Modulation and intracellular transport of CD20 and CD21 antigens induced by B1 and B2 monoclonal antibodies in RAJI and JOK-1 cells—An immunofluorescence and immunoelectron microscopy

~ Pergamon

0145-2126(94)E0045-B

Leukemia Research Vol. 18, No. 7, pp. 541-552, 1994 Copyright (~) 1994 Elsevier Science Ltd

Printed in Great Britain. All rights reserved 0145-2126/94 $7.00 + 0.00

MODULATION AND INTRACELLULAR TRANSPORT OF CD20 AND CD21 ANTIGENS INDUCED BY B1 AND B2 MONOCLONAL

ANTIBODIES IN RAJI AND JOK-1 CELLS--AN IMMUNOFLUORESCENCE AND IMMUNOELECTRON MICROSCOPY

STUDY

STANISLAW PULCZYNSKI,* ANNE MARIE BOESENt and OLAF MYHRE JENSEN* *University Department of Pathology; and ?University Department of Medicine and Hematology,

Aarhus Amtssygehus, Aarhus, Denmark

(Received 25 November 1993. Revision accepted 5 March 1994)

Abstract--By fluorescence microscopy (FM), flow cytometry (FCM) and immunoelectron microscopy (IEM) we have shown that B1 and B2 monoclonal antibodies (MoAbs) were able to induce modulation of CD20 and CD21 in RAJI and JOK-1 cell lines. Redistribution and internalization of both antigens (Ags) after binding with MoAbs was readily demonstrated by FM, and by IEM CD20 and CD21 were found to be processed by the pathway of receptor- mediated endocytosis. The rate of intracellular transport varied: CD21 > CD20 and RAJI > JOK-I. Approximately 65 and 55% of CD20 and 60 and 45% of CD21 were cleared from the surface of RAJI and JOK-1 cells, respectively (FCM and IEM). These values, however, clearly exceeded those corresponding to internalization (11, 9, 24 and 16%) indicating shedding of Ag-MoAb complexes. No evidence of recycling was found. The present data support the hypothesis that the kinetics of modulation vary from one Ag to another and probably also reflect the stage of differentiation of the malignant B-cells. The results are discussed in the context of the possible usefulness of B1 and B2 MoAbs in the therapy of B-cell malignancies.

Key words: Antibody-induced antigenic modulation, internalization, receptor-mediated endocytosis, CD20, CD21, immunotherapy, malignant B-cells.

Introduction

ANTIBODY-INDUCED antigenic modulation (AIAM) is defined as a reversible decrease or loss of antigenic expression in cells exposed to specific antibodies (Abs). Interaction with Abs results in redistribution of antigen (Ag)-Ab complexes on the cell surface, often followed by shedding to the extracellular environment or internalization through the pathway closely resembling receptor-mediated endocytosis of hormones, growth factors and other natural ligands

Abbreviations: Abs, antibodies; Ags, antigens; AIAM, antibody-induced antigenic modulation; B-CLL, B- chronic lymphocytic leukemia; EBV, Epstein-Barr virus; FCM, flow cytometry; FITC, fluorescein isothiocyanate; FM, fluorescence microscopy; IEM, immunoelectron microscopy; IF, immunofluorescence; MFI, mean fluorescence intensity; MoAbs, monoclonal antibodies; PE, phycoerythrin; RAM IgG, rabbit antimouse immunoglobulins.

Correspondence to: Stanislaw Pulczynski, MD, Uni- versity Department of Pathology, Aarhus Amtssygehus, Tage Hansensgade DK-8000 Aarhus C, Denmark.

541

[1, 2]. Ultrastructural studies have revealed that various ligands are transferred through plasmalemmal pits to endosomal structures and then released to the cytosol, recycled back to the cell surface or delivered to lysosomal compartments for destruction [1-4].

