CELLULAR
IMMUNOLOGY
23, 232-239 (1976)
The Role of the Thymus in the Establishment Cells in Liver Chimeras1 TEHILA UMIEL Department
of Cell Biology,
AND NATHAN TRAINING
The Weizmann Received
of Suppressor
Institute
December
of Science, Rehovot,
Israel
22, 1975
The role of the thymus in the establishment of specific suppression was studied in an experimental model in which lethally irradiated Fl mice were reconstituted with parental C57BL neonatal liver cells and then challenged with immunocompetent spleen cells syngeneic to the donor. In this model, inhibition #of a graft-versus-host response by these spleen cells s’erved as an indication of the establishment of a suppressive state towards syngeneic antigens in the liver chimeras. It was demonstrated that since thymectomy of the chimeras could not prevent the elicitation of a graft-versushost response by spleen cells, the thymus is essential for the establishment of this specific suppression. Reconstitution of such chimeras with intact thymus grafts of either donor (C57BL) or host (Fl) origin led to the inhibition of the graft-versushost res,ponse and to the reappearance of the suppressive state. Removal of the thymus in intact liver chimeras after establishment of a suppressive state did not affect suppression. Thus, it was concluded that the thymus is needed in the chimeras during a critical period in the development of suppression. Once suppression is established, the presence of the thymus is no longer required.
INTRODUCTION Elucidation of the mechanisms by which self is distinguished from nonself is a major need in the field of immunology. The question of how tolerance is established during embryogenesis and maintained in adulthood is of crucial importance in this respect. Several hypotheses on the nature and mechanism of tolerance have been proposed. Originally, tolerance was thought to be acquired through the elimination of cells capable of mounting immunological responses to self (1) Other hypotheses proposed that, rather than through cell death, tolerance could be established with the aid of active suppressive mechanisms involving serum factors (Z-6), cell factors, or cell-to-cell interactions (7-12)) all of which blocked the reactivity of lymphocytes, thus leading to unresponsiveness. Recently, we studied the mechanism of development of tolerance in an experimental model of tolerance induction in cells derived from fetal livers (13, 14). In this model system, lethally irradiated Fl mice were reconstituted with parental C57BL fetal liver cells and challenged 2 months later with spleen cells syngeneic to the donor liver cells. The rationale of this model was that if liver cells did not 1 Supported by a grant from the Yehudit Segal Fund, Jerusalem. 2Harold Korda Professor of Cancer Research. Established Investigator Scientist’s Bureau, Ministry of Health, Israel. 232 Copyright 1976 by Academic Press, Inc. P reproduction in any form resqved. All rights CI
of the Chief
SUPPRESSOR CELLS OF NEONATAL LIVER
233
elicit a response in the Fl because of lack of competence, the subsequent introduction of competent cells syngeneic to the liver inoculum should induce a response against the host. Alternatively, if the tolerance observed in the liver chimeras were induced by mechanisms preventing reactivity of liver cells, immunocompetent cells syngeneic with the liver should also be blocked and no graft-versus-host (GvH) response should be manifested. In these experiments, no immune reactivity could be produced by cells syngeneic with the liver donor cells. Thus, the tolerance induced in the embryonic liver cells seemed to be due at least in part to a specific suppressive effect on self antigens by the injected liver cells. A preliminary study suggested, in addition, that the presence of the thymus could be critical for the long-lasting suppression observed in this system (13, 15). Using this model, we attempted to analyze the role of the thymus in the establishment of specific suppression. MATERIALS
AND
METHODS
Mice. Inbred C57BL/6 male mice, 2-3 months old, (C3H/eb X C57BL/G)Fl hybrids, and newborn C57BL/6 mice were supplied by the Animal Breeding Center of the Weizmann Institute of Science. Thymectoulzy and irradiation. Thymectomy was performed on 6-8- or 16-weekold (C3H/eb x C57BL/6) Fl mice under Nembutal anesthesia; thymuses were removed by suction. Mice later found to contain thymic remnants were discarded from the experiment. Mice were exposed to 850 R of total body irradiation from a Vo source (gamma beam 150 A, Atomic Energy of Canada), 60 R/min ; focal skin distance, 34 in. Preparation of cell suspensions. Liver cells from newborn mice were prepared as previously described (14). In short, liver cells were suspended by gently pipetting with a 5-ml pipet in phosphate-buffered saline (PBS) supplemented with 10% fetal calf serum (FCS) (D i f co Laboratories, Detroit, Mich.). The cells were then passed through a fine stainless-steel mesh into PBS supplemented with 10% FCS. Cells were washed three times, resuspended in cold PBS and stained in trypan blue solution. The viable nucleated cells were counted. A close of 3-4 x 10’ liver cells were injected into irradiated mice l-3 hr after exposure. Spleens and thymuses from S-lo-week-old mice were aseptically removed and dispersed by pressing through a stainless-steel mesh into Eagle’s medium, and nucleated cells were counted. Unless otherwise indicated, a close of lo7 spleen cells or lOa