Immunology 1976 31 519
Human thymus cells: blastogenic response to mitogens, antigens and allogeneic cells
TIN HAN, JUN MINOWADA, SANBAMURTHY SUBRAMANIAN* & LUCIUS F. SINKS Departments of Medicine B, Immunology and Immunochemistry Research and Pediatrics, Roswell Park Memorial Institute, New York State Department of Health, Buffalo, New York, 14263 and *Department of Cardiovascular Surgery, Children's Hospital, Buffalo, New York, 14222
Received 15 January 1976; accepted for publication 15 March 1976
lymphocytes from nine to ten randomly selected age-matched children responded very well to one or more antigens.
Summary. Over 90 per cent of the thymus cells from each of twenty-six donors were T lymphocytes, identified by E-rosetting and less than 3 per cent of the cells were B lymphocytes identified by EACrosetting. With advancing age, the proportion of T lymphocytes decreased while that of B lymphocytes increased. The degree of [3H]thymidine incorporation of thymus cells was inversely proportional to the age of the thymus-cell donor. The PHA or PWMinduced blastogenic response of thymus cells gradually increased with advancing age when the response was expressed as the stimulation index. However, the actual rate of [3H]thymidine incorporation in all three groups was rather similar when cells were cultured with mitogens. The difference in stimulation index was due to the variation in incorporation rate in cultures without stimulants. The PHA response was approximately four-fold higher than that of PWM response. Thymus cell response to allogeneic lymphocytes, on the other hand, had no correlation with the age of thymus donor. The most surprising result in the present study was that the thymus cells from each of ten donors, aged 1-14 years, were incapable of responding to all four different recall antigens. Peripheral blood
INTRODUCTION Lymphocytes have been identified as either thymusdependent (T) cells or thymus-independent (B) cells in human and other mammalian species. It has been well documented that the thymus cells (T lymphocytes) from small laboratory animals, such as mice or rats, are stimulated by phytohaemagglutinin (PHA), pokeweed mitogen (PWM) and allogeneic lymphoid cells (Ricke & Schwarz, 1964; Han & Bennett, 1972; Mangi & Mardiney, 1970; Blomgren & Svedmyr, 1971). Studies with regard to the ability of human thymus cells to be transformed by the agents are conflicting. Some investigators have observed that human thymus cells do not respond in this way, others showing the reverse (Claman, 1966; McIntyre & Segel, 1966; Schwarz, 1967; Han, Minowada, Subramanian & Sinks, 1976). PHA is considered to stimulate T lymphocytes while PWM is affective for both T and B lymphocytes (Asherson, 1972). The responding cells in allogeneic 'one-way' mixed lymphocyte reaction, in animals and in man are T lymphocytes (Anderson, Nordling
Correspondence: Dr Tin Han, Medicine B, Roswell Park Memorial Institute, 666 Elm Street, Buffalo, New York 14263.
519
520
Tin Han et al.
& Hayry, 1973; Han et al., 1976; Johnston & Wilson, 1970; Plate & McKenzie, 1973). In vitro lymphocyte responses to antigens are thought to be in vitro correlates of delayed hypersensitivity skin test responses. It is likely that the peripheral blood lymphocytes from sensitized individuals, that are transformed into blasts in the presence of specific antigen, are T cells (Han, unpublished observation). There have been no previously reported studies of stimulation of thymus lymphocytes by specific antigens. The present study of human thymus cell responses to mitogens, antigens and allogeneic cells shows that these cells are stimulated by mitogens and allogeneic cells, but do not respond to each and every antigen. MATERIALS AND METHODS Thymus donors Thymus tissue was obtained from twenty-six patients with cardiac abnormality, ranging in age from 1 month to 20 years. There were fifteen males and eleven females. Approximately one-fourth to onethird of thymus gland was obtained from each patient at the time of open-heart surgery. Preparation of thymus cells Suspensions of thymus cells were prepared by mincing the gland in PRMI 1640 culture medium containing 10 per cent pooled human plasma and antibiotics (100 u of penicillin and 50 ,ug of streptomycin per ml). E-rosette 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 0-1 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 magnification x 500. 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 moore sheep erythrocytes were attached.
EAC-rosette 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 rosetting was generally the same as for E rosetting; however, the mixture of test lymphocytes and EAC-indicator erythrocytes was left to settle without centrifugation, at room temperature. After 1-1 5 h, the EAC rosettes were determined.