AIAM has important implications for therapy of human neoplastic diseases with monoclonal antibodies (MoAbs) [5-7]. In particular, the devel- opment of MoAbs directed against Ags present on the surface of malignant hematopoietic cells has provided the basis of immunotherapy in patients with malignant hematologic disorders. Native MoAbs or conjugates with drugs, toxins (immunotoxins), and radioisotopes (radioimmunoconjugates) have been used for in vivo administration or ex vivo purging of bone marrow grafts prior to autologous bone marrow transplantation [8-10]. Yet, malignant cells were often able to escape elimination by unmodified MoAbs modulating target Ags [8, 11, 12]. On the other hand, internalization of immunotoxins and MoAb-drug conjugates is a prerequisite, though not

542 S. PULCZVNSKt et al.

the only requirement , for selective killing of target cells [9, 13, 14].

Despite the fact that a number of MoAbs have previously been applied in serotherapy of B-cell malignancies [8, 15-18], many basic aspects regarding A I A M of different B-cell Ags remain to be eluci- dated. Thus, although previous studies have shown uptake of CD21 induced by its natural ligand and Epstein-Barr virus (EBV) [19-24], antibody-induced internalization of this Ag has not hitherto been reported. Even less is known about internalization of CD20 [25], which has been postulated to lack the ability to undergo modulat ion upon interaction with MoAbs [26-28].

In the present study we demonstrated A I A M of CD20 and CD21 Ags in R A J I and JOK-1 cells. Redistribution, internalization and intracellular t ransport of both Ags were visualized and the kinetics of modulat ion determined using fluorescence microscopy (FM), flow cytometry (FCM) and immunoelectron microscopy (IEM).

Materials and Methods

Cells" The human malignant B-cell lines RAJI and JOK-1 were

kept in exponential growth phase at 37°C in RPMI-1640 medium supplemented with 10% heat-inactivated fetal calf serum (FCS), glutamine, HEPES and antibiotics in a humidified 5% CO2 atmosphere. Before use the cells were chilled on ice to block endocytosis and washed twice in Hanks' balanced salt solution containing 1% bovine serum albumin (BSA) and 2% heat inactivated human AB serum (HBSS-BSA-AB). HL60 (myeloid) and U937 (mono- blastic) cell lines were used as controls for non-specific uptake.

Immunoreagents Murine MoAbs B1 (CD20, IgG2a) [29] and B2 (CD21,

IgM) [30} (kindly provided by Dr L. M. Nadler, Dana- Farber Cancer Institute, Boston, MA) were used for indirect immunofluorescence (IF) and IEM. Murine MY8 ( myeloid, IgG2a) and Mo 1 ( CD 1 lb, IgM) MoAbs (Coulter Electronics, Luton, U.K.) were used as controls for background staining. Reactivity of MoAbs with the cells determined by FCM and expressed as mean fluorescence intensity (MFI) was as follows: RAJI/B1,125; JOK-1/B1, 99; RAJI/B2, 26; JOK-1/B2, 16; controls, 4-6. For direct IF, following fluorescein isothiocyanate (FITC) and phycoerythrin (PE)-conjugated MoAbs were employed: B1-FITC, B1-PE and B2-PE (Coulter Electronics, Luton, U.K.). All MoAbs were used at saturating concentrations. In some control experiments 1:2, 1:4 and 1:8 dilutions were applied.

FITC-conjugated rabbit antimouse immunoglobulins (RAM F(ab')2-FITC) and rabbit antimouse immunoglobulins (DAKO A/S, Copenhagen, Denmark) coupled to 12 nm colloidal gold (RAM IgG G12) were used as second step reagents for IF and IEM, respectively. Col- loidal gold particles were prepared by reduction of gold chloride (HAuC14) (Degussa, Hanau, Germany) with a

mixture of tannic acid and citrate, evaluated for homo- genicity in EM, and coupled to RAM IgG as previously described [31, 32].

