Intervirology / / : 74—81 (1979)
Cellular Immune Response to Human Cytomegalovirus I. Lymphocyte Transformation Studies in the Rabbit Robert N. Lausch, Constance Jones and Ralph C. Christensen Department o f Microbiology and Specialized Cancer Research Center, The Pennsylvania State University College of Medicine, Hcrshey, Pa., and Departments o f Health Radiation Sciences and Radiation Medicine, University o f Kentucky Medical Center, Lexington, Ky.
Key Words. Cytomegalovirus • Herpes simplex virus type 1 • Lymphocyte transformation
Cytomegalovirus (CMV) is a ubiquitous human pathogen with an infectious incidence surpassing 90% in certain populations sur veyed [!]■ While exposure of the healthy child or adult to this herpesvirus frequently results in subclinical disease, very serious sequelae can occur following infection of developing fetuses and immuno-debilitated patients [2]. There are at present no reliable means for preventing or treating CMV infection. Thus, Address inquiries to: Dr. Robert N. Lausch, De partment of Microbiology and Immunology, University o f South Alabama College o f Medicine, Mobile, AL 36688 (USA) Received: March 2, 1978 Revised: April 13, 1978
recovery from infection is dependent upon host defense mechanisms. To date, investiga tions of the host’s immune response to the virus have been largely confined to seroepidemiologic studies. However, it is currently believed that cell-mediated immunity may play an im portant, and perhaps the predominant, role in controlling infections due to herpesviruses [3], and recent studies suggest that CMV is not an exception [4]. Other workers have shown that lympho cyte transformation is a useful method for monitoring cellular immunity to herpesviruses in animals and man [5,6]. As a prelude to studying the latter, it was considered desirable to characterize the stimulating antigen and evaluate its specificity in an experimental
Downloaded by: King's College London 137.73.144.138 - 1/16/2019 5:21:23 AM
Summary. The lymphocyte transformation assay was used to monitor the cellular immune response of rabbits sensitized to cytomegalovirus (CMV). Peripheral blood lymphocytes from animals inoculated with purified virus were specifically stimulated by crude or twice-banded CMV. Blood cells from rabbits immunized to herpes simplex virus type I (HSV-I) responded to that antigen but not to CMV. Viable or heated CMV preparations stimulated unwashed blood cells as efficiently as washed cells. Furthermore, preincubation of stimulating antigen with antiCM V serum did not prevent lymphocyte activation. Lymphoid cells readily stimulated by virus antigen were not stimulated by cells transformed by CMV or HSV-1.
animal model. In this report we describe the results of studies on cell-mediated immunity to CMV using rabbits as the experimental host.
Materials and Methods Cells Primary rabbit kidney (RK)and human embryonic lung (HEL) cultures were prepared by standard methods. Vero cells (a continuous line o f monkey kidney cells) and Flow cells (a continuous line o f HEL cells) were subcultured as required. The development of hamster cell lines transformed by CMV (line Cx90-3B,T-2) and herpes simplex virus type I (HSV-I) (line 14-012-8-1.T-10) has been previously described [7,8], as has the transformation o f HEL cells (line CMV-Mj-HEL-2,T-I) by CMV [9], All cultures were grown on Dulbecco medium supplemented with 10% fetal calf serum (FCS), 0.075% sodium bicarbonate, 100 IU/ml penicillin, and 100 ¡¿g/ml streptomycin. Viruses The ADI69 strain o f CMV was grown in HEL cells and titrated on Flow cells. HSV-1 strain 14-012 was grown in Vero cells and titrated on RK cells. All virus stocks were free of mycoplasma contamination as assessed by the method o f Schneider el al. [10], Preparation o f Virus Antigens CMV was grown by infecting subconfluent monolayers o f HEL cells with a multiplicity o f 0.25-0.5 PFU/cell. After 4 and 7 days the supernatant was col lected and clarified by low-speed centrifugation. Such preparations were designated crude virus and had in fectious titers o f I0 M 0 7 PFU/ml. To purify the virus, crude CMV was first concentrated 100-fold via centri fugation for 3 h at 12,500 rpm in a Sorvall RC2-B centrifuge. The pellet was resuspended and centrifuged at low speed to sediment cell fragments. The super natant containing virus was banded by density gradient centrifugation. Briefly, 10s—109 PFU of CMV were layered onto a 20-60% linear sucrose gradient and centrifuged at 39,000 rpm in an SW4I rotor for 90 min. The resultant band was diluted 1:2 with phosphatebuffered saline (PBS) and sonicated for 15 sec. Then, the virus sample was layered onto a 20-50% linear potassium tartrate gradient and centrifuged as de scribed above. The visible band was recovered, sonic