thymocytes was injected into individual recipients. Tlzys~zzls and spleen i+nplantation. Ten days after reconstitution with parental liver cells, thymectomized, irradiated mice were implanted under the kidney capsule with a whole thymus obtained from a newborn C57BL/6 or (C3H/eb x C57BL/G)Fl donor, or with a whole spleen obtained from a newborn C57BL/G donor. At the end of the experiments the vitality of the thymus and spleen grafts was examined. Response to mitogens. Spleen cell suspensions were prepared in RPMI-1640 (Gibco, USA) enriched with 5% fetal bovine serum (FCS) (Rehatuin, NSF Reheis Chemical Co., Kankakee, Ill.) at a concentration of 10F cells/ml in each test tube culture (Falcon 2001, Los Angeles, Calif.). Triplicate cultures were tested for each group. Phytohemagglutinin (PHA) (Wellcome Research Laboratories, Beckenham, England) and concanavalin A (Con A) (Miles-Yecla, Rehovot,
234
UMIEL
AND
TRAININ
Israel) were used as T-cell mitogens (16). Cell cultures were incubated for 3 days at 37°C in a humidified incubator with air and 5% CO*. Two hours prior to the end of the incubation period, 2 #.Zi of tritiated thymidine (Negev, Irsael) were added to each culture, and the culture tubes were shaken at 37°C. The radioactivity pulse was interrupted by placing the cultures on ice. The contents of each culture tube was poured onto a fiberglass filter (Tamar, Israel), rinsed with saline, precipitated with 5% trichloroacetic acid solution and dried. The uptake of [ 3H] thymidine was measured. Results are presented as counts per min (cpm) of three cultures * standard error (SE). RESULTS Preliminary experiments suggested that the thymus is required for the expression of specific suppression in embryonic liver chimeras (14, 15). Experiments were subsequently designed to analyze further the role of the thymus in this system. The first point was to elucidate whether the presence of the thymus influences the survival rate of fetal liver chimeras. Thus, intact and thymectomized Fl mice were irradiated with 850 R and reconstituted with parental newborn liver cells, and the mortality rate of intact and thymectomized mice was follohred. As shown in Table 1, although thymectomized mice started to die somewhat earlier than intact mice, similar mortality rates (11.6%) were observed in both groups 2 months after liver reconstitution. Two months following liver reconstitution, thymectomized and intact chimeras were challenged with different doses of C57BL spleen cells (ranging between 10G and 1Os cells) syngeneic with the donor liver cells, and the mortality rate of each group was followed. As seen in Fig. 1, while thymectomized mice reconstituted with parental liver cells died as a consequence of GvH reaction even when challenged with low doses ( 106) of spleen cells, intact mice treated similarly did not suffer as a result of the GvH reaction to the inoculated spleen, even when high doses ( 108) of cells were injected. It can thus be concluded that liver chimeras can prevent specifically lymphoid cells of their own make-up from eliciting a GvH response in their host and that this inhibition depends on the presence of the thymus. In the next experiment we tested whether restoration of thymic function in thymectomized chimeras leads to the repair of the specific inhibitory function against the GvH reaction of lymphocytes, as observed in intact liver chimeras. For this purpose thymectomized and intact Fl mice were irradiated and reconstituted TABLE
1
Mortality Rate of Intact and Thymectomized (C3H/eb X C57BL)Fl Lethally Irradiated Mice after Reconstitution with Parental C57BL Newborn Liver Cells= Percentage
Treatment 6
Thymectomy
1.6 1.6
of mortality
at dayb
12
24
50
60
1.6
10 11.6
11.6 11.6
11.6 11.6
10
5 3 X 107 liver cells were injected into each mouse. b Each group consisted of 60 mice.
SUPPRESSOR
CELLS
OF
NEONATAL
235
LIVER
100 -
80 a z c1 = co;” z
I
40 -
20 -
FIG 1. Mortality rate of intact and thymectomizcd liver chimeras following challenge with different doses of parental C57BL spleen cells. Open symbols, intact chimeras; closed symbols, thymectomized chimeras. (o), 106; (V), 5X108; (W), 10’; (+), 5X10’; (A), lOa spleen cells.
with parental (C57BL) newborn liver cells. Ten days later, the thymectomized mice were grafted with a newborn thymus syngeneic either to the liver (C57BL) or to the host (Fl). I n addition, thymic suspensions were prepared and cells in amounts equivalent to single thymuses were injected iv to other thymectomized chimeras. Additional thymectomized controls were either grafted with newborn C57BL spleens or sham grafted. Each group consisted of 10-13 animals in each experiment. Two months following liver reconstitution all the mice were challenged with 2 X lo7 C57BL spleen cells and ‘the mortality rate was followed for 90 days. Figure 2 summarizes the results of a representative experiment. Thymectomized mice grafted with newborn C57BL spleens started to die 15 days after spleen challenge, reaching a 100% mortality rate 40 days after challenge. Thymectomized control mice reached a 100% mortality rate 75 days after chal-
FIG. 2. Mortality rate of thymectomized chimeras following restoration with thymic grafts, spleen grafts, or thymic cells and challenged with parental (C57BL), spleen cells. (n---•), C57BL thymus graft; (m-m), (CSH/eb X C57BL)Fl thymus graft; (A-A), C57BL spleen graft; (O-O), C57BL thymus cells; (O-O), (CSH/eb X C57BL)Fl thymus cells; (+- - -+), sham grafted.
