Scand. ]. Immunol., Vol. 5, 1976.
Surface Markers on Human B and T Lymphocytes VIII. Association Between Complement and Epstein-Barr Virus Receptors on Human Lymphoid Cells M. JONDAL, G. KLEIN, M. B. A. OLDSTONE, V. BOKISH & E. YEFENOF Department of Tumor Biology, Karolinska Institute, Stockholm, Sweden, and Department of Immunopathology, Scripps Clinic and Research Foundation, La Jolla, California, USA
Jondal, M., Klein, G., Oldstone, M. B. A., Bokish, V. & Yefenof, E. Surface Markers on Human B and T Lymphocytes. VIII. Association Between Complement and Epstein-Barr Virus Reseptors on Human Lymphoid Cells. Scand. ]. Immunol. 5, 401-410, 1976. An association has been found between the Epstein-Barr virus (HBV) and complement (C3d) receptors on human lymphoid cells. The evidence •was fourfold: there was a correlation between the expression of these two receptors; inhibition experiments showed that the binding sites probably are close to each other in the cell membrane, although not identical; EBV and complement receptors have been found to co-cap in either order; and lymphoid cell lines lacking complement receptors could not be superinfected with EBV. M. Jondat, Department of Tumor Biology, Karotinska Institute, S-104 Ol Stockhotm, Sweden
Epstein-Barr virus (EBV) is the causative agent of infectious mononucleosis (18). It can transform human and simian lymphocytes, •which normally have a limited life span in vitro, into permanently established cell lines that carry multiple copies of the viral genome per cell (19, 34, 38, 41, 45). Some of these lines have a malignant potential, as demonstrated by their progressive gro^wth in heterotransplanted, immunodeficient animals, such as nude mice, anti-lymphocyte antiserum-treated hamsters, and autologous simian hosts (4, 28, 32, 35, 40, 51). In two human tumors, anaplastic carcinoma of the nasopharynx and African Burkitt's lymphoma, EBV DNA can be directly demonstrated in the proliferating tumor cells (17, 28, 43). An etiologic role for EBV in the genesis of these two tumors, alone or together with co-
factors, is therefore being seriously considered (35). A detailed understanding of the hostvirus interaction is thus important. In an earlier paper of this series we studied the surface characteristics of EBV-transformed cell lines (22). It was found that all lymphoid cell lines that carried the viral genome had surface characteristics typical of bursa-equivalent (B) lymphocytes. Moreover, peripheral B lymphocytes, but not thymus-derived (T) lymphocytes, expressed •virus-binding sites. These findings have been confirmed by an independent technique and were extended to show that most, if not all, normal B lymphocytes have the capacity to bind EBV (14). In the present work we have analyzed the relationship of the viral receptors to other receptors on the surfaces of normal and transformed B lymphocytes. This study was initiated
402
M. Jondal, G. Klein, M. B. A. Oldstone, V. Bokish & E. Yefenof
Table I. Simultaneous expression of Epstein-Barr virus (EBV) and complement receptors on various human cell types Cell type Raji Daudi Maku Namalwa
JHTC-33 Seraphine NC-37 LY-28 SKL-1
BJAB Ramos Molt-4 Kaplan Jijoye Akuba Sulubo Rael 698 75 CCRF-CEM CORF-H-SB 1301 K-562 Human B lymphocytes
Ref.
(10, 27, 46) (30)
(53) (27) (23) (27) (9b) (27) (8)
(31) (29) (39) (26) (20) (27) (27) (27) (42) (5)
(24) (24) (11) (33)
Classification B B B B B B B B B B B T
cell cell cell cell cell cell cell cell cell cell cell cell
B cell B cell B cell B cell B cell B cell B cell T cell T cell Undefined Undefined
EACn,* receptors
EBV+ receptors
EBNA antigen**
Superinfecta-
Pos. Pos. Pos. Pos. Pos. Pos. Pos. Pos. Pos. Pos. Pos. Pos.
