AJEBAK 53 (Pt. 5) 399-411 (1975)
RADIOSENSITIVITY OF SUPPRESSOR CELLS IN NEWBORN RATS by PETER McCULLAGH (From the Department of Immunology, John Curtin School of Medical Research, Australian National University, P.O. Box 334, Canberra City, A.C.T., Australia 2601.) {Accepted for puhUcation November 3, 1975.) Summary. The resistance of neonatal rats to sustaining adoptive immune responses against heterologous erythrocytes following the transfer of normal thoracic duct lymphocytes was examined. Irradiation of the neonatal rat at levels as low as 350 rad was found to be effective in overcoming this resistance, although preliminary exposure to antigen could interfere with facilitation of adoptive responses by irradiation. It is suggested that the failure of the neonate to sustain adoptive immune responses is explicable on the basis of an active suppression and, as a corollary, unresponsiveness resulting either from macrophage immaturity or the transfer of maternal antibody is discounted as a likely explanation for the immunological behaviour of the newborn rat towards the antigens examined.
INTRODUCTION. While the inability of newborn rats and mice to mount immune responses of the magnitude observed in adult animals has been readily accepted as a consequence of the immaturity of their lymphoid system, reports of the failure of lymphoid cells transferred from normal, adult donors to mount adoptive immune responses in neonatal hosts are less readily explained. Impairment of adoptive responses in neonates was first noted by Dixon and Weigle (1957). Antibody was not produced following antigenic challenge of 6-day old rabbits which had received lymph node cells from adult animals unless immune donors were used. Similarly, Nossal (1959) reported that spleen cells from normal adult rats and mice responded poorly to challenge after transfer to intact or irradiated neonates, whereas cells from immune donors could respond, provided they had been incubated with antigen before transfer. These poor adoptive responses following the transfer of cells from immunocompetent adults to neonates have most commonly been ascribed to immaturity of the phagocytic cells of the host. Thus, Argyris (1968) was able to augment the immune response of newborn mice to sheep erythrocytes by transferring peritoneal cells, provided that the cell dosage and time of administration were appropriate. She surmised
400
PETER McCULLAGH
that, during development of the neonate, an initial period of deficiency in both immunocompetent cells and macrophages was succeeded by a phase when macrophages alone were inadequate. A similar conclusion was drawn by Bendinelh, Senesi and Falcone (1971), whilst Hardy, Globerson and Danon (1973) found that peritoneal exudate cells from neonatal mice were markedly inferior to similar cells from adults in reconstituting the reactivity of irradiated adult mice to antigen. The aim of this and the succeeding paper (McCullagh, 1975) was to examine the immunological reactivity of neonatal rats to heterologous erythrocytes in the light of the present understanding of the nature of immunological tolerance of such antigens. Accordingly, evidence is presented for the existence of a dominant state responsible for suppressing immune responses in neonatal rats. The first paper is concerned with the facilitation of adoptive immune responses in neonates by preliminary irradiation of the host and the infiuence of earlier exposure to antigen on the effects of irradiation. The second paper (McCullagh, 1975) delineates the origin and some of the properties of the cells which enable the neonate to suppress adoptive responses and establishes the presence of antibody-forming cell precursors in the neonate. MATERIALS AND METHODS. Rats used in the preliminary experiments were from an outbred albino colony. The remaining experiments utilized (DA x PvG/c)F^ hybrid rats. Thoracic duct lymph was collected from each donor in 5 ml of medium 199 containing 100 units of heparin at room temperature, cannulation of the thoracic duct having been performed by the method of Gowans (1959). In the preliminary group of experiments peritoneal cells which were collected from previously unstimulated peritoneal cavities injected with sheep erythrocytes shortly before irrigation were incubated with thoracic duct lymphocytes as previously described (McGullagh, 1970). Injections of both lymphocytes and sheep erythrocytes were invariably administered by the intravenous route unless an alternative is indicated. The volume injected was generally 0-15 ml. Spleens were disrupted on stainless steel wire grids to produce single cell suspensions and direct plaque-forming cell estimations were performed by means of Gunningham and Szenberg's (1968) modification of the technique of Jerne, Nordin and Henry (1963). Haemolysin titres of sera were measured as described in a previous paper (McGullagh, 1970). Rats received whole body irradiation in rotating drums placed 25 cm from a 100 curie ••^Go source. PRELIMINARY E X P E R I M E N T S .
