Intervirology 6: 249-257 (1975/76)
In vitro Stimulation of Human Lymphocytes by Purified Cytomegalovirus A nne M oller-L arsen, H ans K erzel A ndersen, I ver H eron and Israel Sarov Institute of Medical Microbiology, University o f Aarhus, Aarhus, and Chanock Centre for Virology, Hebrew University, Hadassah Medical School, Jerusalem
Key Words. Lymphocyte stimulation • Cytomegalovirus ■Cellular immunity Summary. Lymphocytes from 19 healthy donors were tested against purified cyto megalovirus (CMV), strain Ad 169, in a lymphocyte-transformation test. The test was performed in microcultures using various preparations and concentrations of antigen. These included, besides purified CMV, CMV-infected cells, herpes simplex virus type 1 antigen, and control antigens. Lymphocytes from CMV-scropositive donors were stimulated by purified virus and infected cells, whereas lymphocytes from seronegative donors did not respond. Similarly, only lymphocytes from herpes-seropositive donors did respond to herpes antigen. With these preparations of antigen the test was found to be sensitive and specific.
Stimulation of sensitized lymphocytes by viral antigens has been suggested to be an expression of cell-mediated immunity [1,2]. This technique has been applied to a number of viral systems [3-11], but not properly in the case of cytomegalovirus (CMV). In many of these studies the viral antigen was only partially purified, and occasionally blast transformation was seen in persons who were seronegative [7,10,12]. Recently a method has been developed for preparation of highly purified human CMV from the supernatants of CMV-infected cells [13]. The present study was undertaken to examine whether peripheral lymphocytes from healthy human adults would be stimulated by the highly purified CMV antigen. Address inquiries to: Dr. A nne M oller-L arsen, Institute o f Medical Microbiology, Bartholin Building, University of Aarhus, Aarhus (Denmark)
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Received: December4, 1975; revised: January27, 1976.
250
M oller-L arsen/A ndersen/H eron/S arov
Materials and Methods
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Purified C M V antigen. Highly purified CMV, strain Ad 169, was obtained from the supernatant fluids of infected human embryonic fibroblasts by successive zone and density centrifugation as described by Sarov and A bady [13]. The concentration of the virus was adjusted to 0.3 OD 260 nm. The infectivity o f the viral preparations was determined by the method of P lummer and Benyesh-M elnick [14]. 1 OD 260 nm gave 1 0 M 0 8 PFU/ml. The viral band was dialyzed overnight against phosphate-buffered saline (PBS), pH 7.2, and inactivated at 56° for 1 h. This treatment resulted in complete inactivation o f the virus. Supernatant from uninfected fibroblasts of the same line, cultured for 10 days, was treated in the same way as the purified CMV antigen and served as control antigen. In one experiment CMV was inactivated by UV irradiation as follows: 1 ml o f the purified virus (0.3 OD 260 nm) was placed in a 35-mm Petri dish and irradiated by a short wave UV lamp, UVS-54, from a distance of 28 cm for 10 min with continual shaking. This treatment resulted in complete inactivation of the virus. CMV-infected cells were produced according to a method by T hurman et al. [11]. Monolayers of human fibroblasts in 250-ml Falcon bottles containing 4 x 106 cells were infected with CMV strain Ad 169 at a multiplicity o f 2 PFU/cell. The virus was adsorbed for 2 h at 37°, and Eagle’s MEM - supplemented with 2% calf serum and 2 0 0 IU penicillin, 200 ug streptomycin and 2.5 ¡xg mycostatin per ml - was added to the cultures. Following incubation at 37° in 5% CO2 atmosphere for 5-6 days, the medium was removed and the cultures were washed once with PBS, pH 7.2. RPMI-I640 medium containing 40 ¡xg/ml mitomycin-C (Sigma Chemical Co.) was added and the cultures were incubated for 1 h at 37°. The cells were then washed five times with PBS and removed from the bottles with trypsin. The cells were then washed twice in RPMI-1640 and adjusted to contain 2 * 104 cells/ml of RPMI-1640. 