Scand. ]. Immunol., Vol, 6, 1977.
Enhanced Cytotoxicity of Human Lymphocytes Against Rabies-Infected Cells by Rabies-Specific Antibodies B. HARFAST, T, ANDERSSON & M, GRANDIEN Department of Immunology, University of Stockholm; Department of Infectious Diseases, RoslagstuUs Hospital; and Department of Virology, National Bacteriological Laboratory; Stockholm, Sweden
Harfast, B,, Andersson, T. & Grandien, M. Enhanced Cytotoxicity of Human Lymphocytes Against Rabies-Infected Cells by Rabies-Specific Antibodies, Scand. ]. Immunol. 6, 1107-1112, 1977, Human anti-rabies immune sera enhanced the in vitro cytotoxicity of human lymphocytes against rabies virus-infected green-monkey kidney cells. The immune se/a were collected from patients immunized with rabies vaccine produced either in human diploid cells or in nervous tissue. Significant cytotoxicity was observed even with high serum dilutions, indicating that the K-cell assay might be a sensitive tool for detection of anti-rabies antibodies. B. Harfast, Department Stockholm, Sweden
In vitro studies of immunity to viruses have shown that cells infected with viruses can be killed by a variety of different mechanisms (1, 3). In the mouse, antibody-independent T-cellmediated lysis of virus-infected cells occurs only when effector cells and target cells share at least one set of antigenic specificities determined by the H-2 genome complex (5), In contrast, other immune mechanisms, such as antibody-initiated, complement-dependent lysis (C-lysis) and antibody-dependent lymphocytemediated lysis (K-cell lysis), damage infected target cells without such restrictions with regard to expression of major transplantation antigens (22). These two mechanisms (C-lysis and Kcell lysis) involve antibodies that specifically recognize virus-induced antigens (1, 3), The C-lysis is mediated by complement components, whereas K-cell lysis is mediated by Fc-receptorbearing lymphocytes (1, 3, 13), Wiktor et al, (21) found that C-lysis is operative in a model in vitro system of rabies infection.
of Immunology,
University
of Stockholm,
S.106 91
The purpose of the present study was to establish whether vaccination with rabies vaccine elicits formation of antibodies capable of lysing rabies virus-infected target cells in the presence of unsensitized human lymphocytes. The reasons were twofold. First, the K-cell assay, because of its high sensitivity, would be expected to constitute a useful tool for monitoring anti-rabies antibodies arising after vaccination or in the course of infection. Second, assessment of K-cell-inducing antibodies in the blood of patients could be informative as to the possible protective (or pathogenic) role of different immune effector mechanisms after infection or passive and active immunization against rabies. MATERIALS AND METHODS Virus. The Flury HEP strain of rabies virus, passaged in human diploid cells, originally derived from the Wistar Institute, Philadelphia,
1108 B. Harfast, T. Andersson & M. Grandien Table I. Antibody titers after vaccination Serum,
1 2 3 4 5
Type of vaccine* NTV HDCV HDCV HDCV HDCV
Immunization schedule 14 daily doses Day 0, 3, 7, 14, 21 Day 0, 3, 7, 14, 21 Day 0, 3, 7, 14, 21 Day 0, 30
Days after ' last vaccination 20 15 30 30 15
Antibody titerst IF
N T (IU/ml)
1:243 1:27 1:27 1:27 1:27
il.6 9.4 18.8 18.8
2.4
'.
MH (mm)
o' 21 , 21 26 21
* NTV = nervous tissue vaccine; HDCV = human diploid cell vaccine. t IF = immunofluorescence test; NT = neutralization test; IU = international units; and MH mixed hemadsorption test, which is given as the diameters of the hemadsorption zones in millimeters.
