Immunology 1976 30 361
Human thymus cells EXCELLENT RESPONDERS BUT POOR STIMULATORS IN 'ONE-WAY' MIXED LYMPHOCYTE REACTION
TIN HAN,* J. MINOWADA,* S. SUBRAMANIANt & L. F. SINKS* * Departments of Medicine B, Immunology and Immunochemistry, Research and Pediatrics Roswell Park Memorial Institute, New York State Department of Health, and t Department of Cardiovascular Surgery, Children's Hospital, Buffalo, New York, U.S.A.
Received 13 August 1975; acceptedfor publication 29 August 1975
Summary. Human allogeneic 'one-way' mixed lymphocyte reactions between thymus cells and thymus cells were entirely absent. Of twenty-one mixed lymphocyte reactions between peripheral blood lymphocytes as responding cells and thymus cells as stimulating cells, only eleven had a weak but significant reaction. In contrast, a highly significant response was observed in each of eighteen mixed lymphocyte reactions between thymus cells as responding cells and peripheral blood lymphocytes as stimulating cells and in each of eleven mixed lymphocyte reactions between peripheral blood lymphocytes and peripheral blood lymphocytes. These findings indicate that the thymus cells (T lymphocytes) possess excellent proliferative capacity, with little or no stimulating capacity, while peripheral blood lymphocytes (T and B lymphocytes), on the other hand, are good responders, as well as good stimulators, in the mixed lymphocyte reaction.
independent (B) cells in the animal system as well as in the human system. Human T lymphocytes can be identified by their ability to form rosettes with unsensitized sheep red blood cells (Jondal, Holm and Wigzell, 1972; Minowada, Ohnuma and Moore, 1972), and by the lack of receptor for immunoglobulin or complement. Human B lymphocytes, on the other hand, can be identified by their ability to form rosettes with sensitized sheep red blood cells (antigen-antibody-complement complex) (Bianco, Patrick and Nussenzweig, 1970; Jondal et al., 1972) and by the presence of receptors for aggregated immunoglobulin (Dickler and Kunkel, 1972). When allogeneic lymphocytes from two genetically different donors are cultured together in the mixed lymphocyte reaction, some of the responding cells begin to transform into blasts by stimulating cells (inactivated cells). Mixed lymphocyte reaction generally represents a test for in vitro cell-mediated immunity and it is thought to be an in vitro correlate of homograft rejection in vivo, specifically. It is generally believed that the responding cells are T lymphocytes (Plate and McKenzie, 1973; Anderson, Nordling and Hiyry, 1973; Johnston and Wilson, 1970). There have been conflicting studies with regard to which cell type (T or B lymphocyte) acts as stimulator in the mixed lymphocyte reaction. It
INTRODUCTION It is now well recognized that the lymphocytes can be separated into thymus-dependent (T) and thymusCorrespondence: Dr Tin Han, Roswell Park Memorial Institute, Department of Health, 666 Elm Street, Buffalo, New York 14263, U.S.A.
361
Tin Han et al.
362
has been reported that the T and B cells possess equal stimulatory capacities in mouse mixed lymphocyte reaction (von Boehmer, 1 974a; Cheers and Sprent, 1973). It has also been reported that the stimulatory capacity in mouse mixed lymphocyte reaction is almost exclusively attributed to B lymphocytes (Plate and McKenzie, 1973; Harrison, 1973). Lohrmann, Novikovs and Graw (1974) recently demonstrated that the proliferative response of peripheral blood lymphocytes in human mixed lymphocyte reaction is largely due to the stimulation by B lymphocytes. We have completed a study of human allogeneic 'one-way' mixed lymphocyte reactions between thymus cells and peripheral blood lymphocytes and found that the thymus cells (T lymphocytes) possess excellent proliferative capacity with little or no stimulating capacity and the peripheral blood lymphocytes (T and B lymphocytes), on the other hand, are good responders as well as good stimulators. MATERIALS AND METHODS
Preparation of thymus cells Portions of thymus were obtained from eleven nonmalignant patients at the time of open-heart surgery and whole thymuses were obtained from one patient with malignant thymoma and from one patient with myasthenia gravis. They ranged in age from 6 months to 24 yr. There were five males and eight females. Suspensions of thymus cells were prepared by mincing the thymus tissue in RPMI 1640 culture medium containing 10 per cent pooled human plasma and antibiotics (100 u of penicillin and 50 pug of streptomycin per millilitre). Preparation of peripheral blood lymphocytes Peripheral blood lymphocytes were prepared from heparinized venous blood of healthy donors by centrifugation over a Ficoll-Hypaque gradient (Boyum, 1968). This preparation usually contained more than 90 per cent lymphocytes, the remaining being monocytes and granulocytes.
