Immunology 1978 34 51
Incidental
of suppressor T cells in the induction of immunological tolerance
appearance
M. FUJIWARA & Al KARIYONE Department of Immunology, Institute of Medical Science, University of Tokyo, P.O. Takanawa, Tokyo 108, Japan
Received 21 March 1977; acceptedfor publication 12 April 1977
INTRODUCTION
Summary. Adult C3H mice were rendered tolerant to human serum albumin (HSA) by weekly injections of the ultracentrifuged, soluble form of the antigen. Following these injections, generation of tolerance and of suppressor cells were examined to see if these phenomena were in any way correlated. On the weekly administration of 1-0 mg of soluble HSA (sHSA), suppressor cells were detected after 15 successive injections. At a dose of 2-5 mg of sHSA, suppressor cells were found earlier, i.e. after 3 and 6 injections but disappeared after 10 injections. Suppressor cells were also detected after increasing doses of sHSA, 0-1 mg, 0-5 mg and then 2-5 mg or 12-5 mg, given at intervals of 1 week. The kinetics of the appearance of suppressor cells was examined after these increasing doses of sHSA, and it was shown that they were found between 1 and 6 days after the last tolerogen injection. The suppressive activity of tolerized spleen cells was shown to be dependent on T cells and not due to carry-over of the tolerogen. From these experimental data, it is proposed that the generation of suppressor cells is not essential to tolerance induction by protein antigens, but that their appearance is concerned with regulation of the immune response which often accompanies tolerance induction.
Immunological tolerance is one of the central problems in immunology (Howard & Mitchison, 1975). At the cellular level, the specific tolerant state was shown to be manifest in both T and B cells (Weigle, 1973). Although tolerance of B cell lineage has been extensively analysed the mechanisms of T cell tolerance are much less well understood. A few proposals have been made: classically clonal elimination (Burnet, 1959), production of serum blocking factors in transplantation immunity (Hellstrom, Hellstr6m & Allison, 1971) or recently generation of suppressor T cells (Zembala & Asherson, 1973; Basten, Miller, Sprent & Cheers, 1974; Phanuphak, Moorhead & Claman, 1974). It seems still a debatable problem whether appearance of suppressor cells is essential for the induction and maintenance of immunological tolerance. It was reported previously (Fujiwara, 1976) that suppressor T cells were detected in a profoundly tolerant state induced to human serum albumin (HSA) in adult C3H mice, and the suggestion was made that suppressor cells might regulate the appearance of auto-reactive cells. Following the experimental systems reported previously (Fujiwara, 1976), we have investigated the kinetics of generation of suppressor cells and the effect of varying the schedules and doses of the tolerogen.
Correspondence: Dr M. Fujiwara, Department of Immunology, Institute of Medical Science, University of Tokyo, P.O. Takanawa, Tokyo 108, Japan.
51
52
M. Fujiwara & Ai Kariyone
MATERIALS AND METHODS Mice Female C3H/HeJms mice and AKR/Jms mice of both sexes were supplied from the Breeding Unit of the Institute of Medical Science, University of Tokyo.
Antigens Crystalline human serum albumin (HSA) was obtained commercially (Pentex, Kankakee, Ill.). Horse red blood cells (HRBC) were used as a control antigen for checking the specificity of tolerance.
800 r under Telecovalt y-emitter (Shimazu Ltd., Tokyo). Recipients were challenged intraperitoneally with 0 5 mg of alum precipitated HSA (alHSA) plus 1 x 109 Bordetella pertussis vaccine on the day of cell transfer and with 0-1 mg of alHSA 11 days later. In some experiments, 0 05 ml of 10% HRBC was injected with alHSA. They were bled 10 days after the second challenge immunization. Titration of serum antibodies Levels of serum antibodies to HSA were determined by the passive haemagglutination (HA) test, using glutaraldehyde-fixed, HSA-conjugated goat red blood cells as described by Avrameas, Taudou & Chuilon (1969). Antibodies to HRBC were also titrated by direct HA test. Titers were expressed as mean log2 reciprocal of the maximal serum dilution giving positive HA.