Immunofluorescence and flow cytometry analysis To evaluate modulation of CD20 and CD21 Ags the cells

were incubated for 30 min at 4°C with MoAbs, washed twice, cultured at 37°C for periods of time varying from 15 min to 24h, washed, fixed in a buffered 1.5% paraformaldehyde solution and analyzed by FCM as previously described [33]. In some experiments saturating concentrations of respective MoAbs were added to the culture medium. In brief, the experiments were set up in two different ways. (1) After incubation with MoAbs the cells were cultured at 37°C for various periods of time, washed, incubated with 50 ~tl RAM F(ab')2-FITC for 30 min at 4°C, washed again and fixed. The clearance of Ag-MoAb complexes from the cell surface due to modulation was assessed by FCM measurements of surface-associated IF reflecting the amount of complexes remaining on the cell surface at given time intervals. (2) The incubation with RAM F(ab')2-FITC was performed immediately after the wash following labeling with MoAbs, that is, before culture at 37°C. In some experiments direct IF was used as one step labeling with FITC- or PE-conjugated MoAbs. Subsequent FCM analysis permitted measurements of cell-associated IF reflecting both surface-bound and internalized complexes at given time intervals. This approach enabled an evaluation of Ag loss due to externalization.

FCM analysis was performed using an EPICS Profile flow cytometer (Coulter Electronics, Luton, U.K.) At each time point the MFI of 10,000 ceils was calculated over 256 channels in a linear scale and the percentage of cells positive for each MoAb determined. Subtraction of background fluorescence was performed channel by channel using Cyto- logic software (Coulter Electronics). The degree of modulation (M) was calculated as the percentage of MFI in relation to unmodulated cells using the following equation:

MFI(sample) - MFI(control) M(%) = 100 -

MFI(unmodulated cells) - MFI(control)'

For FM, the cells were labeled with direct or indirect IF technique and cultured at 37°C as described above. Observation of viable cells was performed at the above mentioned time intervals using a Leica Diaplan fluorescence microscope (Wetzlar, Germany).

Immunoelectron microscopy The cells were washed, incubated with MoAbs, washed,

stained with RAM IgG G12 under gentle shaking, washed again and incubated in culture medium at 37°C as described [33, 341. After 0.5, 1, 2, 3, 6, 12 and 24 h, aliquots of 5 x 10 ° cells were removed from the cultures, washed, and processed for EM according to our standard method. Briefly, fixation in 2.5% glutaraldehyde and 1% osmium tetroxide was followed by prestaining with 2% uranyl acetate and centrifugation in warm agar. The resulting pellet was cut into 1 mm blocks, dehydrated in a graded series of ethanol, embedded in epon and cut on a LKB ultra- microtome III (LKB Biotechnologi AB, Broma, Sweden). Ultrathin sections were picked up on Formvar-coated copper grids, stained in uranyl acetate and lead citrate and examined with a Philips EM 201 (Eindhoven, The Netherlands) or a Zeiss EM910 (Oberkochen, Germany) electron microscope operating at 60 kV.

Modulation of CD20 and CD21 543

Quantitation of colloidal gold distribution Quantitative analysis was carried out by counting gold

particles on micrographs at a final magnification of × 20.160 using a × 4 stand magnifier. In order to evaluate the clearance from the cell surface, the uptake and the intracellular distribution of the Ags within different intracellular compartments, separate quantitation of gold particles attached to the plasma membrane, cytoplasmic vesicles, multivesicular bodies and lysosomes was performed at given time intervals. Approximately 50 randomly selected equa- torial cell sections were examined, and the mean values per cell section calculated. The degree of internalization and intracellular distribution of gold tracer at given time intervals were expressed as a percentage of the total number of gold particles at the starting point of each experiment.

The reproducibility of samples was ensured as described previously [33, 34] by the homogeneity of cell suspensions, uniform distribution and random orientation of cells in agar pellets as well as by random selection of blocks, ultrathin sections and cell profiles used for analysis. Repeated counting of selected ultrathin sections, as well as counting performed on several sections obtained from the same specimen, yielded similar results indicating the reproducibility of the quantitative method.

Controls To determine background staining, B1 and B2 MoAbs

were omitted or replaced by isotype-matched irrelevant Abs. Various dilutions of MoAbs yielded results similar to those obtained with saturating concentrations. No internalization was observed after incubation of cells with RAM IgG G12 only. The possibility of non-specific uptake of B 1 and B2 was excluded, as HL60 and U937 cells incubated in culture medium at saturating concentrations of respective MoAbs did not show any sign of internalization, The modulation of CD20 and CD21 was not seen in control experiments including: (1) fixation of cells in 1.5 % solution of paraformaldehyde prior to immunolabeling; (2) addition of 0.2% (30 mMol/1) sodium azide to the immunoreagents, washing buffers and culture medium; and (3) incubation of labeled cells in culture medium at 0°C.