Pa., USA, and passaged in green-monkey kidney cells (GMK), served to infect the target cells. The infectious titer of the virus preparation used was 1 X 10^-5 mean tissue culture infective dose (TCIDso/ml). Imnmnization. Laboratory workers or individuals exposed to rabies virus-infected animals were vaccinated with rabies vaccine produced in human diploid cells (HDCV). This vaccine is produced by Institute Merieux, Lyon, France, from the Pitman Moore strains of fixed rabies vims grown in a culture of human diploid cells (WI-38) and inactivated with ^-propiolactone. The vaccination schedule is given in Table I. A serum from a patient (a subject attacked by a rabid dog) immunized with the nervous tissue vaccine (NTV) was also tested. All sera used in the. antibody-dependent cellmediated cytotoxicity test were heat-inactivated at 56°C for 60 min. Antibody-dependent lymphocyte-mediated cytotoxicity assay. Confluent monolayers of GMK cells, grown on coverslips in Leighton tubes, were infected with 0.1 ml rabies virus. Two days after infection, cell monolayers were trypsinized and then washed twice in TrisHanks' (TH). Cells (1-2 X lO^) were suspended in 1 ml TH and incubated with 0.1 ml 5iNa2CrO4 (specific activity, 1 mCi/ml, 5-11 li.g Cr/ml) for 60 min. The labeled cells were then washed an additional three times and adjusted to the desired concentration in Parker medium supplemented with 7.5% fetal calf serum (FCS). Uninfected GMK cell mono-
layers were always cultured in parallel. Serum dilutions, target cells (5 X lO^), and lymphocytes were then added to conical plastic tubes in a total volume of 0.75 ml. The cells were sedimented before incubation for 6 h at 37°C. Augmentation of cytotoxicity above background was expressed as percentage isotope release observed with lymphocytes and test sera minus the percentage isotope release with lymphocytes alone. Immune serum in the absence of lymphocytes was not more cytotoxic than culture medium for the target cells. Lymphocytes alone were only occasionally more cytotoxic for the infected cells than medium alone after 6 h. Condition of infected cells. After 6 h of incubation, the isotope release (mean ± SD) from infected and uninfected target cells in medium alone was 13% ± 5.0% and 9.8% ± 3.8%, respectively. At the start of the cytotoxicity assay, 80% of cells were found, by investigation with the immunofluorescence technique, to be infected with rabies. Isolation of lymphocytes. Lymphocytes were isolated from human peripheral blood as previously described (9). Unless otherwise stated, lymphocytes were derived from nonimmune subjects. Serologieal tests. Serum specimens were assayed for antibodies by the following three tests: 1. Immunofluorescence (IF). The indirect fluorescence technique in accordance with standard methods was used to detect antibodies reacting with fixed rabies virus-infected cells.
K-Cell Activity to Rabies-Infected Cells 1109 Table II. Variation of lymphocyte to target cell ratio Lymphocyte to target cell ratio 100:1 100:1 50:1 50:1 10:1 10:1
Serum
Uninfected
Infected No serum Immune serum* No serum Immune serum No serum Immune serum
% Augmentation
% 5iCr release
28,2 41,9 21,8 36,3 17.9 22.1
-(- 0.8 ± 2.9 -4- 1,4 + 2,9 + 1,9 ± 0.5
9.9 ± 0,7 9.6 ± 0,2 8,8 9,0 8,9 8,5
+ + ± ±
0.2 0,8 0,7 0,4
Infected
Uninfected
14
0
15
0
4
0
* The serum was diluted 1:100,
Sera were tested in serial threefold dilution, and the highest dilution giving bright green, distinct, cytoplasmic inclusions was defined as the "IF titer'. 2. Neutralization test (NT). A rabies-neutralizing antibody assay was performed as described by Debbie et al. (4). Sera were tested in fourfold dilutions starting from 1:6,25, and the highest serum dilution neutralizing 50% of the virus was defined as the 'NT titer'. These were then converted into international units (IU) (19), 3. Mixed hemadsorption (MH) test. The method described by Espmark & Fagraeus (7) was used. Briefly, test sera were applied to paper discs and were allowed to diffuse through an agar layer to the virus-infected cells. Antibodies bound to the virus-infected cells were traced by adding sheep erythrocytes coated with an indicator system previously described (7), The diameters of the hemadsorption zones were measured. It has been shown (7) that there exists a linear relationship between the log of the serum dilution and the diameter of the zone. Test zones smaller than 8 mm were regarded as negative (8), Absorption of serum. Equal volumes of human anti-rabies serum and rabies-infected GMK cells were incubated for 2 h at 37 °C and for 48 h at 4°C, The mixture was centrifuged, and the supernatant was absorbed once more under the same conditions. Absorption with uninfected GMK cells was carried out in parallel. Fractionation of serum. Rabbit antiserum to human IgG was coupled to cyanogen bromide
(CNBr)-activated Sepharose 4B (Pharmacia Fine Chemicals, Uppsala, Sweden) (6), Human anti-rabies antiserum was passed twice through a column containing such Sepharose-anti-IgG gel. The effluent (IgG-depleted fraction) was concentrated to the starting volume and tested for its capacity to induce cytotoxicity.
