Immunology, 1975, 29, 749.
Lymphocyte Reactivity to Influenza Virus in Man P. J. COLE* AND M. E. MOLYNEUX Division of Communicable Diseases, Clinical Research Centre, Watford Road, Harrow, Middlesex
(Received 20th February 1975; accepted for publication 17th March 1975) Summary. Dermal delayed hypersensitivity and in vitro lymphocyte reactivity, both to purified influenza antigens, have been observed in man. A correlation between these two indices of delayed hypersensitivity was found in subjects without known recent exposure to the virus, but neither correlated with levels of circulating haemagglutination-inhibiting (HAI) antibody. Results suggest that in man immunological sensitization of lymphocytes to influenza virus antigen is long-lived. INTRODUCTION It was shown by Beveridge and Burnet in 1944 that in many people the intradermal injection of influenza virus was followed by a skin lesion which clinically resembled a tuberculin reaction and which occured more often in those with raised haemagglutinationinhibiting (HAI) antibody titres to influenza virus (Beveridge and Burnet, 1944). Recently Habershon et al. confirmed this observation using purified influenza virus, and showed that the histology of the lesion was compatible with a delayed hypersensitivity reaction (Habershon, Molyneux, Slavin, Loewi and Tyrrell, 1973). However, they did not exclude the possibility that such lesions might be, at least in part, Arthus reactions, since they only examined the histology and stained for immunoglobulins and complement in the late stages of the reaction. It has also been shown in a small study of nine people that human lymphocytes may be transformed in vitro by influenza virus antigen (Ruben, Jackson and Gotoff, 1973). But in this work and in other studies (Cate and Kelly, 1969; Denman, Denman, Greenwood, Gall and Heath, 1970) appreciable lymphocyte transformation was only obtained after recent natural infection or immunization with influenza virus. However, we have found no report in the literature of a study of the relationship in man between skin reactivity and the immunological sensitization of lymphocytes to influenza virus. In this study we have, therefore, looked at skin reactions and lymphocyte transformation in volunteers without a known recent history of exposure to influenza virus in an attempt to clarify the nature of the skin response and to determine any relationship between it and the ability of lymphocytes to be transformed. MATERIALS AND METHODS Volunteers Twenty-five millilitres of venous blood was obtained from forty-eight adult volunteers, * Present address and correspondence: Dr P. J. Cole, Host Defence Unit, Department of Medicine, Cardiothoracic Institute, Brompton Hospital, Fulham Road, London SW3.
749
750 P. J. Cole and M. E. Molyneux at the Clinical Research Centre, Harrow, and the Common Cold Unit, Salisbury, for serological and lymphocyte transformation tests. On all forty-eight volunteers skin tests were then performed and read at 48 hours.
Skin tests The inactivated influenza viruses A/Aichi/68(H3N2) and B/Mass/3/66 were used. These antigens were kindly prepared and safety tested as for influenza vaccine production by Richardson Merell Incorporated, Cincinnati, Ohio, the viruses being grown in the allantoic cavity of chick embryos, zonally ultracentrifuged, formaldehyde-inactivated and stored at 40. Four chick cell agglutinating (CCA) units (as estimated by the makers) of antigen in 0-1 ml isotonic saline were injected intradermally. Control skin tests were performed with uninfected chick allantoic fluid, treated identically to the skin test antigen. Purified protein derivative (PPD) diluted 1/1000 (Evans Medical Ltd) was also used as a skin test antigen. Quantification of the skin test was achieved by calculating the area of induration as the product of two diameters at right angles. The skin test had been shown previously to be dose-dependent (Habershon et al., 1973) and a dose of 4 CCA units was found to give positive reactions in 45-55 per cent of subjects, whilst not provoking significant rise in circulating HAI antibody titre. Six-hourly biopsies were taken of positive reactions in seven volunteers for 48 hours from the time of injection of antigen. These were examined histologically and by immunofluorescent staining, with antisera (Wellcome Research Laboratories) directed against whole immunoglobulins, IgG, IgA, IgM and the third component of complement (C3). The sensitivity of this staining was not defined but it has been used successfully to stain immunoglobulin deposits and C3 in skin biopsies from patients with lupus erythematosus and dermatitis herpetiformis. It is also being used routinely for staining immunoglobulin in plasma cells and deposits of immunoglobulin on renal glomeruli.