Statistical analysis

Mean and standard deviation (S.D.) values of results from several independent experiments were calculated. The significance of differences between mean values was calculated using the correlation coefficient (r) and the Student's t-test at 0.05 significance level.

a time 0

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m FIG. 1. Redistribution and internalization of CD20 and CD21 Ags in RAJI and JOK-1 cells. The cells were stained with B1 or B2 MoAbs + RAM F(ab')2-FITC, cultured at 37°C and examined by FM. (a) Uniform surface fluorescence at time 0. (b) Capping and intracellular localization of IF after 1 h. (c) Persistent capping in RAJI cells compared with patchy surface fluorescence in JOK-1 cells after 24 h. In some cells intracellular IF was visible. The fluorescence intensities on the photographs are not comparable

because different exposure times were used.

544

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S. PtrLCZYNSrd et al.

FIG. 2. Redistribution of CD20 Ag on the cell surface of JOK-1 cells labeled with B1 MoAb + RAM IgG G12. A. Uniform distribution of single gold particles and small aggregates at time 0. B. Cap-like staining pattern after 6 h of incubation in culture medium at 37°C. Magnification

×18,200.

Results

Distribution and internalization of CD20 and CD21

Observation of viable cells showed that, after binding with B1 and B2 MoAbs, the CD20 and CD21 Ags were redistributed on the cell surface and internalized in both RAJI and JOK-1 cells. Initially, a homogeneous, circumferential distribution of surface fluorescence was observed, rapidly changing into a patchy pattern (Fig. l(a)). Migration of fluorescence patches resulted in a polar staining pattern (capping) (Fig. l(b)), pronounced after 1 h of incubation at 37°C. The uptake of some patches into the cells was observed in all experiments. Exocytosis of internalized fluorescent grains was often seen in RAJI, but

not in JOK-1 cells. The course of modulation was the same for CD20 and CD21 Ags. Also, both cell lines showed similar patterns of surface redistribution and internalization during the first 3 h of incubation. Later, RAJI cells still showed fine-grained caps, whereas coarse grains of surface fluorescence were commonly found in JOK-1 cells (Fig. l(c)). The course of events described was the same with direct and indirect IF techniques.

IEM enabled visualization of CD20 and CD21 Ags at the ultrastructural level by tracing the MoAbs with RAM IgG G12. Labeling density was several times higher with CD20 than with CD21 in both cell lines. Initially, the cells showed diffuse distribution of single gold particles and small clusters attached to

I L _

CD20 CD21 545

FIG. 3. Internalization of CD20 and CD21 Ags in JOK-1 cells. The cells were labeled with B1 or B2 MoAbs + RAM IgG G12 prior to incubation in culture medium at 37°C. Uptake of gold particles through plasmalemmal pits (A and C) and macropinocytosis (B) at time O. Gold-labeled endosomes after 30 min (D-F) and after 1 h (G). Accumulation of gold particles within multivesicular bodies (H and I) after 3 h and in lysosomes (J and K) after 6 h. Plasmalemmal pits were usually not coated (A and C). Note the close relation of gold particles to each other and to vesical membranes (A-G), aggregation in multivesicular bodies (H and I), and pronounced flocculation of colloidal gold in lysosomes (J and K). A

similar route of internalization was found in RAJI cells. Magnification x71,800.

546 S. PULCZYNSKI et al.

the plasma membrane (Fig. 2(A)). Upon incubation at 37°C, clustering gradually became more pronounced resulting in a cap-like staining pattern at later time intervals (Fig. 2(B)). Although labeling of the cell surface decreased during the time, gold tracer was present on the plasmalemma at the end of each experiment indicating incomplete modulation of both Ags.

