 1989). A variety of methods for the detection of IgGFc~,R interactions have been described (for review see Kerbel and Elliott, 1983). Of these, EA rosette assays are well established and sensitive. To overcome the problems associated with visual quantitation, assays have been developed to measure rosette formation by other means. For example,

0022-1759/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)

208 EA binding can be scored either using radioactively labelled indicator cells (Jungi, 1985), photometrically employing the haemoglobin absorbance of E directly (Rummage and Leu, 1985), or using the pseudoperoxidase activity of human erythrocytes (Van de Winkel et al., 1987a). None of these assays, however, can discriminate between individual cells. In order to be able to study IgG-Fc receptor interactions on a subpopulation of cells, we have developed a new method to quantitate EA rosetting. Cells are incubated with indicator erythrocytes labelled with fluorescein and sensitized with calibrated amounts of antibody, and rosette formation is evaluated flow cytometrically. Analysis of the samples is rapid, reproducible and permits the use of a second fluorescence parameter, by which a subpopulation of cells can be analysed for rosette-forming capacity.

Materials and methods

Cell lines Human monocytic (U937) and erythromyeloid (K562) cell lines were cultured at low density (maximally 3 × 105 cells/ml) in RPMI 1640, supplemented with 5% heat-inactivated FCS, 2 mM glutamine, 1 mM sodium pyruvate and 5 0 / t g / m l gentamicin. Viability was assessed either microscopically using trypan blue exclusion, or flow cytometrically by staining with propidium iodide (Sigma, St. Louis, MO), and always exceeded 95%. A n tibodies A murine IgG1 mAb against glycophorin A was obtained from M. Bos, CLB, Amsterdam. Heavy chain isotype switch variants of this mAb, of mlgG2a and mlgG2b isotypes, were prepared by Dr. Boot, CLB, Amsterdam. Removal of contaminating Ig isotypes from the different antibody preparations was performed by incubation with an excess of rat mAb against mlG2a or IgG2b (Boot et ai., 1988) or with a goat anti-mlgG1 antiserum (Southern Biotechnology, Birmingham, AL), all coupled to Sepharose 4B (Pharmacia, Uppsala, Sweden). F(ab')2 fragments of the mlgG1 mAb were prepared as described previously and contained no uncleaved molecules as revealed by

SDS-PAGE (Van de Winkel et al., 1987a). Human antiserum against Rhesus D was obtained from Merz and Dade (Diidingen, Switzerland). Monoclonal antibodies KB61 (Pulford et al., 1986) and IV.3 (Looney et al., 1986) against FcTRII were gifts from Drs. Pulford and Anderson, respectively. Monoclonal antibody VB2 was produced in our own laboratory after immunization of B A L B / c mice with monocytes and fusion with S P 2 / 0 cells. This mlgG2a mAb reacts with an antigen which is expressed in high density on most human leukocytes including U937 cells, but is absent on K562 cells. VB2 was purified by affinity chromatography with protein G Sepharose (Pharmacia) and biotinylated using biotin-succinimide ester (Sigma). Streptavidine-PE was obtained from Tago (Burlingame, CA). Fluorescein labelling and sensitization of erythro~ytes For labelling of indicator E, a 5% suspension of Rhesus D-positive human red blood cells in PBS was incubated with 0.1 m g / m l FITC in PBS (BBL, Cockeysville, MD) for 30 min at 37°C. For microscopic evaluation of rosette formation indicator E were left unlabelled. After washing once with PBS, a 0.5% suspension of FITC-labelled, or unlabelled, E was incubated with various subhaemagglutinating dilutions of anti-Rhesus D antiserum, various subclasses of anti-glycophorin A mAb or PBS for 30 min at 37°C. During the incubation cells were kept in suspension by gentle agitation. EA were washed twice with PBS, resuspended in RPMI 1640 with 5% FCS, and stored at 4 ° C until use (within 30 min). EA rosette formation and analysis Rosettes were formed by mixing EA and cells at a ratio of 8 : 1, except when indicated otherwise in the results section, centrifuged (5 rain, 20 x g) and incubated for 1 h at 4 ° C . In rosette inhibition experiments, cells were incubated with anti-FcyR mAb for 30 rain at 4 ° C prior to addition of EA. Samples were carefully resuspended before analysis. For double fluorescence experiments using PE as a second parameter, cells were stained prior to the addition of EA. Cells were therefore incubated