10 ¡xl o f this cell suspension was used per well in lymphocyte stimulation experiments. Control fibroblasts were obtained from uninfected cultures prepared for stimulation in exactly the same manner as the infected ones. Herpes simplex type 1 (HSV-1) antigen. HSV-1 antigens were prepared from human embryonic lung fibroblasts (HEL cells) grown in Eagle’s MEM with 2% calf serum. The cells were infected by HSV-1, 2-4 PFU/cell. 24-48 h after infection the infected cultures were frozen and thawed twice and centrifuged for 10 min at 2,000 rpm. The virus in the supernatant was partially purified by two 1-hour centrifugations at 100,000 g. The pelleted viruses were resuspended in PBS, pH 7.2. The virus preparation was inactivated by UV light [4], Before UV treatment the titer was 4 x IO7 PFU/ml. After UV treatment the titer was 0. HEL antigen was prepared from uninfected HEL cells treated in the same manner. Leucocyte cultures. Venous blood from 19 volunteers was taken into glass bottles containing preservative-free heparin at a final concentration o f 20 IU/ml o f blood. The mononuclear cells were obtained following isopaque-Ficoll flotation [15]. The mononuclear cells were washed twice in RPMI-1640 containing 15-20 IU heparin, 100¡xg of streptomycin per ml, 100 IU of penicillin per mi, 10 niM HEPES buffer, and 5% heat-inactivated human serum from individuals who were seronegative against both CMV and HSV, as determined by complement fixation (CF) and neutralization (Nt) tests [16]. After washing, the cells were resuspended in RPMI-1640 with streptomycin, penicillin, HEPES buffer, and 15% human serum. The cells were counted, and the cell suspension was diluted to contain 1 x 106 lymphocytes per ml. The cell suspension was dispensed in 0.2-ml
251
Lymphocyte Stimulation by Cytomegalovirus
Table l. Incorporation of 14C-thymidine in lymphocytes from a seropositive and a sero negative individual, stimulated by various antigen preparations1 Lymphocyte donor
Dilutions CMV antigens and control antigens
R. A. (CMV-CF titer 32)
cone. 1:5 1:25
1,827 3,775 3,317
1,812 3,000 4,165
N .H . cone. CMV-CF titer < 4 1:5 and Nt titer < 2 1:25
76 61 111
345 194 236
purified CMV antigen infected --------------------------------------------- cells untreated heatUVinactivated inactivated not done 3,000 3,876
uninfected cells
768 4,047 3,000
84 52 48
71 147 467
545 944 447
81 276 377
1 In this experiment CMV was used in concentration 1.0 OD 260 nm.
aliquots in the wells of a Linbro microplate No. IS-MRC-96. Cultures were added to 10 |xl medium containing the different antigens. Phytohemagglutinin (PHA) was used in a solu tion containing 0.1 p.g/p.1. Triplicate determinations were performed. The plates were closed tightly by a plastic cover and incubated in 37° for a total of 6 days. Cultures stimulated with PHA were incubated for a total o f 3 days. Thymidine incorporation. For the final 24 h o f incubation, 1 p.1 of 14C-thymidine from a thymidine solution containing 0.02 ¡xCi/ptl was added to each well. The cells were harvested with a Skatron-harvester POB 28 (Lierbyen, Norway) on Whatman Glass Fiber Paper GF 81, thickness 0.25 mm. In the harvester the filters were washed with demineralized water. The filters were placed in plastic vials with 5 ml o f triton toluol scintillation fluid and counted in aTri-carb liquid scintillation spectrometer N o .2450. Results are given in tables I and II as the calculated medium values in disintegrations per minute (dpm). Detection o f C M V and H SV antibodies. The sera were tested for antibodies to CMV by neutralization and complement fixation tests [16], Antibodies to HSV were detected in a similar way.
