Scand. J. Immunol. 8, 339-346, 1978
DNA Synthesis in Subpopulations of Blood Mononuclear Leucocytes in Human Subjects after Vaccination against Yellow Fever A. EHRNST, B. LAMBERT & A. FAGRAEUS Department of Immunology, National Bacteriological Laboratory and Department of Clinical Genetics, Karolinska Hospital, Stockholm, Sweden
Ehrnst, A., Lambert, B. & Fagraeus, A. DNA Synthesis in Subpopulations of Blood Mononuclear Leucocytes in Human Subjects after Vaccination against Yellow Fever. Scand. J. Immunol. 8, 339-346, 1978. After vaccination of five volunteers with yellow fever live vaccine, blood mononuclear cells were isolated and labelled with 'H-thymidine at intervals. DNA synthesis was measured by scintillation counting and autoradiography of rosetted cells. Rosetting with sheep erythrocytes (E-RFC) identified T cells, and such erythrocytes coated with IgM antibodies and complement (EAC-RFC) identified B cells and monbcytes. DNA synthesis in the total mononuclear cell fraction, as well as in subfractions enriched in or deprived of E-RFC, displayed a sharp increase on day 10-11 after vaccination, remained high on day 13-14, and then returned to the prevaccination level. There was a corresponding morphological transformation, measured by size distribution and number of nucleoli per cell. The major fraction of DNA-synthesizing cells before, during and after the peak of activity was found among non-rosette-forming cells. However, during the activity peak the numbers and proportion of DNA-synthesizing E-RFC were increased while the response with regard to EAC-RFC was not obvious. Thus within a complex cellular response a transient T-cell response was identified. Anneka Ehrnst, Department of Immunology, National Bacteriological Laboratory, S-IOS 21 Stockholm. Sweden.
A raised labelling index of mononuclear cells in the peripheral blood is found during many types of viral infections [12]. Crowther et al. [4] used 'H-thymidine-labelling of peripheral lymphocytes in combination with autoradiography and scintillation counting to study the cellular response to vaccination with killed vaccines. Cell proliferation, detected as DNA synthesis, was highest 1 week after vaccination. In normal blood less than 0.2% of the cells were labelled with "H-thymidine. After vaccination the proportion of large mononuclear cells and plasma cells increased significantly. Medium-sized lym-
phocytes also became more numerous. At the time of this detailed study techniques of identifying human T and B cells were not available. In the present study DNA-synthesizing cells among subgroups of mononuclear blood leucocytes in volunteers, vaccinated against yellow fever, were identified by their capacity to form rosettes with sheep erythrocytes (E-rosettes) or sheep erythrocytes coated with IgM antibodies and complement (EAC-rosettes). DNA synthesis was studied using incorporation of 'Hthymidine, measured-by scintillation counting and autoradiography. The data indicate a
0300-9475/78/1000-0339 $02.00 © 1978 Blackwell Scientific Publications
339
340
A. Ehrnst, B. Lambert & A. Fagraeus
complex response involving different cell types, including a transient T-cell response. These data have been preliminarily reported [5].
MATERIALS AND METHODS Volunteers of vaccination and isolation of mononuclear cells from the blood. Five persons, A (§, 29 years), B ((J, 28 years), C (tj, 30 years), D (cJ, 56 years) and E ((?, 40 years), were given a subcutaneous injection of yellow fever vaccine (17 D vaccine. Burroughs Wellcome). They were apparently healthy on the day of vaccination and during the subsequent 3-4 weeks except E, who later on the day of vaccination presented with a slight cold which lasted for a few days. B suffers from pollen allergy but was in remission at the time of vaccination. Mononuclear cell suspensions were obtained by centrifugation of heparinized blood on Ficoll-Isopaque gradient according to Thorsby & Bratlie [13]. Microscopic inspection revealed less than 2% polymorphonuclear cells. Cell viability, as determined by trypan blue dye exclusion, was over 98%. Uptake of 'H-thymidine. Mononuclear cells were suspended in RPMI 1640 medium, supplemented with 25% fetal calf serum and 10 (iCi/ml of [methyl-^H]thymidine (5 Ci/mM, The Radiochemical Centre, Amersham) and incubated at 37°C. After 1 h the cells were extracted three times with 5% TCA, resuspended in cold 70% ethanol and filtered onto glass fibre filter. The activity was measured in a scintillation counter at an efficiency of about 35% and a background of 50 counts/ min. Control filters containing