209 with a biotinylated antibody for 45 rain at 4 ° C . After washing with PBS containing 0.2% BSA (PBS-BSA), the streptavidin-PE conjugate was added and incubated for 45 min at 4 ° C . The cells were washed again and rosetted as above. PE fluorescence was measured above 585 nm. For double fluorescence using PI as a second parameter rosettes were formed as above. 5 min before analysis 10 /tg of PI in PBS were added to the sample (150/~1) and emission was measured above 630 nm. For microscopic quantitation of rosette formation 300 cells were examined in each sample and scored positive when at least one erythrocyte was bound. Unsensitized E were included as a control in each experiment, and never showed any binding. Flow cytometric quantitation of rosettes was performed using FITC-labelled EA. In each sample 5000 cells were examined with a FACStar (Becton Dickinson, Mountain View, CA) equipped with an argon laser emitting at 488 nm. Windows were set in the scatter patterns in such a way that free EA were not recorded. Quantitation of the fraction of cells that bound at least one indicator E, was based on the F I T C fluorescence, being either of high (rosette), or low intensity (unconjugated ceil). A marker was placed between these two populations and the percentage of rosette forming cells calculated. Background rosetting with unsensitized erythrocytes amounted to 3.6 + 2% (mean + SD, n = 20). Measurements were performed at low sheath pressure (5 psi) in order to minimize mechanical forces which might disturb interactions between rosette-forming cells and EA. The flow rate was set at 1000 particles/s (including unbound EA) by adjustment of the sample pressure. Higher flow rates resulted in significantly increased background rosetting.

Quantitation of the degree of erythrocyte sensitization The number of IgG molecules bound per indicator erythrocyte at given dilutions of mAb preparations was determined flow cytometrically as described by Poncelet and Carayon (1985). In each experiment the flow cytometer was calibrated using quantitative fluorescein standards (Flow Cytometry Standards Corporation, Research Trian-

gle Park, NC) as described by Le Bouteiller et al. (1983). The mean fluorescence intensity of eight different latex beads was determined and a standard curve was constructed, which was linear over the whole range (P(least s q u a r e s ) > 0.9994, (n = 4)). E sensitized as described above, were incubated with FITC-conjugated F(ab')2 fragments of goat anti-mouse Ig (Tago) for 45 min at 4 ° C . After washing with PBS-BSA, the mean fluorescence intensity was measured. The relative number of mAb molecules detected was determined after correction for non-specific binding and autofluorescence of E, using the calibration curve. In order to be able to correct for quenching and variations in binding of the conjugate to the different sensitizing Abs, an internal standard was used. This consisted of beads with a known number of lg binding sites (Flow Cytometry Standards Corporation). These were incubated with saturating amounts of various anti-glycophorin A m A b for 45 rain at 4 °C, while gently shaking. After washing with PBS-BSA, the fluorescence intensity was measured and the relative binding ratio of the F I T C - F ( a b ' ) z conjugate to each of the mAbs was determined. The number of anti-glycophorin A mAb bound per erythrocyte at various dilutions of the mAbs was then calculated by dividing the relative number of mAb molecules detected, by the above ratio.