Results
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Inactivation procedures with CM V for preparation o f the antigen. Initial experiments were performed to compare different preparations of CMV antigen. Table I shows the results of such an experiment. CMV particles inactivated either by UV or by heat (56°, 1 h) stimulate 14C-thymidine uptake in peripheral
252
M oller-L arsen/A ndersen/H eron/S arov
CONCENTRATION OF ANTIGEN
lymphocytes of a CMV-positive individual essentially to the same degree as the untreated purified viruses and the CMV-infected cells. The control antigen from the medium of uninfected fibroblast cultures did not give any stimulation. The heat-inactivated, purified CMV and CMV-infected cells were chosen for the following experiments. Dose response curve. As shown in figure 1, the lymphocytes from the three seropositive individuals responded somewhat differently. With the dilution 1:4, maximal stimulation was obtained in two of these subjects, and a submaximal but considerable stimulation was seen in the third, whereas lympho cytes from seronegative donors were not stimulated by the purified CMV at any amounts. Repeated experiments confirmed this, so the 1:1 dilution as well as 1:4 was chosen for the further experiments. Response o f lymphocytes from seronegative and seropositive adults. Lymphocytes were obtained from 19 healthy donors; 10 were CMV-seropositive, 9 were seronegative.
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Fig. I. Dose-response curves with purified CMV. Lymphocytes from five donors were tested with various amounts o f purified virus. The antigen (0.3 O D 260 nm) was diluted from 1:1 to 1:256 and added in 10-ul aliquots. Higher concentrations o f antigen (2:1 and 3:1) were also tested by adding 20 and 30 ;xl o f antigen to the wells.
253
Lymphocyte Stimulation by Cytomegalovirus Table II. Antigen stimulation of lymphocytes from 19 individuals Donor
Serology CMV
Lymphocyte stimulation, dpm1, with antigens indicated : HSV
CF
Nt
CF
G.E.P. I. H. A .M .L. H.J. B.P. R.A. B.S. G.L. T.S. G.K .
16 32 32 4 16 32 32 32 64 64
+ + + + + + + + + +
AC2
In vitro Stimulation of Human Lymphocytes by Purified Cytomegalovirus A nne M oller-L arsen, H ans K erzel A ndersen, I ver H eron and Israel Sarov Institute of Medical Microbiology, University o f Aarhus, Aarhus, and Chanock Centre for Virology, Hebrew University, Hadassah Medical School, Jerusalem
Key Words. Lymphocyte stimulation • Cytomegalovirus ■Cellular immunity Summary. Lymphocytes from 19 healthy donors were tested against purified cyto megalovirus (CMV), strain Ad 169, in a lymphocyte-transformation test. The test was performed in microcultures using various preparations and concentrations of antigen. These included, besides purified CMV, CMV-infected cells, herpes simplex virus type 1 antigen, and control antigens. Lymphocytes from CMV-scropositive donors were stimulated by purified virus and infected cells, whereas lymphocytes from seronegative donors did not respond. Similarly, only lymphocytes from herpes-seropositive donors did respond to herpes antigen. With these preparations of antigen the test was found to be sensitive and specific.
Stimulation of sensitized lymphocytes by viral antigens has been suggested to be an expression of cell-mediated immunity [1,2]. This technique has been applied to a number of viral systems [3-11], but not properly in the case of cytomegalovirus (CMV). In many of these studies the viral antigen was only partially purified, and occasionally blast transformation was seen in persons who were seronegative [7,10,12]. Recently a method has been developed for preparation of highly purified human CMV from the supernatants of CMV-infected cells [13]. The present study was undertaken to examine whether peripheral lymphocytes from healthy human adults would be stimulated by the highly purified CMV antigen. Address inquiries to: Dr. A nne M oller-L arsen, Institute o f Medical Microbiology, Bartholin Building, University of Aarhus, Aarhus (Denmark)
Downloaded by: Nagoya University 133.6.82.173 - 1/15/2019 5:13:04 AM
Received: December4, 1975; revised: January27, 1976.