Pos.' Pos. Pos. Pos. Pos. Pos. Pos. Pos. Pos. Pos. Pos. Pos.
Pos. Pos. Pos. Pos. Pos. Pos. Pos. Pos. Pos. Neg. Neg. Neg.
-f
Neg. Neg. Neg. Neg. Neg. Neg. Neg. Neg. Neg. Neg. Neg.
Neg. Neg. Neg. Neg. Neg. Neg. Neg. Neg. Neg. Neg. Neg.
Pos. Pos. Pos. Pos. Pos. Neg. Neg. Neg.
Pos.
Pos.
Neg. Neg.
bilitytt
+ -1+ -
.
tt tt tt tt tt tt tt
tt
* Indicator cells prepared with l:2-diluted fresh mouse serum. i" Cell lines were scored as positive or negative as defined in Materials and Methods. Most of the positive lines contained more than 80% of receptor-positive cells with the exception of SKL-1, BJAB, and MoIt-4, which were 4 0 % - 5 0 % positive. ** The EBV-determined Epstein-Barr nuclear antigen (EBNA) was detected as previously described (47). t t Superinfectability of cell lines as reported by us and others (26, 29, 31). BJAB and Ramos were susceptible to primary infection (29, 31), in contrast to the Molt-4, CCRF-CEM, 698, 75, 1301, and K-562 cell lines (25, 37).
after one of us (Dr. Oldstone) had observed that there was a correlation between the expression of EBV and complement receptors on several cultured cell lines. We now present evidence strongly indicating that EBV-binding sites and complement-binding sites are closely associated on the surface of human B lymphocytes and established lymphoid lines. MATERIALS AND METHODS Concentration of EBV from P3HR-1 cell cultures. The EBV preparation was kindly
supplied by Dr. Alice Adams. It was prepared (1) by culturing the Burkitt-lymphoma-derived P3HR-1 line (20) in RPMI 1640 with 2% fetal calf serum for 14 days at 33°C with an initial cell concentration of 2 X lO^/ml. Cells and debris were removed by low-speed centrifugation. Solid, dry-heat-sterilized NaCl (20 g/1) and polyethylene glycol 6000 (AB Kebo, Stockholm, Sweden), 50% (wt/vol) sterile solution in 0.5M NaCl, were added. The mixture was incubated for 1 h at 4°C. The virus was collected by centrifugation at 7000 g for 15 min in a Sorvall GS 3 rotor and resus-
EBV and Complement Receptors
pended in 1% of the initial volume. The virus preparation induced 22% early-antigen-positive cells in superinfected Raji cells after 48 h. Detection of EBV receptors. RPMI with 5% fetal calf serum was used as medium throughout. EBV receptors were detected by the immunofluorescence assay described previously (26, 27). 10^ cells were incubated for 1 h at 4°C in 50 fjd l:2-diluted virus preparation. After two washes, 50 /xl fluoresceinconjugated anti-EBV membrane antigen antiserum (Mutua) was added for 30 min at 4°C. After two final washes the cells were immediately counted and the number of membrane-fluorescence-positive cells scored. Cell lines with more than 30% positive cells were considered positive and lines with less than 5% negative. Detection of complement receptors. Four different indicator cells were used. C3b receptors were detected with antibody-covered SRBC treated either with l:50-diluted fresh human serum (EACh) or stepwise with purified complement components (EACj^23(b) )• ^^^ receptors were detected with indicator cells prepared either with l:2-diluted fresh mouse serum (EACm) or by enzymatic cleavage of
403
binding more than three complement indicator cells was determined. Neutralization of EBV inhibition of rosette formation. Twenty-five /xl of the EBV preparation was preincubated with 25 /xl of medium, anti-EBV antisera, or control sera for 1 h at 4°C. Thereafter the procedure was the same as for the inhibition of complement rosette formation. Additional inhibition studies. C3 was purified as described (6) and used at a concentration of 1 mg/ml. Antisera were purchased from Hyland and Dakopatts (Hyland Div., Travenol Laboratories Inc., Costa Mesa, Calif., USA; Dakopatts A/S Copenhagen, Denmark). Inhibition of rosette formation was performed as described above.