Results

Parameters affecting EA rosette formation quantitared by the flow cytometric method Flow cytometry was used to analyse EA rosette formation with FITC-labelled indicator erythrocytes. The low affinity interaction between human F c y R I l on K562 cells and E sensitized with a mlgG1 mAb against glycophorin A (EA-mlgG1) was employed as a model. Using optimally sensitized EA-mlgG1, two populations with different fluorescence intensity were observed by flowcytometry (Fig. 1). The ratio of particles having a high fluorescence intensity relative to those with low intensity, correlated well with the microscopically determined number of cells binding at least one indicator erythrocyte. This binding of EAmlgG1 to K562 cells was 90% inhibitable by anti-

210 F c ' / R I I m A b IV.3 a n d KB61. Rosette f o r m a t i o n of E sensitized with a s a t u r a t i n g a m o u n t of F ( a b ' ) 2 fragments of the m l g G 1 a n t i - g l y c o p h o r i n A m A b was observed to be 3.1 + 1.1% ( m e a n + SD, n = 3), which is similar to rosetting unsensitized E (3.6 + 2%, m e a n + SD, n = 20). T h e n u m b e r of rosettes formed between K562 cells and E A - m l g G 1 was strongly influenced by the ratio of i n d i c a t o r EA to cells (Fig. 1). F l u o r e s cence histograms show that this ratio p r o f o u n d l y affected b o t h the p e r c e n t a g e of rosettes, as well as the fluorescence intensity. Increasing fluorescence intensity c o r r e l a t e d well with an increased n u m b e r of E A b o u n d per cell, q u a n t i t a t e d m i c r o s c o p i c a l l y (not shown). A n o t h e r p a r a m e t e r influencing the degree of rosette f o r m a t i o n was the n u m b e r of IgG molecules b o u n d to i n d i c a t o r E. W e d e t e r m i n e d the n u m b e r of m l g G 1 molecules b o u n d p e r e r y t h r o cyte at various d i l u t i o n s of sensitizing a n t i s e r u m using c a l i b r a t e d indirect i m m u n o f l u o r e s c e n c e . T h e degree of i n d i c a t o r E sensitization e s t i m a t e d by this m e t h o d c o r r e l a t e d exactly with the level of b i n d i n g d e t e r m i n e d previously b y a r a d i o m e t r i c a l m e t h o d , e m p l o y i n g the same batch of a n t i b o d y (Van de W i n k e i et al., 1988). Using this c a l i b r a t e d antiserum, F I T C - l a b e l l e d e r y t h r o c y t e s were c o a t e d

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390 140 .50 13 0 mlgG1 molecules / erythrocyte (x 10-3) Fig. 2. Rosette formation between K562 cells and E sensitized with different numbers of murine IgG1 molecules per indicator cell. Five different ratios of EA to K562 are shown; 32 : 1 (O), 16:1 (v), 8:1 (e), 4:1 (zx) and 2:1 (E]). Each point represents the mean of four individual experiments. The conditions used for the remaining studies are indicated by the closed symbols. For clarity SD are only shown for these conditions. with k n o w n a m o u n t s of m l g G 1 a n t i - g l y c o p h o r i n A m A b a n d tested for rosetting at different E A to K562 ratios (from 2 : 1 up to 3 2 : 1 , Fig. 2). T h e results show that with increasing a n t i b o d y d e n s i t y a p l a t e a u value was reached a b o v e 1 4 0 x 10 3 molecules p e r E, but the level of this p l a t e a u was d e p e n d e n t on the ratio of E A to K562 used. F o r practical reasons (e.g., the a m o u n t of time n e e d e d to measure a sample) a ratio of 8 : 1 ( i n d i c a t e d with closed s y m b o l s in the figure) was used for the r e m a i n i n g studies.

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Fig. 1. Fluorescence histogram showing rosette formation between K562 cells and FITC-labelled human erythrocytes, optimally sensitized with a murine IgG1 anti-glycophorin A antibody. K562 cells were incubated with EA at ratios of 1:32 ( ), 1:16 ( . . . . . . ), 1:8 (. . . . . . ) and 1:4 ( . . . . ). The percentage of EA-binding K562 cells was 86, 80, 61 and 49% under these conditions respectively.

