Clin. exp. Immunol. (1975) 22, 121-125
B- AND T-CELL MARKERS ON HUMAN LYMPHOBLASTS AFTER STIMULATION WITH MITOGEN OR ANTIGENS J. G. KREEFTENBERG, M. F. LEERLING AND H. G. LOGGEN Rijks Instituut voor de Volksgezondheid, Postbox 1, Bilthoven, The Netherlands (Received revised 24 March 1975)
SUMMARY
Receptors for sheep erythrocytes and for the third component of complement were demonstrated on human lymphoblasts after stimulation with various antigens and mitogens. With these markers B- and T-cell stimulation could be differentiated. Phytohaemagglutinin (PHA) and purified protein derivative (PPD) proved to activate predominantly T cells, whereas foetal calf serum (FCS) was shown to be a B-cell stimulator. Candida albicans and allogeneic cells, on the other hand, stimulated both B and T cells.
INTRODUCTION It has long been considered that induction of blast transformation of sensitized lymphocytes by antigen in vitro is a measure of specific cellular immunity (Roitt et al., 1969). However, data have been presented which suggest that bone-marrow-derived cells (B cells) may also proliferate in vitro after non-specific and specific stimulation (Hellstr6m, Zeromski & Perlmann, 1971; Elfenbein, Harrison & Green, 1973; Philips & Roitt, 1973). Recently it has been shown that lymphocytes of various species including man, may be divided into two populations on the basis of whether or not they possess receptors for the third component of complement or sheep red cells on their surface (Lay et al., 1971; Wybran, Chantler & Fudenberg, 1973; Bianco, Patrick & Nussenzweig, 1970; Shevach et al., 1972). With these markers, direct identification of stimulated B cells and thymus-derived (T cells) would be possible (Elfenbein et al., 1973; Hellstrbm et al., 1971; Mendes et al., 1974; Janossy et al., 1973; Philips & Roitt, 1973). The purpose of our experiments was to find out if these identification methods will give more information about the role of B and T cells in the lymphocyte-stimulation test. In this study we have differentiated B- and T-cell activation after stimulation of lymphocytes with phytohaemagglutinin (PHA), foetal calf serum (FCS), tuberculin (PPD), Candida albicans antigen and allogeneic cells in the mixed lymphocyte culture (MLC). Correspondence: Dr J. G. Kreeftenberg, Rijks Instituut voor de Volksgezondheid, Postbox 1, Bilthoven, The Netherlands.
121
122
J. G. Kreeftenberg, M. F. Leerling and H. G. Loggen MATERIALS AND METHODS
Minimum essential medium (MEM) for spinner cultures (Gibco) buffered with Tris (0 025 M) was used. Purified protein derivative (PPD) without preservatives prepared by the Rijks Instituut voor de Volksgezondheid was dissolved in MEM until a concentration of 200 #g/ml was obtained. Fifty microlitres of this solution was added to 1 ml of lymphocyte suspension. Candida albicans antigen was prepared by Hollister-Stier Laboratories (Dallas, Texas, U.S.A.). Fifty microlitres of a 1/10 diluted solution was added to 1 ml of lymphocyte suspension. Phytohaemagglutinin (PHA) was prepared by Wellcome Laboratories, Beckenham, Kent. One ampoule containing 5 ml of dried material was dissolved in 20 ml of MEM. Fifty microlitres of this solution was added to 1 ml of lymphocyte suspension. Foetal calf serum (FCS) was prepared by the Rijks Instituut voor de Volksgezondheid. A selected serum batch with a good culture quality in the lymphocyte stimulation test and no demonstrable endotoxin was used in a concentration of 20%. Preparation of autologous plasma. Autologous plasma was prepared by centrifuging heparinized blood from healthy volunteers at 500 g for 15 min at room temperature. The plasma was pipetted off and the cell sediment was resuspended in MEM to the original blood volume. To eliminate the thrombocytes the plasma was centrifuged once more for 15 min at 1900 g at room temperature. Preparation of lymphocytes. The resuspended cell sediment was layered on a Ficoll Isopaque solution (specific density 1-078) followed by centrifugation during 15 min at 10oog in the interphase. The lymphocyterich interphase was obtained and washed with MEM + 5% FCS. The final suspension of lymphocytes was adjusted to a concentration of 0 3 x 106 viable lymphocytes per millilitre in MEM supplemented with FCS (20%), streptomycin (100 #g/ml) and