75
ated for 15 sec, and dialyzed against 2 liters o f PBS. Aiiquots of the virus were then frozen at -80 in PBS. The infectivity o f the banded virus preparations was I04-I0'! PFU/ml. Monolayers o f Vero cells were infected with HSV-I at a multiplicity o f approximately 0.1 PFU/cell. Cells were harvested by scraping when CPE was maximal and disrupted by two cycles o f freeze-thawing. After brief (15 sec) sonication, the preparation was clarified by low-speed centrifugation, and the supernatant was centrifuged for 45 min at 14,000 rpm in an SW27 rotor onto a 40% potassium tartrate pad. The layer of virus was drawn off and banded by flotation in a 15-30% potassium tartrate gradient. Centrifugation was for approximately 2 h al 24.000 rpm using an SW27 rotor. The virus band was dialyzed against PBS and then frozen in dry ice. Virus stocks banded in this manner had an infectivity o f 3 x |0 7 to 3 x |0 8 PFU/ml. Animals Adult New Zealand White and Dutchland rabbits (5-7 lb) and Hartley guinea pigs (350-400 g) were purchased from a local supplier. Animals were in oculated intramuscularly or subcutaneously with virus in complete Freund's adjuvant, and intradcrmally when virus was given alone. Lymphocyte Transformation Assay Blood was drawn into syringes containing pre servative-free heparin at a final concentration of approximately 80 IU/ml o f blood. The whole blood was then diluted 1:10 in RPM1-I640 with 10 ¡zg/ml non-essential amino acids, 300 ug/ml ¿-glutamine, 100 jzg/ml streptomycin, 100 IU/ml penicillin, 0.225% NaHCOii and 10 miM Hepes buffer (LTA medium). Triplicate glass tube ( I 0 x 75 mm) cultures containing 0.2 ml o f diluted whole blood and 10 ¡zl of the appro priate antigen were incubated in a COa (5%) Wedco incubator at 37 for 4-6 days. For the final 18 h o f in cubation, I pCi of [3H]-thymidine was added to each tube. The cultures were transferred to glass-fiber filter discs (Whatman GS-A, 2.4 cm) and dried. The discs were submerged in cold 5% trichloroacetic acid for 15 min, then washed in cold 95% ethanol 3 times. They were decolorized using 3% hydrogen peroxide and washed once with 95% ethanol. The filters were ovendried and placed in scintillation vials with 6 ml OCS (Amersham/Searle, Arlington Heights, III.). Radio activity was monitored using an LS-9000 Beckman scintillation counter and expressed as the mean cpm
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Cellular Immunity to Human CMV
Lausch/Jones/Christensen
of three replicates. Results were expressed as the stimulation index (SI), which is the ratio o f cpm in the presence o f antigen and lymphoid cells to the cpm with lymphoid cells only. In order to determine the number o f lymphoid cells present in whole blood samples, the red cells were lysed by addition o f 0.9 ml 3% acetic acid to 0.1 ml cells. Cells were counted and adjusted to the concentration desired. In certain experiments the results with heparinized whole blood were compared with results obtained using mononuclear blood cells prepared by Ficoll-paque gradients. Whole blood (I I ml) was diluted with 13 ml LTA medium. Aliquots (12 ml) were layered on top o f 8.5 ml Ficoll-paque (Pharmacia Fine Chemicals, Piscataway, N.J.) in mincing tubes. The tubes were centrifuged for 40 min at room temperature at 1,000 rpm in a Sorvall GLC-I centrifuge. The band o f lymphoid cells at the interface was collected, washed twice with LTA medium, then resuspended in the same medium supplemented with 5% rabbit immune fresh plasma, and counted. The assay was then completed as described above.
