Immunology 1978 34 721
Active E rosette formation by human lymphoblasts
G. SEMENZATO, G. AMADORI, P. SARASIN & G. GASPAROTTO Istituto di Medicina Clinica dell'Universita di Padova, Clinica Medica 20 e Cattedra di Immunopatologia, Padova, Italy
Received 14 July 1977; acceptedfor publication 25 August 1977
Summary. The percentage of active and total E rosettes for lymphocytes cultured for 72h both with and without phytohaemagglutinin (PHA) was evaluated in 20 normal subjects. A clear increase in the formation of active E rosettes by PHA-stimulated lymphocytes was demonstrated with respect to unstimulated lymphocytes. Our data gives further proof that the active E rosettes constitute a specific subpopulation of lymphocytes which represent functionally active T cells in the blood.
T cell population (Fudenberg, Wybran & Robbins, 1975; Kerman, Smith, Stefani & Ezdinli, 1976). The aim of the present study was to determine the percentage of active E rosettes for lymphocytes cultured for 72 h both with and without phytohaemagglutinin and thus better define the nature of this cellular subpopulation.
MATERIALS AND METHODS Lymphocyte separation Lymphocytes were separated from heparinized peripheral blood of 20 healthy subjects (between 24 and 51 years of age) by centrifugation on a FicollHypaque density gradient (Boyum, 1968). The cells were washed three times in Hank's balanced salt solution and resuspended in TC medium 199 (Difco Laboratories, Detroit, U.S.A.).
INTRODUCTION
Sheep red blood cell rosette formation has been demonstrated to be a specific property of human thymus derived lymphocytes (Jondal, Holm & Wigzell, 1972; Lay, Mendes, Bianco & Nussenzweig, 1971). These rosettes (E) were made in vitro by light centrifugation of a mixture of the two types of cells. Rosettes that were formed immediately after centrifugation have been termed active rosettes; the rosettes that required incubation at 40 for optimal formation were designated as total rosettes (Wybran & Fudenberg, 1973b). Clinical studies suggest that the active T cells represent a subpopulation of T lymphocytes more actively involved in cellular immunity than the total
Lymphocyte culture Mitogen cultures were prepared in 16 x 95 mm sterile tubes by adding 1 x 106 lymphocytes to 1 ml of solution containing TC medium 199 supplemented with pooled human AB serum (20%), 100 units of penicillin, 100 4ug of streptomycin and 50pl of PHA-M (Difco). The cultures were incubated at 370 in a 5 % CO2 atmosphere for 72 h. Control cultures were identical except that PHA was omitted. For each subject 16 samples were tested. Of these 16 samples four were studied, after 72 h, both with
Correspondence: Dr Gianpietro Semenzato, Istituto di Medicina Clinica dell'Universiti, Via Giustiniani, 2-35100 Padova, Italy.
0019-2805/78/0400-0721 $02.00 (©1978 Blackwell Scientific Publications
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and without PHA in order to measure the lymphocytes' blastic capacity, using the 3H-Thymidine incorporation method, previously described (Semenzato, Amadori, Tosato & Gasparotto, 1977). The remaining samples, with and without PHA were suspended in TC medium 199 at a concentration of 4 x 106 cells/ml for the rosette tests. Cellular viability was tested by the trypan blue exclusion test.
resuspended. Resuspension and counting of active rosette-forming lymphocytes (A-RFC) was similar to that of the total rosettes. The results are expressed as the mean ± standard error of the mean. Student's 't'-test was used to estimate the significance. RESULTS
Total rosette forming cells (T-RFC) The total rosette assay was performed according to the method of Stjermsward, Jondal, Vanky, Wigzell & Sealy (1972). Sheep red blood cells (SRBC) were stored at 40 in Alsever's solution (1/1) and used within 1 week of being drawn; immediately before use they were washed 3 times with Hank's solution. 1 x 106 lymphocytes were incubated with SRBC (lymphocyte: erythrocyte ratio 1: 80) for 10 min, centrifuged at 200 g for 5 min and incubated at 40 overnight. The next day, the pellet was gently resuspended and 200 lymphocytes were counted in each sample; all determinations were made in duplicate. A rosette-forming lymphocyte (T-RFC) was defined as one that bound three or more SRBC. Aggregates of more than three lymphoid cells were not scored. Active rosette forming cells (A-RFC) The active rosette test was performed according to the method of Wybran & Fudenberg (1973b). Briefly the lymphocytes were incubated in TC medium 199 containing 50% foetal calf serum (FCS) (Difco) at 370 for 60 min. The FCS had previously been heat inactivated (560 for 30 min) and adsorbed against both sheep and human AB erythrocytes (at 370 and at 40). After incubation, SRBC in saline was added to obtain a final ratio of 8 red cells to 1 lymphocyte (Chisholm & Tubergen, 1976). The tubes were centrifuged for 5 min at 200 g and
As shown in Table 1, the percentage of A-RFC after stimulation with PHA is greatly increased with respect to the cultures without stimulant (41-7+ 1*6% vs 23-3±1 0%; P
Active E rosette formation by human lymphoblasts
G. SEMENZATO, G. AMADORI, P. SARASIN & G. GASPAROTTO Istituto di Medicina Clinica dell'Universita di Padova, Clinica Medica 20 e Cattedra di Immunopatologia, Padova, Italy
Received 14 July 1977; acceptedfor publication 25 August 1977
Summary. The percentage of active and total E rosettes for lymphocytes cultured for 72h both with and without phytohaemagglutinin (PHA) was evaluated in 20 normal subjects. A clear increase in the formation of active E rosettes by PHA-stimulated lymphocytes was demonstrated with respect to unstimulated lymphocytes. Our data gives further proof that the active E rosettes constitute a specific subpopulation of lymphocytes which represent functionally active T cells in the blood.