penicillin (100 i.u./ml). Lymphocyte culture. The total volume per culture tube was 1 ml. The cells were cultured in polystyrene tubes (Nunc, Denmark) (Schellekens & Eysvoogel, 1968). Thymidine incorporation. After 40-48 or 112-120 hr cultivation 0075 uCi of [2-14C]thymidine (specific activity 6*25 mCi/mM was added). Twenty-four hours later the cells were collected and washed on glass fibre filters (Millipore). After drying, the filters were placed into scintillation vials and 2 ml of scintillation fluid was added. Counting was done in a Packard liquid scintillation counter. Sheep red cell suspension (E). Sheep red cells (SRC) were obtained from sheep blood stored in Alsever's solution. The red cells were washed three times with normal saline and adjusted to a final concentration of 100 x 106 red cells per millilitre of Hanks's medium. Sensitized sheep red cell suspension (EAC). Five millilitres of a 5% suspension of washed sheep red cells was incubated for 30 min at 37°C with 5 ml amboceptor diluted 1/500 in saline. The cells were washed four times and resuspended in 5 ml saline. Five millilitres of monkey (Macaca fascicularis) complement (serum stored at - 70°C) diluted 1/40 in saline was then added and the suspension was incubated for 30 min at 37°C. The cells were finally washed four times and adjusted to a concentration of 100 x 106 red cells per millilitre. E-receptor lymphoblasts. The lymphoblast suspension was prepared in the same way as will be mentioned under complement receptor lymphoblasts. To the suspended lymphoblast sediment one drop of a SRC suspension was added. After centrifugation for 5 min at 200 g the tubes were placed for 1 hr at+20°C. Then the sediment was resuspended gently and the rosette-forming lymphoblasts were counted in a haemocytometer after adding one drop of Toluidine Blue. The lymphoblasts could be identified by their typical staining with Toluidine Blue. The percentage of T blast cells was the number of rosette-forming blasts per 100 blast cells. Complement receptor lymphoblasts (CRL). After 64-72 or 136-144 hr the tubes with lymphocyte suspensions were centrifuged for 10 min at 200 g. The supernatant was discarded and the sediment was resuspended vigorously to disrupt the clumps of blast cells. Cultures stimulated with PHA were washed once. Then one drop of sensitized SRC was added and the mixture was incubated for J hr at 37°C. Under these conditions a negligible number of rosettes were formed with SRC or SRC amboceptor complex. The percentage of B blast cells was the number of rosette-forming blasts per 100 blast cells.
RESULTS After stimulation of human lymphocytes with FCS, PHA, Candida, PPD or allogeneic cells
B- and T-cell markers on human lymphoblasts
123
TABLE 1. B- and T-cell markers on lymphoblasts after stimulation with mitogen or antigens Antigen or mitogen
[V4CjThymidine incorporated per 0-3 x 106 cells (ct/10 min)
Autologous plasma FCS PHA
Candida PPD
MLC
C receptor
E receptor
lymphoblasts
lymphoblasts
(%)
(%)
374 (302-450) 2422 (1858-3179) 32773 (25153-39449) 9084 (8572-9919)
83
4
(74-95)
(2-7)
74 (71-82) 12 (10-16) 46 (37-54)
13550 (9349-19537) 12758 (10550-15714)
(21-30) 44 (41-46)
12 (11-14) 84 (81-86) 52 (48-54) 84 (75-90) 56 (49-64)
27
The values given are the averages of three experiments. The lowest and highest values are given in brackets. All values were estimated in 6-day cultures except for PHA stimulation which was estimated in 3-day cultures.
both the E- and EAC-binding capacities of the lymphocytes are still intact. The number of red cells per lymphocyte is increased evenly. It appears to be possible to make use of the receptors for the third component of complement or sheep red cells to identify B- and T-cell stimulation in the lymphocyte-stimulation test (Table 1). As expected, PHA is mainly a T-cell stimulator, although 1-20% of the blasts are B-cell derived. In contradistinction to PHA, FCS stimulates mainly B cells. Allogeneic cells are both T- and B-cell stimulators. PPD is mainly a T-cell stimulator. The B-cell stimulation observed is probably a background stimulation by FCS although some direct or indirect stimulation of B cells by PPD cannot be excluded. As could be expected, Candida is a good T-cell stimulator, but some B stimulation also takes place.