RESULTS Serological studies Serological tests were carried out to ensure the presence of anti-rabies virus antibodies in the sera tested In the K-cell assay. All immune sera tested had serum titers of 1:27 or higher in the indirect IF test. The titers of the preimmune sera were lower than 1:3. The finding that the serum from the donor vaccinated with NTV had a very high IF titer but not a correspondingly high NT titer and MH titer has been observed and discussed previously (8). Demonstration of K-cell cytotoxicity against rabies virus-infected GMK cells Experiments were designed to establish whether immune sera could induce cytotoxicity against rabies virus-infected target cells in the presence of human lymphocytes. The five human immune sera presented in Table I were used. Attempts were not made to compare the relative strength of the various sera since their history varied with regard to immunization schedule, time of storage, source of vaccine, and days after last immunization.
1110 B, Harfast, T. Andersson & M. Grandien Table III. Antibody-dependent kidney (GMK) cells
lymphocyte-mediated cytotoxicity against rabies virus-infected green-monkev
% 5iCr release from GMK cells Expt,
Serum (no.)
1
None Immune (1) Immune (1) Immune (1) Immune (2) Immune (2) Immune (3 + 4) Immune (3 + 4) Neg.pool
Dilution
1:100. 1:400 1:1600 1:200 1:1000 1:200 1:1000 1:100 1:1600
Neg,pooltt 2
-
None Immune (5)
1:100 1:1000 1:10,000 1:100 1:1000 1:10,000
Pre-immune (5)
3
4
-
None Immune (2) Immune (2) Neg. None Immune (3 + 4)
1:100 1:1600 1:100 -
1:200
Infected 10.3 ± 33,9 ± 28,9 ± 26,4 + 40,7 + 28.3 ± 33.0 + 22,8 ± 13,7 ± 13,2 ± 21,8 ± 36,3 + 25.3 ± 23,0 + 24,4 + 22.8 + 23,0 + 15.4 + 25,6 ± 29,8 + 12,0 Jr 35,1 ± 51,0 -H
1,0 0,2*** 0,7*** 1.5*** 1,0*** 2,0** 2,7** 1,5** 1,5 0,9 1.4 2.9* 1,2 0,6 2,2 1,7 0,8 0,6 3,5* 2,4** 0,4 0,6 1,5***
Uninfected 15,1 ± 13,5 ± 14,3 + 14,3 + 13,5 ± 15,1 + 15,7 ± 13,5 ± 17,8 ± 13,6 ±
± ± ± ± ± + ± 12,9 ± 14,0 ± 8,8 9,0 8,1 9.5 9,5 8,8 9,8
0,6 0,7 0,2 0,2 0,6 0,1
0,3 0,3 0,2 0,3 0,2 0,8 0,6 0,3 0,8 0.4 0.9 2,0 2,5
13,6 ± 0,7 10,4 + 0,0 22,7 ± 1-1 20,1 ± 1,5
% Augmentation! Infected
Uninfected
24 19 16 30 18 23 13 3 3
-2 -1 -1 -2 0 1 -2
15 4
0 -1 1 1 0 1
1
3 1 1
3 2
10 14 -3
-3
16
-3
1 1
Statistical difference from lymphocytes alone: *** P
Enhanced Cytotoxicity of Human Lymphocytes Against Rabies-Infected Cells by Rabies-Specific Antibodies B. HARFAST, T, ANDERSSON & M, GRANDIEN Department of Immunology, University of Stockholm; Department of Infectious Diseases, RoslagstuUs Hospital; and Department of Virology, National Bacteriological Laboratory; Stockholm, Sweden