T-cell assay For E rosettes as a T-cell marker, 01 ml of the lymphocyte suspension at a concentration of 5 x 106 per ml was mixed with 01 ml of 1 per cent (v/v) sheep erythrocyte suspension in a small test tube
(7 x 50 mm). Mixed-cell suspensions were then centrifuged at 200 g for 3 min at room temperature and were left undisturbed at 40 for 3 h, or overnight. A few drops of 0-1 per cent Trypan Blue Solution in phosphate-buffered saline (PBS) were then added to the tube and the cell pellet was gently resuspended, following which a drop of the cell resuspension was examined under a microscope at x 500 magnification. Each sample was tested in duplicate, and at least 200 cells were observed to determine the percentage of E rosettes. A positive E rosette was defined as a lymphocyte to which three or more sheep erythrocytes were attached.
B-cell assay For EAC rosettes as a B-cell marker, bovine erythrocytes were used as indicator erythrocytes since unsensitized bovine erythrocytes do not bind either T cells or B cells. Bovine erythrocytes were first sensitized with appropriate dilutions of rabbit antibovine erythrocyte serum (an IgM class antibody) at 370 for 30 min, and the erythrocytes were washed three times with PBS. One millilitre of a 2-5 per cent (v/v) suspension of the sensitized erythrocytes was incubated with 1 0 ml of 1/10 dilution of fresh mouse serum (DBA/2 Ha strain) as a complement source at 370 for 30 min. The cells were then washed three times with PBS and were resuspended at 1 per cent (v/v) suspension in PBS for EAC rosetting. The procedure for EAC resetting was generally the same as for E resetting; however, the mixture of test lymphocytes and EAC indicator erythrocytes was left to settle without centrifugation, at room temperature. After 1-15 h, the EAC rosettes were determined. 'One-way' mixed lymphocyte reaction Both thymus cells and peripheral blood lymphocytes were suspended in RPMI 1640 culture medium containing 10 per cent pooled human plasma of donors with blood type AB Rh + and antibiotics at a concentration of 106/ml. Stimulating cells were inactivated by mitomycin C treatment. Responding cells were untreated. To each tube, 1 ml of responding cell suspension and 1 ml of stimulating cell suspension were added. Single cell cultures were also set up. In most of the experiments, the 1:1 ratio of responding cells and stimulating cells was used. In a few experiments, various ratios of responding cells and stimulating cells were used with constant concentrations of responding cells and varying concentrations
Human thymus cells
of stimulating cells. The experiments were carried out in duplicate. Thymus cells and peripheral blood lymphocytes were utilized as either responding cells or stimulating cells; thus, there were four different combinations of responding and stimulating cells in 'one-way' mixed lymphocyte reactions. Culture tubes with loose-fitting caps were incubated at 370 in a humidified atmosphere of 5 per cent CO2 in air for 6 days. One microcurie of [3H]thymidine (specific activity, 2-0 Ci/mmole) was added to each tube 24 h prior to harvesting the cells. In one experiment, the kinetics of mixed lymphocyte reaction (peripheral blood lymphocytes as responding cells and thymus cells as stimulating cells) were determined by harvesting the cells on days 4, 6 and 8 of incubation. Incorporation of [3H]thymidine was measured according to the method previously described (Pauly, Sokal and Han, 1973). 'One-way' mixed lymphocyte reaction was expressed as the blastogenic index (BI), which is counts per minute (c.p.m.) in mixed-cell culture minus c.p.m. in mitomycin C-treated stimulating cell culture, divided by c.p.m. in untreated responding cell cultures. The BI of two or more was considered to be significant for mixed lymphocyte reaction. Statistical analyses were performed by Student's ttest.
363
Table 1. The clinical data of thymus cell donors and the results of E and EAC rosettes of thymus cells
Thymus cell donor
E rosettes
(per cent) (1) (2) (3) (4) (5) (6) (7)
(8) (9) (10) (11) (12) (13)
R.B., normal M.K., normal R.C., normal S.P., normal S.O., normal M.M., normal W.K., normal C.G., normal C.D., normal T.F., normal S.J., normal R.K., thymoma K.A., myasthenia gravis
EAC rosettes (per cent)
95 0 98-0 93-4 99-2 99-2 93-9 98-2 97 1 98-1 94-2 97-6 96-7
15 2-8
Human thymus cells EXCELLENT RESPONDERS BUT POOR STIMULATORS IN 'ONE-WAY' MIXED LYMPHOCYTE REACTION
TIN HAN,* J. MINOWADA,* S. SUBRAMANIANt & L. F. SINKS* * Departments of Medicine B, Immunology and Immunochemistry, Research and Pediatrics Roswell Park Memorial Institute, New York State Department of Health, and t Department of Cardiovascular Surgery, Children's Hospital, Buffalo, New York, U.S.A.
