Int. J. Cancer: 17, 693-700 (1976)

SURFACE MARKERS ON HUMAN B- AND T-LYMPHOCYTES. TX. TWO-COLOR IMMUNOFLUORESCENCE STUDIES ON THE ASSOCIATION BETWEEN EBV RECEPTORS AND COMPLEMENT RECEPTORS ON THE SURFACE OF LYMPHOID CELL LINES Eitan YEFENOF I, George KLEIN Mikael JONDAL and Michael B. A. OLDSTONE Department of Tumor Biology, Karolinska Institutet, S-104 01 Stockholm 60, Sweden: and Department of Immunopathology, Scripps Clinic and Research Foundation, 476 Prospect Street, La Jo Ila, CaliJ, USA

SUMMARY

Receptors for the third component of complement ( C 3 ) were demonstrated on the surface of established human lymphoid cell lines by a membrane fluorescence test with FITC- or TRITC-conjugated antibodies against human C3. Two-color fluorescence staining of EBV receptors and C3 receptors showed complete overlapping of green and red fluorescence. Capping of the EBV receptor induced co-capping of the C3 receptor und vice versa. There was neither overlapping nor co-capping when EBV or C3 receptors were examined in relation to Fc receptors, surface IgM or p2 microglobulin. The kinetic pattern of EBV receptor capping was identical with the pattern of C3 receptor capping but differed from the pattern of IgM capping. These results suggest a close association between EBV and C3 receptors on the human B-lyniphocyte.

Epstein-Barr virus (EBV) receptors were demonstrated on the surface of human B-lymphocytes from normal peripheral blood (Jondal and Klein, 1973), and it was suggested that the EBV receptor is a reliable B-lymphocyte marker (Greaves et al., 1975). EBV receptors are also present on the majority of established, EBV-carrying lymphoid cell lines. Such lines carry B cell markers, in agreement with the fact that only B cells can be infected with and transformed by EBV into permanent lines (Jondal and Klein, 1973). Some virus producer lines lack detectable EBV receptors, presumably due to negative selection against receptor-carrying and therefore reinfectable cells (Jondal and Klein, 1973). C3 receptors were detected on normal B-lymphocytes (Bianco et al., 1970) and OR the majority of EBV-carrying established lines (Jondal and Klein, 1973). Some of these lines, such as Daudi and Raji, were found to aciivate complement by the alternative pathway (Okada and Baba, 1974; Theofilopoulos et al., 1974). Recently, a close association was found between EBV receptors and C3 receptors in a collection of established human lymphoid lines (Jondal et al., 1976). EBV receptor-positive lines were also C3

receptor-positive whereas EBV receptor-negative lines had no detectable C3 receptors, although they carried other B-cell markers. Moreover, EAC rosette formation by C3 receptor-positive lines was partially inhibited by concentrated EBV preparations. This inhibition could be neutralized by EBV antibodypositive but not by negative sera. Binding of EBV to virus receptor-positive cells was prevented by active mouse complement but not by heat-inactivated complement. EBV membrane antigen (MA)-positive material, collected from the supernatant of virus-producing cultures, can bind to EBV receptors and " cap ", i.e. redistribute and aggregate into clusters and caps on the cell surface, with or without the addition of antibodies (Yefenof and Klein, 1974; Hinuma et al., 1975). Capping was successfully used to demonstrate the independence or interdependence of various membrane constituents, e.g. different components of the H-2 (Neauport-Sautes et al., 1973) or the HL-A complex (Mayr et al., 1973), p2 microglobulin (prim) and surface immunoglobulins (Poulik et a f . , 1973). In order to visualize the postulated relationship between EBV and C3 receptors, we performed two color staining and capping studies involving not only these two receptors, but also Fc receptors, surface IgM and pzm for comparison. To visualize the C3 receptor, we utilized its demonstrated ability to bind soluble C3 and to activate it through the indirect pathway. A brilliant membrane fluorescence was obtained after the addition of fresh human complement, followed by a n anti-C3 conjugate. This reaction is probably identical with the corresponding reaction obtained by Floyd et al. (1971) and by Okada and Baba (1974). The indirectly stained C3 receptors readily capped upon incubation at 37" C. Our data lend further support to the idea that EBV receptors and the C3 binding sites are closely associated and represent one redistribution and capping unit within the membrane of the human B-lymphocyte. Received: February 13, 1976.

694

YEFENOF ET AL.