Lymphocyte stimulation Thymus cells were suspended in culture medium at a concentration of 106 per ml. Two ml of thymus cell suspension were transferred to 16 x 125 mm Falcon disposable plastic tubes. The following mitogens or antigens were added to each tube: 10 jug purified PHA (Burroughs Wellcome Company, Tuckahoe, New York); 0 1 ml of reconstituted PWM (Grand Island Biological Company, Grand Island, New York); 7 5 ug purified protein derivative (PPD, Parke Davis Company, Detroit, Michigan); 0 1 ml of 1:100 diluted mumps vaccine (Eli Lilly and Company, Indianapolis, Indiana); 01 ml of 1:250 diluted monilia extract (Hollister-Stier Laboratories, Spokane, Washington); and 10 u streptokinase and 2-5 u streptodornase (Varidase, Lederle Laboratories Division, Pearl River, New York). The control cultures received no mitogen or antigen. For 'one-way' mixed lymphocyte reaction, 1 ml of thymus-cell suspension (106 per ml, responding cells) and 1 ml of peripheral blood lymphocytes (106 per ml, stimulating cells inactivated by mitomycin C treatment) were added to each culture tube. Single cell cultures were also set up. The experiments were carried out in duplicate. Culture tubes with loose-fitting caps were incubated at 370 in a humidified atmosphere of 5 per cent CO2 in air for 3 days (mitogen response) or 6 days (antigen response and mixed lymphocyte reaction)
Human thymus cells in most of the experiments. In a few experiments, control cultures or mitogen-stimulated cultures were incubated for 1, 2, 3 and 6 days, or for 3 and 6 days, respectively. [3H]thymidine (1 #Ci, specific activity, 2 0 Ci/mmole) was added to each tube 24 h
Table 2. [3H]thymidine incorporation of thymus cells Day of No. of culture cultures 1 2 3 6
prior to harvesting the cells. Incorporation of [3H]thymidine into DNA was measured according to the
method previously described (Pauly, Sokal & Han, 1973). The lymphocyte response to mitogens and antigens was expressed as stimulation index (SI), which is a ratio of counts per minute (c.p.m.) in stinrulated culture and ct/min in unstimulated culture. 'One-way' mixed lymphocyte reaction was also expressed as the SI, which is ct/min in mixed-cell culture minus ct/min in mitomycin-C treated stimulating cell culture, divided by ct/min in untreated responding cell culture. An SI of two or more was considered to be significant for antigen response or 'one-way' mixed lymphocyte reaction.
521
[3H]-thymidine incorporation (c.p.m.) mean+ s.e.
58,056+ 13,938 42,897+ 12,210 10,812+ 2704 405+91
10 11 26 20
Table 3. Correlation of the [3H]thymidine incorporation day 3 of control culture with age of the thymus donor Age group
on
No. of cultures
[3H]Thymidine incorporation (c.p.m.)
17 4 5 26
13,850+ 3922
I II III
Total
mean+ s.e. 6654+ 1774 3812+ 1549 10,812+ 2704
RESULTS 58,000. The [3H]thymidine incorporation gradually decreased with time, and it was insignificant on day 6 of culture. Correlation of the [3H] thymidine incorporation on day 3 of control culture with age of the thymus donor is shown in Table 3. The highest incorporation of [3H]thymidine in thymus cells was observed from age-group I, the lowest incorporation in thymus cells from age-group III, and the intermediate incorporation in thymus cells from age-group II. Thymus-cell responses to PHA and PWM are presented in Table 4. The PHA response was nearly four-fold higher than that of the PWM response. There was no difference of PHA or PWM responses between male and female donors. When the response was expressed as the SI, it was found that was over
Viability of the thymus cells at the completion of isolation ranged from 95-99 per cent. The results of E and EAC rosettes of thymus cells obtained from twenty-six donors are shown according to age group in Table 1. Over 90 per cent of the thymus cells from each donor were proven to be T lymphocytes identified as E rosetting; less than 3 per cent of the cells, being EAC rosetting (B lymphocytes). There was a fairly good correlation between the number of T or B cells and the age of thymus donor. The proportion of T lymphocytes decreased, while that of B lymphocytes increased with advancing age. Table 2 shows the [3H]thymidine incorporation of thymus cells (control cultures) at various days of incubation. On day 1 of culture, the mean c.p.m.
Table 1. The results of E and EAC rosettes of thymus cells according to the age group
Group
Age
(ranges)* 1 nm-5 y 6 y-10 y III11 y-20 y 1 m-20 y Total 1 11
No. of pts
97-5 95-5 94-5 96-6
17 4 5 26 *m
Percentage of E rosettes mean (ranges)
=
(934-99 2) (94-7-97-1) (94 2-96 7) (93-4-99 2)
month, y = year.
Percentage of EAC rosettes mean (ranges)
0-5 0-8 2-0 0-9
(0-1 3) (0 1-1-8) (0 1-2 9) (0-2 9)
522
Tin Han et al. Table 4. Thymus-cell responses to phytohemagglutinin (PHA) and pokeweed mitogen (PWM)
[3H]thymidine Age group I
11
No. of cultures
Composition of cultures
incorporation (c.p.m.) mean+ s.e.