250
M oller-L arsen/A ndersen/H eron/S arov
Materials and Methods
Downloaded by: Nagoya University 133.6.82.173 - 1/15/2019 5:13:04 AM
Purified C M V antigen. Highly purified CMV, strain Ad 169, was obtained from the supernatant fluids of infected human embryonic fibroblasts by successive zone and density centrifugation as described by Sarov and A bady [13]. The concentration of the virus was adjusted to 0.3 OD 260 nm. The infectivity o f the viral preparations was determined by the method of P lummer and Benyesh-M elnick [14]. 1 OD 260 nm gave 1 0 M 0 8 PFU/ml. The viral band was dialyzed overnight against phosphate-buffered saline (PBS), pH 7.2, and inactivated at 56° for 1 h. This treatment resulted in complete inactivation o f the virus. Supernatant from uninfected fibroblasts of the same line, cultured for 10 days, was treated in the same way as the purified CMV antigen and served as control antigen. In one experiment CMV was inactivated by UV irradiation as follows: 1 ml o f the purified virus (0.3 OD 260 nm) was placed in a 35-mm Petri dish and irradiated by a short wave UV lamp, UVS-54, from a distance of 28 cm for 10 min with continual shaking. This treatment resulted in complete inactivation of the virus. CMV-infected cells were produced according to a method by T hurman et al. [11]. Monolayers of human fibroblasts in 250-ml Falcon bottles containing 4 x 106 cells were infected with CMV strain Ad 169 at a multiplicity o f 2 PFU/cell. The virus was adsorbed for 2 h at 37°, and Eagle’s MEM - supplemented with 2% calf serum and 2 0 0 IU penicillin, 200 ug streptomycin and 2.5 ¡xg mycostatin per ml - was added to the cultures. Following incubation at 37° in 5% CO2 atmosphere for 5-6 days, the medium was removed and the cultures were washed once with PBS, pH 7.2. RPMI-I640 medium containing 40 ¡xg/ml mitomycin-C (Sigma Chemical Co.) was added and the cultures were incubated for 1 h at 37°. The cells were then washed five times with PBS and removed from the bottles with trypsin. The cells were then washed twice in RPMI-1640 and adjusted to contain 2 * 104 cells/ml of RPMI-1640. 10 ¡xl o f this cell suspension was used per well in lymphocyte stimulation experiments. Control fibroblasts were obtained from uninfected cultures prepared for stimulation in exactly the same manner as the infected ones. Herpes simplex type 1 (HSV-1) antigen. HSV-1 antigens were prepared from human embryonic lung fibroblasts (HEL cells) grown in Eagle’s MEM with 2% calf serum. The cells were infected by HSV-1, 2-4 PFU/cell. 24-48 h after infection the infected cultures were frozen and thawed twice and centrifuged for 10 min at 2,000 rpm. The virus in the supernatant was partially purified by two 1-hour centrifugations at 100,000 g. The pelleted viruses were resuspended in PBS, pH 7.2. The virus preparation was inactivated by UV light [4], Before UV treatment the titer was 4 x IO7 PFU/ml. After UV treatment the titer was 0. HEL antigen was prepared from uninfected HEL cells treated in the same manner. Leucocyte cultures. Venous blood from 19 volunteers was taken into glass bottles containing preservative-free heparin at a final concentration o f 20 IU/ml o f blood. The mononuclear cells were obtained following isopaque-Ficoll flotation [15]. The mononuclear cells were washed twice in RPMI-1640 containing 15-20 IU heparin, 100¡xg of streptomycin per ml, 100 IU of penicillin per mi, 10 niM HEPES buffer, and 5% heat-inactivated human serum from individuals who were seronegative against both CMV and HSV, as determined by complement fixation (CF) and neutralization (Nt) tests [16]. After washing, the cells were resuspended in RPMI-1640 with streptomycin, penicillin, HEPES buffer, and 15% human serum. The cells were counted, and the cell suspension was diluted to contain 1 x 106 lymphocytes per ml. The cell suspension was dispensed in 0.2-ml
251
Lymphocyte Stimulation by Cytomegalovirus
Table l. Incorporation of 14C-thymidine in lymphocytes from a seropositive and a sero negative individual, stimulated by various antigen preparations1 Lymphocyte donor
Dilutions CMV antigens and control antigens
R. A. (CMV-CF titer 32)
cone. 1:5 1:25
1,827 3,775 3,317
1,812 3,000 4,165
N .H . cone. CMV-CF titer < 4 1:5 and Nt titer < 2 1:25
76 61 111
345 194 236
purified CMV antigen infected --------------------------------------------- cells untreated heatUVinactivated inactivated not done 3,000 3,876