During the first 2 h of incubation at 37°C gold particles were commonly found in plasmalemmal pits (Fig. 3(A) and (C)) and in cytoplasmic vesicles localized in the peripheral cytoplasm (Fig. 3(D) and (F)) indicating uptake of CD20-B1 and CD21-B2 complexes by RAJI and JOK-1 cells. Membrane coating was usually not found in these structures. Uptake of larger areas of gold-labeled plasmalemma (macropinocytosis) was sometimes seen (Fig. 3(B)). Later, most of the intracellular labeling was found in larger vesicles and tubular structures (Fig. 3(E)) often located within perinuclear cytoplasm (endosomes) and in multivesicular bodies (Fig. 3(G)-(I)). Fusion of these endosomal structures was seen in all experiments. Labeling of endosomes and multivesicular bodies decreased at later time intervals followed by the presence of gold particles in tran- sitional forms between multivesicular bodies and lysosomes. Increasing accumulation of gold tracer in lysosomes was observed after 3 h (Fig. 3(J) and (K)). Golgi cisternes were always free of labeling. Mor- phological features of externalization including fusion of vesicles and multivesicular bodies with plasmalemma followed by discharge of their content to the environment were seen in RAJI cells.

The location of gold particles in relation to the membranes and to each other was also evaluated providing information about the intracellular processing of the immunocomplexes. Thus, a thin layer of moderately electron-dense material (protein coat) surrounded the particles attached to the cell surface and to the inner membrane of endosomal structures (Fig. 3(A)-(G)), but was not present in lysosomes, where the particles always appeared aggregated (Fig. 3(J) and (K)). A minor degree of aggregation was observed in endosomes and gradually increased during the time indicating ongoing destruction of the protein coat and subsequent flocculation of gold colloid.

Kinetics of modulation and intracellular transport FCM analysis clearly showed that B1 and B2

MoAbs induced rapid modulation of CD20 and CD21 Ags in both RAJI and JOK-1 cells (Fig. 4). To determine the clearance of Ag-MoAb complexes from the cell surface, the cells were incubated with

MoAbs, cultured at 37°C for varying periods of time, collected and stained with RAM F(ab')2-FITC. Sur- face-associated IF was then measured by FCM, providing information about Ag-MoAb complexes present on the cell surface at given time intervals: i.e. immunocomplexes that have not been internalized and immunocomplexes that possibly have been recycled. A decrease in fluorescence intensity was found in all experiments, most rapidly during the first 2-3 h (Fig. 4, curves (a)). After 24 h 68 and 55% of CD20-B1 complexes and 60 and 51% CD21-B2 complexes were cleared from the cell surface in RAJI and JOK-1 cells, respectively. In parallel experiments the cells were labeled with B1 or B2 MoAbs + RAM F(ab')2-FITC (or with directly conjugated MoAbs), cultured at 37°C, collected and analyzed by FCM to measure cell-associated IF. This approach enabled simultaneous measurements of surface-bound and internalized Ag-MoAb complexes at given time intervals yielding information about the loss of immunocomplexes due to externalization. The results showed that approximately 60 and 45% of CD20 complexes and 40 and 35% of CD21 complexes were lost after 24 h in RAJI and JOK-1 cells, respectively (Fig. 4, curves (b)). Similar results were obtained with directly conjugated MoAbs indicating that second Ab did not alter the kinetics of modulation of Ag-MoAb complexes and excluding the possibility that only second Ab was externalized. Furthermore, addition of the respective MoAbs to the culture medium did not influence the described course of modulation (data not shown), indicating that clearance of Ag-MoAb complexes was an active process rather than a result of simple dissociation owing to a concentration gradient. Thus, in both types of experiments CD20 was modulated to a greater extent than CD21. Moreover, modulation of both Ags was more pronounced in RAJI than in JOK-1 cells.