236
UMIEL
AND
TRAININ
TABLE
2
Response to T Mitogens of Spleen Cells from Thymectomizcd Liver Chimcms Grafted Thymus Implants or Thymocytcs and Challenged with C57BL Spleen Cells Origin of spleen cells tested
Origin of graft
Response to mitogenR No mitogen (cpm f SE)
Untreatedc Intact chimera Thymectomized chimera fthymus implant Thymectomizcd chimera +thymocytes
with
Con A (cpm f SE)
C57BL Fl
7,946 8,219 5,862 8,202
f 456 f 429 f 558 * 1,180
74,586 57,684 36,468 42,565
CS7BL Fl
7,463 f 2.58 6,418 zk 1,051
zk f f &
@I)*
1,295 4,435 3,161 1,842
9.3 7.0 6.2 5.1
14,930 * 1,007 19,891 f 1,024
2.0 3.0
a Each figure represents the average of a pool of three cultures. *SI = stimulation index (counts per minute in mitogen-treated in cultures without mitogen). e Spleens obtained from C57BL mice.
(cpm f 53,390 20,070 15,790 14,221
PHA SE)
(SI)
III + f f
873 913 799 577
6.70 2.44 2.60 1.70
58 632
0.48 0.68
3,602 f 4,419 f
cultures/counts
per minute
lenge; mice grafted with thymocytes reached 80% mortality 90 days after spleen challenge. In contrast, mice grafted with a thymus from either C57BL or Fl origin reached a 27% mortality rate 90 days after spleen challenge. From this it can be concluded that the thymus plays an essential role in the establishment of a suppressive state in liver chimeras and that this function can be restored in thymectomized mice by transplanting a whole thymus from either host or donor origin. Thymectomized mice treated either with thymocytes or with spleen grafts did not show any improvement in their survival rates as compared to control thymectomized mice. The mortality rate of animals grafted with spleens and TABLE Mortality
3
Rate of Liver Chimeras Thymectomized at Different Time Intervals after Challenge with Parental C57BL Spleen Cells Number of mice
Treatment
Thymectomy before irradiation and liver reconstitution Thymectomy 1 day before spleen challenge Thymectomy 10 days after spleen challenge Thymectomy 20 days after spleen challenge Thymectomy 30 days after spleen challenge Intact chimera a Mice died within
7
Perccntnge
of mortality
at day
6
12
24
50
60
14.2
14.2
57.1
71.4
85.0
12.5
12.5
12.5
12.5
12.5
12.5
12.5
-
100
8
-
8
-
8
-
-
12.54
12.5
12.5
12.5
8 20
-
-
12.5a -
12.5 -
12.5 -
12.5 -
2 days after thymectomy.
-
90
summssoK
cents
0~ NEONATAL
LIVER
237
thymuses of parental (C57BL) origin was faster than that of mice grafted with Fl tissues. This may be clue to a GvH reaction developed by grafted parental cells. In addition, T-cell function of thymectomized mice restored by thymus implants or treated with thymic cell suspensions was evaluated. For this purpose, the spleens of these animals were tested for their mitogenic reactivity. Table 2 summarizes the results. It can be noted that thymectomized chimeras grafted with thymic implants manifested a reactivity to Con A and to PHA similar to that of intact liver chimeras while thymectomized mice injected with thymus cells had a lower reactivity. Next we tested whether the thymus is needed only during an initial period after liver reconstitution for the maturation of suppressive functions or permanently for maintentance of suppression. Thymuses were removed from various groups of intact liver chimeras at weekly intervals after challenge with parental spleen cells, and the mortality rate was followed. As seen in Table 3, no mortality due to GvH reaction was noted in these mice, in contrast to control mice which had been thymectomized prior to liver reconstitution. This showed that the thymus is required in the liver chimeras during a critical period in which liver cells seem to gain suppressive properties. Once this process is established, thymic requirement is no longer critical, at least under the experimental conditions we employed. DISCUSSION The results of this study suggest that the establishment of a specific suppressive state requires the presence of the thymus. We demonstrated that in contrast to intact liver chimeras, thymectomized chimeras could not prevent the GvH response elicited by immunocompetent cells syngeneic with the donor liver cells. Moreover, reconstitution of such thymectomized mice with thymus grafts led to inhibition of this GvH response. It was suggested that the presence of an intact thymus was required, since neither isolated thymic cells nor intact spleen grafts could compensate for thymic function. The role of the thymus appears to be limited to a critical period during the initial stage of establishment of suppression. Once this is achieved, the thymus can be removed without affecting the continuity of this phenomenon. It could be argued that the role of the thymus in the establishment of longlasting