A s s a y reliability was e v a l u a t e d by q u a n t i t a t i n g E A b i n d i n g b o t h by c o n v e n t i o n a l m i c r o s c o p i c exa m i n a t i o n a n d by flow c y t o m e t r y . I n d i c a t o r E A m l g G 1 a n d K562 cells, which express a high n u m ber of F c T R I I a n d no o t h e r k n o w n F c receptor, were used as a m o d e l for low-affinity interactions. N o rosetting was o b s e r v e d b e t w e e n these cells and h u m a n I g G - s e n s i t i z e d erythrocytes, which readily interact with F c y R I (Van de W i n k e l et al., 1987b). In o r d e r to s t u d y high affinity rosetting interactions, i n d i c a t o r E sensitized with h u m a n antirhesus D a n t i b o d y a n d U937 cells which express b o t h F c - / R I a n d F c , / R I I ( F a n g e r et al., 1989) were used. I n d i c a t o r E were sensitized with various a m o u n t s of IgG, rosettes were f o r m e d a n d after resuspension, s a m p l e s were e x a m i n e d b o t h

211 visually a n d by flow c y t o m e t r y . A n excellent correlation was o b s e r v e d for b o t h types of F c ~ , R - E A i n t e r a c t i o n s (Fig. 3). T h e degree of i n d i c a t o r cell sensitization at which h a l f - m a x i m a l rosetting was o b s e r v e d was m u c h smaller for F c y R I - than for F c ~ , R I l - m e d i a t e d rosette formation.

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W e used a mixed cell p o p u l a t i o n to test the a p p l i c a b i l i t y of the flow c y t o m e t r i c assay for the q u a n t i t a t i o n of rosetting on a s u b p o p u l a t i o n of cells. Again, U937 a n d K562 cells were e m p l o y e d . U937 forms rosettes with E A - m l g G 1 less readily than K562 cells (Van de W i n k e l et al., 1987b). S u b o p t i m a l l y sensitized E A - m l g G 1 ( + 5 0 , 0 0 0 IgG1 m o l e c u l e s / E ) were used for these experiments. These i n d i c a t o r cells scarcely interact with U937 cells, while K562 readily forms rosettes. T h e s e c o n d fluorescence p a r a m e t e r was used to disc r i m i n a t e between b o t h types of cell. A new m A b , VB2, recognizing an antigen with a high level of expression of U937 cells but which is a b s e n t on K562 cells, was e m p l o y e d . U937 cells were identified using b i o t i n y l a t e d VB2 a n d s t r e p t a v i d i n - P E . A f t e r washing, the F I T C - l a b e l l e d i n d i c a t o r E A m l g G 1 were a d d e d a n d rosette f o r m a t i o n was analysed. Fig. 4A illustrates that b o t h cell types were effectively s e p a r a t e d using the PE fluo75

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Fig. 3. Comparison of flow cytometric (open symbols) and microscopic (closed symbols) evaluation of rosette formation. Rosetting between either K562 ceils and E sensitized with different amounts of routine IgGl molecules (o, e), or between U937 cells and E sensitized with different numbers of human IgG molecules per E ([3, I), was quantitated. The data represent the mean ± SD of four individual experiments.