DISCUSSION This study has shown that after specific and non-specific stimulation of human lymphocytes both the E- and the EAC-binding capacities of the cell membrane are considerably enhanced. This finding provided a direct method for studying the B- and T-cell stimulating capacity of antigens and mitogens in human lymphocyte cultures. Separate estimations of the percentage of respectively E- and EAC-binding lymphoblasts were made and it was found that the sum of the percentages of B and T cells was practically
124
J. G. Kreeftenberg, M. F. Leerling and H. G. Loggen
always about 100%. It can therefore be inferred that most of the stimulated lymphoblasts are of either T-cell or B-cell origin. Recently Mendes et al. (1974) used similar methods to assess the extent in which E- and EAC-binding lymphocytes were stimulated by a number of mitogens. As a criterion for stimulation, these authors used positive radioautography instead of a typical staining with Toluidin Blue. Although their results are, on the whole, similar to ours, the sum of the percentages of E- and EAC-binding stimulated lymphocytes found by them was usually smaller than the total number of labelled cells. This can be explained by assuming that some small lymphocytes, although labelled, have receptors which are more difficult to detect than those on the Toluidin Blue-positive blast cells. In our experiments we confirmed the observations of Philips et al. (1973); Mendes et al. (1974); Hellstrdm et al. (1971) and Janossy et al. (1973) pertaining to the B-cell stimulating activity of PHA. However, these authors used an immunofluorescence staining with antiimmunoglobulin serum to identify stimulated B lymphocytes. Under the conditions by which we prepared E and EAC rosettes, a negligible number of very small rosettes were formed by fresh unstimulated PHA-treated lymphocytes and rabbit erythrocytes. Therefore, rosette formation by binding of E or EAC to lymphocytes through PHA cannot be excluded completely, but this phenomenon does not seriously interfere with our observations. In our hands FCS is a B-cell stimulator. The serum used had been prepared in our institute under aseptic conditions and was devoid of demonstrable endotoxin in the limulus lysate test (Reinhold & Fine, 1971). In spite of this stimulation we prefer the use of a selected lot of FCS instead of human plasma or serum in the lymphocyte-stimulation test for the following reasons. (1) Large quantities of FCS are available per lot and therefore many experiments can be done with one single lot. This increases the uniformity of these experiments. (2) A considerable variability of different human plasmas or sera in their ability to support transformation has been observed (Knoke, Kanaide & Powell, 1974; Hinze & Chickosky, 1972). (3) The use of human plasma or serum in the lymphocyte stimulation test might give misleading results because antibodies present in the plasma or serum would interfere with the lymphocyte stimulation (Lee & Sigel, 1974). PPD mainly stimulates T cells from donors immunized with BCG vaccine. Some Bcell stimulation (as described by Yoshida, Sonozaki & Cohen, 1973) cannot be excluded, because of the background B-cell stimulation by FCS. The mixed lymphocyte culture is generally believed to represent a T-cell activity. However, in our hands 44%0 of the blasts are B-cell derived. Although part of this stimulation is caused by FCS it appears that allogeneic lymphocytes can stimulate B cells. However, some B-cell stimulation may be T celldependent. These results confirm the data presented by Vischer (1972) and Hellstrdm et al. (1971). Candida antigen is a mixture of different antigens which may stimulate both B and T cells. The results suggest that both lymphocyte populations are activated by Candida
antigen. In conclusion it appears to be possible to identify B and T blasts after antigen or mitogen stimulation of human lymphocytes. With these techniques we started to investigate the lymphocyte stimulation in man after vaccination with various virus vaccines. Preliminary studies suggest that the techniques described here are useful. The authors wish to thank Drs H. Cohen, J. Nagel and J. D. van Ramshorst for their helpful suggestions and criticism of the manuscript.