uninfected cells
768 4,047 3,000
84 52 48
71 147 467
545 944 447
81 276 377
1 In this experiment CMV was used in concentration 1.0 OD 260 nm.
aliquots in the wells of a Linbro microplate No. IS-MRC-96. Cultures were added to 10 |xl medium containing the different antigens. Phytohemagglutinin (PHA) was used in a solu tion containing 0.1 p.g/p.1. Triplicate determinations were performed. The plates were closed tightly by a plastic cover and incubated in 37° for a total of 6 days. Cultures stimulated with PHA were incubated for a total o f 3 days. Thymidine incorporation. For the final 24 h o f incubation, 1 p.1 of 14C-thymidine from a thymidine solution containing 0.02 ¡xCi/ptl was added to each well. The cells were harvested with a Skatron-harvester POB 28 (Lierbyen, Norway) on Whatman Glass Fiber Paper GF 81, thickness 0.25 mm. In the harvester the filters were washed with demineralized water. The filters were placed in plastic vials with 5 ml o f triton toluol scintillation fluid and counted in aTri-carb liquid scintillation spectrometer N o .2450. Results are given in tables I and II as the calculated medium values in disintegrations per minute (dpm). Detection o f C M V and H SV antibodies. The sera were tested for antibodies to CMV by neutralization and complement fixation tests [16], Antibodies to HSV were detected in a similar way.
Results
Downloaded by: Nagoya University 133.6.82.173 - 1/15/2019 5:13:04 AM
Inactivation procedures with CM V for preparation o f the antigen. Initial experiments were performed to compare different preparations of CMV antigen. Table I shows the results of such an experiment. CMV particles inactivated either by UV or by heat (56°, 1 h) stimulate 14C-thymidine uptake in peripheral
252
M oller-L arsen/A ndersen/H eron/S arov
CONCENTRATION OF ANTIGEN
lymphocytes of a CMV-positive individual essentially to the same degree as the untreated purified viruses and the CMV-infected cells. The control antigen from the medium of uninfected fibroblast cultures did not give any stimulation. The heat-inactivated, purified CMV and CMV-infected cells were chosen for the following experiments. Dose response curve. As shown in figure 1, the lymphocytes from the three seropositive individuals responded somewhat differently. With the dilution 1:4, maximal stimulation was obtained in two of these subjects, and a submaximal but considerable stimulation was seen in the third, whereas lympho cytes from seronegative donors were not stimulated by the purified CMV at any amounts. Repeated experiments confirmed this, so the 1:1 dilution as well as 1:4 was chosen for the further experiments. Response o f lymphocytes from seronegative and seropositive adults. Lymphocytes were obtained from 19 healthy donors; 10 were CMV-seropositive, 9 were seronegative.
Downloaded by: Nagoya University 133.6.82.173 - 1/15/2019 5:13:04 AM
Fig. I. Dose-response curves with purified CMV. Lymphocytes from five donors were tested with various amounts o f purified virus. The antigen (0.3 O D 260 nm) was diluted from 1:1 to 1:256 and added in 10-ul aliquots. Higher concentrations o f antigen (2:1 and 3:1) were also tested by adding 20 and 30 ;xl o f antigen to the wells.
253
Lymphocyte Stimulation by Cytomegalovirus Table II. Antigen stimulation of lymphocytes from 19 individuals Donor
Serology CMV
Lymphocyte stimulation, dpm1, with antigens indicated : HSV
CF
Nt
CF
G.E.P. I. H. A .M .L. H.J. B.P. R.A. B.S. G.L. T.S. G.K .
16 32 32 4 16 32 32 32 64 64
+ + + + + + + + + +
AC2