samples incubated at 4°C gave activities ranging between 14 and 69 cpm, which was less than 10% of the activity of the samples incubated at 37°C. Duplicate samples varied by less than 5% and the results presented are arithmetic means of duplicate samples. Morphology of mononuclear cells. Mononuclear cells were smeared on glass slides, air-dried, and fixed in acetone at —20°C for 20 min. Some slides were stained with May-Grunwald Giemsa, and 300 cells per slide were examined in a light microscope for size and morphological characteristics [14]. Other slides were first treated for 30 min at room temperature with a human serum containing autoantibodies to nucleoli and then stained with a sheep anti-human FITC conjugate (National Bacteriological Laboratory) for another 30 min at room temperature. These slides were examined in a Zeiss immunofluorescence microscope. 300 cells per slide were counted and the numbers of nucleoli per cell were recorded. Detection and separation of rosette-forming cells (RFC). E-rosettes [6] were prepared according to Mendes et al. [9]. Antibody-complement-coated erythrocytes, 19S EAC [11], were obtained by mixing equal volumes of 1% E suspension and rabbit 19S anti-E in a subagglutination titre and incubating 45 min at 37°C. After two washes rabbit C-6-deficient serum, diluted 1:5, was added for 60 min at 37°C. Equal volumes of 19S EAC and lymphocytes, 2-3x10" cells/ml, were incubated for 30 min at 37°C. The cells were resuspended vigorously and counted immediately in a warm
Burker chamber to avoid E-rosette formation [9, 11]. Separation of E-RFC from non-E-RFC was performed according to Mendes et al. [9]. Autoradiography on E-RFC and EAC-RFC. Lymphocytes labelled by 'H-thymidine incorporation (S-phase cells) were subjected to E- or EAC-rosetting. The cells were smeared on gelatinized glass slides by application and removal of a droplet by a Pasteur pipette, air-dried and fixed in an ethanol/acetone mixture (3:1) for 30 min. After rehydration in stepwise diluted ethanol, the slides were rinsed in running water for 30 min and then immersed in 5% TCA for 30 min at -t-4°C to remove any acid-soluble radioactivity. The slides were rinsed in running water for 30 min, dehydrated in ethanol, airdried and covered with autoradiographic film (Kodak AR. 10). Exposure was for 7 days. After development and fixation, the cells were stained according to Giemsa. Labelled E-RFC, EAC-RFC and nonrosette-forming cells were identified under the light microscope. Calculations of the percentage of E-RFC and EACRFC in solution and on smears, respectively, generally revealed no significant difference at the 5% level by Student's / test. When the rosette numbers in solution were small (.$5%) there was a tendency to receive higher rosette numbers of smeared cells than of cells in solution (0.01
DNA Synthesis in Subpopulations of Blood Mononuclear Leucocytes in Human Subjects after Vaccination against Yellow Fever A. EHRNST, B. LAMBERT & A. FAGRAEUS Department of Immunology, National Bacteriological Laboratory and Department of Clinical Genetics, Karolinska Hospital, Stockholm, Sweden
Ehrnst, A., Lambert, B. & Fagraeus, A. DNA Synthesis in Subpopulations of Blood Mononuclear Leucocytes in Human Subjects after Vaccination against Yellow Fever. Scand. J. Immunol. 8, 339-346, 1978. After vaccination of five volunteers with yellow fever live vaccine, blood mononuclear cells were isolated and labelled with 'H-thymidine at intervals. DNA synthesis was measured by scintillation counting and autoradiography of rosetted cells. Rosetting with sheep erythrocytes (E-RFC) identified T cells, and such erythrocytes coated with IgM antibodies and complement (EAC-RFC) identified B cells and monbcytes. DNA synthesis in the total mononuclear cell fraction, as well as in subfractions enriched in or deprived of E-RFC, displayed a sharp increase on day 10-11 after vaccination, remained high on day 13-14, and then returned to the prevaccination level. There was a corresponding morphological transformation, measured by size distribution and number of nucleoli per cell. The major fraction of DNA-synthesizing cells before, during and after the peak of activity was found among non-rosette-forming cells. However, during the activity peak the numbers and proportion of DNA-synthesizing E-RFC were increased while the response with regard to EAC-RFC was not obvious. Thus within a complex cellular response a transient T-cell response was identified. Anneka Ehrnst, Department of Immunology, National Bacteriological Laboratory, S-IOS 21 Stockholm. Sweden.