The kinetics of internalization and intracellular transport of CD20 and CD21 Ags was evaluated by quantitative analysis of gold tracer distribution in RAJI and JOK-1 cells at various time points. The rates and extent of surface clearing and internalization are shown in Fig. 5. During 1-3 hours, a rapid decrease of cell surface labeling occurred in all experiments, followed by a slight further reduction (Fig. 5, curves (a)). Finally, 63 and 54% of CD20 and 60 and 45% of CD21 disappeared from the surface of RAJI and JOK-1 cells, respectively. These data were fully consistent with the respective FCM- data (Fig. 4, curves (a)) yielding significant correlation (r > 95%). Eleven and 9% of CD20 under- went internalization, whereas 24 and 16% of CD21 was taken up by RAJI and JOK-1 cells, respectively

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FIG. 4. Modulation of CD20 and CD21 Ags in RAJI and JOK-1 cells analysed by FCM, (a). Surface-associated IF in cells labeled with B1 or B2 MoAbs, incubated in culture medium at 37°C and then labeled with RAM F(ab')2-FITC. The decrease in fluorescence intensity indicates clearance of Ag-MoAb complexes from the cell surface. The results reflect both unmodulated and recycled Ag-MoAb complexes at given time points. (b). Cell-associated IF rep- resenting the simultaneous measurements of surface-bound and internalized Ag-MoAb complexes at given time intervals. The cells were labeled with B1 or B2 MoAbs and RAM F(ab')2-FITC, or directly conjugated MoAbs, prior to incubation at 37°C. Decrease in fluorescence intensity indicates that Ag-MoAb complexes were externalized by shedding and/or exocytosis. Time of incubation and mean fluorescence intensity expressed as a percentage in relation to unmodulated cells are indicated on the x and y axis, respectively. Data shown represent mean values of at least seven experiments. S.D. at each time point did not exceed

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(Fig. 5, curve (b)). Internalization occurred faster with CD21 than CD20 and was more rapid in R A J I than in J O K cells. Also, a slight decrease of intracellular labeling was found at later time intervals in RAJI , but not in JOK-1 cells indicating exocytosis of immunocomplexes . The differences in the kinetics of A I A M were statistically significant, except those regarding surface clearance of CD20 and CD21 in R A J I cells.

To determine the kinetics of intracellular transport , a detailed analysis of intracellular labeling was

performed. The data clearly showed, that both Ags were transferred through cytoplasmic vesicles and multivesicular bodies to lysosomes (Fig. 6). A fast transfer of both Ags through the endosomal com- par tment was found in R A J I cells compared with prolonged retention in vesicles and, especially, in multivesicular bodies in JOK-1 cells. In consequence, a maximum accumulation of gold tracer in lysosomes was reached after 6 and 12-24 hours in R A J I and JOK-1 cells, respectively. Also, the transfer of CD21 was faster than that of CD20.

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FIG. 5. Internalization of CD20 and CD21 Ags in RAJI and JOK-1 cells evaluated by quantitative IEM. Cells were labeled with B1 or B2 MoAbs + RAM IgG G12 before incubation in culture medium at 37°C. Time of incubation and number of gold particles found on cell surface (a) and intracellularly (b) expressed as percentages in relation to time 0 are indicated on the x and y axis, respectively. The data represent the mean values of two experiments. S.D. around the highest values did not exceed 10%, increasing to 50% around the lowest values. Note differences in extent

and rate of internalization.

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Discussion

Our results demonstrate for the first time that B1 and B2 MoAbs are able to induce modulation of CD20 and CD21 Ags in malignant B-cells. Redis- tribution on the cell surface, internalization and intracellular transport were readily visualized by FM and IEM indicating that both Ags used the same pathway closely resembling receptor-mediated endocytosis. The kinetics of AIAM, however, varied significantly regarding the extent and rate of internalization and intracellular transport (CD21 > CD20 and RAJI > JOK-1) as well as clearance of immunocomplexes from the cell surface (CD20 > CD21 and RAJI > JOK-1). These findings are consistent with the results of our previous studies on AIAM and CD10 and CD19 Ags in B-cell lines [33], in which we demonstrated that AIAM was more pronounced in less differentiated cells. Thus, the present results

further support the hypothesis that the kinetics of internalization and of intracellular transport of a given surface molecule in malignant B-cells vary from one type of cell to another possibly depending on the stage of cell differentiation. In addition, various B-cell Ags are internalized to a different extent in the same type of malignant B-cells. However, it cannot be ruled out that the different isotype of MoAbs used might have contributed to some of the differences observed. In our studies the degree of internalization was not cor- related with initial Ag density. These matters are pres- ently being further investigated in our laboratory using fresh B-cell material.