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Fig. 4. Quantitation of rosette formation in a mixed cell population of unlabeUed K562 cells and PE-labelled U937 cells. Indicator E were labelled with FITC and sensitised with + 50,000 mlgG1 molecules per cell. A: fluorescence distribution of a 60 : 40 mixture of K562 cells and U937 cells, labelled with PE using mAb VB2 and rosetted with FlTC-labelled EA-mlgG1. The percentages of events were 36%, 4%, 44$ and 177o for quadrants 1, 2, 3 and 4 respectively, in which 1 represents non-rosetting U937 cells, 2 represents rosening U937 cells, 3 represents non-rosetting K562 cells and 4 represents K562 cells forming rosettes. B: comparison of cytofluoromettic quantitation in mixed and homogeneous cell suspensions. Values were either obtained from A (hatched bars), or from samples containing only one unlabelled cell type, rosetted with the same FITC-labeUed EA-mlgGI (shaded bars). Data are presented as the mean + SD of three individual experiments. rescence. EA b i n d i n g in this m i x e d cell p o p u l a t i o n was consistent with that o b s e r v e d with u n l a b e l l e d cells a n a l y s e d in parallel (Fig. 4B). T h e P E - s t a i n ing of U937 cells itself had no influence on rosette formation, as e v a l u a t e d b y testing E A - m l g G 2 a rosette f o r m a t i o n b o t h on s t a i n e d a n d u n s t a i n e d cells. N o significant difference in E A b i n d i n g was found (results not shown). In a d d i t i o n to a n a l y s i n g the r o s e t t e - f o r m i n g c a p a c i t y within a mixed cell p o p u l a t i o n , we also p e r f o r m e d d o u b l e fluorescence e x p e r i m e n t s on K562 cells using PI staining as a second p a r a m e ter. Viable cells d i d not stain, whereas d e a d cells acquired a strong red fluorescence (not shown). T h e a d d i t i o n of PI 5 min b e f o r e e x a m i n a t i o n ensured that o n l y rosette f o r m a t i o n involving viable cells was estimated.

Flow cytometric analysis of the binding of murine isotype switch variants to F c y R l l on 1(562 cells T h e c o m b i n e d use of the flow c y t o m e t r i c c a l i b r a t i o n of i n d i c a t o r cell sensitization (see

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Fig. 5. Comparison of the binding profiles of indicator erythrocytes, sensitized with different murine IgG subclasses, to FcyRII o . K562 cells. A: quantitation of the number of antibody molecules bound per erythrocyte. E were sensitized with various dilutions of anti-glycophorin A heavy chain isotype switch variant mAb of the subclasses IgG1 (o), IgG2a (rl) or lgG2b (a,). Quantitation was performed using calibrated indirect immunofluorescence as described in the materials and methods section. Data are expressed as mean :1:SD of four separate experiments. B: rosette formation between K562 cells and indicator E, sensitized with different murine IgG subclasses. FITC-labelled E were sensitized with calibrated amounts of either mlgG1 (o), mlgG2a (El) or mlgG2b (zx) antibody and incubated with K562 cells at a ratio of 8 : 1. Each point represents the mean of four individual experiments.

above) and quantitation of rosetting permitted analyses of ligand specificity of Fc receptors. The interaction between F c y R l I on K562 cells and E sensitized with switch variants of anti-glycophorin A mAb of isotypes mlgG1, mlgG2a and mlgG2b were analysed. Quantitating the number of IgG molecules per indicator E by cytofluorimetry for each subclass, permitted a correct comparison between these isotypes (Fig. 5A). Remarkably, the binding patterns of all three types of indicator cells were similar (Fig. 5B). This suggests that F c y R I I on K562 cells has a similar affinity for murine IgG1, IgG2a and IgG2b. Inhibition experiments with specific antiF c y R l l mAb were performed in order to ensure that the observed EA binding was mediated by FcyRII. Anti-FcyRII mAb IV.3 and KB61 inhibited the binding of EA, optimally sensitized with all three isotypes of anti glycophorin A mAb, by more than 90%. Control mAbs of the same isotypes, reactive with antigens present in high density on K562 cells, never showed inhibition above 20% (n = 3). Monoclonal antibodies against

FcyRI, FcyRIII and several other antigens did not influence EA-binding (not shown).