A raised labelling index of mononuclear cells in the peripheral blood is found during many types of viral infections [12]. Crowther et al. [4] used 'H-thymidine-labelling of peripheral lymphocytes in combination with autoradiography and scintillation counting to study the cellular response to vaccination with killed vaccines. Cell proliferation, detected as DNA synthesis, was highest 1 week after vaccination. In normal blood less than 0.2% of the cells were labelled with "H-thymidine. After vaccination the proportion of large mononuclear cells and plasma cells increased significantly. Medium-sized lym-
phocytes also became more numerous. At the time of this detailed study techniques of identifying human T and B cells were not available. In the present study DNA-synthesizing cells among subgroups of mononuclear blood leucocytes in volunteers, vaccinated against yellow fever, were identified by their capacity to form rosettes with sheep erythrocytes (E-rosettes) or sheep erythrocytes coated with IgM antibodies and complement (EAC-rosettes). DNA synthesis was studied using incorporation of 'Hthymidine, measured-by scintillation counting and autoradiography. The data indicate a
0300-9475/78/1000-0339 $02.00 © 1978 Blackwell Scientific Publications
339
340
A. Ehrnst, B. Lambert & A. Fagraeus
complex response involving different cell types, including a transient T-cell response. These data have been preliminarily reported [5].
MATERIALS AND METHODS Volunteers of vaccination and isolation of mononuclear cells from the blood. Five persons, A (§, 29 years), B ((J, 28 years), C (tj, 30 years), D (cJ, 56 years) and E ((?, 40 years), were given a subcutaneous injection of yellow fever vaccine (17 D vaccine. Burroughs Wellcome). They were apparently healthy on the day of vaccination and during the subsequent 3-4 weeks except E, who later on the day of vaccination presented with a slight cold which lasted for a few days. B suffers from pollen allergy but was in remission at the time of vaccination. Mononuclear cell suspensions were obtained by centrifugation of heparinized blood on Ficoll-Isopaque gradient according to Thorsby & Bratlie [13]. Microscopic inspection revealed less than 2% polymorphonuclear cells. Cell viability, as determined by trypan blue dye exclusion, was over 98%. Uptake of 'H-thymidine. Mononuclear cells were suspended in RPMI 1640 medium, supplemented with 25% fetal calf serum and 10 (iCi/ml of [methyl-^H]thymidine (5 Ci/mM, The Radiochemical Centre, Amersham) and incubated at 37°C. After 1 h the cells were extracted three times with 5% TCA, resuspended in cold 70% ethanol and filtered onto glass fibre filter. The activity was measured in a scintillation counter at an efficiency of about 35% and a background of 50 counts/ min. Control filters containing samples incubated at 4°C gave activities ranging between 14 and 69 cpm, which was less than 10% of the activity of the samples incubated at 37°C. Duplicate samples varied by less than 5% and the results presented are arithmetic means of duplicate samples. Morphology of mononuclear cells. Mononuclear cells were smeared on glass slides, air-dried, and fixed in acetone at —20°C for 20 min. Some slides were stained with May-Grunwald Giemsa, and 300 cells per slide were examined in a light microscope for size and morphological characteristics [14]. Other slides were first treated for 30 min at room temperature with a human serum containing autoantibodies to nucleoli and then stained with a sheep anti-human FITC conjugate (National Bacteriological Laboratory) for another 30 min at room temperature. These slides were examined in a Zeiss immunofluorescence microscope. 300 cells per slide were counted and the numbers of nucleoli per cell were recorded. Detection and separation of rosette-forming cells (RFC). E-rosettes [6] were prepared according to Mendes et al. [9]. Antibody-complement-coated erythrocytes, 19S EAC [11], were obtained by mixing equal volumes of 1% E suspension and rabbit 19S anti-E in a subagglutination titre and incubating 45 min at 37°C. After two washes rabbit C-6-deficient serum, diluted 1:5, was added for 60 min at 37°C. Equal volumes of 19S EAC and lymphocytes, 2-3x10" cells/ml, were incubated for 30 min at 37°C. The cells were resuspended vigorously and counted immediately in a warm
Burker chamber to avoid E-rosette formation [9, 11]. Separation of E-RFC from non-E-RFC was performed according to Mendes et al. [9]. Autoradiography on E-RFC and EAC-RFC. Lymphocytes labelled by 'H-thymidine incorporation (S-phase cells) were subjected to E- or EAC-rosetting. The cells were smeared on gelatinized glass slides by application and removal of a droplet by a Pasteur pipette, air-dried and fixed in an ethanol/acetone mixture (3:1) for 30 min. After rehydration in stepwise diluted ethanol, the slides were rinsed in running water for 30 min and then immersed in 5% TCA for 30 min at -t-4°C to remove any acid-soluble radioactivity. The slides were rinsed in running water for 30 min, dehydrated in ethanol, airdried and covered with autoradiographic film (Kodak AR. 10). Exposure was for 7 days. After development and fixation, the cells were stained according to Giemsa. Labelled E-RFC, EAC-RFC and nonrosette-forming cells were identified under the light microscope. Calculations of the percentage of E-RFC and EACRFC in solution and on smears, respectively, generally revealed no significant difference at the 5% level by Student's / test. When the rosette numbers in solution were small (.$5%) there was a tendency to receive higher rosette numbers of smeared cells than of cells in solution (0.01