B- and T-cell markers on human lymphoblasts
125
REFERENCES LEE, J.C. & SIGEL, M.M. (1974). A differential effect of IgM and IgG antibodies on the blastogenic response of lymphocytes to rubella virus. Cell. Immunol. 13, 22. MENDES, N.F., ZENHA, M.J., MUSATTI, C.C. & NASPITZ, C. (1974) Lymphocyte membrane receptors in cultures treated with mitogens. Cell. Immunol. 12, 331. PHILIPS, B. & RoiTT, I.M. (1973) Evidence for transformation of human B lymphocytes by PHA. Nature: New Biology, 241, 254. REINHOLD, R.B. & FINE, J. (1971) A technique for quantitative measurement of endotoxin in human plasma. Proc. Soc. exp. Biol. (N.Y.), 137, 334. ROITT, I.M., GREAVES, M.F., TORRIGIANI, G., BROSTOFF, J. & PLAYFAIR, J.H.L. (1969) The cellular basis of immunological responses. A synthesis of some current views. Lancet, ii, 367. SCHELLEKENS, P.T.A. & EYSVOOGEL, V.P. (1968) Lymphocyte transformation in vitro. I. Tissue culture conditions and quantitative measurements. Clin. exp. Immunol. 3, 571. SHEVACH, E.M., HERBERMAN, R., FRANK, M.M. & GREEN, J. (1972) Receptors for complement and immunoglobulin on human leukemic cells and human lymphoblastoid cell lines. J. clin. Invest. 51, 1933. VISCHER, T.L. (1972) Immunoglobulin-like surface molecules and theta antigen during the specific and non-specific stimulation of mouse spleen cells in vitro. Clin. exp. Immunol. 11, 523. WYBRAN, J., CHANTLER, S. & FUDENBERG, H.H. (1973) Isolation of normal T cells in chronic lymphatic leukaemia. Lancet, i, 126. YOSHIDA, T., SONOZAKI, H. & COHEN, S. (1973) The production of migration inhibition factor by B and T cells of the guinea pig. J. exp. Med. 138, 784.
BIANCO, C., PATRICK, R. & NUSSENZWEIG, V. (1970) A population of lymphocytes bearing a membrane receptor for antigen-antibody-complement complexes. J. exp. Med. 132, 702. ELFENBEIN, G.J., HARRISON, M.R. & GREEN, J. (1973) Demonstration of proliferation by bone marrow derived lymphocytes of guinea pigs, mice and rabbits in response to mitogen stimulation in vitro. J. Immunol. 110, 1334. HELLSTROM, U., ZEROMSKI, J. & PERLMANN, P. (1971) Immunoglobulin light chain determinants on unstimulated and stimulated human blood lymphocytes, assayed by indirect immunofluorescence. Immunology, 20, 1099. HINZ, C.F. & CHICKOSKY, J.F. (1972) Factors influencing the stimulation of human lymphocytes by antigen during culture in vitro. Int. Arch. Allergy, 42, 836. JANOSSY, G., GREAVES, M.F., DOENHOFF, M.J. & SNAJDR, J. (1973) Lymphocyte activation. V. Quantitation of the proliferative responses to mitogens using defined T and B cell populations. Clin. exp. Immunol. 14, 581. JONDAL, M., HOLM, G. & WIGZELL, H. (1972) Surface markers on human T and B lymphocytes. 1. A large population of lymphocytes forming non immune rosettes with sheep red blood cells. J. exp. Med. 136, 207. KNOKE, J., KANAIDE, A. & POWELL, A.E. (1974) Modulation of lymphocytic responses by factors in human plasma. I. The effects on normal, stimulated lymphocytes of various normal plasmas in microplate culture. Int. Arch. Allergy, 46, 584. LAY, W.H., MENDES, N.F., BIANCO, C. & NUSSENZWEIG, V. (1971) Binding of sheep red blood cells to a large population of human lymphocytes. Nature (Lond.), 230, 531.