Anti-O sera Anti-0 antibodies were produced in AKR mice by injecting 2 x 107 C3H thymocytes weekly from 5 to 6 times. Treatment of spleen cells with anti-0 plus complement was performed as described previously (Fujiwara, 1976).
RESULTS Tolerance induction One percent HSA solution was centrifuged at 123,000 g for 180 min and the upper third of the supernatant was collected. An appropriate amount of ultracentrifuged, soluble HSA (sHSA) was injected intravenously into 5-week-old mice, followed by several weekly injections.
Generation of suppressor cells by repeated injections of sHSA C3H mice were injected weekly with either 0 1, 1-0 or 2-5 mg of sHSA. Spleen cells were prepared from tolerized mice 7 days after the last tolerogen injection or from age-matched untreated mice and 5 x 107 cells were injected into lethally irradiated syngeneic mice. To detect the presence of suppressor cells, irradiated mice were reconstituted with a mixture of normal spleen cells and spleen cells from tolerized mice (5 x 107 cells each) and their antibody response was compared with controls which received either
Cell transfer experiment Spleen cells were prepared in Eagle's minimum essential medium buffered with 20 mm HEPES and 5-10 x 107 cells were transferred into syngeneic mice, 15-20 weeks old, which were irradiated at
Table 1. The effect of weekly repeated injections of 10 mg of sHSA on tolerance induction and the generation of suppressor cells Mean HA titres ± s.e.*
Normal
Normal ± tolerized
Tolerizedt
No. of
injections
HSA
HRBC
7 10
3-2 ±036 3 2 0-36 3-4 ± 10
12-2 ±0 19
15t
12-2 ±O019 9-6 ±0-25
HSA
HRBC
3-4 ±0 47 12-8 ±0 61 3-8 ±0-97 119 ±0-18 0-67 ±0-67 9 5 ±0 50
HSA
HRBC
1 6 ±0 40 0 1.1 ±0-61
11 3 ±0 71 110 ±0-54 8-7 ±0-76
* Each value represents the mean of 5-6 samples. t C3H mice were injected weekly with 1 0 mg of sHSA beginning at the age of 5 weeks. Spleen cells were prepared 1 week after the last injection and cell transfer experiments were done as described in Materials and Methods. t Separate experiment from the groups receiving 7 or 10 injections.
Suppressors in tolerance induction normal or tolerized spleen cells separately. In those mice injected weekly with 0 1 mg of sHSA for 10 or 15 weeks, neither tolerance nor suppressive activity was induced. When injected with 1 0 mg of sHSA, mice were rendered tolerant with seven injections; but suppressor cells were not detected until fifteen successive injections (Table 1). When mice were tolerized with weekly administration of 2 5 mg of sHSA, suppressor cells were detected after three and seven injections. After ten injections, however, suppressor cells were lost from spleen cell populations despite the continued existence of the tolerant state (Table 2). These data indicate two points: (a) generation of suppressor cells is not necessarily related to tolerance induction; and (b) suppressor cells tend to be generated late at a low dose and early at a high dose of sHSA. These points might imply some regulatory role of suppressor cells. Kinetics of appearance of suppressor cells in relation to antigen dose As preliminary experiments had indicated efficient
53
tolerance induction by injecting weekly with increasing doses of sHSA, the following protocol was employed. Mice were injected first with 0-1 mg sHSA, then 1 week later with 0*5 mg. Finally, after a further week the mice were divided into three groups: the first group received 0 5 mg, the second 2 5 mg and the third 12-5 mg of sHSA. (The tolerogen for the third injection was prepared by taking the top half instead of the top third after centrifugation since the latter did not contain sufficient protein.) The spleens were examined 4 days after the last tolerogen injection for the presence of suppressor cells. As shown in Table 3, suppressor cells were detected in the mice injected with 2-5 or 12-5 mg but not 0 5 mg sHSA. Next the kinetics of the appearance of suppressor cells was examined after the experimental protocol: 0 1 mg 05 mg -12-5 mg. As shown in Fig. 1, suppressor cells were present 1 day after the tolerogen injection and remained active until 6 days, but had disappeared by 13 days. These results were in accord with relatively early appearance of suppressor cells in mice injected successively with 2-5 mg of sHSA.