Results Comparison o f Ficoll-Paque-Banded Peripheral Blood Lymphocytes with Whole Blood Cell Cultures in the Lymphocyte Transformation Assay In the initial experiments the responsiveness of CMV-sensitized rabbit peripheral blood lymphocytes to virus antigen was tested using whole blood diluted 1:10 or cells prepared by Ficoll-paque gradients. Both types of pre parations yielded cells responsive to CMV (table I). Higher stimulation indexes were ob served more frequently with unfractionated blood cells. Table I also shows that the degree of responsiveness to CMV was related to the dilution of antigen used and the number of mononuclear cells per culture. Whole blood cells were also more responsive on a per cell basis to concanavalin A stimulation. In the mitogen studies, the highest stimulation in dexes were observed with I05 cells per culture. In tests to determine the optimal incubation
period, the 4-day assay gave results equal to or better than those obtained at 6 days in 8 of 12 trials (data not shown). Accordingly, the 4-day assay was used in most subsequent tests. Effect o f Hyperimmune Serum on Peripheral Blood Lymphocyte Response The foregoing results were obtained in the presence of immune serum, and it was im portant to determine whether CMV antibodies present in the rabbit blood affected the results. Table II shows that preincubation of CMV with hyperimmune CMV antiserum did not significantly reduce or increase the antigen’s capacity to stimulate sensitized rabbit effector cells. Therefore, all further experiments were performed using whole blood. Characterization o f CM V Antigen Preparations The ADI69 strain of CM V, banded first on a sucrose gradient and then on a potassium tartrate gradient, was used for host immuni zation and also served as the stimulating antigen. Characteristics of two such pre parations are shown in table III. The purified virus stocks containing lO'-lO6 PFU/ml were capable of evoking significant stimulation of sensitized lymphocytes at dilutions as high as 1:80. The inciting antigen was heat-stable and associated with material sedimentable by high speed centrifugation. Medium containing FCS or extracts of cells in which the virus was grown did not stimulate the virus-sensitized cells. Thus, the immune reaction appeared to be specific for CMV antigen. Kinetics o f Immune Response Following CM V Sensitization The capacity of different human CMV pre parations to induce cell-mediated immunity in adult rabbits was investigated. Basically, it
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76
Cellular Immunity to Human CMV
77
Table I. Stimulation of CMV-sensitized rabbit peripheral blood lymphocytes by virus and concanavalin A: comparison of washed whole blood versus Ficoll-paque preparation o f responding cells Stimulating agent
CMV, 2 x banded (1 :5 dilution) CMV, 2 x banded (1:20 dilution) Concanavalin A (50 gg/ml) None
2 x IO3 1 xlO 3 0.5 x |0 3 2 x io3 1 x |0 3 0.5 x IO3 2 x 10» 1 x |0 3 0.5 x 103 2 x 10s 1 x |0 3 0.5 x IO3
Lymphocyte preparation whole blood
Ficoll-paque
cpm ± SE1
SI
cpm ± SE
SI
5,818 ± 345 4,043 ± 1,102 3,325 ± 132 2,301 ± 257 1,639 ± 306 708± 167 118,139 ± 583 196.977 ±3,432 176,620 ±2,952 246± 18 220 ± 18 211 ± 31
24 18 16 9.4 7.5 3.4 480 895 837
9,378 ± 4,735 2,063 ± 49 825 ± 75 4.292 ± 1,868 520 ± 150 985 ± 267 70,363 ±15,560 96,510 ± 5,402 36,504 ± 2,037 405 ± 33 342 ± 20 598 ± 78
23 6 1.4 11 1.5 1.6 174 282 61
-
-
-
-
SE = Standard error.
was found that inoculation of crude or banded virus could immunize the host, but responsive ness was modest and transient in nature. On the other hand, repeated immunization with banded virus in complete Freund's adjuvant resulted in long-lasting sensitization. Figure I depicts examples of these two types of re sponse. It is evident that viable CMV im munization in the presence or absence of ad juvant did not lead to sensitization to HSV-I antigen. Other tests established that HSV-1immunized rabbits did not respond to CMV (fig.2). As noted above (table III) no signifi cant DNA synthesis occurred when peripheral blood lymphocytes from hosts sensitized to banded CMV were incubated with HEL ex tract or FCS. However, low but significant responses to these control antigens were ob served after repeated crude virus immunization (data not shown).