Lymphocyte transformation Preparation of lymphocytes. A standard method was used in which 20 ml of heparinized venous blood was sedimented with 10 ml of freshly prepared 3 per cent fine-grain pig-skin gelatin in isotonic saline at 370 for 30 minutes. Leucocyte-rich plasma was removed and centrifuged at 200 g for 10 minutes, the serum being retained and the sedimented leucocytes resuspended in 2 ml of RPMI 1640 Medium (Gibco) using HEPES buffer (Gibco) (N-2-hydroxyethylpiperazine-N-ethanesulphonic acid) at a final 20 M molar concentration with added 0 5 per cent bicarbonate, 0-292 mg/ml L-glutamine, 100 units/ml penicillin and streptomycin, and 10 per cent human serum. This cell suspension was layered over an equal volume of freshly prepared Ficoll-Triosil mixture (specific gravity 1.078) comprising 4 parts of 8 per cent (weight/volume) Ficoll (Pharmacia Fine Chemicals, Uppsala, Sweden) mixed with 1 part of 50 per cent Triosil (Nyegaard & Company, Oslo, Norway). After centrifugation for 40 minutes at 500 g (room temperature) the cell band was aspirated with a Pasteur pipette, washed three times in medium alone, and resuspended at 1 x 106 lymphocytes per millilitre in the 10 per cent serum medium described above. A trypan blue exclusion test showed that in the resulting cell suspension more than 98 per cent of the cells were viable lymphocytes. Mitogen and antigens. Phytohaemagglutinin (PHA) (Wellcome purified grade) was used as mitogen at a concentration of 50 ug/ml and preservative-free PPD (Central Veterinary
751 Lymphocyte Reactivity to Influenza Virus Laboratory, Weybridge, Surrey) was used as control specific antigen at a concentration of 25 Mg/ml. Influenza antigens A2/Aichi/68 (H3N2) and B/Mass/3/66 were prepared as for skin testing and exhaustively dialysed against distilled water. A dose of 2-5 x 103 CCA units/ml of each viral antigen was found to give optimal lymphocyte transformation in 144-hour cultures, as determined from preliminary dose-response and time-response experiments on ten volunteers. Culture and serum. Quadruplicate cultures were established in microtitre plates (Cooke Microtitre) using 10 1l of antigen or mitogen and 200 pi of cell suspension. Ten microlitres of antigen or mitogen were added to control cultures after the incubation period (immediately before harvesting) to ensure that each test and its control culture consisted of identical materials-the test cultures only differing by reason of their incubation with mitogen or antigen. Quadruplicate cultures in 10 per cent autologous serum were all duplicated in 10 per cent pooled human AB Rhesus-negative serum, previously screened for absence of HAI antibodies. Cultures with mitogen were harvested after 72 hours at 370 in a moist atmosphere of 5 per cent carbon dioxide in air, whilst those with antigen were harvested after 144 hours. The last 24 hours of each culture were in the presence of 10 pI of tritiated thymidine (Radiochemical Centre, Amersham, Bucks) containing 1 iCi specific activity. Harvesting. The bottom of each microtitre culture well was thoroughly scraped, and washed twice, with an Eppendorf pipette tip to detach adherent cells. The cell suspension with washings was transferred to a glass fibre filter paper (Whatman GF/C 2-5 cm) on a vacuum manifold, where it was washed twice with saline and once with 3 per cent acetic acid to lyse red blood cells. Cold 5 per cent trichloracetic acid (TCA) was used for precipitation on the filter paper and for washing the precipitate when formed. After washing with 100 per cent methanol the filter paper was allowed to dry overnight and counted in (a)
(c)
(b)
7
66
JEEE C0
E ~5_
23
O2 E
-I 0
2
3
4
0
2 3 Skin test area score
4
0
2
3
4
FIG. 1. Correlation of area of skin test reaction and lymphocyte reactivity, both to influenza virus. The area of induration of skin was calculated as the product of two diameters at right angles and scored as 0 = absent or erythema only; 1 = 0-25 sq. mm; 2 = 25-100 sq. mm; 3 = 100-400 sq. mm; 4 = >400 sq. mm. (a) PPD. (b) Influenza virus B/Mass/3/66. (c) Influenza virus A2/Aichi/68 (H3N2).