Received 13 August 1975; acceptedfor publication 29 August 1975
Summary. Human allogeneic 'one-way' mixed lymphocyte reactions between thymus cells and thymus cells were entirely absent. Of twenty-one mixed lymphocyte reactions between peripheral blood lymphocytes as responding cells and thymus cells as stimulating cells, only eleven had a weak but significant reaction. In contrast, a highly significant response was observed in each of eighteen mixed lymphocyte reactions between thymus cells as responding cells and peripheral blood lymphocytes as stimulating cells and in each of eleven mixed lymphocyte reactions between peripheral blood lymphocytes and peripheral blood lymphocytes. These findings indicate that the thymus cells (T lymphocytes) possess excellent proliferative capacity, with little or no stimulating capacity, while peripheral blood lymphocytes (T and B lymphocytes), on the other hand, are good responders, as well as good stimulators, in the mixed lymphocyte reaction.
independent (B) cells in the animal system as well as in the human system. Human T lymphocytes can be identified by their ability to form rosettes with unsensitized sheep red blood cells (Jondal, Holm and Wigzell, 1972; Minowada, Ohnuma and Moore, 1972), and by the lack of receptor for immunoglobulin or complement. Human B lymphocytes, on the other hand, can be identified by their ability to form rosettes with sensitized sheep red blood cells (antigen-antibody-complement complex) (Bianco, Patrick and Nussenzweig, 1970; Jondal et al., 1972) and by the presence of receptors for aggregated immunoglobulin (Dickler and Kunkel, 1972). When allogeneic lymphocytes from two genetically different donors are cultured together in the mixed lymphocyte reaction, some of the responding cells begin to transform into blasts by stimulating cells (inactivated cells). Mixed lymphocyte reaction generally represents a test for in vitro cell-mediated immunity and it is thought to be an in vitro correlate of homograft rejection in vivo, specifically. It is generally believed that the responding cells are T lymphocytes (Plate and McKenzie, 1973; Anderson, Nordling and Hiyry, 1973; Johnston and Wilson, 1970). There have been conflicting studies with regard to which cell type (T or B lymphocyte) acts as stimulator in the mixed lymphocyte reaction. It
INTRODUCTION It is now well recognized that the lymphocytes can be separated into thymus-dependent (T) and thymusCorrespondence: Dr Tin Han, Roswell Park Memorial Institute, Department of Health, 666 Elm Street, Buffalo, New York 14263, U.S.A.
361
Tin Han et al.
362
has been reported that the T and B cells possess equal stimulatory capacities in mouse mixed lymphocyte reaction (von Boehmer, 1 974a; Cheers and Sprent, 1973). It has also been reported that the stimulatory capacity in mouse mixed lymphocyte reaction is almost exclusively attributed to B lymphocytes (Plate and McKenzie, 1973; Harrison, 1973). Lohrmann, Novikovs and Graw (1974) recently demonstrated that the proliferative response of peripheral blood lymphocytes in human mixed lymphocyte reaction is largely due to the stimulation by B lymphocytes. We have completed a study of human allogeneic 'one-way' mixed lymphocyte reactions between thymus cells and peripheral blood lymphocytes and found that the thymus cells (T lymphocytes) possess excellent proliferative capacity with little or no stimulating capacity and the peripheral blood lymphocytes (T and B lymphocytes), on the other hand, are good responders as well as good stimulators. MATERIALS AND METHODS
Preparation of thymus cells Portions of thymus were obtained from eleven nonmalignant patients at the time of open-heart surgery and whole thymuses were obtained from one patient with malignant thymoma and from one patient with myasthenia gravis. They ranged in age from 6 months to 24 yr. There were five males and eight females. Suspensions of thymus cells were prepared by mincing the thymus tissue in RPMI 1640 culture medium containing 10 per cent pooled human plasma and antibiotics (100 u of penicillin and 50 pug of streptomycin per millilitre). Preparation of peripheral blood lymphocytes Peripheral blood lymphocytes were prepared from heparinized venous blood of healthy donors by centrifugation over a Ficoll-Hypaque gradient (Boyum, 1968). This preparation usually contained more than 90 per cent lymphocytes, the remaining being monocytes and granulocytes.