Table 2. The effect of weekly repeated injections of 2-5 mg of sHSA on tolerance induction and the generation of suppressor cells Mean HA titres ± s.e.
Normal No. of injections 3 6 10
HSA
Normal + tolerized
HRBC
4-2±1-0 4-2±1-0
*
3-9 0-48
7-8 ± 0-48
HSA
HRBC
HSA
HRBC
8-3 ±0-63
0 0 0
8-8 ± 0-25
1 1+1
1-6±1-0 3-3 ±12
Tolerized
For experimental details see legend of Fig. 1. Not tested.
*
Table 3. The effect of dose of sHSA on tolerance induction and the generation of suppressor cells Mean HA titres ± s.e. Normal
Normal + tolerized
Tolerized
Doses of sHSA* (mg)
HSA
HRBC
HSA
HRBC
HSA
HRBC
0-5 25 12-5
5-0 ±1-3 5-0 ± 1*3 5-0 ±1-3
8-3 ±0-66 8-3 ±0-66 8-3 ±0-66
4-5 ±0 74 1*8 ±1 0 1-0 ±0 77
7-4 ±0-51 8-3 ±0-63 8-2 ±0 85
0 0 0
8-8 ±0-38 7-9 ±0 50 8-2 ±0-85
* Five-week old mice were injected with 0 1 mg and 7 days later with 0 5 mg of sHSA. The doses for the third injection given 7 days later are shown.
54
M. Fujiwara & Ai Kariyone Control level
5-
t
T
6
r I
4
_
0
5 SI
3
2
Incidental
of suppressor T cells in the induction of immunological tolerance
appearance
M. FUJIWARA & Al KARIYONE Department of Immunology, Institute of Medical Science, University of Tokyo, P.O. Takanawa, Tokyo 108, Japan
Received 21 March 1977; acceptedfor publication 12 April 1977
INTRODUCTION
Summary. Adult C3H mice were rendered tolerant to human serum albumin (HSA) by weekly injections of the ultracentrifuged, soluble form of the antigen. Following these injections, generation of tolerance and of suppressor cells were examined to see if these phenomena were in any way correlated. On the weekly administration of 1-0 mg of soluble HSA (sHSA), suppressor cells were detected after 15 successive injections. At a dose of 2-5 mg of sHSA, suppressor cells were found earlier, i.e. after 3 and 6 injections but disappeared after 10 injections. Suppressor cells were also detected after increasing doses of sHSA, 0-1 mg, 0-5 mg and then 2-5 mg or 12-5 mg, given at intervals of 1 week. The kinetics of the appearance of suppressor cells was examined after these increasing doses of sHSA, and it was shown that they were found between 1 and 6 days after the last tolerogen injection. The suppressive activity of tolerized spleen cells was shown to be dependent on T cells and not due to carry-over of the tolerogen. From these experimental data, it is proposed that the generation of suppressor cells is not essential to tolerance induction by protein antigens, but that their appearance is concerned with regulation of the immune response which often accompanies tolerance induction.