RESULTS
Parallel expression of complement and EBV receptors All 12 cell lines that were positive for EACm receptors also expressed EBV receptors (Table I). Eleven of them were classified as B-cell EAC.423(a) ^'^ •^•'^^i423(b) • ^^^ ^y^^jct procedure lines and nine carried the EBV genome. has been described elsewhere (7, 21). C3 Surprisingly, Molt-4 a T-cell line, expressed receptors were also detected by an immuno- EACm and EBV receptors. Four of nine EBVfluorescence assay. Purified C3 (6), 1 mg/ml, genome- and EBV-receptor-positive cell lines was adsorbed to receptor-positive cells and could be superinfected (26, 29, 31). BJAB subsequently stained with a rabbit anti-C3 and Ramos were susceptible to primary infecantiserum (Dakopatts A/S, Copenhagen, Den- tion (25, 29, 31), in contrast to the Molt-4 mark) conjugated with tetramethyl-rhodamine line (37). (BBL, Cockeysville, Md., USA). The same All complement-receptor-negative lines were labeling technique was used as for the detection also negative for EBV receptors. None of five of EBV receptors. EBV-genome-positive and EBV-receptor-negaThe same criteria, for positive or negative tive cell lines were susceptible to superinfection cells, as those applied in the EBV receptor (25, 26, 29), 698, 75, CCRF-CEM, 1301, test, were adopted for scoring C-binding cells. and K-562 could not be infected (25, 37). Inhibition of EAC rosette formation by EBV. The undefined cell lines 1301 and K-562, Twenty-five /il cell suspension (10^/ml) was derived from patients with acute lymphatic mixed with 25 yX oi the virus preparation, at leukemia and chronic myelocytic leukemia, various dilutions, and incubated for 1 h at respectively, lacked both EACm and EBV 4°C. Twenty-five /xl of complement indicator receptors. Human B lymphocytes, isolated from cells (0.25%) was added and the mixture peripheral blood, expressed EACm and EBV incubated for an additional 1 h at 4°C. After receptors as well, as described earlier (14, 21, thorough resuspension, the number of cells 22).
404
M. Jondal, G. Klein, M. B. A. Oldstone, V. Bokish & E. Yefenof
nonproducer cell line (control II). The control preparations were treated in exactly the same way as the EBV-containing preparation. In further control experiments we investigated whether the EBV preparations influenced the binding of EACm to monocytes (although EBV did not bind to these cells). These experiments were done in such a way that FicollIsopaque-purified cells, containing monocytes, were allowed to ingest IgG-covered sheep erythrocytes (SRBC) and were then subjected to an osmotic shock in order to lyse the extracellular SRBC, and finally the inhibiting effect of the EBV preparations on the rosetteforming ability of the phagocytic monocytes, with internal SRBC, was investigated. As expected, no inhibition was found. Cells washed free of unbound viral supernatant were blocked, as were cells in the presence of the viral preparation. The Daudi cell line, which also expresses binding sites for IgG (Fc receptors), was tested for its capacity to bind IgG-covered indicator cells, before and after treatment with viral concentrate. There was no inhibition of Fcindicator-cell-binding at viral concentrations that completely inhibited the binding of EAC indicator cells. Furthermore, whereas complete
Table II. Complement and Epstein-Barr virus (EBV) receptors on Daudi and Raji cells % rosette-forming cells
EBV fluorescent cells
EACi423(b)' Daudi Raji
2 96
72 95
97