suppression in the chimeric state is through the provision of functional suppressor cells of thymic origin (12). This possibility is rather remote, since in the present experimental conditions administration of isolated thymus cells to thymectomized chimeras did not lead to suppression. It is therefore more plausible that thymic function is exerted by the provision of an adequate microenvironment or by means of thymic humoral factors, or by both, since thymus grafts of either donor or host type were effective in reconstitution of suppressive function of the chimera against the immunological attack of competent cells. Accordingly, suppression could be related to cells of liver origin which differentiate or proliferate in the thymic microenvironment. Indeed, an inductive function of the thymus was demonstrated in earlier experiments in which thymectomized mice were reconstituted with unrelated thymic grafts (17-19). It was found in those experiments that the thymus was repopulated by cells of host origin and that the host cells performed the thymic function. Another possibility which ought to be considered is that the thymus functions through the secretion of factors which either prevent
238
UMIEL
AND
TRAININ
the response of competent cells (20) or induce reactivity of suppressors. This last alternative would be analogous to the activation of T cells by thymic hormones (21) . The observation that suppression is not expressed in the thymectomized chimeras raised the question of what prevents reactivity of the parental liver cells against their thymectomized, irradiated Fl hosts. It may be argued that in the absence of the thymus, liver cells do not differentiate to acquire reactivity (22, 23). Accordingly, low mortality rates in the intact and thymectomized chimeras following the injection of parental liver cells may be related to different mechanisms. In the intact chimeras low mortality may be due to active suppression of reactive cells, while in the thymectomized mice it may result from a lack of maturation of these cells. It should be noted, though, that embryonic liver cells have a priori the potential to suppress certain immune responses (24, 25), and thus suppression could be expected to manifest itself in thymectomized as well as in intact mice. However, suppression was not expressed in the thymectomized mice after challenge with parental spleen cells, suggesting that the thymus is required for the maintenance or for the proliferation of suppressor cells. Furthermore, suppression by liver cells as detected in a mixed lymphocyte culture in vitro (24, 25) did not reflect the specificity manifested in the intact chimeras. Hence, the thymus may also contribute to further differentiation of primitive suppressors of liver origin. Accordingly, the thymus may be essential for the differentiation of suppressor cells within the liver into T suppressors or for the proliferation of suppressor cells to achieve a more defined function. Still, it seems that thymus is needed at the early stages of establishment of specific suppression, since thymectomy after maturation of liver cells did not interfere with this process. ACKNOWLEDGMENTS The authors wish for excellent technical
to thank Mrs. Ruth Goldman, Mr. Noah Kuller,
and Mr.
Itzhak
Serussi
assistance.
REFERENCES Vanderbilt University 1. Burnet, M., “The Clonal Selection Theory of Acquired Immunity.” Press, Nashville, Tenn., 1959. 2. Voisin, G. A., Kinsky, R., and Maillord, J., Ann. Inst. Pasteur Paris 115, 855, 1968. 3. HellstrGm, I., Hellstrgm, K. E., Srob, R., and Thomas, E. D., Proc. Nut. Acad. Sci. USA 66, 65, 1970. 4. Cohen, I. R., Globerson, A., and Feldman, M., Transplant. Proc. 3, 891, 1971. 5. Wekerle, H., Cohen, I. R., and Feldman, M., Nature New Biol. 241, 25, 1973. 6. Auerbach, R., In “Cellular Selection and Regulation in Immune Response” (G. M. Edelman, Ed.), Raven Press, New York, 1974. 7. Asherson, G. L., Zambala, M., and Barnes, R. M. R., Cl&z. Exp. Immunol. 9, 111, 1971. 8. Feldmann, M., Nature (London) 242, 84, 1973. 9. Gershon, R. K., 1% “Contemporary Topics in Immunobiology” (M. D. Cooper and L. N. Werner, Eds.), pp. l-40, Plenum Press, New York, 1974. 10. Zan-Bar, I., Nachtigal, D., and Feldman, M., Cell. Immunol. 17, 202, 1975. 11. Ha, T. Y., and Waksman, B. H., J. Immunol. 110, 1290, 1973. 12. Folch, H., and Waksman, B. H., J. Immunol. 113, 140, 1974. 13. Umiel, T., Bach, F. H., and Auerbach, R., In “Joint Meeting of European Societies for Immunology”, p. 80, Strasbourg, France, 1973. 14. Umiel, T., Tralwplantation 19, 485, 1975. A&an. Exp. Med. BioZ. 66, p. 565, 15. Umiel, T., Iti “Immune Reactivity of Lymphocytes,” Plenum Press, New York, 1976.