MATERIAL AND METHODS

Ten human lymphoid lines were used for the C3 receptor test, of which six were EBV-genomepositive and four negative (Table 1). All lines were maintained on RPMI-I640 medium, with the addition of 10% fetal calf serum (FCS). For the capping experiments, the non-producer Raji, the low virus producer Daudi and the EBV-negative Ra No. 1 lines were selected. All three have EBV and C3 receptors (Jondal et al., 1976).In addition, Daudi and Ra No. 1 cells have a high concentration of membrane-associated IgM molecules (Klein et al., 1968; Yefenof and Klein, 1976). Detection of C3 receptors by EAC rosettes

The method used was as described by Jondal(l974) with slight modifications. Indicator cells were prepared by exposing sheep red blood cells (SRBC) to 19s rabbit anti-SRBC antibodies and fresh mouse serum. Target cells were mixed with indicator cells at a 1:lOO ratio, spun down and incubated as a pellet for 30 min at 37" C. The cells were resuspended and the rosettes counted at low power. Rernoval of rosette-forming cells ( R F C )

Three ml of a rosetted cell suspension was layered on 3 ml Ficoll Isopaque gradients in glass tubes. The tubes were spun for 12 min at 680 x g . Cells from the interphase were collected, counted for rosettes and tested further for C3 receptors by the fluorescence test described below. Detection of C3 receptors b y indirect membrane fluorescence ( M F )

Five hundred thousand washed fresh cells were incubated for 15 min at 37" C with 0.1 ml EBVnegative serum (" Larsson ", " Ingemar " or *' BA 002/2 ") diluted 1 :25, used as the source of human complement. Controls were incubated at 4" C, and at 37" C with heat-inactivated sera. The cells were washed twice and incubated for a further 30 min at 4" C either with a fluorescein isothiocyanate (FITC)conjugated goat anti-human / 3 l ~ / B l anti-serum ~ (FaPlc) diluted I :I0 (Hyland Laboratories, Los Angeles, Calif., USA), or with a tetramethylrodamine isothiocyanate (TR1TC)-conjugated rabbit anti-C3 anti-serum (RaC3) diluted 1 :4 (Dakopatts, Copenhagen, Denmark). The cells were washed twice, fixed in 1 : I BSS:glycerol, and MF-positive and negative cells were differentially counted in a Leitz Ortholux fluorescence microscope under oil immersion. In some experiments, cells were first stained for EBV receptors by FITC conjugate as described below. For the C3 receptor counterstain, they were

subsequently incubated with 1 mg/ml purified C3 (Bokish et al., 1975) followed by incubation with RaC3. EBV receptor staining Five hundred thousand washed cells were incubated with 0.5 ml P3HR-1-derived EBV concentrate, prepared by the method of Adams (1973). The virus was the gift of Dr. Alice Adams. The EBV-concentrate was diluted 1 : 5 andsubsequently incubated with the cells for 1 h at 4" C. I n infectivity tests, this virus preparation was capable of inducing 23% early antigen (EA)-positive cells among lo6 superinfected Raji cells in 48 h. The cells were washed twice and incubated for 30 min at 4" C with 0.1 ml FITC or TRlTC conjugated Abwao IgG, known to contain a high titer of anti-EBV antibodies. The cells were washed and fixed with 1:l BSS:glycerol. Detection of ISM, p,ni and Fc receptors by MF

Membrane-associated IgM molecules were stained directly with FITC- or TRITC-conjugated rabbit anti-human IgM (p chain specific, Dakopatts), for 30min. For plm staining, the cells were incubated with rabbit anti-human psm (Dakopatts), followed by FITC-conjugated goat anti-rabbit immunoglobulin (Hyland Laboratories). Fc receptors were detected by incubating the cells with heat-aggregated human immunoglobulin prepared from heatinactivated, EBV-negative serum, followed by FITCconjugated rabbit anti-human F c (FaFc, 7' chainspecific, Dakopatts). The FaFc reagent gave no background staining with untreated cells in any of the cell lines used to detect Fc receptors. All reactions were carried out at 4 ° C in order to minimize redistribution. Capping arid co-capping experitvents

To induce capping, cells stainedfor agivenreceptor were incubated at 37" C for different times. After washing they were mounted in BSS-glycerol whereafter cap-positive and -negative cells were differentially counted. Only cells that were MF-positive within less than half of their total circumference were considered cap-positive. For capping experiments, cells were stained for the first receptor, incubated at 37" C over periods required for cap induction and subsequently chilled to 4" C . Thereafter, the second receptor was stained with the opposite color, but without increasing the temperature, in order to avoid further redistribution. The cells were washed, fixed and examined for red and green fluorescence in a Leitz Ortholux microscope, with a Ploem-type " Opakilluminator " system. For FITC fluorescence, 470 nm blue excitation light was used, with 4 mm BG38-t1.5 mm BGIZiAL470 (Schott., Mainz, West Germany) as

695

EBV AND COMPLEMENT RECEPTORS TABLE I DETECTION OF C3 RECEPTORS BY EAC ROSETTES AND BY MEMBRANE FLUORESCENCE (MF)

EBV genome status

Cell line

Percentage EAC binding cells

Percentage MF-positive cells

References

95 93 25 2 2 0 24 54 0

97 94 29 36 8 0 25 59 0 0

Epstein et al., 1966 Klein el a/., 1968 Klein e t a / . ,in press Klein e r a / . , in press Klein et a/., in press Foley et al., 1965 Menezes et at., 1975 Clenients e t a / . , 1975 Ben Bassat et al., 1976 Klein e f a]., I972