17
Thymus cells alone Thymus cells+ PHA Thymus cells+PWM Thymus cells alone Thymus cells+ PHA Thymus cells+PWM Thymus cells alone Thymus cells+ PHA Thymus cells+ PWM Thymus cells alone Thymus cells+ PHA Thymus cells+ PWM
13,850+ 3922 85,390+ 20,698 24,339+ 6270 6654+ 1774 103,891 + 57,199 25,502+ 13,298 3812+ 1549 74,400+ 17,620 16,321+ 3160 10,812+ 2704 86,123+ 15,900 22,922+ 4503
4
III
5
Total
26
both PHA and PWM response improved with advancing age. However, it should be emphasized that the actual rate of incorporation of [3H]thymidine in all three groups was rather similar when cells were cultured with stimulants. The difference in SI was due to the variation in incorporation rate in cultures without stimulants. Table 5 shows the comparison of PHA and PWM responses of thymus cells, according to two different culture periods (3 and 6 days). Again, the [3H]thymidine incorporation in control cultures on day 3 of incubation was rather high, while that on day 6 of incubation was very low. The [3H]thymidine incorporations of PHA-stimulated cultures, incubated for 3 and 6 days, were about the same. However, the PWM-stimulated cultures on day 6 of incubation was higher than those incubated for only 3 days. When the response was expressed as the SI, it was found that both PHA and PWM responses were significantly
Stimulation index mean+ s.e. 9 3+ 2-4 2-3+0-6 13-0+ 6-0 3-2+ 1-3
62-2+ 30 4 20-9+ 14-4 20-1 + 7 0 6-2+ 3 0
higher in cultures incubated for 6 days as compared to those incubated for 3 days.
'One-way' mixed lymphocyte reactions between thymus cells (responding cells) and peripheral blood lymphocytes (stimulating cells) are shown in Table 6. Thymus cells from each donor were found to be capable of responding to allogeneic cells. When the mixed lymphocyte reactions were analysed, according to the age of thymus donors, no correlation was noted. Table 7 shows the results of thymus cell responses to antigens. It is of great interest that the thymus cells from each of ten donors, aged 1-14 years were incapable of responding to all four different antigens. It is highly unlikely that the thymus donors were not sensitized against one or more of these microbial antigens, although such possibility cannot be entirely ruled out, since we did not study the antigeninduced blastogenic response of peripheral blood
Table 5. Comparison of PHA and PWM responses of thymus cells according to two different culture periods
Day of culture
No. of cultures
Composition of
3
5
Thymus cells alone Thymus cells+PHA Thymus cells+PWM Thymus cells alone Thymus cells+ PHA Thymus cells+PWM
6
5
cultures
[3H]Thymidine incorporation (c.p im.) mean+ s.e.
Stimulation index mean+ s.e.
6688+ 1532
103,834+ 42,830 21,892+ 10,418 617+
16 4+ 4-8 3-3+ 1.0
159
106,850± 19,244 32,413+ 17,583
208-0+ 59-6 41-8+ 10-8
Human thymus cells
523
Table 6. Thymus cell responses to allogeneic peripheral blood lymphocytes ('one-way' mixed lymphocyte reactions)
[3H]Thymidine Age group
12
II
6
III
4
Total
Composition of culture
No. of cultures
22
incorporation (c.p.m.) mean+ s.e.
Thymus cells Peripheral blood lymphocytes* Thymus cells and peripheral blood lymphocytes* Thymus cells Peripheral blood lymphocytes* Thymus cells and peripheral blood lymphocytes* Thymus cells Peripheral blood lymphocytes* Thymus cells and peripheral blood lymphocytes* Thymus cells Peripheral blood lymphocytes* Thymus cells and peripheral blood lymphocytes* *
714+ 152 285+ 30 19,093+ 3662 404+ 110 204+ 24 27,722+ 9934 731+ 426 332+ 124 15,350+ 8315 641+ 125 271 + 30 20,766+ 3773
Treated with nitomycin C.
Table 7. Lack of thymus cell responses to recall antigens*
[3H]thymidine Composition of culture
incorporation (c.p.m.)
Stimulation Index mean (ranges)
mean (ranges)
Thymus cells alone Thymus cells+ PPD Thymus cells+mumps Thymus cells+ monilia Thymus cells+ Varidase *
435 (335-659) 360 (200-465) 365 (208-623) 414 (216-549) 300 (195-431)
0-8 (0-4-1-2) 0-8 (0 5-1l1) 1 0 (0-5-1-6) 0 7 (0 4-09)
Thymus cells from ten donors, aged 1-14 years were used.
Table 8. Peripheral blood lymphocyte (PBL) responses to recall antigens*
Composition of culture PBL alone PBL+PPD PBL+ mumps PBL+ monilia PBL+ Varidase
[3H]Thymidine incorporation (c.p.m.) mean (ranges)
Stimulation index mean (ranges)
748 (112-2508) 2529 (481-11,929) 3532 (399-10,557) 1823 (357-3956) 3635 (352-11,524)
4 0 (0 8-9 7) 6-7 (1P8-19-0) 3-6 (1-3-13-2) 5-3 (1 1-10-3)
* Peripheral blood lymphocytes from ten donors, aged 1-14 years were used.
Stimulation index mean+ s.e. -
30-0+ 5 8 54-7+ 17-8 30 5+ 15 0
37 0+ 6-7
Tin Han et al.
524
lymphocytes from these donors. We have studied the in vitro antigen response of peripheral blood lymphocytes from ten age-matched children (randomly selected) and the results are presented in Table 8. Peripheral blood lymphocytes from nine of ten donors responded to one or more antigens, and such lymphocytes from only one donor failed to respond to all antigens, indicating that children aged 1-14 years are usually sensitized against one or more of the microbial antigens.