Discussion

Studies on FcR are complicated by the existence of different types, which are generally coexpressed and which show variation in expression during culture (Gandour et al., 1983). Furthermore, large affinity differences exist between the three types of receptor (for reviews see: Anderson and Looney, 1986; Unkeless et al., 1988; Fanger et al., 1989). This complexity emphasises the need for reliable test systems for the study of FcR-ligand interactions. Rosette formation with antibody coated erythrocytes is a commonly used and sensitive FcR assay. Visual quantitation of rosette formation, however, is very time consuming, while useful information about individual cells is lost with several other methods (for review see: Coombs and Wilson, 1982). Previously, fluorochromes (e.g., FITC or

213 rhodamine) have been employed for the labelling of indicator E to facilitate analytical rosetting tests (for review see: Ling and Richardson, 1981). In a further variation Brown et al. (1979) used a Coulter channelyzer to quantitate EA rosettes merely on the basis of particle size. In this study we have combined the advantages of both methods in a new flow cytometric assay. The low-affinity interaction of FcTRII on K562 cells and mlgGl-sensitized E was used as a model. Using optimally sensitized EA-mlgG1, rosette formation was observed, but when unsensitized E or E saturated with F(ab')2 fragments were employed no rosettes were obtained. This confirmed the Fc dependency of EA binding in the assay. This was further supported by the absence of rosettes in the presence of FcTRII-blocking mAb. Both the ratio of EA to rosette-forming cells, as well as the degree of indicator cell sensitization affected the number of rosettes profoundly. These two parameters easily account for the contradictory results reported in previous rosetting studies, where the estimated EA-mlgG1 binding by K562 cells range from no binding (Ichiki et al., 1985) to profound rosetting (Lemke et al., 1985; Van de Winkel et al., 1987b). The degree of EA binding mediated by F c T R I I on K562 cells, as well as that mediated by FcTRI on U937 cells, was assessed both by visual examination and by flow cytometry. A perfect correlation was observed between these methods, indicating that neither F I T C labelling of EA, nor flow cytometric evaluation interfere with rosette formation. The observed number of U937 cells forming rosettes with EA-hlgG was consistent with the data of others (Dougherty et al., 1987). The availability of a second fluorescence parameter permitted the application of a variety of staining procedures. For example, PE labelling was used to investigate rosette formation on a subpopulation of cells while PI staining was used for the simultaneous assessment of cell viability. To illustrate the sensitivity of our assay we performed binding studies of E sensitized with a murine IgG heavy chain switch variant mAb against glycophorin A, to FcTRII on K562 cells. Although rosette formation between K562 cells and indicator E sensitized with either mlgG1, mlgG2a or m l g G 2 b has been reported previously (Ichiki et al., 1985; Lemke et al., 1985), the indica-

tor EA were poorly characterised. For an objective evaluation of the binding of different subclasses, a well-defined indicator system is essential. For this purpose we applied calibrated flow cytometry which has been claimed to be an accurate method for the quantitation of antigen expression (Le Bouteiller et al., 1983; Poncelet and Carayon, 1985). Remarkably, we observed a comparable affinity of FcTRII on K562 cells for murine IgG1, IgG2a and IgG2b. Based on a variety of functional and binding studies it has been reported that FcTRII on other cell types bind mlgG1 and m l g G 2 b better than mlgG2a (Jones et al., 1985; Looney et al., 1986; Boot et al., 1989). A possible explanation for this apparent discrepancy might be differences between the cell types used. In fact it has been shown by Chiofalo et al. (1988) that the binding characteristics of FcRII on K562 cells for human IgG isotypes differ from those reported for FcRII on platelets (Karas et ai., 1982). In conclusion, we have developed a sensitive, simple and reliable assay for the measurement of FcyR-ligand interactions simultaneously with a second fluorescence parameter.

Acknowledgements The authors wish to thank Drs. Van Kessel and Van Strijp for excellent assistance with the flow cytometry, and Dr. Boot for generously supplying us with the heavy chain isotype switch variant mAb against glycophorin A.

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