* Sheep erythrocytes treated stepwise with purified complement components (EACi423(b)) m d by enzymatic cleavage of EACi423(b)to EACi423(d) •
• As shown in Table II, Daudi cells, which are known to express C3d but not C3b receptors, also carry EBV receptors. Raji cells express both C3b and C3d receptors and carry EBV receptors as well. EBV inhibition of rosette formation Both EACh and EACm rosette formation was inhibited by preincubation with different virus dilutions (Table III). Different cell lines were blocked to different degrees. There was no inhibition with the control supernatants, derived from a 0.5-h-old P3HR-1 supernatant (control I) and a l4-day-old supernatant from Raji, a
Table III. Inhibition of EACm and EACh rosette formation by Epstein-Barr virus (EBV)*
% rosette-forming cells Cell line
EBV dilutions
cell
Raji Daudi Maku Namalwa Human lymphocytes
EACm EACh EACm EACh
Control I Control II Medium
1:1
1:2
1:4
1:8
1:16
1:32
1:64
1:1
1:1
5
59 5
75
89
1
3
14
95 92 86
95 93 89
87
72
96 89 41
97
21
2
2
1
2 1
91 92
90 79
98 93
EACm EACh EACm EACh
26
37
87
96
95
96
91
98
83 21
82
87
86 81
29
78
75'
83 80 71
87
73
87 85
9
86 47 48
97 86 63 73
95
71 2
EACm EACh
• 1 0
2 2
3 5
7
10
14
8
15
15
83 76
75
14 15
14 15
13
13
84 76 14 15
* Inhibition of rosette formation was performed as described in Materials and Methods. Control I and II are described in the Results section. EACm and EACh are antibody-covered sheep erythrocytes treated with l:2-diluted fresh mouse serum and l:50-diluted fresh human serum, respectively.
EBV and Complement Receptors
405
Table IV. Neutralization of Epstein-Barr virus (EBV) inhibition of EAC^* rosette formation by EBV antisera EBV preincubated
% rosette-forming cells
withj
Medium Anti-EBV I Anti-EBV II Control serum I Control serum II
Raji
Namalwa
Daudi
Lymphocytes
1:1**
1:2
0
1:1
1:2
0
1:1
1:2
0
1:1
8 58 79 7 8
48 89 93 46 39
98 96 96 92 97
2 48 81 1
8 79 86 3
91 89 90 90 87
0 9 12
2
8 68 75 7 3
49
1
82 92 87 94 89
83 87 35 37
1:2
1
2 12 13 2
0
1
0 13 14 14 14 13
* EACn, ^= antibody-covered sheep erythrocytes treated with l:2-diluted fresh mouse serum, t Sera used in this experiment are described in Table V. ** EBV dilutions preincubated with the different sera as described in Materials and Methods.
crisscross co-cappitig was found between EBV and complement receptors, Fc and EBV receptors were found to cap independently in the cell membrane (54). Neutralization of EBV inhibition of rosette formation Preincubation of the viral preparation with anti-EBV-positive sera neutralized the EACmrosette-inhibiting capacity of the latter (Table IV). EBV-antibody-negative antisera did not influence the reaction. The EBV antibody titers of the sera are shown in Table V.
Additional control experiments were performed to exclude the possibility of complement contamination of the EBV preparation. Viral preparations were treated with immune complexes in the form of antibody-covered SRBC, known to react strongly with soluble complement components. There was no difference in the ability of immune-complex-treated and untreated viral preparations to inhibit EACm rosette formation. In another experiment we tested antibody-treated SRBC for complement fixation in the course of incubation with the viral preparation at 37 °C for 30 min. Following incubation, the SRBC failed to adhere to
Table V. Epstein-Barr virus (EBV) antibody titers of the sera used for the neutralization tests in Table IV* Anti-VCA titer Anti-EBV serum I P.T. KCC 1582 Anti-EBV serum II O.O. KCC 1349 EBV-negative control serum I LE.
EBV-negative control serum II E.K.