Shedding, which is another aspect of AIAM, has for a long time been known as an important mechanism involved in the regulation of cell activation and growth as well as in escape of tumors from immunologic destruction [35]. A spontaneous shedding of CD21 by RAJI cells was previously described

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FIG. 6. Distribution of gold particles within subcellular compartments during internalization of CD20 and CD2I Ags. Labeling of the cells and the axes as in Fig. 5. Data indicate transient accumulation of the gold tracer in cytoplasmic vesicles and multivesicular bodies followed by final accumulation in lysosomes. S.D. around the highest values were <25% increasing to >50% around the lowest values. Note. that transfer of gold tracer to lysosomes was significantly faster in RAJI than in JOK-1 cells and that endosomal accumulation occurred earlier with CD21 than CD20. C)~(3 vesicles; A--A multivesicular bodies:

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[36], but few studies have addressed antibody- induced shedding of surface molecules in human leukocytes [37, 38]. Even though the measurement of soluble immunocomplexes in culture medium was not possible with the methods used in this study, our data indicate that CD20 and CD21 Ags were not only internalized, but also rapidly shed from the surface of RAJI and JOK-1 cells, since the clearance of Ag- MoAb complexes from the cell surface during the first hours of modulation experiments (FCM and IEM) clearly exceeded the degree of internalization. Our data suggest that the loss of immunocomplexes was not just a result of simple dissociation owing a concentration gradient because: (1) it was completely abrogated when cell metabolism was blocked by chill- ing on ice, addition of sodium azide or fixation; and (2) it was not significantly reduced by addition of the respective MoAbs to the culture medium.

The fate of the internalized immunocomplexes could be only partly determined owing to the limi- tation of the methods used in this study. Never- theless, IEM allowed identification of the endosomal compartment as a site of degradation of the complexes, since the protein coat that initially surrounded the gold particles gradually disappeared within endosomes and multivesicular bodies, whereas the gold particles in lysosomes were clustered in dense patches indicating flocculation of gold colloid due to digestion of the protein coat. These observations do not rule out the possibility that Ag-MoAb complexes were, at least to some extent, released from the endosomes and recycled back to the cell surface, but our quantitative data provided no evidence of recycling of either CD20- B1 or CD21-B2 complexes. Recycled complexes should--together with non-modulated complexes--

550 S. PULCZVNSKt et al.

be detected by FCM on the surface of cells labeled with MoAbs, and not stained with secondary Ab till after culture. Thus, if recycling of Ag-MoAb complexes occurred, the values of surface-associated IF (Fig. 4, curves (a)) would be higher than the respective values obtained by counting of surface- bound gold particles (Fig. 5, curves (a)) because in the IEM experiments (cells labeled with secondary Ab prior to modulation) the recycled complexes would not be detected. In consequence, subtraction of surface-associated IF (Fig. 4, curves (a)) from cell-associated IF (Fig. 4, curves (b)) at given time intervals would cause an underestimation of intracellularly located Ag complexes in comparison with IEM (Fig. 5, curves (b)), which would enable direct tracing and quantitation of these complexes. In this study, FCM and quantitative IEM showed similar values with regard to surface clearance and internalization of CD20 and CD21 indicating that these Ags, unlike CD10 and CD19 [33, 34] were not recycled in the malignant B-cell lines. However, a more complete understanding of the intracellular processing of these internalized immunocomplexes requires further studies aimed at separate visualization of Ags and MoAbs on the cell sections.

The results of previous studies on modulation of CD20 and CD21 Ags in various cells of B-lineage have been in contradiction with each other and with our results. B1 and 2H7 anti-CD20 MoAbs were reported not to induce modulation of CD20 Ag in various malignant B-cell lines [26, 28] and in splenic B-lymphocytes [27]. In these studies either indirect IF assay was used [27, 28] or lack of cytotoxicity of B 1-immunotoxin [26] and 2H7-adriamycin immuno- conjugate [28] was attributed to the lack of internalization of CD20-MoAb complexes. Also, Bergui et al. [39] found that B1 MoAb did not induce capping in normal B-lymphocytes and B-CLL cells. In contrast, Press et al. [25] obtained results similar to ours using 125j-labeled IF5, another anti-CD20 MoAb, which induced approximately 40% surface clearing and 15-20% internalization of CD20 in Daudi cells. Even though IF5 and B1 bind to different sites of the CD20 molecule [40] and they play different roles in B-cell activation [41], the course of AIAM induced by each MoAb seems to be very similar. This observation suggests that internalization is not the main mechanism accounting for regulation of expression and accessibility of the CD20 molecule to its unknown ligand.