Table II. Failure of anti-CMV serum to inhibit sensitized rabbit peripheral blood lymphocyte re sponse to virus antigen1 Test Serum N o.2
Serum dilution tested
Average cpm ±SE3
1
_
3,719 ± 2 9 3 3,116 ± 3 7 7 2,557 ± 700 3,043 ± 478 4,639 ± 109 1,553 ± 4 3 4 1,740 ± 7 3 2
2
none rabbit 435, prebleed rabbit 435, anti-CMV rabbit 435, prebleed rabbit 435, anti-CMV hamster, normal hamster, anti-CMV
1 1 1 1 1 1
2.5 2.5 10 10 5 5
1 The rabbit and hamster anti-CMV sera had virus neutralization titers o f > 1: 1,000. Antigen and serum were incubated for 30 min at 37 prior to testing. 2 The average cpm in the absence o f antigen was 590 ± 2 0 in test No. I and 253 ± 40 in test No. 2. 3 SE = Standard error.
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1
Number of leukocytes per culture
Lausch/Jones,'Christensen
78
Table III. Stimulation of sensitized rabbit peripheral blood lymphocytes by various preparations o f CMV antigen Experiment N o.1
Test antigen
1
CMV, twice-banded untreated heated, 56 for 2 h ultracentrifugation supernatant pellet
2
CMV, twice-banded crude CMV Dulbecco medium containing 5% FCS HEL cell extract
PFU/ml
Average cpm ± SE2
SI
1 x 10« < I02
16,071 ± 16.481 ±
35 36
Cellular Immune Response to Human Cytomegalovirus I. Lymphocyte Transformation Studies in the Rabbit Robert N. Lausch, Constance Jones and Ralph C. Christensen Department o f Microbiology and Specialized Cancer Research Center, The Pennsylvania State University College of Medicine, Hcrshey, Pa., and Departments o f Health Radiation Sciences and Radiation Medicine, University o f Kentucky Medical Center, Lexington, Ky.
Key Words. Cytomegalovirus • Herpes simplex virus type 1 • Lymphocyte transformation
Cytomegalovirus (CMV) is a ubiquitous human pathogen with an infectious incidence surpassing 90% in certain populations sur veyed [!]■ While exposure of the healthy child or adult to this herpesvirus frequently results in subclinical disease, very serious sequelae can occur following infection of developing fetuses and immuno-debilitated patients [2]. There are at present no reliable means for preventing or treating CMV infection. Thus, Address inquiries to: Dr. Robert N. Lausch, De partment of Microbiology and Immunology, University o f South Alabama College o f Medicine, Mobile, AL 36688 (USA) Received: March 2, 1978 Revised: April 13, 1978
recovery from infection is dependent upon host defense mechanisms. To date, investiga tions of the host’s immune response to the virus have been largely confined to seroepidemiologic studies. However, it is currently believed that cell-mediated immunity may play an im portant, and perhaps the predominant, role in controlling infections due to herpesviruses [3], and recent studies suggest that CMV is not an exception [4]. Other workers have shown that lympho cyte transformation is a useful method for monitoring cellular immunity to herpesviruses in animals and man [5,6]. As a prelude to studying the latter, it was considered desirable to characterize the stimulating antigen and evaluate its specificity in an experimental
Downloaded by: King's College London 137.73.144.138 - 1/16/2019 5:21:23 AM
Summary. The lymphocyte transformation assay was used to monitor the cellular immune response of rabbits sensitized to cytomegalovirus (CMV). Peripheral blood lymphocytes from animals inoculated with purified virus were specifically stimulated by crude or twice-banded CMV. Blood cells from rabbits immunized to herpes simplex virus type I (HSV-I) responded to that antigen but not to CMV. Viable or heated CMV preparations stimulated unwashed blood cells as efficiently as washed cells. Furthermore, preincubation of stimulating antigen with antiCM V serum did not prevent lymphocyte activation. Lymphoid cells readily stimulated by virus antigen were not stimulated by cells transformed by CMV or HSV-1.
animal model. In this report we describe the results of studies on cell-mediated immunity to CMV using rabbits as the experimental host.