Harfast, B,, Andersson, T. & Grandien, M. Enhanced Cytotoxicity of Human Lymphocytes Against Rabies-Infected Cells by Rabies-Specific Antibodies, Scand. ]. Immunol. 6, 1107-1112, 1977, Human anti-rabies immune sera enhanced the in vitro cytotoxicity of human lymphocytes against rabies virus-infected green-monkey kidney cells. The immune se/a were collected from patients immunized with rabies vaccine produced either in human diploid cells or in nervous tissue. Significant cytotoxicity was observed even with high serum dilutions, indicating that the K-cell assay might be a sensitive tool for detection of anti-rabies antibodies. B. Harfast, Department Stockholm, Sweden
In vitro studies of immunity to viruses have shown that cells infected with viruses can be killed by a variety of different mechanisms (1, 3). In the mouse, antibody-independent T-cellmediated lysis of virus-infected cells occurs only when effector cells and target cells share at least one set of antigenic specificities determined by the H-2 genome complex (5), In contrast, other immune mechanisms, such as antibody-initiated, complement-dependent lysis (C-lysis) and antibody-dependent lymphocytemediated lysis (K-cell lysis), damage infected target cells without such restrictions with regard to expression of major transplantation antigens (22). These two mechanisms (C-lysis and Kcell lysis) involve antibodies that specifically recognize virus-induced antigens (1, 3), The C-lysis is mediated by complement components, whereas K-cell lysis is mediated by Fc-receptorbearing lymphocytes (1, 3, 13), Wiktor et al, (21) found that C-lysis is operative in a model in vitro system of rabies infection.
of Immunology,
University
of Stockholm,
S.106 91
The purpose of the present study was to establish whether vaccination with rabies vaccine elicits formation of antibodies capable of lysing rabies virus-infected target cells in the presence of unsensitized human lymphocytes. The reasons were twofold. First, the K-cell assay, because of its high sensitivity, would be expected to constitute a useful tool for monitoring anti-rabies antibodies arising after vaccination or in the course of infection. Second, assessment of K-cell-inducing antibodies in the blood of patients could be informative as to the possible protective (or pathogenic) role of different immune effector mechanisms after infection or passive and active immunization against rabies. MATERIALS AND METHODS Virus. The Flury HEP strain of rabies virus, passaged in human diploid cells, originally derived from the Wistar Institute, Philadelphia,
1108 B. Harfast, T. Andersson & M. Grandien Table I. Antibody titers after vaccination Serum,
1 2 3 4 5
Type of vaccine* NTV HDCV HDCV HDCV HDCV
Immunization schedule 14 daily doses Day 0, 3, 7, 14, 21 Day 0, 3, 7, 14, 21 Day 0, 3, 7, 14, 21 Day 0, 30
Days after ' last vaccination 20 15 30 30 15
Antibody titerst IF
N T (IU/ml)
1:243 1:27 1:27 1:27 1:27
il.6 9.4 18.8 18.8
2.4
'.
MH (mm)
o' 21 , 21 26 21
* NTV = nervous tissue vaccine; HDCV = human diploid cell vaccine. t IF = immunofluorescence test; NT = neutralization test; IU = international units; and MH mixed hemadsorption test, which is given as the diameters of the hemadsorption zones in millimeters.