P. J. Cole and M. E. Molyneux 752 a liquid scintillation vial (Johnsen & Jorgenson) containing 1 ml of methanol and 10 ml of Triton-xylene scintillation fluid comprising 1 part of Triton X-100 (Lennig Chemicals) to 2 parts of scintillant xylene with 0-012 g per cent POPOP (1,4-bis-2- (5-phenyloxazolyl)benzene) and 0-6 g per cent PPO (2,5-Diphenyloxazole) (Nuclear Enterprises Ltd., Sighthill, Edinburgh). Samples were counted at 40 in a Packard Liquid Scintillation Counter to obtain counts per minute (ct/min) which were then corrected for background and quenching. Preliminary controls. Human cord-blood lymphocytes, which might be assumed not to have been exposed to the antigens PPD and influenza previously, were cultured with the latter antigens. In such cultures there was no stimulation, which excluded a non-specific mitogenic effect of these antigens; yet these lymphocytes were stimulated by PHA, proving the cells' capability to respond. Expression of results. The stimulation ratio (ct/min in test cultures divided by ct/min in control cultures) and increment (ct/min in test cultures minus ct/min in control cultures) of the lymphocyte cultures, were calculated after subtraction of background. As the control mitogen-free and antigen-free culture counts were similar and did not approach zero, ratio and increment were equally satisfactory. Results here have been expressed as increments.
Serology HAI tests were performed by a standard method (Tyrrell, Peto and King, 1967). RESULTS SKIN TESTS
Forty-two per cent of the forty-eight subjects gave a positive skin reaction to the intradermal injection of influenza antigen. These reactions consisted of erythema and indura12000 900_
800 700 600 _ 500 _
a)
400_
Lymphocyte Reactivity to Influenza Virus in Man P. J. COLE* AND M. E. MOLYNEUX Division of Communicable Diseases, Clinical Research Centre, Watford Road, Harrow, Middlesex
(Received 20th February 1975; accepted for publication 17th March 1975) Summary. Dermal delayed hypersensitivity and in vitro lymphocyte reactivity, both to purified influenza antigens, have been observed in man. A correlation between these two indices of delayed hypersensitivity was found in subjects without known recent exposure to the virus, but neither correlated with levels of circulating haemagglutination-inhibiting (HAI) antibody. Results suggest that in man immunological sensitization of lymphocytes to influenza virus antigen is long-lived. INTRODUCTION It was shown by Beveridge and Burnet in 1944 that in many people the intradermal injection of influenza virus was followed by a skin lesion which clinically resembled a tuberculin reaction and which occured more often in those with raised haemagglutinationinhibiting (HAI) antibody titres to influenza virus (Beveridge and Burnet, 1944). Recently Habershon et al. confirmed this observation using purified influenza virus, and showed that the histology of the lesion was compatible with a delayed hypersensitivity reaction (Habershon, Molyneux, Slavin, Loewi and Tyrrell, 1973). However, they did not exclude the possibility that such lesions might be, at least in part, Arthus reactions, since they only examined the histology and stained for immunoglobulins and complement in the late stages of the reaction. It has also been shown in a small study of nine people that human lymphocytes may be transformed in vitro by influenza virus antigen (Ruben, Jackson and Gotoff, 1973). But in this work and in other studies (Cate and Kelly, 1969; Denman, Denman, Greenwood, Gall and Heath, 1970) appreciable lymphocyte transformation was only obtained after recent natural infection or immunization with influenza virus. However, we have found no report in the literature of a study of the relationship in man between skin reactivity and the immunological sensitization of lymphocytes to influenza virus. In this study we have, therefore, looked at skin reactions and lymphocyte transformation in volunteers without a known recent history of exposure to influenza virus in an attempt to clarify the nature of the skin response and to determine any relationship between it and the ability of lymphocytes to be transformed. MATERIALS AND METHODS Volunteers Twenty-five millilitres of venous blood was obtained from forty-eight adult volunteers, * Present address and correspondence: Dr P. J. Cole, Host Defence Unit, Department of Medicine, Cardiothoracic Institute, Brompton Hospital, Fulham Road, London SW3.