SUPPRESSOR
CELLS
OF
NEONATAL
LIVER
239
16. Andersson, J., Sjoberg, O., and Mijller, G., Transplant. Rev. 11, 131, 1972. 17. Feldman, M., and Globerson, A., Am. N.Y. Acad. Sci. 120, 182, 1964. 18. Miller, J. F. A. P., J&‘&w Inst. Moltogv. No. 2, 99, 1964. 19. Leuchars, E., Cross, A. M., and Dukor, P., Transplantation 3, 28, 1964. 20. Szent-Gyorgyi, A., Hegyeli, A., and McLaughlin, J. A., Biochcvz. J. 48, 149, 1962. 21. Van Bekkum, D. W., “The Biological Activity of Thymic Hormones.” Kooyker Scientific, Rotterdam, 1975. 22. Tyan, M. L., Science 145, 934, 1964. 23. Umiel, T. Globerson, A., and Auerbach, R., Proc. Sot. Exp. Biol. Med. 129, 598, 1968. 24. Globerson, A., Zinkernagel, R. M., and Umiel, T., Trunsplantatiolz, 20, 480, 1975. 25. Umiel, T., and Globerson, A., In “Proceedings 10th Leucocyte Culture Confer’ence, Amsterdam,” in press.
IMMUNOLOGY
23, 232-239 (1976)
The Role of the Thymus in the Establishment Cells in Liver Chimeras1 TEHILA UMIEL Department
of Cell Biology,
AND NATHAN TRAINING
The Weizmann Received
of Suppressor
Institute
December
of Science, Rehovot,
Israel
22, 1975
The role of the thymus in the establishment of specific suppression was studied in an experimental model in which lethally irradiated Fl mice were reconstituted with parental C57BL neonatal liver cells and then challenged with immunocompetent spleen cells syngeneic to the donor. In this model, inhibition #of a graft-versus-host response by these spleen cells s’erved as an indication of the establishment of a suppressive state towards syngeneic antigens in the liver chimeras. It was demonstrated that since thymectomy of the chimeras could not prevent the elicitation of a graft-versushost response by spleen cells, the thymus is essential for the establishment of this specific suppression. Reconstitution of such chimeras with intact thymus grafts of either donor (C57BL) or host (Fl) origin led to the inhibition of the graft-versushost res,ponse and to the reappearance of the suppressive state. Removal of the thymus in intact liver chimeras after establishment of a suppressive state did not affect suppression. Thus, it was concluded that the thymus is needed in the chimeras during a critical period in the development of suppression. Once suppression is established, the presence of the thymus is no longer required.
INTRODUCTION Elucidation of the mechanisms by which self is distinguished from nonself is a major need in the field of immunology. The question of how tolerance is established during embryogenesis and maintained in adulthood is of crucial importance in this respect. Several hypotheses on the nature and mechanism of tolerance have been proposed. Originally, tolerance was thought to be acquired through the elimination of cells capable of mounting immunological responses to self (1) Other hypotheses proposed that, rather than through cell death, tolerance could be established with the aid of active suppressive mechanisms involving serum factors (Z-6), cell factors, or cell-to-cell interactions (7-12)) all of which blocked the reactivity of lymphocytes, thus leading to unresponsiveness. Recently, we studied the mechanism of development of tolerance in an experimental model of tolerance induction in cells derived from fetal livers (13, 14). In this model system, lethally irradiated Fl mice were reconstituted with parental C57BL fetal liver cells and challenged 2 months later with spleen cells syngeneic to the donor liver cells. The rationale of this model was that if liver cells did not 1 Supported by a grant from the Yehudit Segal Fund, Jerusalem. 2Harold Korda Professor of Cancer Research. Established Investigator Scientist’s Bureau, Ministry of Health, Israel. 232 Copyright 1976 by Academic Press, Inc. P reproduction in any form resqved. All rights CI
of the Chief
SUPPRESSOR CELLS OF NEONATAL LIVER
233
elicit a response in the Fl because of lack of competence, the subsequent introduction of competent cells syngeneic to the liver inoculum should induce a response against the host. Alternatively, if the tolerance observed in the liver chimeras were induced by mechanisms preventing reactivity of liver cells, immunocompetent cells syngeneic with the liver should also be blocked and no graft-versus-host (GvH) response should be manifested. In these experiments, no immune reactivity could be produced by cells syngeneic with the liver donor cells. Thus, the tolerance induced in the embryonic liver cells seemed to be due at least in part to a specific suppressive effect on self antigens by the injected liver cells. A preliminary study suggested, in addition, that the presence of the thymus could be critical for the long-lasting suppression observed in this system (13, 15). Using this model, we attempted to analyze the role of the thymus in the establishment of specific suppression. MATERIALS
AND
METHODS