DISCUSSION There have been conflicting reports with regard to the ability of human thymus cells to proliferate in the presence of mitogens or allogeneic cells (Claman, 1966; Han et al., 1976; McIntyre & Segel, 1966). The present study shows that cells of human thymus can be transformed by PHA, PWM and allogeneic peripheral blood lymphocytes. It is of interest that a fairly good correlation existed between the proportion of T or B cells and the age of thymus donor. The proportion of T lymphocytes decreased and that of B lymphocytes increased with advancing age. These findings suggest that the incidence of B-cell contamination in thymus was more frequent in older donors. It has recently been reported that the proportion of E rosettes of human thymus cells decreased gradually with advancing age (Galli & Schlesinger, 1975). A significant [3H]thymidine incorporation of thymus cells, at the beginning of culture was observed in the present study. A good correlation between the age of thymus donor and the [3H]thymidine incorporation of thymus cells was also observed, indicating that the proportion of cells active in this respect decreased with advancing age. It has been reported that the thymus cells from individuals over 36 years of age responded well to PHA or Con A but that lymphocytes from the thymus of children up to 14 years of age did not respond to these mitogens (Galli & Schlesinger, 1975). In contrast, the present data demonstrate that the thymus cells from individuals of different ages responded to both PHA and PWM. The lack of mitogen response of thymus cells from younger donors (Galli & Schlesinger, 1975) could have been due to measuring this on day 3 of culture. In contrast to PHA- or PWM-induced blastogenesis of thymus cells, their responses to allogeneic peri-
pheral blood lymphocytes showed no correlation with the age of thymus donor. Of interest is the lack of response by cells of the thymus to the four antigens used here. This has not been previously reported. It is highly unlikely that the thymus donors in the present study were not sensitized to any of these antigens. This is supported by the observation that peripheral blood lymphocytes from nine of ten randomly selected children were capable of responding in vitro to one or more of these antigens. The possibility that the donors of thymic cells are anergic to all antigens, however, cannot be ruled out entirely, since we did not study the antigen response of peripheral blood lymphocytes from these donors. The thymus cells may be incapable of responding, but the peripheral blood T lymphocytes, on the other hand, are capable of responding to a specific antigen, to which the donor of both cell-types is sensitized. ACKNOWLEDGMENTS The authors wish to thank Mrs Ellen Cray and Mrs Barbara Dadey for their technical assistance. This study was supported in part by United States Public Health Service Grants CA-12318, CA-14323 and CA-14413. REFERENCES ANDERSON L.C., NORDLING S. & HAYRY P. (1973) Proliferation of B and T cells in mixed lymphocyte cultures. J. exp. Med. 138, 324. ASHERSON G.L. (1972) The development of lymphocyte function tests. Brit. J. Hosp. Med. 8, 665. BLOMGREN H. & SVEDMYR E. (1971) In vitro stimulation of mouse thymus cells by PHA and allogeneic cells. Cell. Immunol. 2, 285. CLAMAN H.N. (1966) Human thymus cell cultures-Evidence for two functional populations. Proc. Soc. exp. biol. (N. Y.) 121, 236. GALLI U. & SCHLESINGER M. (1975) Subpopulations of human thymus cells differing in their capacity to form stable E-rosettes and in their immunologic reactivity. J. Immunol., 115, 827. HAN T. & BENNETT M. (1972) Response of mouse thymocytes to mitogens and allogeneic cells. Seventh Leukocyte Culture Conference (Abstract no. 85). Pointe-Au-Pic, Quebec, Canada. HAN T., MINOWADA J., SUBRAMANIAN S. & SINKS L.F. (1976) Human thymus cells: Excellent responders but poor stimulators in 'one-way' mixed lymphocyte reaction. Immunology, 30, 361.
Human thymus cells JOHNSTON J.M. & WILSON D.B. (1970) Origin of immunoreactive lymphocytes in rats. Cell. Immunol. 1, 430. MANGI R. & MARDINEY M.R., Jr. (1970) Transformation of mouse lymphocytes to allogeneic lymphocytes and phytohemagglutinin. J. Immunol. 105, 90. McINTYRE R.O. & SEGEL W.D. (1966) PHA and thymic lymphocytes. Lancet, i, 1265. PAULY J.L., SOKAL J.E. & HAN T. (1973) Whole blood culture technique for functional studies of lymphocyte reactivity to mitogens, antigens and homologous lymphocytes. J. Lab. clin. Med. 82, 500.
B
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PLATE, J.M.D. and MCKENZIE, I.F.C. (1973) B-cell stimulation of allogeneic T-cell proliferation in mixed lymphocyte cultures. Nature: New Biology, 26, 247. RICKE W.O. & SCHWARZ M.R. (1964) The culture and karyotype of rat lymphocytes stimulated with phytohemagglutinin. Anat. Rec. 150, 383. SCHWARZ, M.R. (1967) Response of thymus and other human lymphoid tissues to PHA, PWM and genetically dissimilar lymphoid cells. Proc. Soc. exp. biol. (N. Y.) 125, 701.