749
750 P. J. Cole and M. E. Molyneux at the Clinical Research Centre, Harrow, and the Common Cold Unit, Salisbury, for serological and lymphocyte transformation tests. On all forty-eight volunteers skin tests were then performed and read at 48 hours.
Skin tests The inactivated influenza viruses A/Aichi/68(H3N2) and B/Mass/3/66 were used. These antigens were kindly prepared and safety tested as for influenza vaccine production by Richardson Merell Incorporated, Cincinnati, Ohio, the viruses being grown in the allantoic cavity of chick embryos, zonally ultracentrifuged, formaldehyde-inactivated and stored at 40. Four chick cell agglutinating (CCA) units (as estimated by the makers) of antigen in 0-1 ml isotonic saline were injected intradermally. Control skin tests were performed with uninfected chick allantoic fluid, treated identically to the skin test antigen. Purified protein derivative (PPD) diluted 1/1000 (Evans Medical Ltd) was also used as a skin test antigen. Quantification of the skin test was achieved by calculating the area of induration as the product of two diameters at right angles. The skin test had been shown previously to be dose-dependent (Habershon et al., 1973) and a dose of 4 CCA units was found to give positive reactions in 45-55 per cent of subjects, whilst not provoking significant rise in circulating HAI antibody titre. Six-hourly biopsies were taken of positive reactions in seven volunteers for 48 hours from the time of injection of antigen. These were examined histologically and by immunofluorescent staining, with antisera (Wellcome Research Laboratories) directed against whole immunoglobulins, IgG, IgA, IgM and the third component of complement (C3). The sensitivity of this staining was not defined but it has been used successfully to stain immunoglobulin deposits and C3 in skin biopsies from patients with lupus erythematosus and dermatitis herpetiformis. It is also being used routinely for staining immunoglobulin in plasma cells and deposits of immunoglobulin on renal glomeruli.
Lymphocyte transformation Preparation of lymphocytes. A standard method was used in which 20 ml of heparinized venous blood was sedimented with 10 ml of freshly prepared 3 per cent fine-grain pig-skin gelatin in isotonic saline at 370 for 30 minutes. Leucocyte-rich plasma was removed and centrifuged at 200 g for 10 minutes, the serum being retained and the sedimented leucocytes resuspended in 2 ml of RPMI 1640 Medium (Gibco) using HEPES buffer (Gibco) (N-2-hydroxyethylpiperazine-N-ethanesulphonic acid) at a final 20 M molar concentration with added 0 5 per cent bicarbonate, 0-292 mg/ml L-glutamine, 100 units/ml penicillin and streptomycin, and 10 per cent human serum. This cell suspension was layered over an equal volume of freshly prepared Ficoll-Triosil mixture (specific gravity 1.078) comprising 4 parts of 8 per cent (weight/volume) Ficoll (Pharmacia Fine Chemicals, Uppsala, Sweden) mixed with 1 part of 50 per cent Triosil (Nyegaard & Company, Oslo, Norway). After centrifugation for 40 minutes at 500 g (room temperature) the cell band was aspirated with a Pasteur pipette, washed three times in medium alone, and resuspended at 1 x 106 lymphocytes per millilitre in the 10 per cent serum medium described above. A trypan blue exclusion test showed that in the resulting cell suspension more than 98 per cent of the cells were viable lymphocytes. Mitogen and antigens. Phytohaemagglutinin (PHA) (Wellcome purified grade) was used as mitogen at a concentration of 50 ug/ml and preservative-free PPD (Central Veterinary