T cell population (Fudenberg, Wybran & Robbins, 1975; Kerman, Smith, Stefani & Ezdinli, 1976). The aim of the present study was to determine the percentage of active E rosettes for lymphocytes cultured for 72 h both with and without phytohaemagglutinin and thus better define the nature of this cellular subpopulation.
MATERIALS AND METHODS Lymphocyte separation Lymphocytes were separated from heparinized peripheral blood of 20 healthy subjects (between 24 and 51 years of age) by centrifugation on a FicollHypaque density gradient (Boyum, 1968). The cells were washed three times in Hank's balanced salt solution and resuspended in TC medium 199 (Difco Laboratories, Detroit, U.S.A.).
INTRODUCTION
Sheep red blood cell rosette formation has been demonstrated to be a specific property of human thymus derived lymphocytes (Jondal, Holm & Wigzell, 1972; Lay, Mendes, Bianco & Nussenzweig, 1971). These rosettes (E) were made in vitro by light centrifugation of a mixture of the two types of cells. Rosettes that were formed immediately after centrifugation have been termed active rosettes; the rosettes that required incubation at 40 for optimal formation were designated as total rosettes (Wybran & Fudenberg, 1973b). Clinical studies suggest that the active T cells represent a subpopulation of T lymphocytes more actively involved in cellular immunity than the total
Lymphocyte culture Mitogen cultures were prepared in 16 x 95 mm sterile tubes by adding 1 x 106 lymphocytes to 1 ml of solution containing TC medium 199 supplemented with pooled human AB serum (20%), 100 units of penicillin, 100 4ug of streptomycin and 50pl of PHA-M (Difco). The cultures were incubated at 370 in a 5 % CO2 atmosphere for 72 h. Control cultures were identical except that PHA was omitted. For each subject 16 samples were tested. Of these 16 samples four were studied, after 72 h, both with
Correspondence: Dr Gianpietro Semenzato, Istituto di Medicina Clinica dell'Universiti, Via Giustiniani, 2-35100 Padova, Italy.
0019-2805/78/0400-0721 $02.00 (©1978 Blackwell Scientific Publications
721
722
G. Semenzato et al.
and without PHA in order to measure the lymphocytes' blastic capacity, using the 3H-Thymidine incorporation method, previously described (Semenzato, Amadori, Tosato & Gasparotto, 1977). The remaining samples, with and without PHA were suspended in TC medium 199 at a concentration of 4 x 106 cells/ml for the rosette tests. Cellular viability was tested by the trypan blue exclusion test.
resuspended. Resuspension and counting of active rosette-forming lymphocytes (A-RFC) was similar to that of the total rosettes. The results are expressed as the mean ± standard error of the mean. Student's 't'-test was used to estimate the significance. RESULTS
Total rosette forming cells (T-RFC) The total rosette assay was performed according to the method of Stjermsward, Jondal, Vanky, Wigzell & Sealy (1972). Sheep red blood cells (SRBC) were stored at 40 in Alsever's solution (1/1) and used within 1 week of being drawn; immediately before use they were washed 3 times with Hank's solution. 1 x 106 lymphocytes were incubated with SRBC (lymphocyte: erythrocyte ratio 1: 80) for 10 min, centrifuged at 200 g for 5 min and incubated at 40 overnight. The next day, the pellet was gently resuspended and 200 lymphocytes were counted in each sample; all determinations were made in duplicate. A rosette-forming lymphocyte (T-RFC) was defined as one that bound three or more SRBC. Aggregates of more than three lymphoid cells were not scored. Active rosette forming cells (A-RFC) The active rosette test was performed according to the method of Wybran & Fudenberg (1973b). Briefly the lymphocytes were incubated in TC medium 199 containing 50% foetal calf serum (FCS) (Difco) at 370 for 60 min. The FCS had previously been heat inactivated (560 for 30 min) and adsorbed against both sheep and human AB erythrocytes (at 370 and at 40). After incubation, SRBC in saline was added to obtain a final ratio of 8 red cells to 1 lymphocyte (Chisholm & Tubergen, 1976). The tubes were centrifuged for 5 min at 200 g and
As shown in Table 1, the percentage of A-RFC after stimulation with PHA is greatly increased with respect to the cultures without stimulant (41-7+ 1*6% vs 23-3±1 0%; P