Anti-EA titer
Anti-MA titer
•' 1:1280-2560
. 1:320-640
Not done
1:2560-5120
1:2560
l/lOO
Surface Markers on Human B and T Lymphocytes VIII. Association Between Complement and Epstein-Barr Virus Receptors on Human Lymphoid Cells M. JONDAL, G. KLEIN, M. B. A. OLDSTONE, V. BOKISH & E. YEFENOF Department of Tumor Biology, Karolinska Institute, Stockholm, Sweden, and Department of Immunopathology, Scripps Clinic and Research Foundation, La Jolla, California, USA
Jondal, M., Klein, G., Oldstone, M. B. A., Bokish, V. & Yefenof, E. Surface Markers on Human B and T Lymphocytes. VIII. Association Between Complement and Epstein-Barr Virus Reseptors on Human Lymphoid Cells. Scand. ]. Immunol. 5, 401-410, 1976. An association has been found between the Epstein-Barr virus (HBV) and complement (C3d) receptors on human lymphoid cells. The evidence •was fourfold: there was a correlation between the expression of these two receptors; inhibition experiments showed that the binding sites probably are close to each other in the cell membrane, although not identical; EBV and complement receptors have been found to co-cap in either order; and lymphoid cell lines lacking complement receptors could not be superinfected with EBV. M. Jondat, Department of Tumor Biology, Karotinska Institute, S-104 Ol Stockhotm, Sweden
Epstein-Barr virus (EBV) is the causative agent of infectious mononucleosis (18). It can transform human and simian lymphocytes, •which normally have a limited life span in vitro, into permanently established cell lines that carry multiple copies of the viral genome per cell (19, 34, 38, 41, 45). Some of these lines have a malignant potential, as demonstrated by their progressive gro^wth in heterotransplanted, immunodeficient animals, such as nude mice, anti-lymphocyte antiserum-treated hamsters, and autologous simian hosts (4, 28, 32, 35, 40, 51). In two human tumors, anaplastic carcinoma of the nasopharynx and African Burkitt's lymphoma, EBV DNA can be directly demonstrated in the proliferating tumor cells (17, 28, 43). An etiologic role for EBV in the genesis of these two tumors, alone or together with co-
factors, is therefore being seriously considered (35). A detailed understanding of the hostvirus interaction is thus important. In an earlier paper of this series we studied the surface characteristics of EBV-transformed cell lines (22). It was found that all lymphoid cell lines that carried the viral genome had surface characteristics typical of bursa-equivalent (B) lymphocytes. Moreover, peripheral B lymphocytes, but not thymus-derived (T) lymphocytes, expressed •virus-binding sites. These findings have been confirmed by an independent technique and were extended to show that most, if not all, normal B lymphocytes have the capacity to bind EBV (14). In the present work we have analyzed the relationship of the viral receptors to other receptors on the surfaces of normal and transformed B lymphocytes. This study was initiated
402
M. Jondal, G. Klein, M. B. A. Oldstone, V. Bokish & E. Yefenof
Table I. Simultaneous expression of Epstein-Barr virus (EBV) and complement receptors on various human cell types Cell type Raji Daudi Maku Namalwa
JHTC-33 Seraphine NC-37 LY-28 SKL-1
BJAB Ramos Molt-4 Kaplan Jijoye Akuba Sulubo Rael 698 75 CCRF-CEM CORF-H-SB 1301 K-562 Human B lymphocytes
Ref.
(10, 27, 46) (30)
(53) (27) (23) (27) (9b) (27) (8)
(31) (29) (39) (26) (20) (27) (27) (27) (42) (5)
(24) (24) (11) (33)
Classification B B B B B B B B B B B T
cell cell cell cell cell cell cell cell cell cell cell cell
B cell B cell B cell B cell B cell B cell B cell T cell T cell Undefined Undefined
EACn,* receptors
EBV+ receptors
EBNA antigen**
Superinfecta-
Pos. Pos. Pos. Pos. Pos. Pos. Pos. Pos. Pos. Pos. Pos. Pos.