Mice. Inbred C57BL/6 male mice, 2-3 months old, (C3H/eb X C57BL/G)Fl hybrids, and newborn C57BL/6 mice were supplied by the Animal Breeding Center of the Weizmann Institute of Science. Thymectoulzy and irradiation. Thymectomy was performed on 6-8- or 16-weekold (C3H/eb x C57BL/6) Fl mice under Nembutal anesthesia; thymuses were removed by suction. Mice later found to contain thymic remnants were discarded from the experiment. Mice were exposed to 850 R of total body irradiation from a Vo source (gamma beam 150 A, Atomic Energy of Canada), 60 R/min ; focal skin distance, 34 in. Preparation of cell suspensions. Liver cells from newborn mice were prepared as previously described (14). In short, liver cells were suspended by gently pipetting with a 5-ml pipet in phosphate-buffered saline (PBS) supplemented with 10% fetal calf serum (FCS) (D i f co Laboratories, Detroit, Mich.). The cells were then passed through a fine stainless-steel mesh into PBS supplemented with 10% FCS. Cells were washed three times, resuspended in cold PBS and stained in trypan blue solution. The viable nucleated cells were counted. A close of 3-4 x 10’ liver cells were injected into irradiated mice l-3 hr after exposure. Spleens and thymuses from S-lo-week-old mice were aseptically removed and dispersed by pressing through a stainless-steel mesh into Eagle’s medium, and nucleated cells were counted. Unless otherwise indicated, a close of lo7 spleen cells or lOa thymocytes was injected into individual recipients. Tlzys~zzls and spleen i+nplantation. Ten days after reconstitution with parental liver cells, thymectomized, irradiated mice were implanted under the kidney capsule with a whole thymus obtained from a newborn C57BL/6 or (C3H/eb x C57BL/G)Fl donor, or with a whole spleen obtained from a newborn C57BL/G donor. At the end of the experiments the vitality of the thymus and spleen grafts was examined. Response to mitogens. Spleen cell suspensions were prepared in RPMI-1640 (Gibco, USA) enriched with 5% fetal bovine serum (FCS) (Rehatuin, NSF Reheis Chemical Co., Kankakee, Ill.) at a concentration of 10F cells/ml in each test tube culture (Falcon 2001, Los Angeles, Calif.). Triplicate cultures were tested for each group. Phytohemagglutinin (PHA) (Wellcome Research Laboratories, Beckenham, England) and concanavalin A (Con A) (Miles-Yecla, Rehovot,
234
UMIEL
AND
TRAININ
Israel) were used as T-cell mitogens (16). Cell cultures were incubated for 3 days at 37°C in a humidified incubator with air and 5% CO*. Two hours prior to the end of the incubation period, 2 #.Zi of tritiated thymidine (Negev, Irsael) were added to each culture, and the culture tubes were shaken at 37°C. The radioactivity pulse was interrupted by placing the cultures on ice. The contents of each culture tube was poured onto a fiberglass filter (Tamar, Israel), rinsed with saline, precipitated with 5% trichloroacetic acid solution and dried. The uptake of [ 3H] thymidine was measured. Results are presented as counts per min (cpm) of three cultures * standard error (SE). RESULTS Preliminary experiments suggested that the thymus is required for the expression of specific suppression in embryonic liver chimeras (14, 15). Experiments were subsequently designed to analyze further the role of the thymus in this system. The first point was to elucidate whether the presence of the thymus influences the survival rate of fetal liver chimeras. Thus, intact and thymectomized Fl mice were irradiated with 850 R and reconstituted with parental newborn liver cells, and the mortality rate of intact and thymectomized mice was follohred. As shown in Table 1, although thymectomized mice started to die somewhat earlier than intact mice, similar mortality rates (11.6%) were observed in both groups 2 months after liver reconstitution. Two months following liver reconstitution, thymectomized and intact chimeras were challenged with different doses of C57BL spleen cells (ranging between 10G and 1Os cells) syngeneic with the donor liver cells, and the mortality rate of each group was followed. As seen in Fig. 1, while thymectomized mice reconstituted with parental liver cells died as a consequence of GvH reaction even when challenged with low doses ( 106) of spleen cells, intact mice treated similarly did not suffer as a result of the GvH reaction to the inoculated spleen, even when high doses ( 108) of cells were injected. It can thus be concluded that liver chimeras can prevent specifically lymphoid cells of their own make-up from eliciting a GvH response in their host and that this inhibition depends on the presence of the thymus. In the next experiment we tested whether restoration of thymic function in thymectomized chimeras leads to the repair of the specific inhibitory function against the GvH reaction of lymphocytes, as observed in intact liver chimeras. For this purpose thymectomized and intact Fl mice were irradiated and reconstituted TABLE
1
Mortality Rate of Intact and Thymectomized (C3H/eb X C57BL)Fl Lethally Irradiated Mice after Reconstitution with Parental C57BL Newborn Liver Cells= Percentage
Treatment 6
Thymectomy
1.6 1.6
of mortality
at dayb
12
24
50
60
1.6
10 11.6
11.6 11.6
11.6 11.6
10
5 3 X 107 liver cells were injected into each mouse. b Each group consisted of 60 mice.