Immunological tolerance is one of the central problems in immunology (Howard & Mitchison, 1975). At the cellular level, the specific tolerant state was shown to be manifest in both T and B cells (Weigle, 1973). Although tolerance of B cell lineage has been extensively analysed the mechanisms of T cell tolerance are much less well understood. A few proposals have been made: classically clonal elimination (Burnet, 1959), production of serum blocking factors in transplantation immunity (Hellstrom, Hellstr6m & Allison, 1971) or recently generation of suppressor T cells (Zembala & Asherson, 1973; Basten, Miller, Sprent & Cheers, 1974; Phanuphak, Moorhead & Claman, 1974). It seems still a debatable problem whether appearance of suppressor cells is essential for the induction and maintenance of immunological tolerance. It was reported previously (Fujiwara, 1976) that suppressor T cells were detected in a profoundly tolerant state induced to human serum albumin (HSA) in adult C3H mice, and the suggestion was made that suppressor cells might regulate the appearance of auto-reactive cells. Following the experimental systems reported previously (Fujiwara, 1976), we have investigated the kinetics of generation of suppressor cells and the effect of varying the schedules and doses of the tolerogen.
Correspondence: Dr M. Fujiwara, Department of Immunology, Institute of Medical Science, University of Tokyo, P.O. Takanawa, Tokyo 108, Japan.
51
52
M. Fujiwara & Ai Kariyone
MATERIALS AND METHODS Mice Female C3H/HeJms mice and AKR/Jms mice of both sexes were supplied from the Breeding Unit of the Institute of Medical Science, University of Tokyo.
Antigens Crystalline human serum albumin (HSA) was obtained commercially (Pentex, Kankakee, Ill.). Horse red blood cells (HRBC) were used as a control antigen for checking the specificity of tolerance.
800 r under Telecovalt y-emitter (Shimazu Ltd., Tokyo). Recipients were challenged intraperitoneally with 0 5 mg of alum precipitated HSA (alHSA) plus 1 x 109 Bordetella pertussis vaccine on the day of cell transfer and with 0-1 mg of alHSA 11 days later. In some experiments, 0 05 ml of 10% HRBC was injected with alHSA. They were bled 10 days after the second challenge immunization. Titration of serum antibodies Levels of serum antibodies to HSA were determined by the passive haemagglutination (HA) test, using glutaraldehyde-fixed, HSA-conjugated goat red blood cells as described by Avrameas, Taudou & Chuilon (1969). Antibodies to HRBC were also titrated by direct HA test. Titers were expressed as mean log2 reciprocal of the maximal serum dilution giving positive HA.
Anti-O sera Anti-0 antibodies were produced in AKR mice by injecting 2 x 107 C3H thymocytes weekly from 5 to 6 times. Treatment of spleen cells with anti-0 plus complement was performed as described previously (Fujiwara, 1976).
RESULTS Tolerance induction One percent HSA solution was centrifuged at 123,000 g for 180 min and the upper third of the supernatant was collected. An appropriate amount of ultracentrifuged, soluble HSA (sHSA) was injected intravenously into 5-week-old mice, followed by several weekly injections.
Generation of suppressor cells by repeated injections of sHSA C3H mice were injected weekly with either 0 1, 1-0 or 2-5 mg of sHSA. Spleen cells were prepared from tolerized mice 7 days after the last tolerogen injection or from age-matched untreated mice and 5 x 107 cells were injected into lethally irradiated syngeneic mice. To detect the presence of suppressor cells, irradiated mice were reconstituted with a mixture of normal spleen cells and spleen cells from tolerized mice (5 x 107 cells each) and their antibody response was compared with controls which received either
Cell transfer experiment Spleen cells were prepared in Eagle's minimum essential medium buffered with 20 mm HEPES and 5-10 x 107 cells were transferred into syngeneic mice, 15-20 weeks old, which were irradiated at
Table 1. The effect of weekly repeated injections of 10 mg of sHSA on tolerance induction and the generation of suppressor cells Mean HA titres ± s.e.*
Normal
Normal ± tolerized
Tolerizedt
No. of
injections
HSA
HRBC
7 10
3-2 ±036 3 2 0-36 3-4 ± 10
12-2 ±0 19
15t
12-2 ±O019 9-6 ±0-25
HSA
HRBC
3-4 ±0 47 12-8 ±0 61 3-8 ±0-97 119 ±0-18 0-67 ±0-67 9 5 ±0 50
HSA
HRBC
1 6 ±0 40 0 1.1 ±0-61
11 3 ±0 71 110 ±0-54 8-7 ±0-76
* Each value represents the mean of 5-6 samples. t C3H mice were injected weekly with 1 0 mg of sHSA beginning at the age of 5 weeks. Spleen cells were prepared 1 week after the last injection and cell transfer experiments were done as described in Materials and Methods. t Separate experiment from the groups receiving 7 or 10 injections.