Pa., USA, and passaged in green-monkey kidney cells (GMK), served to infect the target cells. The infectious titer of the virus preparation used was 1 X 10^-5 mean tissue culture infective dose (TCIDso/ml). Imnmnization. Laboratory workers or individuals exposed to rabies virus-infected animals were vaccinated with rabies vaccine produced in human diploid cells (HDCV). This vaccine is produced by Institute Merieux, Lyon, France, from the Pitman Moore strains of fixed rabies vims grown in a culture of human diploid cells (WI-38) and inactivated with ^-propiolactone. The vaccination schedule is given in Table I. A serum from a patient (a subject attacked by a rabid dog) immunized with the nervous tissue vaccine (NTV) was also tested. All sera used in the. antibody-dependent cellmediated cytotoxicity test were heat-inactivated at 56°C for 60 min. Antibody-dependent lymphocyte-mediated cytotoxicity assay. Confluent monolayers of GMK cells, grown on coverslips in Leighton tubes, were infected with 0.1 ml rabies virus. Two days after infection, cell monolayers were trypsinized and then washed twice in TrisHanks' (TH). Cells (1-2 X lO^) were suspended in 1 ml TH and incubated with 0.1 ml 5iNa2CrO4 (specific activity, 1 mCi/ml, 5-11 li.g Cr/ml) for 60 min. The labeled cells were then washed an additional three times and adjusted to the desired concentration in Parker medium supplemented with 7.5% fetal calf serum (FCS). Uninfected GMK cell mono-
layers were always cultured in parallel. Serum dilutions, target cells (5 X lO^), and lymphocytes were then added to conical plastic tubes in a total volume of 0.75 ml. The cells were sedimented before incubation for 6 h at 37°C. Augmentation of cytotoxicity above background was expressed as percentage isotope release observed with lymphocytes and test sera minus the percentage isotope release with lymphocytes alone. Immune serum in the absence of lymphocytes was not more cytotoxic than culture medium for the target cells. Lymphocytes alone were only occasionally more cytotoxic for the infected cells than medium alone after 6 h. Condition of infected cells. After 6 h of incubation, the isotope release (mean ± SD) from infected and uninfected target cells in medium alone was 13% ± 5.0% and 9.8% ± 3.8%, respectively. At the start of the cytotoxicity assay, 80% of cells were found, by investigation with the immunofluorescence technique, to be infected with rabies. Isolation of lymphocytes. Lymphocytes were isolated from human peripheral blood as previously described (9). Unless otherwise stated, lymphocytes were derived from nonimmune subjects. Serologieal tests. Serum specimens were assayed for antibodies by the following three tests: 1. Immunofluorescence (IF). The indirect fluorescence technique in accordance with standard methods was used to detect antibodies reacting with fixed rabies virus-infected cells.
K-Cell Activity to Rabies-Infected Cells 1109 Table II. Variation of lymphocyte to target cell ratio Lymphocyte to target cell ratio 100:1 100:1 50:1 50:1 10:1 10:1
Serum
Uninfected
Infected No serum Immune serum* No serum Immune serum No serum Immune serum
% Augmentation
% 5iCr release
28,2 41,9 21,8 36,3 17.9 22.1
-(- 0.8 ± 2.9 -4- 1,4 + 2,9 + 1,9 ± 0.5
9.9 ± 0,7 9.6 ± 0,2 8,8 9,0 8,9 8,5
+ + ± ±
0.2 0,8 0,7 0,4
Infected
Uninfected
14
0
15
0
4
0
* The serum was diluted 1:100,
Sera were tested in serial threefold dilution, and the highest dilution giving bright green, distinct, cytoplasmic inclusions was defined as the "IF titer'. 2. Neutralization test (NT). A rabies-neutralizing antibody assay was performed as described by Debbie et al. (4). Sera were tested in fourfold dilutions starting from 1:6,25, and the highest serum dilution neutralizing 50% of the virus was defined as the 'NT titer'. These were then converted into international units (IU) (19), 3. Mixed hemadsorption (MH) test. The method described by Espmark & Fagraeus (7) was used. Briefly, test sera were applied to paper discs and were allowed to diffuse through an agar layer to the virus-infected cells. Antibodies bound to the virus-infected cells were traced by adding sheep erythrocytes coated with an indicator system previously described (7), The diameters of the hemadsorption zones were measured. It has been shown (7) that there exists a linear relationship between the log of the serum dilution and the diameter of the zone. Test zones smaller than 8 mm were regarded as negative (8), Absorption of serum. Equal volumes of human anti-rabies serum and rabies-infected GMK cells were incubated for 2 h at 37 °C and for 48 h at 4°C, The mixture was centrifuged, and the supernatant was absorbed once more under the same conditions. Absorption with uninfected GMK cells was carried out in parallel. Fractionation of serum. Rabbit antiserum to human IgG was coupled to cyanogen bromide
(CNBr)-activated Sepharose 4B (Pharmacia Fine Chemicals, Uppsala, Sweden) (6), Human anti-rabies antiserum was passed twice through a column containing such Sepharose-anti-IgG gel. The effluent (IgG-depleted fraction) was concentrated to the starting volume and tested for its capacity to induce cytotoxicity.