SUPPRESSOR
CELLS
OF
NEONATAL
235
LIVER
100 -
80 a z c1 = co;” z
I
40 -
20 -
FIG 1. Mortality rate of intact and thymectomizcd liver chimeras following challenge with different doses of parental C57BL spleen cells. Open symbols, intact chimeras; closed symbols, thymectomized chimeras. (o), 106; (V), 5X108; (W), 10’; (+), 5X10’; (A), lOa spleen cells.
with parental (C57BL) newborn liver cells. Ten days later, the thymectomized mice were grafted with a newborn thymus syngeneic either to the liver (C57BL) or to the host (Fl). I n addition, thymic suspensions were prepared and cells in amounts equivalent to single thymuses were injected iv to other thymectomized chimeras. Additional thymectomized controls were either grafted with newborn C57BL spleens or sham grafted. Each group consisted of 10-13 animals in each experiment. Two months following liver reconstitution all the mice were challenged with 2 X lo7 C57BL spleen cells and ‘the mortality rate was followed for 90 days. Figure 2 summarizes the results of a representative experiment. Thymectomized mice grafted with newborn C57BL spleens started to die 15 days after spleen challenge, reaching a 100% mortality rate 40 days after challenge. Thymectomized control mice reached a 100% mortality rate 75 days after chal-
FIG. 2. Mortality rate of thymectomized chimeras following restoration with thymic grafts, spleen grafts, or thymic cells and challenged with parental (C57BL), spleen cells. (n---•), C57BL thymus graft; (m-m), (CSH/eb X C57BL)Fl thymus graft; (A-A), C57BL spleen graft; (O-O), C57BL thymus cells; (O-O), (CSH/eb X C57BL)Fl thymus cells; (+- - -+), sham grafted.
236
UMIEL
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TRAININ
TABLE
2
Response to T Mitogens of Spleen Cells from Thymectomizcd Liver Chimcms Grafted Thymus Implants or Thymocytcs and Challenged with C57BL Spleen Cells Origin of spleen cells tested
Origin of graft
Response to mitogenR No mitogen (cpm f SE)
Untreatedc Intact chimera Thymectomized chimera fthymus implant Thymectomizcd chimera +thymocytes
with
Con A (cpm f SE)
C57BL Fl
7,946 8,219 5,862 8,202
f 456 f 429 f 558 * 1,180
74,586 57,684 36,468 42,565
CS7BL Fl
7,463 f 2.58 6,418 zk 1,051
zk f f &
@I)*
1,295 4,435 3,161 1,842
9.3 7.0 6.2 5.1
14,930 * 1,007 19,891 f 1,024
2.0 3.0
a Each figure represents the average of a pool of three cultures. *SI = stimulation index (counts per minute in mitogen-treated in cultures without mitogen). e Spleens obtained from C57BL mice.
(cpm f 53,390 20,070 15,790 14,221
PHA SE)
(SI)
III + f f
873 913 799 577
6.70 2.44 2.60 1.70
58 632
0.48 0.68
3,602 f 4,419 f
cultures/counts
per minute
lenge; mice grafted with thymocytes reached 80% mortality 90 days after spleen challenge. In contrast, mice grafted with a thymus from either C57BL or Fl origin reached a 27% mortality rate 90 days after spleen challenge. From this it can be concluded that the thymus plays an essential role in the establishment of a suppressive state in liver chimeras and that this function can be restored in thymectomized mice by transplanting a whole thymus from either host or donor origin. Thymectomized mice treated either with thymocytes or with spleen grafts did not show any improvement in their survival rates as compared to control thymectomized mice. The mortality rate of animals grafted with spleens and TABLE Mortality
3
Rate of Liver Chimeras Thymectomized at Different Time Intervals after Challenge with Parental C57BL Spleen Cells Number of mice
Treatment
Thymectomy before irradiation and liver reconstitution Thymectomy 1 day before spleen challenge Thymectomy 10 days after spleen challenge Thymectomy 20 days after spleen challenge Thymectomy 30 days after spleen challenge Intact chimera a Mice died within
7
Perccntnge
of mortality
at day
6
12
24
50
60
14.2
14.2
57.1
71.4
85.0
12.5
12.5
12.5
12.5
12.5
12.5
12.5
-
100
8
-
8
-
8
-
-
12.54
12.5
12.5
12.5
8 20
-
-
12.5a -
12.5 -
12.5 -
12.5 -
2 days after thymectomy.