++ -

Raji Daudi Ra No. 1 AW Ra No. 1 I1 WA Ra No. 1 1301

BJAB G C-BJ A B 75

+

Rae1

0

As judged by the presence of EBV-DNA detected by molecular hybridization and/or the EBNA test (Reedman and Klein, 1973).

primary filters, 495 nm interference plate and K510 barrier filter. For TRITC fluorescence, 546 nm green excitation light was used, with a 4 nm BG38+ 2 nm BG36fAL546 as primary filters, 580 nm interference plate and K590 barrier filter. The cells were examined under oil immersion at a magnification of x 341. RESULTS

C3 receptor detection by MF in comparison with EAC rosettes

Table I summarizes the results obtained with 10 lines. EAC rosette and M F tests were performed on parallel aliquots of the same culture. The EBVnegative " Larsson " serum was used as the complement source. Three cell lines were completely negative in both tests. In six cell lines the percentage of positive cells detected by M F was slightly higher than the frequency of EAC-positive cells.

As shown in Table I1 the M F reaction for C3 receptor on Raji or Daudi cells was only obtained if the human complement was incubated with the target cells at 37" C but not at 4" C. Heat inactivation of the complement completely prevented the reaction. To exclude the possibility that the EBV-negative complement source contained antibodies against the target cells, the cells were incubated with the serum and subsequently exposed to FaFc diluted 1 :lo. They were completely negative. Highly positive M F reactions were obtained when the same cells were treated with a polyvalent heat-inactivated anti-HL-A serum (Uguen), followed by FaFc (Table 11). In three cell lines, Ra No. 1 , BJAB and GCBJAB, C3 receptor tests were also carried out by M F after the removal of the EAC-positive cells. As shown in Table 111, the proportion of MF-positive cells decreased considerably, but did not disappear

TABLE I1 C3 RECEPTOR DETECTION BY M F ON RAJI AND DAUDl CELLS. DEPENDENCE ON ACTIVE COMPLEMENT AND ON TEMPERATURE First incubation

E BV-negative serum Larsson Heat-inactivated Larsson serum -

Larsson serum Larsson serum Heat-inactivated Uguen serum Heat-inactivated Uguen serum

Temperature

Second incubation

37" c

FaBlc

4"

4" C or 37" C 4" C or 37" C 4" c 4" C or 37" C

FaB1c

FaAc FaBI c FaFc

4" c 4" c 4" c 4" c

Negative Negative Negative Negative

4" C or 37" C

FaBtc

4" c

Negative

4" C or 37" C

FaFc

4" c

Positive

FITGconjugated goat anti-human PIC. - ' FITC conjugated anti-human Fc ( y ) . donor.

Temperature

c

M F reaction

Positive

- a Polvvalent anti-HL-A serum of

a multitransfused

696

YEFENOF ET AL. TABLE I11

Surface IgM capping on Daudi cells

DETECTlON OF C3 RECEPTORS BY MEMBRANE FLUORESCENCE BEFORE A N D AFTER DEPLETION OF EAC BINDING CELLS ~~

Cells

Percentage Percentage Percentage Percentage M FMFEACEACpositive positive positive positive cells before cells before cells after cells after depletion depletion depletion depletion

Ra No. I

25

29

0

BJAB

24

25

0

9

GC-BJAB

54

59

0

14

10

completely. This is in line with the data in Table I suggesting that the M F test is more sensitive than the EAC rosette test. In addition, the complement used for detecting C3 receptor may be able to recognize C3b in addition to the C3d receptor identified by EAC mouse. Capping of C3 receptors

Figure 1 demonstrates the kinetics of C3 receptor capping on Raji cells. Incubation of stained cells at 37" C for 10 to 40 rnin turned the ring-like fluorescence first into dots and later into caps involving about 40% of the cells after 30min. Kinetics of capping the C3 and EBV receptors closely paralleled each other.

IgM capping on Daudi cells in the indirect test was described in detail elsewhere (Yefenof and Klein, 1974). In the present direct test, capping occurred according to the same pattern but in a lower proportion of the cells (Fig. 2). About 20% of the cells were capped after 90min incubation at 37" C . Kinetics of capping IgM could be clearly dissociated from the capping EBV receptor. Cap formation of EBV receptors on Raji and Daitdi cells

Incubation of Raji or Daudi cells after exposure to EBV and a direct anti-EBV conjugate at 37" C has led to the formation of dot spot-like fluorescence, merging into caps on 40 % of the cells after 30 min as illustrated in Figures 1 and 2. Double staining of EBV and C3 receptors

In order to detect whether EBV and C3 receptors were localized in the same areas and capped together, FITC staining and redistribution of C3 receptors were followed by TRITC staining for EBV receptors and vice versa, as described in " Material and Methods ". As shown in Figures 3-5 and in Table IV, green and red staining was localized in exactly the same dots, spots and caps. I n the photographs, the field was slightly shifted between the exposure of green vs red fluorescence, in order to confirm the

FIGURE 1 Kinetics of EBV and C3 receptor capping on Raji cells.