Human thymus cells: blastogenic response to mitogens, antigens and allogeneic cells
TIN HAN, JUN MINOWADA, SANBAMURTHY SUBRAMANIAN* & LUCIUS F. SINKS Departments of Medicine B, Immunology and Immunochemistry Research and Pediatrics, Roswell Park Memorial Institute, New York State Department of Health, Buffalo, New York, 14263 and *Department of Cardiovascular Surgery, Children's Hospital, Buffalo, New York, 14222
Received 15 January 1976; accepted for publication 15 March 1976
lymphocytes from nine to ten randomly selected age-matched children responded very well to one or more antigens.
Summary. Over 90 per cent of the thymus cells from each of twenty-six donors were T lymphocytes, identified by E-rosetting and less than 3 per cent of the cells were B lymphocytes identified by EACrosetting. With advancing age, the proportion of T lymphocytes decreased while that of B lymphocytes increased. The degree of [3H]thymidine incorporation of thymus cells was inversely proportional to the age of the thymus-cell donor. The PHA or PWMinduced blastogenic response of thymus cells gradually increased with advancing age when the response was expressed as the stimulation index. However, the actual rate of [3H]thymidine incorporation in all three groups was rather similar when cells were cultured with mitogens. The difference in stimulation index was due to the variation in incorporation rate in cultures without stimulants. The PHA response was approximately four-fold higher than that of PWM response. Thymus cell response to allogeneic lymphocytes, on the other hand, had no correlation with the age of thymus donor. The most surprising result in the present study was that the thymus cells from each of ten donors, aged 1-14 years, were incapable of responding to all four different recall antigens. Peripheral blood
INTRODUCTION Lymphocytes have been identified as either thymusdependent (T) cells or thymus-independent (B) cells in human and other mammalian species. It has been well documented that the thymus cells (T lymphocytes) from small laboratory animals, such as mice or rats, are stimulated by phytohaemagglutinin (PHA), pokeweed mitogen (PWM) and allogeneic lymphoid cells (Ricke & Schwarz, 1964; Han & Bennett, 1972; Mangi & Mardiney, 1970; Blomgren & Svedmyr, 1971). Studies with regard to the ability of human thymus cells to be transformed by the agents are conflicting. Some investigators have observed that human thymus cells do not respond in this way, others showing the reverse (Claman, 1966; McIntyre & Segel, 1966; Schwarz, 1967; Han, Minowada, Subramanian & Sinks, 1976). PHA is considered to stimulate T lymphocytes while PWM is affective for both T and B lymphocytes (Asherson, 1972). The responding cells in allogeneic 'one-way' mixed lymphocyte reaction, in animals and in man are T lymphocytes (Anderson, Nordling
Correspondence: Dr Tin Han, Medicine B, Roswell Park Memorial Institute, 666 Elm Street, Buffalo, New York 14263.
519
520
Tin Han et al.
& Hayry, 1973; Han et al., 1976; Johnston & Wilson, 1970; Plate & McKenzie, 1973). In vitro lymphocyte responses to antigens are thought to be in vitro correlates of delayed hypersensitivity skin test responses. It is likely that the peripheral blood lymphocytes from sensitized individuals, that are transformed into blasts in the presence of specific antigen, are T cells (Han, unpublished observation). There have been no previously reported studies of stimulation of thymus lymphocytes by specific antigens. The present study of human thymus cell responses to mitogens, antigens and allogeneic cells shows that these cells are stimulated by mitogens and allogeneic cells, but do not respond to each and every antigen. MATERIALS AND METHODS Thymus donors Thymus tissue was obtained from twenty-six patients with cardiac abnormality, ranging in age from 1 month to 20 years. There were fifteen males and eleven females. Approximately one-fourth to onethird of thymus gland was obtained from each patient at the time of open-heart surgery. Preparation of thymus cells Suspensions of thymus cells were prepared by mincing the gland in PRMI 1640 culture medium containing 10 per cent pooled human plasma and antibiotics (100 u of penicillin and 50 ,ug of streptomycin per ml). E-rosette 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 0-1 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 magnification x 500. 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 moore sheep erythrocytes were attached.
EAC-rosette 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 rosetting was generally the same as for E rosetting; however, the mixture of test lymphocytes and EAC-indicator erythrocytes was left to settle without centrifugation, at room temperature. After 1-1 5 h, the EAC rosettes were determined.