The antigenic structure of the CD21 molecule is complex, since at least four epitopes have been ident- ified [23, 42]. CD21 is the receptor for both the C3d fragment of complement involved in modulation of B-cell growth and EBV [43, 44]. These two ligands

bind to the same epitope, which is distinct from epitopes used by B2 and HB5 MoAbs [23, 42]. Though internalization of CD21 upon interaction with C3d and EBV has been well characterized in normal and malignant B-cells [19-24], antibody- induced internalization of CD21 has not yet been described. HB5 MoAb was shown to induce capping in normal B-lymphocytes [21, 39] and in B-CLL cells [39], but not in lymphoblastic cell lines [21]. Tedder et al. [45] found that HB5-immunotoxins were neither toxic to RAJI, other lymphoblastic cell lines nor normal proliferating B-lymphocytes, unless the last ones were simultaneously treated with EBV. The authors concluded that HB5 MoAb induces internalization of CD21 Ag only in normal B-cells infected with EBV.

Several factors may account for the discrepancies mentioned above. Generally, the results of the different studies are not directly comparable, since different cells, MoAbs and methods were used. Also, we found that even minor variations in the exper- imental procedures, i.e. changes of temperature, concentration of reagents and washing procedures influenced the results (own unpublished observations). Moreover, the lack of efficacy of MoAb- toxin-drug conjugates does not necessarily exclude internalization, because in some cases intracellular processing of these agents may lead to destruction instead of release to the cytosol resulting in lack of cytotoxicity [9, 13, 14].

In conclusion, the methods used in this study proved sufficiently sensitive to detect even a low degree of internalization, as we did in the case of CD20 and CD21 Ags. The therapeutic efficacy of unmodified anti-CD20 MoAbs, which has been demonstrated in previous studies [15, 17, 46-48], is consistent with a low degree of modulation of CD20. Similar promising results have been reported with regard to CD21, although the experience is more limited [16, 49]. On the other hand, the few studies on immunotoxins targeting CD20 and CD21 have up to now provided disappointing results. Despite these results and despite the fact that therapeutic efficacy of immunotoxins often correlates with the degree of internalization [50, 51], the present results should encourage further studies on immunotoxins derived from B1 and B2 MoAbs because (1) it has been postulated that one toxin molecule released into the cytosol may be enough to kill the target cell [6, 52]; (2) different toxins guided by the same MoAb may be processed differently by the target cells and, there- fore, vary in their ability to be released into the cytosol and kill the cells [53]; and finally (3) efficacy of slowly internalized immunotoxins may be con- siderably improved by altering the immunotoxin molecule [54-56].

Modulation of CD20 and CD21 551

Acknowledgements--The authors thank Dr Peter Hokland, University Department of Medicine and Hema- tology, Aarhus County Hospital, Denmark, for growing cell lines, advice on flow cytometry methods, and critical reading of the manuscript; Mrs Majken Sand for excellent technical assistance and Mr Poul Erik Nielsen for expert help with the photography. This work was supported by the Danish Medical Research Council, the Ferd. & Ellen Hindsgauls Foundation (Denmark), The Danish Medical Association Research Fund (Hojmosegaard Foundation), the Foundation of 1870 (Denmark), the Director Ib Hen- riksens Foundation (Denmark), the Anders Hasselbalchs Foundation (Denmark), the Mimi and Victor Larsens Foundation (Denmark) and the P. Carl Petersens Foun- dation (Denmark).

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Documents

Modulation and intracellular transport of CD20 and CD21 antigens induced by B1 and B2 monoclonal antibodies in RAJI and JOK-1 cells—An immunofluorescence and immunoelectron microscopy