751 Lymphocyte Reactivity to Influenza Virus Laboratory, Weybridge, Surrey) was used as control specific antigen at a concentration of 25 Mg/ml. Influenza antigens A2/Aichi/68 (H3N2) and B/Mass/3/66 were prepared as for skin testing and exhaustively dialysed against distilled water. A dose of 2-5 x 103 CCA units/ml of each viral antigen was found to give optimal lymphocyte transformation in 144-hour cultures, as determined from preliminary dose-response and time-response experiments on ten volunteers. Culture and serum. Quadruplicate cultures were established in microtitre plates (Cooke Microtitre) using 10 1l of antigen or mitogen and 200 pi of cell suspension. Ten microlitres of antigen or mitogen were added to control cultures after the incubation period (immediately before harvesting) to ensure that each test and its control culture consisted of identical materials-the test cultures only differing by reason of their incubation with mitogen or antigen. Quadruplicate cultures in 10 per cent autologous serum were all duplicated in 10 per cent pooled human AB Rhesus-negative serum, previously screened for absence of HAI antibodies. Cultures with mitogen were harvested after 72 hours at 370 in a moist atmosphere of 5 per cent carbon dioxide in air, whilst those with antigen were harvested after 144 hours. The last 24 hours of each culture were in the presence of 10 pI of tritiated thymidine (Radiochemical Centre, Amersham, Bucks) containing 1 iCi specific activity. Harvesting. The bottom of each microtitre culture well was thoroughly scraped, and washed twice, with an Eppendorf pipette tip to detach adherent cells. The cell suspension with washings was transferred to a glass fibre filter paper (Whatman GF/C 2-5 cm) on a vacuum manifold, where it was washed twice with saline and once with 3 per cent acetic acid to lyse red blood cells. Cold 5 per cent trichloracetic acid (TCA) was used for precipitation on the filter paper and for washing the precipitate when formed. After washing with 100 per cent methanol the filter paper was allowed to dry overnight and counted in (a)
(c)
(b)
7
66
JEEE C0
E ~5_
23
O2 E
-I 0
2
3
4
0
2 3 Skin test area score
4
0
2
3
4
FIG. 1. Correlation of area of skin test reaction and lymphocyte reactivity, both to influenza virus. The area of induration of skin was calculated as the product of two diameters at right angles and scored as 0 = absent or erythema only; 1 = 0-25 sq. mm; 2 = 25-100 sq. mm; 3 = 100-400 sq. mm; 4 = >400 sq. mm. (a) PPD. (b) Influenza virus B/Mass/3/66. (c) Influenza virus A2/Aichi/68 (H3N2).
P. J. Cole and M. E. Molyneux 752 a liquid scintillation vial (Johnsen & Jorgenson) containing 1 ml of methanol and 10 ml of Triton-xylene scintillation fluid comprising 1 part of Triton X-100 (Lennig Chemicals) to 2 parts of scintillant xylene with 0-012 g per cent POPOP (1,4-bis-2- (5-phenyloxazolyl)benzene) and 0-6 g per cent PPO (2,5-Diphenyloxazole) (Nuclear Enterprises Ltd., Sighthill, Edinburgh). Samples were counted at 40 in a Packard Liquid Scintillation Counter to obtain counts per minute (ct/min) which were then corrected for background and quenching. Preliminary controls. Human cord-blood lymphocytes, which might be assumed not to have been exposed to the antigens PPD and influenza previously, were cultured with the latter antigens. In such cultures there was no stimulation, which excluded a non-specific mitogenic effect of these antigens; yet these lymphocytes were stimulated by PHA, proving the cells' capability to respond. Expression of results. The stimulation ratio (ct/min in test cultures divided by ct/min in control cultures) and increment (ct/min in test cultures minus ct/min in control cultures) of the lymphocyte cultures, were calculated after subtraction of background. As the control mitogen-free and antigen-free culture counts were similar and did not approach zero, ratio and increment were equally satisfactory. Results here have been expressed as increments.
Serology HAI tests were performed by a standard method (Tyrrell, Peto and King, 1967). RESULTS SKIN TESTS
Forty-two per cent of the forty-eight subjects gave a positive skin reaction to the intradermal injection of influenza antigen. These reactions consisted of erythema and indura12000 900_
800 700 600 _ 500 _
a)
400_