Suppressors in tolerance induction normal or tolerized spleen cells separately. In those mice injected weekly with 0 1 mg of sHSA for 10 or 15 weeks, neither tolerance nor suppressive activity was induced. When injected with 1 0 mg of sHSA, mice were rendered tolerant with seven injections; but suppressor cells were not detected until fifteen successive injections (Table 1). When mice were tolerized with weekly administration of 2 5 mg of sHSA, suppressor cells were detected after three and seven injections. After ten injections, however, suppressor cells were lost from spleen cell populations despite the continued existence of the tolerant state (Table 2). These data indicate two points: (a) generation of suppressor cells is not necessarily related to tolerance induction; and (b) suppressor cells tend to be generated late at a low dose and early at a high dose of sHSA. These points might imply some regulatory role of suppressor cells. Kinetics of appearance of suppressor cells in relation to antigen dose As preliminary experiments had indicated efficient
53
tolerance induction by injecting weekly with increasing doses of sHSA, the following protocol was employed. Mice were injected first with 0-1 mg sHSA, then 1 week later with 0*5 mg. Finally, after a further week the mice were divided into three groups: the first group received 0 5 mg, the second 2 5 mg and the third 12-5 mg of sHSA. (The tolerogen for the third injection was prepared by taking the top half instead of the top third after centrifugation since the latter did not contain sufficient protein.) The spleens were examined 4 days after the last tolerogen injection for the presence of suppressor cells. As shown in Table 3, suppressor cells were detected in the mice injected with 2-5 or 12-5 mg but not 0 5 mg sHSA. Next the kinetics of the appearance of suppressor cells was examined after the experimental protocol: 0 1 mg 05 mg -12-5 mg. As shown in Fig. 1, suppressor cells were present 1 day after the tolerogen injection and remained active until 6 days, but had disappeared by 13 days. These results were in accord with relatively early appearance of suppressor cells in mice injected successively with 2-5 mg of sHSA.
Table 2. The effect of weekly repeated injections of 2-5 mg of sHSA on tolerance induction and the generation of suppressor cells Mean HA titres ± s.e.
Normal No. of injections 3 6 10
HSA
Normal + tolerized
HRBC
4-2±1-0 4-2±1-0
*
3-9 0-48
7-8 ± 0-48
HSA
HRBC
HSA
HRBC
8-3 ±0-63
0 0 0
8-8 ± 0-25
1 1+1
1-6±1-0 3-3 ±12
Tolerized
For experimental details see legend of Fig. 1. Not tested.
*
Table 3. The effect of dose of sHSA on tolerance induction and the generation of suppressor cells Mean HA titres ± s.e. Normal
Normal + tolerized
Tolerized
Doses of sHSA* (mg)
HSA
HRBC
HSA
HRBC
HSA
HRBC
0-5 25 12-5
5-0 ±1-3 5-0 ± 1*3 5-0 ±1-3
8-3 ±0-66 8-3 ±0-66 8-3 ±0-66
4-5 ±0 74 1*8 ±1 0 1-0 ±0 77
7-4 ±0-51 8-3 ±0-63 8-2 ±0 85
0 0 0
8-8 ±0-38 7-9 ±0 50 8-2 ±0-85
* Five-week old mice were injected with 0 1 mg and 7 days later with 0 5 mg of sHSA. The doses for the third injection given 7 days later are shown.
54
M. Fujiwara & Ai Kariyone Control level
5-
t
T
6
r I
4
_
0
5 SI
3
2