Lymphocyte stimulation Thymus cells were suspended in culture medium at a concentration of 106 per ml. Two ml of thymus cell suspension were transferred to 16 x 125 mm Falcon disposable plastic tubes. The following mitogens or antigens were added to each tube: 10 jug purified PHA (Burroughs Wellcome Company, Tuckahoe, New York); 0 1 ml of reconstituted PWM (Grand Island Biological Company, Grand Island, New York); 7 5 ug purified protein derivative (PPD, Parke Davis Company, Detroit, Michigan); 0 1 ml of 1:100 diluted mumps vaccine (Eli Lilly and Company, Indianapolis, Indiana); 01 ml of 1:250 diluted monilia extract (Hollister-Stier Laboratories, Spokane, Washington); and 10 u streptokinase and 2-5 u streptodornase (Varidase, Lederle Laboratories Division, Pearl River, New York). The control cultures received no mitogen or antigen. For 'one-way' mixed lymphocyte reaction, 1 ml of thymus-cell suspension (106 per ml, responding cells) and 1 ml of peripheral blood lymphocytes (106 per ml, stimulating cells inactivated by mitomycin C treatment) were added to each culture tube. Single cell cultures were also set up. The experiments were carried out in duplicate. Culture tubes with loose-fitting caps were incubated at 370 in a humidified atmosphere of 5 per cent CO2 in air for 3 days (mitogen response) or 6 days (antigen response and mixed lymphocyte reaction)
Human thymus cells in most of the experiments. In a few experiments, control cultures or mitogen-stimulated cultures were incubated for 1, 2, 3 and 6 days, or for 3 and 6 days, respectively. [3H]thymidine (1 #Ci, specific activity, 2 0 Ci/mmole) was added to each tube 24 h
Table 2. [3H]thymidine incorporation of thymus cells Day of No. of culture cultures 1 2 3 6
prior to harvesting the cells. Incorporation of [3H]thymidine into DNA was measured according to the
method previously described (Pauly, Sokal & Han, 1973). The lymphocyte response to mitogens and antigens was expressed as stimulation index (SI), which is a ratio of counts per minute (c.p.m.) in stinrulated culture and ct/min in unstimulated culture. 'One-way' mixed lymphocyte reaction was also expressed as the SI, which is ct/min in mixed-cell culture minus ct/min in mitomycin-C treated stimulating cell culture, divided by ct/min in untreated responding cell culture. An SI of two or more was considered to be significant for antigen response or 'one-way' mixed lymphocyte reaction.
521
[3H]-thymidine incorporation (c.p.m.) mean+ s.e.
58,056+ 13,938 42,897+ 12,210 10,812+ 2704 405+91
10 11 26 20
Table 3. Correlation of the [3H]thymidine incorporation day 3 of control culture with age of the thymus donor Age group
on
No. of cultures
[3H]Thymidine incorporation (c.p.m.)
17 4 5 26
13,850+ 3922
I II III
Total
mean+ s.e. 6654+ 1774 3812+ 1549 10,812+ 2704
RESULTS 58,000. The [3H]thymidine incorporation gradually decreased with time, and it was insignificant on day 6 of culture. Correlation of the [3H] thymidine incorporation on day 3 of control culture with age of the thymus donor is shown in Table 3. The highest incorporation of [3H]thymidine in thymus cells was observed from age-group I, the lowest incorporation in thymus cells from age-group III, and the intermediate incorporation in thymus cells from age-group II. Thymus-cell responses to PHA and PWM are presented in Table 4. The PHA response was nearly four-fold higher than that of the PWM response. There was no difference of PHA or PWM responses between male and female donors. When the response was expressed as the SI, it was found that was over
Viability of the thymus cells at the completion of isolation ranged from 95-99 per cent. The results of E and EAC rosettes of thymus cells obtained from twenty-six donors are shown according to age group in Table 1. Over 90 per cent of the thymus cells from each donor were proven to be T lymphocytes identified as E rosetting; less than 3 per cent of the cells, being EAC rosetting (B lymphocytes). There was a fairly good correlation between the number of T or B cells and the age of thymus donor. The proportion of T lymphocytes decreased, while that of B lymphocytes increased with advancing age. Table 2 shows the [3H]thymidine incorporation of thymus cells (control cultures) at various days of incubation. On day 1 of culture, the mean c.p.m.
Table 1. The results of E and EAC rosettes of thymus cells according to the age group
Group
Age
(ranges)* 1 nm-5 y 6 y-10 y III11 y-20 y 1 m-20 y Total 1 11
No. of pts
97-5 95-5 94-5 96-6
17 4 5 26 *m
Percentage of E rosettes mean (ranges)
=
(934-99 2) (94-7-97-1) (94 2-96 7) (93-4-99 2)
month, y = year.
Percentage of EAC rosettes mean (ranges)
0-5 0-8 2-0 0-9
(0-1 3) (0 1-1-8) (0 1-2 9) (0-2 9)
522
Tin Han et al. Table 4. Thymus-cell responses to phytohemagglutinin (PHA) and pokeweed mitogen (PWM)
[3H]thymidine Age group I
11
No. of cultures
Composition of cultures
incorporation (c.p.m.) mean+ s.e.