Pos.' Pos. Pos. Pos. Pos. Pos. Pos. Pos. Pos. Pos. Pos. Pos.
Pos. Pos. Pos. Pos. Pos. Pos. Pos. Pos. Pos. Neg. Neg. Neg.
-f
Neg. Neg. Neg. Neg. Neg. Neg. Neg. Neg. Neg. Neg. Neg.
Neg. Neg. Neg. Neg. Neg. Neg. Neg. Neg. Neg. Neg. Neg.
Pos. Pos. Pos. Pos. Pos. Neg. Neg. Neg.
Pos.
Pos.
Neg. Neg.
bilitytt
+ -1+ -
.
tt tt tt tt tt tt tt
tt
* Indicator cells prepared with l:2-diluted fresh mouse serum. i" Cell lines were scored as positive or negative as defined in Materials and Methods. Most of the positive lines contained more than 80% of receptor-positive cells with the exception of SKL-1, BJAB, and MoIt-4, which were 4 0 % - 5 0 % positive. ** The EBV-determined Epstein-Barr nuclear antigen (EBNA) was detected as previously described (47). t t Superinfectability of cell lines as reported by us and others (26, 29, 31). BJAB and Ramos were susceptible to primary infection (29, 31), in contrast to the Molt-4, CCRF-CEM, 698, 75, 1301, and K-562 cell lines (25, 37).
after one of us (Dr. Oldstone) had observed that there was a correlation between the expression of EBV and complement receptors on several cultured cell lines. We now present evidence strongly indicating that EBV-binding sites and complement-binding sites are closely associated on the surface of human B lymphocytes and established lymphoid lines. MATERIALS AND METHODS Concentration of EBV from P3HR-1 cell cultures. The EBV preparation was kindly
supplied by Dr. Alice Adams. It was prepared (1) by culturing the Burkitt-lymphoma-derived P3HR-1 line (20) in RPMI 1640 with 2% fetal calf serum for 14 days at 33°C with an initial cell concentration of 2 X lO^/ml. Cells and debris were removed by low-speed centrifugation. Solid, dry-heat-sterilized NaCl (20 g/1) and polyethylene glycol 6000 (AB Kebo, Stockholm, Sweden), 50% (wt/vol) sterile solution in 0.5M NaCl, were added. The mixture was incubated for 1 h at 4°C. The virus was collected by centrifugation at 7000 g for 15 min in a Sorvall GS 3 rotor and resus-
EBV and Complement Receptors
pended in 1% of the initial volume. The virus preparation induced 22% early-antigen-positive cells in superinfected Raji cells after 48 h. Detection of EBV receptors. RPMI with 5% fetal calf serum was used as medium throughout. EBV receptors were detected by the immunofluorescence assay described previously (26, 27). 10^ cells were incubated for 1 h at 4°C in 50 fjd l:2-diluted virus preparation. After two washes, 50 /xl fluoresceinconjugated anti-EBV membrane antigen antiserum (Mutua) was added for 30 min at 4°C. After two final washes the cells were immediately counted and the number of membrane-fluorescence-positive cells scored. Cell lines with more than 30% positive cells were considered positive and lines with less than 5% negative. Detection of complement receptors. Four different indicator cells were used. C3b receptors were detected with antibody-covered SRBC treated either with l:50-diluted fresh human serum (EACh) or stepwise with purified complement components (EACj^23(b) )• ^^^ receptors were detected with indicator cells prepared either with l:2-diluted fresh mouse serum (EACm) or by enzymatic cleavage of
403
binding more than three complement indicator cells was determined. Neutralization of EBV inhibition of rosette formation. Twenty-five /xl of the EBV preparation was preincubated with 25 /xl of medium, anti-EBV antisera, or control sera for 1 h at 4°C. Thereafter the procedure was the same as for the inhibition of complement rosette formation. Additional inhibition studies. C3 was purified as described (6) and used at a concentration of 1 mg/ml. Antisera were purchased from Hyland and Dakopatts (Hyland Div., Travenol Laboratories Inc., Costa Mesa, Calif., USA; Dakopatts A/S Copenhagen, Denmark). Inhibition of rosette formation was performed as described above.