T-cell assay For E rosettes as a T-cell marker, 01 ml of the lymphocyte suspension at a concentration of 5 x 106 per ml was mixed with 01 ml of 1 per cent (v/v) sheep erythrocyte suspension in a small test tube
(7 x 50 mm). Mixed-cell suspensions were then centrifuged at 200 g for 3 min at room temperature and were left undisturbed at 40 for 3 h, or overnight. A few drops of 0-1 per cent Trypan Blue Solution in phosphate-buffered saline (PBS) were then added to the tube and the cell pellet was gently resuspended, following which a drop of the cell resuspension was examined under a microscope at x 500 magnification. Each sample was tested in duplicate, and at least 200 cells were observed to determine the percentage of E rosettes. A positive E rosette was defined as a lymphocyte to which three or more sheep erythrocytes were attached.
B-cell assay For EAC rosettes as a B-cell marker, bovine erythrocytes were used as indicator erythrocytes since unsensitized bovine erythrocytes do not bind either T cells or B cells. Bovine erythrocytes were first sensitized with appropriate dilutions of rabbit antibovine erythrocyte serum (an IgM class antibody) at 370 for 30 min, and the erythrocytes were washed three times with PBS. One millilitre of a 2-5 per cent (v/v) suspension of the sensitized erythrocytes was incubated with 1 0 ml of 1/10 dilution of fresh mouse serum (DBA/2 Ha strain) as a complement source at 370 for 30 min. The cells were then washed three times with PBS and were resuspended at 1 per cent (v/v) suspension in PBS for EAC rosetting. The procedure for EAC resetting was generally the same as for E resetting; however, the mixture of test lymphocytes and EAC indicator erythrocytes was left to settle without centrifugation, at room temperature. After 1-15 h, the EAC rosettes were determined. 'One-way' mixed lymphocyte reaction Both thymus cells and peripheral blood lymphocytes were suspended in RPMI 1640 culture medium containing 10 per cent pooled human plasma of donors with blood type AB Rh + and antibiotics at a concentration of 106/ml. Stimulating cells were inactivated by mitomycin C treatment. Responding cells were untreated. To each tube, 1 ml of responding cell suspension and 1 ml of stimulating cell suspension were added. Single cell cultures were also set up. In most of the experiments, the 1:1 ratio of responding cells and stimulating cells was used. In a few experiments, various ratios of responding cells and stimulating cells were used with constant concentrations of responding cells and varying concentrations
Human thymus cells
of stimulating cells. The experiments were carried out in duplicate. Thymus cells and peripheral blood lymphocytes were utilized as either responding cells or stimulating cells; thus, there were four different combinations of responding and stimulating cells in 'one-way' mixed lymphocyte reactions. Culture tubes with loose-fitting caps were incubated at 370 in a humidified atmosphere of 5 per cent CO2 in air for 6 days. One microcurie of [3H]thymidine (specific activity, 2-0 Ci/mmole) was added to each tube 24 h prior to harvesting the cells. In one experiment, the kinetics of mixed lymphocyte reaction (peripheral blood lymphocytes as responding cells and thymus cells as stimulating cells) were determined by harvesting the cells on days 4, 6 and 8 of incubation. Incorporation of [3H]thymidine was measured according to the method previously described (Pauly, Sokal and Han, 1973). 'One-way' mixed lymphocyte reaction was expressed as the blastogenic index (BI), which is counts per minute (c.p.m.) in mixed-cell culture minus c.p.m. in mitomycin C-treated stimulating cell culture, divided by c.p.m. in untreated responding cell cultures. The BI of two or more was considered to be significant for mixed lymphocyte reaction. Statistical analyses were performed by Student's ttest.
363
Table 1. The clinical data of thymus cell donors and the results of E and EAC rosettes of thymus cells
Thymus cell donor
E rosettes
(per cent) (1) (2) (3) (4) (5) (6) (7)
(8) (9) (10) (11) (12) (13)
R.B., normal M.K., normal R.C., normal S.P., normal S.O., normal M.M., normal W.K., normal C.G., normal C.D., normal T.F., normal S.J., normal R.K., thymoma K.A., myasthenia gravis
EAC rosettes (per cent)
95 0 98-0 93-4 99-2 99-2 93-9 98-2 97 1 98-1 94-2 97-6 96-7
15 2-8