17
Thymus cells alone Thymus cells+ PHA Thymus cells+PWM Thymus cells alone Thymus cells+ PHA Thymus cells+PWM Thymus cells alone Thymus cells+ PHA Thymus cells+ PWM Thymus cells alone Thymus cells+ PHA Thymus cells+ PWM
13,850+ 3922 85,390+ 20,698 24,339+ 6270 6654+ 1774 103,891 + 57,199 25,502+ 13,298 3812+ 1549 74,400+ 17,620 16,321+ 3160 10,812+ 2704 86,123+ 15,900 22,922+ 4503
4
III
5
Total
26
both PHA and PWM response improved with advancing age. However, it should be emphasized that the actual rate of incorporation of [3H]thymidine in all three groups was rather similar when cells were cultured with stimulants. The difference in SI was due to the variation in incorporation rate in cultures without stimulants. Table 5 shows the comparison of PHA and PWM responses of thymus cells, according to two different culture periods (3 and 6 days). Again, the [3H]thymidine incorporation in control cultures on day 3 of incubation was rather high, while that on day 6 of incubation was very low. The [3H]thymidine incorporations of PHA-stimulated cultures, incubated for 3 and 6 days, were about the same. However, the PWM-stimulated cultures on day 6 of incubation was higher than those incubated for only 3 days. When the response was expressed as the SI, it was found that both PHA and PWM responses were significantly
Stimulation index mean+ s.e. 9 3+ 2-4 2-3+0-6 13-0+ 6-0 3-2+ 1-3
62-2+ 30 4 20-9+ 14-4 20-1 + 7 0 6-2+ 3 0
higher in cultures incubated for 6 days as compared to those incubated for 3 days.
'One-way' mixed lymphocyte reactions between thymus cells (responding cells) and peripheral blood lymphocytes (stimulating cells) are shown in Table 6. Thymus cells from each donor were found to be capable of responding to allogeneic cells. When the mixed lymphocyte reactions were analysed, according to the age of thymus donors, no correlation was noted. Table 7 shows the results of thymus cell responses to antigens. It is of great interest that the thymus cells from each of ten donors, aged 1-14 years were incapable of responding to all four different antigens. It is highly unlikely that the thymus donors were not sensitized against one or more of these microbial antigens, although such possibility cannot be entirely ruled out, since we did not study the antigeninduced blastogenic response of peripheral blood
Table 5. Comparison of PHA and PWM responses of thymus cells according to two different culture periods
Day of culture
No. of cultures
Composition of
3
5
Thymus cells alone Thymus cells+PHA Thymus cells+PWM Thymus cells alone Thymus cells+ PHA Thymus cells+PWM
6
5
cultures
[3H]Thymidine incorporation (c.p im.) mean+ s.e.
Stimulation index mean+ s.e.
6688+ 1532
103,834+ 42,830 21,892+ 10,418 617+
16 4+ 4-8 3-3+ 1.0
159
106,850± 19,244 32,413+ 17,583
208-0+ 59-6 41-8+ 10-8
Human thymus cells
523
Table 6. Thymus cell responses to allogeneic peripheral blood lymphocytes ('one-way' mixed lymphocyte reactions)
[3H]Thymidine Age group
12
II
6
III
4
Total
Composition of culture
No. of cultures
22
incorporation (c.p.m.) mean+ s.e.
Thymus cells Peripheral blood lymphocytes* Thymus cells and peripheral blood lymphocytes* Thymus cells Peripheral blood lymphocytes* Thymus cells and peripheral blood lymphocytes* Thymus cells Peripheral blood lymphocytes* Thymus cells and peripheral blood lymphocytes* Thymus cells Peripheral blood lymphocytes* Thymus cells and peripheral blood lymphocytes* *
714+ 152 285+ 30 19,093+ 3662 404+ 110 204+ 24 27,722+ 9934 731+ 426 332+ 124 15,350+ 8315 641+ 125 271 + 30 20,766+ 3773
Treated with nitomycin C.
Table 7. Lack of thymus cell responses to recall antigens*
[3H]thymidine Composition of culture
incorporation (c.p.m.)
Stimulation Index mean (ranges)
mean (ranges)
Thymus cells alone Thymus cells+ PPD Thymus cells+mumps Thymus cells+ monilia Thymus cells+ Varidase *
435 (335-659) 360 (200-465) 365 (208-623) 414 (216-549) 300 (195-431)
0-8 (0-4-1-2) 0-8 (0 5-1l1) 1 0 (0-5-1-6) 0 7 (0 4-09)
Thymus cells from ten donors, aged 1-14 years were used.
Table 8. Peripheral blood lymphocyte (PBL) responses to recall antigens*
Composition of culture PBL alone PBL+PPD PBL+ mumps PBL+ monilia PBL+ Varidase
[3H]Thymidine incorporation (c.p.m.) mean (ranges)
Stimulation index mean (ranges)
748 (112-2508) 2529 (481-11,929) 3532 (399-10,557) 1823 (357-3956) 3635 (352-11,524)
4 0 (0 8-9 7) 6-7 (1P8-19-0) 3-6 (1-3-13-2) 5-3 (1 1-10-3)
* Peripheral blood lymphocytes from ten donors, aged 1-14 years were used.
Stimulation index mean+ s.e. -
30-0+ 5 8 54-7+ 17-8 30 5+ 15 0
37 0+ 6-7
Tin Han et al.
524
lymphocytes from these donors. We have studied the in vitro antigen response of peripheral blood lymphocytes from ten age-matched children (randomly selected) and the results are presented in Table 8. Peripheral blood lymphocytes from nine of ten donors responded to one or more antigens, and such lymphocytes from only one donor failed to respond to all antigens, indicating that children aged 1-14 years are usually sensitized against one or more of the microbial antigens.