RESULTS
Parallel expression of complement and EBV receptors All 12 cell lines that were positive for EACm receptors also expressed EBV receptors (Table I). Eleven of them were classified as B-cell EAC.423(a) ^'^ •^•'^^i423(b) • ^^^ ^y^^jct procedure lines and nine carried the EBV genome. has been described elsewhere (7, 21). C3 Surprisingly, Molt-4 a T-cell line, expressed receptors were also detected by an immuno- EACm and EBV receptors. Four of nine EBVfluorescence assay. Purified C3 (6), 1 mg/ml, genome- and EBV-receptor-positive cell lines was adsorbed to receptor-positive cells and could be superinfected (26, 29, 31). BJAB subsequently stained with a rabbit anti-C3 and Ramos were susceptible to primary infecantiserum (Dakopatts A/S, Copenhagen, Den- tion (25, 29, 31), in contrast to the Molt-4 mark) conjugated with tetramethyl-rhodamine line (37). (BBL, Cockeysville, Md., USA). The same All complement-receptor-negative lines were labeling technique was used as for the detection also negative for EBV receptors. None of five of EBV receptors. EBV-genome-positive and EBV-receptor-negaThe same criteria, for positive or negative tive cell lines were susceptible to superinfection cells, as those applied in the EBV receptor (25, 26, 29), 698, 75, CCRF-CEM, 1301, test, were adopted for scoring C-binding cells. and K-562 could not be infected (25, 37). Inhibition of EAC rosette formation by EBV. The undefined cell lines 1301 and K-562, Twenty-five /il cell suspension (10^/ml) was derived from patients with acute lymphatic mixed with 25 yX oi the virus preparation, at leukemia and chronic myelocytic leukemia, various dilutions, and incubated for 1 h at respectively, lacked both EACm and EBV 4°C. Twenty-five /xl of complement indicator receptors. Human B lymphocytes, isolated from cells (0.25%) was added and the mixture peripheral blood, expressed EACm and EBV incubated for an additional 1 h at 4°C. After receptors as well, as described earlier (14, 21, thorough resuspension, the number of cells 22).
404
M. Jondal, G. Klein, M. B. A. Oldstone, V. Bokish & E. Yefenof
nonproducer cell line (control II). The control preparations were treated in exactly the same way as the EBV-containing preparation. In further control experiments we investigated whether the EBV preparations influenced the binding of EACm to monocytes (although EBV did not bind to these cells). These experiments were done in such a way that FicollIsopaque-purified cells, containing monocytes, were allowed to ingest IgG-covered sheep erythrocytes (SRBC) and were then subjected to an osmotic shock in order to lyse the extracellular SRBC, and finally the inhibiting effect of the EBV preparations on the rosetteforming ability of the phagocytic monocytes, with internal SRBC, was investigated. As expected, no inhibition was found. Cells washed free of unbound viral supernatant were blocked, as were cells in the presence of the viral preparation. The Daudi cell line, which also expresses binding sites for IgG (Fc receptors), was tested for its capacity to bind IgG-covered indicator cells, before and after treatment with viral concentrate. There was no inhibition of Fcindicator-cell-binding at viral concentrations that completely inhibited the binding of EAC indicator cells. Furthermore, whereas complete
Table II. Complement and Epstein-Barr virus (EBV) receptors on Daudi and Raji cells % rosette-forming cells
EBV fluorescent cells
EACi423(b)' Daudi Raji
2 96
72 95
97
* Sheep erythrocytes treated stepwise with purified complement components (EACi423(b)) m d by enzymatic cleavage of EACi423(b)to EACi423(d) •