Materials and Methods Cells Primary rabbit kidney (RK)and human embryonic lung (HEL) cultures were prepared by standard methods. Vero cells (a continuous line o f monkey kidney cells) and Flow cells (a continuous line o f HEL cells) were subcultured as required. The development of hamster cell lines transformed by CMV (line Cx90-3B,T-2) and herpes simplex virus type I (HSV-I) (line 14-012-8-1.T-10) has been previously described [7,8], as has the transformation o f HEL cells (line CMV-Mj-HEL-2,T-I) by CMV [9], All cultures were grown on Dulbecco medium supplemented with 10% fetal calf serum (FCS), 0.075% sodium bicarbonate, 100 IU/ml penicillin, and 100 ¡¿g/ml streptomycin. Viruses The ADI69 strain o f CMV was grown in HEL cells and titrated on Flow cells. HSV-1 strain 14-012 was grown in Vero cells and titrated on RK cells. All virus stocks were free of mycoplasma contamination as assessed by the method o f Schneider el al. [10], Preparation o f Virus Antigens CMV was grown by infecting subconfluent monolayers o f HEL cells with a multiplicity o f 0.25-0.5 PFU/cell. After 4 and 7 days the supernatant was col lected and clarified by low-speed centrifugation. Such preparations were designated crude virus and had in fectious titers o f I0 M 0 7 PFU/ml. To purify the virus, crude CMV was first concentrated 100-fold via centri fugation for 3 h at 12,500 rpm in a Sorvall RC2-B centrifuge. The pellet was resuspended and centrifuged at low speed to sediment cell fragments. The super natant containing virus was banded by density gradient centrifugation. Briefly, 10s—109 PFU of CMV were layered onto a 20-60% linear sucrose gradient and centrifuged at 39,000 rpm in an SW4I rotor for 90 min. The resultant band was diluted 1:2 with phosphatebuffered saline (PBS) and sonicated for 15 sec. Then, the virus sample was layered onto a 20-50% linear potassium tartrate gradient and centrifuged as de scribed above. The visible band was recovered, sonic
75
ated for 15 sec, and dialyzed against 2 liters o f PBS. Aiiquots of the virus were then frozen at -80 in PBS. The infectivity o f the banded virus preparations was I04-I0'! PFU/ml. Monolayers o f Vero cells were infected with HSV-I at a multiplicity o f approximately 0.1 PFU/cell. Cells were harvested by scraping when CPE was maximal and disrupted by two cycles o f freeze-thawing. After brief (15 sec) sonication, the preparation was clarified by low-speed centrifugation, and the supernatant was centrifuged for 45 min at 14,000 rpm in an SW27 rotor onto a 40% potassium tartrate pad. The layer of virus was drawn off and banded by flotation in a 15-30% potassium tartrate gradient. Centrifugation was for approximately 2 h al 24.000 rpm using an SW27 rotor. The virus band was dialyzed against PBS and then frozen in dry ice. Virus stocks banded in this manner had an infectivity o f 3 x |0 7 to 3 x |0 8 PFU/ml. Animals Adult New Zealand White and Dutchland rabbits (5-7 lb) and Hartley guinea pigs (350-400 g) were purchased from a local supplier. Animals were in oculated intramuscularly or subcutaneously with virus in complete Freund's adjuvant, and intradcrmally when virus was given alone. Lymphocyte Transformation Assay Blood was drawn into syringes containing pre servative-free heparin at a final concentration of approximately 80 IU/ml o f blood. The whole blood was then diluted 1:10 in RPM1-I640 with 10 ¡zg/ml non-essential amino acids, 300 ug/ml ¿-glutamine, 100 jzg/ml streptomycin, 100 IU/ml penicillin, 0.225% NaHCOii and 10 miM Hepes buffer (LTA medium). Triplicate glass tube ( I 0 x 75 mm) cultures containing 0.2 ml o f diluted whole blood and 10 ¡zl of the appro priate antigen were incubated