DISCUSSION There have been conflicting reports with regard to the ability of human thymus cells to proliferate in the presence of mitogens or allogeneic cells (Claman, 1966; Han et al., 1976; McIntyre & Segel, 1966). The present study shows that cells of human thymus can be transformed by PHA, PWM and allogeneic peripheral blood lymphocytes. It is of interest that a fairly good correlation existed between the proportion of T or B cells and the age of thymus donor. The proportion of T lymphocytes decreased and that of B lymphocytes increased with advancing age. These findings suggest that the incidence of B-cell contamination in thymus was more frequent in older donors. It has recently been reported that the proportion of E rosettes of human thymus cells decreased gradually with advancing age (Galli & Schlesinger, 1975). A significant [3H]thymidine incorporation of thymus cells, at the beginning of culture was observed in the present study. A good correlation between the age of thymus donor and the [3H]thymidine incorporation of thymus cells was also observed, indicating that the proportion of cells active in this respect decreased with advancing age. It has been reported that the thymus cells from individuals over 36 years of age responded well to PHA or Con A but that lymphocytes from the thymus of children up to 14 years of age did not respond to these mitogens (Galli & Schlesinger, 1975). In contrast, the present data demonstrate that the thymus cells from individuals of different ages responded to both PHA and PWM. The lack of mitogen response of thymus cells from younger donors (Galli & Schlesinger, 1975) could have been due to measuring this on day 3 of culture. In contrast to PHA- or PWM-induced blastogenesis of thymus cells, their responses to allogeneic peri-
pheral blood lymphocytes showed no correlation with the age of thymus donor. Of interest is the lack of response by cells of the thymus to the four antigens used here. This has not been previously reported. It is highly unlikely that the thymus donors in the present study were not sensitized to any of these antigens. This is supported by the observation that peripheral blood lymphocytes from nine of ten randomly selected children were capable of responding in vitro to one or more of these antigens. The possibility that the donors of thymic cells are anergic to all antigens, however, cannot be ruled out entirely, since we did not study the antigen response of peripheral blood lymphocytes from these donors. The thymus cells may be incapable of responding, but the peripheral blood T lymphocytes, on the other hand, are capable of responding to a specific antigen, to which the donor of both cell-types is sensitized. ACKNOWLEDGMENTS The authors wish to thank Mrs Ellen Cray and Mrs Barbara Dadey for their technical assistance. This study was supported in part by United States Public Health Service Grants CA-12318, CA-14323 and CA-14413. REFERENCES ANDERSON L.C., NORDLING S. & HAYRY P. (1973) Proliferation of B and T cells in mixed lymphocyte cultures. J. exp. Med. 138, 324. ASHERSON G.L. (1972) The development of lymphocyte function tests. Brit. J. Hosp. Med. 8, 665. BLOMGREN H. & SVEDMYR E. (1971) In vitro stimulation of mouse thymus cells by PHA and allogeneic cells. Cell. Immunol. 2, 285. CLAMAN H.N. (1966) Human thymus cell cultures-Evidence for two functional populations. Proc. Soc. exp. biol. (N. Y.) 121, 236. GALLI U. & SCHLESINGER M. (1975) Subpopulations of human thymus cells differing in their capacity to form stable E-rosettes and in their immunologic reactivity. J. Immunol., 115, 827. HAN T. & BENNETT M. (1972) Response of mouse thymocytes to mitogens and allogeneic cells. Seventh Leukocyte Culture Conference (Abstract no. 85). Pointe-Au-Pic, Quebec, Canada. HAN T., MINOWADA J., SUBRAMANIAN S. & SINKS L.F. (1976) Human thymus cells: Excellent responders but poor stimulators in 'one-way' mixed lymphocyte reaction. Immunology, 30, 361.
Human thymus cells JOHNSTON J.M. & WILSON D.B. (1970) Origin of immunoreactive lymphocytes in rats. Cell. Immunol. 1, 430. MANGI R. & MARDINEY M.R., Jr. (1970) Transformation of mouse lymphocytes to allogeneic lymphocytes and phytohemagglutinin. J. Immunol. 105, 90. McINTYRE R.O. & SEGEL W.D. (1966) PHA and thymic lymphocytes. Lancet, i, 1265. PAULY J.L., SOKAL J.E. & HAN T. (1973) Whole blood culture technique for functional studies of lymphocyte reactivity to mitogens, antigens and homologous lymphocytes. J. Lab. clin. Med. 82, 500.
B
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PLATE, J.M.D. and MCKENZIE, I.F.C. (1973) B-cell stimulation of allogeneic T-cell proliferation in mixed lymphocyte cultures. Nature: New Biology, 26, 247. RICKE W.O. & SCHWARZ M.R. (1964) The culture and karyotype of rat lymphocytes stimulated with phytohemagglutinin. Anat. Rec. 150, 383. SCHWARZ, M.R. (1967) Response of thymus and other human lymphoid tissues to PHA, PWM and genetically dissimilar lymphoid cells. Proc. Soc. exp. biol. (N. Y.) 125, 701.