RESULTS Serological studies Serological tests were carried out to ensure the presence of anti-rabies virus antibodies in the sera tested In the K-cell assay. All immune sera tested had serum titers of 1:27 or higher in the indirect IF test. The titers of the preimmune sera were lower than 1:3. The finding that the serum from the donor vaccinated with NTV had a very high IF titer but not a correspondingly high NT titer and MH titer has been observed and discussed previously (8). Demonstration of K-cell cytotoxicity against rabies virus-infected GMK cells Experiments were designed to establish whether immune sera could induce cytotoxicity against rabies virus-infected target cells in the presence of human lymphocytes. The five human immune sera presented in Table I were used. Attempts were not made to compare the relative strength of the various sera since their history varied with regard to immunization schedule, time of storage, source of vaccine, and days after last immunization.
1110 B, Harfast, T. Andersson & M. Grandien Table III. Antibody-dependent kidney (GMK) cells
lymphocyte-mediated cytotoxicity against rabies virus-infected green-monkev
% 5iCr release from GMK cells Expt,
Serum (no.)
1
None Immune (1) Immune (1) Immune (1) Immune (2) Immune (2) Immune (3 + 4) Immune (3 + 4) Neg.pool
Dilution
1:100. 1:400 1:1600 1:200 1:1000 1:200 1:1000 1:100 1:1600
Neg,pooltt 2
-
None Immune (5)
1:100 1:1000 1:10,000 1:100 1:1000 1:10,000
Pre-immune (5)
3
4
-
None Immune (2) Immune (2) Neg. None Immune (3 + 4)
1:100 1:1600 1:100 -
1:200
Infected 10.3 ± 33,9 ± 28,9 ± 26,4 + 40,7 + 28.3 ± 33.0 + 22,8 ± 13,7 ± 13,2 ± 21,8 ± 36,3 + 25.3 ± 23,0 + 24,4 + 22.8 + 23,0 + 15.4 + 25,6 ± 29,8 + 12,0 Jr 35,1 ± 51,0 -H
1,0 0,2*** 0,7*** 1.5*** 1,0*** 2,0** 2,7** 1,5** 1,5 0,9 1.4 2.9* 1,2 0,6 2,2 1,7 0,8 0,6 3,5* 2,4** 0,4 0,6 1,5***
Uninfected 15,1 ± 13,5 ± 14,3 + 14,3 + 13,5 ± 15,1 + 15,7 ± 13,5 ± 17,8 ± 13,6 ±
± ± ± ± ± + ± 12,9 ± 14,0 ± 8,8 9,0 8,1 9.5 9,5 8,8 9,8
0,6 0,7 0,2 0,2 0,6 0,1
0,3 0,3 0,2 0,3 0,2 0,8 0,6 0,3 0,8 0.4 0.9 2,0 2,5
13,6 ± 0,7 10,4 + 0,0 22,7 ± 1-1 20,1 ± 1,5
% Augmentation! Infected
Uninfected
24 19 16 30 18 23 13 3 3
-2 -1 -1 -2 0 1 -2
15 4
0 -1 1 1 0 1
1
3 1 1
3 2
10 14 -3
-3
16
-3
1 1
Statistical difference from lymphocytes alone: *** P