in a COa (5%) Wedco incubator at 37 for 4-6 days. For the final 18 h o f in cubation, I pCi of [3H]-thymidine was added to each tube. The cultures were transferred to glass-fiber filter discs (Whatman GS-A, 2.4 cm) and dried. The discs were submerged in cold 5% trichloroacetic acid for 15 min, then washed in cold 95% ethanol 3 times. They were decolorized using 3% hydrogen peroxide and washed once with 95% ethanol. The filters were ovendried and placed in scintillation vials with 6 ml OCS (Amersham/Searle, Arlington Heights, III.). Radio activity was monitored using an LS-9000 Beckman scintillation counter and expressed as the mean cpm
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Cellular Immunity to Human CMV
Lausch/Jones/Christensen
of three replicates. Results were expressed as the stimulation index (SI), which is the ratio o f cpm in the presence o f antigen and lymphoid cells to the cpm with lymphoid cells only. In order to determine the number o f lymphoid cells present in whole blood samples, the red cells were lysed by addition o f 0.9 ml 3% acetic acid to 0.1 ml cells. Cells were counted and adjusted to the concentration desired. In certain experiments the results with heparinized whole blood were compared with results obtained using mononuclear blood cells prepared by Ficoll-paque gradients. Whole blood (I I ml) was diluted with 13 ml LTA medium. Aliquots (12 ml) were layered on top o f 8.5 ml Ficoll-paque (Pharmacia Fine Chemicals, Piscataway, N.J.) in mincing tubes. The tubes were centrifuged for 40 min at room temperature at 1,000 rpm in a Sorvall GLC-I centrifuge. The band o f lymphoid cells at the interface was collected, washed twice with LTA medium, then resuspended in the same medium supplemented with 5% rabbit immune fresh plasma, and counted. The assay was then completed as described above.
Results Comparison o f Ficoll-Paque-Banded Peripheral Blood Lymphocytes with Whole Blood Cell Cultures in the Lymphocyte Transformation Assay In the initial experiments the responsiveness of CMV-sensitized rabbit peripheral blood lymphocytes to virus antigen was tested using whole blood diluted 1:10 or cells prepared by Ficoll-paque gradients. Both types of pre parations yielded cells responsive to CMV (table I). Higher stimulation indexes were ob served more frequently with unfractionated blood cells. Table I also shows that the degree of responsiveness to CMV was related to the dilution of antigen used and the number of mononuclear cells per culture. Whole blood cells were also more responsive on a per cell basis to concanavalin A stimulation. In the mitogen studies, the highest stimulation in dexes were observed with I05 cells per culture. In tests to determine the optimal incubation
period, the 4-day assay gave results equal to or better than those obtained at 6 days in 8 of 12 trials (data not shown). Accordingly, the 4-day assay was used in most subsequent tests. Effect o f Hyperimmune Serum on Peripheral Blood Lymphocyte Response The foregoing results were obtained in the presence of immune serum, and it was im portant to determine whether CMV antibodies present in the rabbit blood affected the results. Table II shows that preincubation of CMV with hyperimmune CMV antiserum did not significantly reduce or increase the antigen’s capacity to stimulate sensitized rabbit effector cells. Therefore, all further experiments were performed using whole blood. Characterization o f CM V Antigen Preparations The ADI69 strain of CM V, banded first on a sucrose gradient and then on a potassium tartrate gradient, was used for host immuni zation and also served as the stimulating antigen. Characteristics of two