B- AND T-CELL MARKERS ON HUMAN LYMPHOBLASTS AFTER STIMULATION WITH MITOGEN OR ANTIGENS J. G. KREEFTENBERG, M. F. LEERLING AND H. G. LOGGEN Rijks Instituut voor de Volksgezondheid, Postbox 1, Bilthoven, The Netherlands (Received revised 24 March 1975)
SUMMARY
Receptors for sheep erythrocytes and for the third component of complement were demonstrated on human lymphoblasts after stimulation with various antigens and mitogens. With these markers B- and T-cell stimulation could be differentiated. Phytohaemagglutinin (PHA) and purified protein derivative (PPD) proved to activate predominantly T cells, whereas foetal calf serum (FCS) was shown to be a B-cell stimulator. Candida albicans and allogeneic cells, on the other hand, stimulated both B and T cells.
INTRODUCTION It has long been considered that induction of blast transformation of sensitized lymphocytes by antigen in vitro is a measure of specific cellular immunity (Roitt et al., 1969). However, data have been presented which suggest that bone-marrow-derived cells (B cells) may also proliferate in vitro after non-specific and specific stimulation (Hellstr6m, Zeromski & Perlmann, 1971; Elfenbein, Harrison & Green, 1973; Philips & Roitt, 1973). Recently it has been shown that lymphocytes of various species including man, may be divided into two populations on the basis of whether or not they possess receptors for the third component of complement or sheep red cells on their surface (Lay et al., 1971; Wybran, Chantler & Fudenberg, 1973; Bianco, Patrick & Nussenzweig, 1970; Shevach et al., 1972). With these markers, direct identification of stimulated B cells and thymus-derived (T cells) would be possible (Elfenbein et al., 1973; Hellstrbm et al., 1971; Mendes et al., 1974; Janossy et al., 1973; Philips & Roitt, 1973). The purpose of our experiments was to find out if these identification methods will give more information about the role of B and T cells in the lymphocyte-stimulation test. In this study we have differentiated B- and T-cell activation after stimulation of lymphocytes with phytohaemagglutinin (PHA), foetal calf serum (FCS), tuberculin (PPD), Candida albicans antigen and allogeneic cells in the mixed lymphocyte culture (MLC). Correspondence: Dr J. G. Kreeftenberg, Rijks Instituut voor de Volksgezondheid, Postbox 1, Bilthoven, The Netherlands.
121
122
J. G. Kreeftenberg, M. F. Leerling and H. G. Loggen MATERIALS AND METHODS
Minimum essential medium (MEM) for spinner cultures (Gibco) buffered with Tris (0 025 M) was used. Purified protein derivative (PPD) without preservatives prepared by the Rijks Instituut voor de Volksgezondheid was dissolved in MEM until a concentration of 200 #g/ml was obtained. Fifty microlitres of this solution was added to 1 ml of lymphocyte suspension. Candida albicans antigen was prepared by Hollister-Stier Laboratories (Dallas, Texas, U.S.A.). Fifty microlitres of a 1/10 diluted solution was added to 1 ml of lymphocyte suspension. Phytohaemagglutinin (PHA) was prepared by Wellcome Laboratories, Beckenham, Kent. One ampoule containing 5 ml of dried material was dissolved in 20 ml of MEM. Fifty microlitres of this solution was added to 1 ml of lymphocyte suspension. Foetal calf serum (FCS) was prepared by the Rijks Instituut voor de Volksgezondheid. A selected serum batch with a good culture quality in the lymphocyte stimulation test and no demonstrable endotoxin was used in a concentration of 20%. Preparation of autologous plasma. Autologous plasma was prepared by centrifuging heparinized blood from healthy volunteers at 500 g for 15 min at room temperature. The plasma was pipetted off and the cell sediment was resuspended in MEM to the original blood volume. To eliminate the thrombocytes the plasma was centrifuged once more for 15 min at 1900 g at room temperature. Preparation of lymphocytes. The resuspended cell sediment was layered on a Ficoll Isopaque solution (specific density 1-078) followed by centrifugation during 15 min at 10oog in the interphase. The lymphocyterich interphase was obtained and washed with MEM + 5% FCS. The final suspension of lymphocytes was adjusted to a concentration of 0 3 x 106 viable lymphocytes per millilitre in MEM supplemented with FCS (20%), streptomycin (100 #g/ml) and penicillin (100 