• As shown in Table II, Daudi cells, which are known to express C3d but not C3b receptors, also carry EBV receptors. Raji cells express both C3b and C3d receptors and carry EBV receptors as well. EBV inhibition of rosette formation Both EACh and EACm rosette formation was inhibited by preincubation with different virus dilutions (Table III). Different cell lines were blocked to different degrees. There was no inhibition with the control supernatants, derived from a 0.5-h-old P3HR-1 supernatant (control I) and a l4-day-old supernatant from Raji, a
Table III. Inhibition of EACm and EACh rosette formation by Epstein-Barr virus (EBV)*
% rosette-forming cells Cell line
EBV dilutions
cell
Raji Daudi Maku Namalwa Human lymphocytes
EACm EACh EACm EACh
Control I Control II Medium
1:1
1:2
1:4
1:8
1:16
1:32
1:64
1:1
1:1
5
59 5
75
89
1
3
14
95 92 86
95 93 89
87
72
96 89 41
97
21
2
2
1
2 1
91 92
90 79
98 93
EACm EACh EACm EACh
26
37
87
96
95
96
91
98
83 21
82
87
86 81
29
78
75'
83 80 71
87
73
87 85
9
86 47 48
97 86 63 73
95
71 2
EACm EACh
• 1 0
2 2
3 5
7
10
14
8
15
15
83 76
75
14 15
14 15
13
13
84 76 14 15
* Inhibition of rosette formation was performed as described in Materials and Methods. Control I and II are described in the Results section. EACm and EACh are antibody-covered sheep erythrocytes treated with l:2-diluted fresh mouse serum and l:50-diluted fresh human serum, respectively.
EBV and Complement Receptors
405
Table IV. Neutralization of Epstein-Barr virus (EBV) inhibition of EAC^* rosette formation by EBV antisera EBV preincubated
% rosette-forming cells
withj
Medium Anti-EBV I Anti-EBV II Control serum I Control serum II
Raji
Namalwa
Daudi
Lymphocytes
1:1**
1:2
0
1:1
1:2
0
1:1
1:2
0
1:1
8 58 79 7 8
48 89 93 46 39
98 96 96 92 97
2 48 81 1
8 79 86 3
91 89 90 90 87
0 9 12
2
8 68 75 7 3
49
1
82 92 87 94 89
83 87 35 37
1:2
1
2 12 13 2
0
1
0 13 14 14 14 13
* EACn, ^= antibody-covered sheep erythrocytes treated with l:2-diluted fresh mouse serum, t Sera used in this experiment are described in Table V. ** EBV dilutions preincubated with the different sera as described in Materials and Methods.
crisscross co-cappitig was found between EBV and complement receptors, Fc and EBV receptors were found to cap independently in the cell membrane (54). Neutralization of EBV inhibition of rosette formation Preincubation of the viral preparation with anti-EBV-positive sera neutralized the EACmrosette-inhibiting capacity of the latter (Table IV). EBV-antibody-negative antisera did not influence the reaction. The EBV antibody titers of the sera are shown in Table V.
Additional control experiments were performed to exclude the possibility of complement contamination of the EBV preparation. Viral preparations were treated with immune complexes in the form of antibody-covered SRBC, known to react strongly with soluble complement components. There was no difference in the ability of immune-complex-treated and untreated viral preparations to inhibit EACm rosette formation. In another experiment we tested antibody-treated SRBC for complement fixation in the course of incubation with the viral preparation at 37 °C for 30 min. Following incubation, the SRBC failed to adhere to
Table V. Epstein-Barr virus (EBV) antibody titers of the sera used for the neutralization tests in Table IV* Anti-VCA titer Anti-EBV serum I P.T. KCC 1582 Anti-EBV serum II O.O. KCC 1349 EBV-negative control serum I LE.
EBV-negative control serum II E.K.
Anti-EA titer
Anti-MA titer
•' 1:1280-2560
. 1:320-640
Not done
1:2560-5120
1:2560
l/lOO