such pre parations are shown in table III. The purified virus stocks containing lO'-lO6 PFU/ml were capable of evoking significant stimulation of sensitized lymphocytes at dilutions as high as 1:80. The inciting antigen was heat-stable and associated with material sedimentable by high speed centrifugation. Medium containing FCS or extracts of cells in which the virus was grown did not stimulate the virus-sensitized cells. Thus, the immune reaction appeared to be specific for CMV antigen. Kinetics o f Immune Response Following CM V Sensitization The capacity of different human CMV pre parations to induce cell-mediated immunity in adult rabbits was investigated. Basically, it
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76
Cellular Immunity to Human CMV
77
Table I. Stimulation of CMV-sensitized rabbit peripheral blood lymphocytes by virus and concanavalin A: comparison of washed whole blood versus Ficoll-paque preparation o f responding cells Stimulating agent
CMV, 2 x banded (1 :5 dilution) CMV, 2 x banded (1:20 dilution) Concanavalin A (50 gg/ml) None
2 x IO3 1 xlO 3 0.5 x |0 3 2 x io3 1 x |0 3 0.5 x IO3 2 x 10» 1 x |0 3 0.5 x 103 2 x 10s 1 x |0 3 0.5 x IO3
Lymphocyte preparation whole blood
Ficoll-paque
cpm ± SE1
SI
cpm ± SE
SI
5,818 ± 345 4,043 ± 1,102 3,325 ± 132 2,301 ± 257 1,639 ± 306 708± 167 118,139 ± 583 196.977 ±3,432 176,620 ±2,952 246± 18 220 ± 18 211 ± 31
24 18 16 9.4 7.5 3.4 480 895 837
9,378 ± 4,735 2,063 ± 49 825 ± 75 4.292 ± 1,868 520 ± 150 985 ± 267 70,363 ±15,560 96,510 ± 5,402 36,504 ± 2,037 405 ± 33 342 ± 20 598 ± 78
23 6 1.4 11 1.5 1.6 174 282 61
-
-
-
-
SE = Standard error.
was found that inoculation of crude or banded virus could immunize the host, but responsive ness was modest and transient in nature. On the other hand, repeated immunization with banded virus in complete Freund's adjuvant resulted in long-lasting sensitization. Figure I depicts examples of these two types of re sponse. It is evident that viable CMV im munization in the presence or absence of ad juvant did not lead to sensitization to HSV-I antigen. Other tests established that HSV-1immunized rabbits did not respond to CMV (fig.2). As noted above (table III) no signifi cant DNA synthesis occurred when peripheral blood lymphocytes from hosts sensitized to banded CMV were incubated with HEL ex tract or FCS. However, low but significant responses to these control antigens were ob served after repeated crude virus immunization (data not shown).
Table II. Failure of anti-CMV serum to inhibit sensitized rabbit peripheral blood lymphocyte re sponse to virus antigen1 Test Serum N o.2
Serum dilution tested
Average cpm ±SE3
1
_
3,719 ± 2 9 3 3,116 ± 3 7 7 2,557 ± 700 3,043 ± 478 4,639 ± 109 1,553 ± 4 3 4 1,740 ± 7 3 2
2
none rabbit 435, prebleed rabbit 435, anti-CMV rabbit 435, prebleed rabbit 435, anti-CMV hamster, normal hamster, anti-CMV
1 1 1 1 1 1
2.5 2.5 10 10 5 5
1 The rabbit and hamster anti-CMV sera had virus neutralization titers o f > 1: 1,000. Antigen and serum were incubated for 30 min at 37 prior to testing. 2 The average cpm in the absence o f antigen was 590 ± 2 0 in test No. I and 253 ± 40 in test No. 2. 3 SE = Standard error.
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1
Number of leukocytes per culture
Lausch/Jones,'Christensen
78
Table III. Stimulation of sensitized rabbit peripheral blood lymphocytes by various preparations o f CMV antigen Experiment N o.1
Test antigen
1
CMV, twice-banded untreated heated, 56 for 2 h ultracentrifugation supernatant pellet
2
CMV, twice-banded crude CMV Dulbecco medium containing 5% FCS HEL cell extract
PFU/ml
Average cpm ± SE2
SI
1 x 10« < I02
16,071 ± 16.481 ±
35 36