i.u./ml). Lymphocyte culture. The total volume per culture tube was 1 ml. The cells were cultured in polystyrene tubes (Nunc, Denmark) (Schellekens & Eysvoogel, 1968). Thymidine incorporation. After 40-48 or 112-120 hr cultivation 0075 uCi of [2-14C]thymidine (specific activity 6*25 mCi/mM was added). Twenty-four hours later the cells were collected and washed on glass fibre filters (Millipore). After drying, the filters were placed into scintillation vials and 2 ml of scintillation fluid was added. Counting was done in a Packard liquid scintillation counter. Sheep red cell suspension (E). Sheep red cells (SRC) were obtained from sheep blood stored in Alsever's solution. The red cells were washed three times with normal saline and adjusted to a final concentration of 100 x 106 red cells per millilitre of Hanks's medium. Sensitized sheep red cell suspension (EAC). Five millilitres of a 5% suspension of washed sheep red cells was incubated for 30 min at 37°C with 5 ml amboceptor diluted 1/500 in saline. The cells were washed four times and resuspended in 5 ml saline. Five millilitres of monkey (Macaca fascicularis) complement (serum stored at - 70°C) diluted 1/40 in saline was then added and the suspension was incubated for 30 min at 37°C. The cells were finally washed four times and adjusted to a concentration of 100 x 106 red cells per millilitre. E-receptor lymphoblasts. The lymphoblast suspension was prepared in the same way as will be mentioned under complement receptor lymphoblasts. To the suspended lymphoblast sediment one drop of a SRC suspension was added. After centrifugation for 5 min at 200 g the tubes were placed for 1 hr at+20°C. Then the sediment was resuspended gently and the rosette-forming lymphoblasts were counted in a haemocytometer after adding one drop of Toluidine Blue. The lymphoblasts could be identified by their typical staining with Toluidine Blue. The percentage of T blast cells was the number of rosette-forming blasts per 100 blast cells. Complement receptor lymphoblasts (CRL). After 64-72 or 136-144 hr the tubes with lymphocyte suspensions were centrifuged for 10 min at 200 g. The supernatant was discarded and the sediment was resuspended vigorously to disrupt the clumps of blast cells. Cultures stimulated with PHA were washed once. Then one drop of sensitized SRC was added and the mixture was incubated for J hr at 37°C. Under these conditions a negligible number of rosettes were formed with SRC or SRC amboceptor complex. The percentage of B blast cells was the number of rosette-forming blasts per 100 blast cells.
RESULTS After stimulation of human lymphocytes with FCS, PHA, Candida, PPD or allogeneic cells
B- and T-cell markers on human lymphoblasts
123
TABLE 1. B- and T-cell markers on lymphoblasts after stimulation with mitogen or antigens Antigen or mitogen
[V4CjThymidine incorporated per 0-3 x 106 cells (ct/10 min)
Autologous plasma FCS PHA
Candida PPD
MLC
C receptor
E receptor
lymphoblasts
lymphoblasts
(%)
(%)
374 (302-450) 2422 (1858-3179) 32773 (25153-39449) 9084 (8572-9919)
83
4
(74-95)
(2-7)
74 (71-82) 12 (10-16) 46 (37-54)
13550 (9349-19537) 12758 (10550-15714)
(21-30) 44 (41-46)
12 (11-14) 84 (81-86) 52 (48-54) 84 (75-90) 56 (49-64)
27
The values given are the averages of three experiments. The lowest and highest values are given in brackets. All values were estimated in 6-day cultures except for PHA stimulation which was estimated in 3-day cultures.
both the E- and EAC-binding capacities of the lymphocytes are still intact. The number of red cells per lymphocyte is increased evenly. It appears to be possible to make use of the receptors for the third component of complement or sheep red cells to identify B- and T-cell stimulation in the lymphocyte-stimulation test (Table 1). As expected, PHA is mainly a T-cell stimulator, although 1-20% of the blasts are B-cell derived. In contradistinction to PHA, FCS stimulates mainly B cells. Allogeneic cells are both T- and B-cell stimulators. PPD is mainly a T-cell stimulator. The B-cell stimulation observed is probably a background stimulation by FCS although some direct or indirect stimulation of B cells by PPD cannot be excluded. As could be expected, Candida is a good T-cell stimulator, but some B stimulation also takes place.