-
90
summssoK
cents
0~ NEONATAL
LIVER
237
thymuses of parental (C57BL) origin was faster than that of mice grafted with Fl tissues. This may be clue to a GvH reaction developed by grafted parental cells. In addition, T-cell function of thymectomized mice restored by thymus implants or treated with thymic cell suspensions was evaluated. For this purpose, the spleens of these animals were tested for their mitogenic reactivity. Table 2 summarizes the results. It can be noted that thymectomized chimeras grafted with thymic implants manifested a reactivity to Con A and to PHA similar to that of intact liver chimeras while thymectomized mice injected with thymus cells had a lower reactivity. Next we tested whether the thymus is needed only during an initial period after liver reconstitution for the maturation of suppressive functions or permanently for maintentance of suppression. Thymuses were removed from various groups of intact liver chimeras at weekly intervals after challenge with parental spleen cells, and the mortality rate was followed. As seen in Table 3, no mortality due to GvH reaction was noted in these mice, in contrast to control mice which had been thymectomized prior to liver reconstitution. This showed that the thymus is required in the liver chimeras during a critical period in which liver cells seem to gain suppressive properties. Once this process is established, thymic requirement is no longer critical, at least under the experimental conditions we employed. DISCUSSION The results of this study suggest that the establishment of a specific suppressive state requires the presence of the thymus. We demonstrated that in contrast to intact liver chimeras, thymectomized chimeras could not prevent the GvH response elicited by immunocompetent cells syngeneic with the donor liver cells. Moreover, reconstitution of such thymectomized mice with thymus grafts led to inhibition of this GvH response. It was suggested that the presence of an intact thymus was required, since neither isolated thymic cells nor intact spleen grafts could compensate for thymic function. The role of the thymus appears to be limited to a critical period during the initial stage of establishment of suppression. Once this is achieved, the thymus can be removed without affecting the continuity of this phenomenon. It could be argued that the role of the thymus in the establishment of longlasting suppression in the chimeric state is through the provision of functional suppressor cells of thymic origin (12). This possibility is rather remote, since in the present experimental conditions administration of isolated thymus cells to thymectomized chimeras did not lead to suppression. It is therefore more plausible that thymic function is exerted by the provision of an adequate microenvironment or by means of thymic humoral factors, or by both, since thymus grafts of either donor or host type were effective in reconstitution of suppressive function of the chimera against the immunological attack of competent cells. Accordingly, suppression could be related to cells of liver origin which differentiate or proliferate in the thymic microenvironment. Indeed, an inductive function of the thymus was demonstrated in earlier experiments in which thymectomized mice were reconstituted with unrelated thymic grafts (17-19). It was found in those experiments that the thymus was repopulated by cells of host origin and that the host cells performed the thymic function. Another possibility which ought to be considered is that the thymus functions through the secretion of factors which either prevent
238
UMIEL
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the response of competent cells (20) or induce reactivity of suppressors. This last alternative would be analogous to the activation of T cells by thymic hormones (21) . The observation that suppression is not expressed in the thymectomized chimeras raised the question of what prevents reactivity of the parental liver cells against their thymectomized, irradiated Fl hosts. It may be argued that in the absence of the thymus, liver cells do not differentiate to acquire reactivity (22, 23). Accordingly, low mortality rates in the intact and thymectomized chimeras following the injection of parental liver cells may be related to different mechanisms. In the intact chimeras low mortality may be due to active suppression of reactive cells, while in the thymectomized mice it may result from a lack of maturation of these cells. It should be noted, though, that embryonic liver cells have a priori the potential to suppress certain immune responses (24, 25), and thus suppression could be expected to manifest itself in thymectomized as well as in intact mice. However, suppression was not expressed in the thymectomized mice after challenge with parental spleen cells, suggesting that the thymus is required for the maintenance or for the proliferation of suppressor cells. Furthermore, suppression by liver cells as detected in a mixed lymphocyte culture in vitro (24, 25) did not reflect the specificity manifested in the intact chimeras. Hence, the thymus may also contribute to further differentiation of primitive suppressors of liver origin. Accordingly, the thymus may be essential for the differentiation of suppressor cells within the liver into T suppressors or for the proliferation of suppressor cells to achieve a more defined function. Still, it seems that thymus is needed at the early stages of establishment of specific suppression, since thymectomy after maturation of liver cells did not interfere with this process. ACKNOWLEDGMENTS The authors wish for excellent technical
to thank Mrs. Ruth Goldman, Mr. Noah Kuller,
and Mr.
Itzhak
Serussi
assistance.
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SUPPRESSOR
CELLS
OF
NEONATAL
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16. Andersson, J., Sjoberg, O., and Mijller, G., Transplant. Rev. 11, 131, 1972. 17. Feldman, M., and Globerson, A., Am. N.Y. Acad. Sci. 120, 182, 1964. 18. Miller, J. F. A. P., J&‘&w Inst. Moltogv. No. 2, 99, 1964. 19. Leuchars, E., Cross, A. M., and Dukor, P., Transplantation 3, 28, 1964. 20. Szent-Gyorgyi, A., Hegyeli, A., and McLaughlin, J. A., Biochcvz. J. 48, 149, 1962. 21. Van Bekkum, D. W., “The Biological Activity of Thymic Hormones.” Kooyker Scientific, Rotterdam, 1975. 22. Tyan, M. L., Science 145, 934, 1964. 23. Umiel, T. Globerson, A., and Auerbach, R., Proc. Sot. Exp. Biol. Med. 129, 598, 1968. 24. Globerson, A., Zinkernagel, R. M., and Umiel, T., Trunsplantatiolz, 20, 480, 1975. 25. Umiel, T., and Globerson, A., In “Proceedings 10th Leucocyte Culture Confer’ence, Amsterdam,” in press.