DISCUSSION This study has shown that after specific and non-specific stimulation of human lymphocytes both the E- and the EAC-binding capacities of the cell membrane are considerably enhanced. This finding provided a direct method for studying the B- and T-cell stimulating capacity of antigens and mitogens in human lymphocyte cultures. Separate estimations of the percentage of respectively E- and EAC-binding lymphoblasts were made and it was found that the sum of the percentages of B and T cells was practically
124
J. G. Kreeftenberg, M. F. Leerling and H. G. Loggen
always about 100%. It can therefore be inferred that most of the stimulated lymphoblasts are of either T-cell or B-cell origin. Recently Mendes et al. (1974) used similar methods to assess the extent in which E- and EAC-binding lymphocytes were stimulated by a number of mitogens. As a criterion for stimulation, these authors used positive radioautography instead of a typical staining with Toluidin Blue. Although their results are, on the whole, similar to ours, the sum of the percentages of E- and EAC-binding stimulated lymphocytes found by them was usually smaller than the total number of labelled cells. This can be explained by assuming that some small lymphocytes, although labelled, have receptors which are more difficult to detect than those on the Toluidin Blue-positive blast cells. In our experiments we confirmed the observations of Philips et al. (1973); Mendes et al. (1974); Hellstrdm et al. (1971) and Janossy et al. (1973) pertaining to the B-cell stimulating activity of PHA. However, these authors used an immunofluorescence staining with antiimmunoglobulin serum to identify stimulated B lymphocytes. Under the conditions by which we prepared E and EAC rosettes, a negligible number of very small rosettes were formed by fresh unstimulated PHA-treated lymphocytes and rabbit erythrocytes. Therefore, rosette formation by binding of E or EAC to lymphocytes through PHA cannot be excluded completely, but this phenomenon does not seriously interfere with our observations. In our hands FCS is a B-cell stimulator. The serum used had been prepared in our institute under aseptic conditions and was devoid of demonstrable endotoxin in the limulus lysate test (Reinhold & Fine, 1971). In spite of this stimulation we prefer the use of a selected lot of FCS instead of human plasma or serum in the lymphocyte-stimulation test for the following reasons. (1) Large quantities of FCS are available per lot and therefore many experiments can be done with one single lot. This increases the uniformity of these experiments. (2) A considerable variability of different human plasmas or sera in their ability to support transformation has been observed (Knoke, Kanaide & Powell, 1974; Hinze & Chickosky, 1972). (3) The use of human plasma or serum in the lymphocyte stimulation test might give misleading results because antibodies present in the plasma or serum would interfere with the lymphocyte stimulation (Lee & Sigel, 1974). PPD mainly stimulates T cells from donors immunized with BCG vaccine. Some Bcell stimulation (as described by Yoshida, Sonozaki & Cohen, 1973) cannot be excluded, because of the background B-cell stimulation by FCS. The mixed lymphocyte culture is generally believed to represent a T-cell activity. However, in our hands 44%0 of the blasts are B-cell derived. Although part of this stimulation is caused by FCS it appears that allogeneic lymphocytes can stimulate B cells. However, some B-cell stimulation may be T celldependent. These results confirm the data presented by Vischer (1972) and Hellstrdm et al. (1971). Candida antigen is a mixture of different antigens which may stimulate both B and T cells. The results suggest that both lymphocyte populations are activated by Candida
antigen. In conclusion it appears to be possible to identify B and T blasts after antigen or mitogen stimulation of human lymphocytes. With these techniques we started to investigate the lymphocyte stimulation in man after vaccination with various virus vaccines. Preliminary studies suggest that the techniques described here are useful. The authors wish to thank Drs H. Cohen, J. Nagel and J. D. van Ramshorst for their helpful suggestions and criticism of the manuscript.
B- and T-cell markers on human lymphoblasts
125
REFERENCES LEE, J.C. & SIGEL, M.M. (1974). A differential effect of IgM and IgG antibodies on the blastogenic response of lymphocytes to rubella virus. Cell. Immunol. 13, 22. MENDES, N.F., ZENHA, M.J., MUSATTI, C.C. & NASPITZ, C. (1974) Lymphocyte membrane receptors in cultures treated with mitogens. Cell. Immunol. 12, 331. PHILIPS, B. & RoiTT, I.M. (1973) Evidence for transformation of human B lymphocytes by PHA. Nature: New Biology, 241, 254. REINHOLD, R.B. & FINE, J. (1971) A technique for quantitative measurement of endotoxin in human plasma. Proc. Soc. exp. Biol. (N.Y.), 137, 334. ROITT, I.M., GREAVES, M.F., TORRIGIANI, G., BROSTOFF, J. & PLAYFAIR, J.H.L. (1969) The cellular basis of immunological responses. A synthesis of some current views. Lancet, ii, 367. SCHELLEKENS, P.T.A. & EYSVOOGEL, V.P. (1968) Lymphocyte transformation in vitro. I. Tissue culture conditions and quantitative measurements. Clin. exp. Immunol. 3, 571. SHEVACH, E.M., HERBERMAN, R., FRANK, M.M. & GREEN, J. (1972) Receptors for complement and immunoglobulin on human leukemic cells and human lymphoblastoid cell lines. J. clin. Invest. 51, 1933. VISCHER, T.L. (1972) Immunoglobulin-like surface molecules and theta antigen during the specific and non-specific stimulation of mouse spleen cells in vitro. Clin. exp. Immunol. 11, 523. WYBRAN, J., CHANTLER, S. & FUDENBERG, H.H. (1973) Isolation of normal T cells in chronic lymphatic leukaemia. Lancet, i, 126. YOSHIDA, T., SONOZAKI, H. & COHEN, S. (1973) The production of migration inhibition factor by B and T cells of the guinea pig. J. exp. Med. 138, 784.
BIANCO, C., PATRICK, R. & NUSSENZWEIG, V. (1970) A population of lymphocytes bearing a membrane receptor for antigen-antibody-complement complexes. J. exp. Med. 132, 702. ELFENBEIN, G.J., HARRISON, M.R. & GREEN, J. (1973) Demonstration of proliferation by bone marrow derived lymphocytes of guinea pigs, mice and rabbits in response to mitogen stimulation in vitro. J. Immunol. 110, 1334. HELLSTROM, U., ZEROMSKI, J. & PERLMANN, P. (1971) Immunoglobulin light chain determinants on unstimulated and stimulated human blood lymphocytes, assayed by indirect immunofluorescence. Immunology, 20, 1099. HINZ, C.F. & CHICKOSKY, J.F. (1972) Factors influencing the stimulation of human lymphocytes by antigen during culture in vitro. Int. Arch. Allergy, 42, 836. JANOSSY, G., GREAVES, M.F., DOENHOFF, M.J. & SNAJDR, J. (1973) Lymphocyte activation. V. Quantitation of the proliferative responses to mitogens using defined T and B cell populations. Clin. exp. Immunol. 14, 581. JONDAL, M., HOLM, G. & WIGZELL, H. (1972) Surface markers on human T and B lymphocytes. 1. A large population of lymphocytes forming non immune rosettes with sheep red blood cells. J. exp. Med. 136, 207. KNOKE, J., KANAIDE, A. & POWELL, A.E. (1974) Modulation of lymphocytic responses by factors in human plasma. I. The effects on normal, stimulated lymphocytes of various normal plasmas in microplate culture. Int. Arch. Allergy, 46, 584. LAY, W.H., MENDES, N.F., BIANCO, C. & NUSSENZWEIG, V. (1971) Binding of sheep red blood cells to a large population of human lymphocytes. Nature (Lond.), 230, 531.