The results of an unreported group of experiments performed 7 years previously, being relevant to the experiments described in this paper, are briefly summarized at this point. The aim of the earlier investigation, undertaken in outbred albino rats, had been to detemiine whether exposure to an antigenic challenge in vitro could equip lymphocytes to mount an adoptive response in a neonatal host. To elucidate this point, 3 groups of rats each received 2-5 X 108 leucocytes (thoracic duct lymphocytes with or without peritoneal macrophages) via the intraperitoneai route within 72 h of birth and were exsanguinated 7 days later to provide sera for haemolysin determination. The first group, which, received thoracic duct lymphocytes plus 10^ sheep erythrocytes, attained a mean titre (logo) of 4-3 ± 0-2 (71 rats). A second group injected with freshly mixed antigen-primed peritoneal cells and lymphocytes had a mean titre of 4-7 ± 0-5 (30 rats). In contrast, the third group of
RADIOSENSITIVITY OF SUPPRESSOR CELLS
401
9 rats, which received a cellular mixture similar in composition to that given to the second group with the difference that the component populations had been incubated together for 8 h prior to transfer, developed a mean haemolysin titre of 7-4 ± 1-0 (P < 0-01). It has been demonstrated previously that lymphocytes submitted to incubation in this manner are thereby stimulated for antibody formation (McGullagh, 1970).
RESULTS. Splenic plaque-forming cell response of neonates to heterologous erythrocytes. The mean total splenie plaque-forming cell response on the 4th to the 7th days after challenge of 32 2-day old rats with 10* sheep erythrocytes failed to exeeed 250 on any day. A similar response was observed in 18 rats challenged with 10^ horse erythrocytes. TABLE 1. Splenic plaque-forming cell responses of young rats in receipt of thoracic duct lymphocytes. Recipient's age (days)
Total response (mean ± S.E.) 5 days
6 days
7 days
3
270±120(10)*
42O±I8O(12)
320 ±70 (9)
5
t t t
8 II
270 ±40
(6)
1800 ±600 (6) 2400±1800(12)
t t t
Each recipient was injected with 10" riormal syngeneic thoracic duct lymphocytes plus 10' sheep erythrocytes. This injection was administered to the 3-day old rats intravenously and to older animals by the intraperitoneal route. * Numbers in parentheses refer to the number of rats examined in each group. t Assays were not performed at these times.
Splenic plaque-forming cell response of neonates injected toith thoracic duct lymphocytes and heterologous erythrocytes. The consequence of transferring inocula of 10* normal thoracic duct lymphocytes plus 10* sheep erythrocytes to young rats is summarized in Table 1. The 5-, 8- and 11-day old recipients in this experiment were injected intraperitoneally. The very low response in the first week of life is apparent. As subsequent experiments in this paper utilize rats younger than 3 days, the responses at this time should be eontrasted with the results of those experiments. That the transfer of inadequate numbers of cells was not a major faetor in determining these poor responses was demonstrated by inereasing the number of normal lymphocytes injected. Thus, the transfer of 4 x 10* lymphocytes to 1-day old rats (the largest dose that eould be administered intravenously) resulted in a total spleen response of 2,900 ± 1,000 plaque-forming cells (mean of 12 reeipients). While this response was appreciably larger than those observed with smaller inocula of lymphocytes, it will be seen to be very small in eomparison with those obtained in later experiments.
402
PETER
MCCULLAGH
The capacity of neonatal rats to sustain adoptive responses to horse erythrocytes was superior to that observed in the case of sheep erythrocytes. One-dayold recipients of 10^ thoracic duct lymphocytes plus 10* horse erythrocytes gave mean total splenic plaque-forming cell responses of 1,770 ± 500 (16 rats), 2,760 ± 500 (31 rats) and 1,190 ± 600 (12 rats) on the 5th, 6th and 7th days respectively after challenge. Variation in the challenging dose of antigen did not significantly alter these results. Administration of 10^ horse erythrocytes with 10^ lymphocytes was followed by a peak response of 1,100 ± 200 plaque-forming cells per spleen on the 7th day while substitution of 10" horse erythrocytes produced a peak of 2,500 ± 1,600 on the 6th day. Splenic plaque-forming cell response of irradiated neonates injected with lijmphocytes. Neonatal rats resemble adult rats tolerant of sheep erythrocytes in that neither type of animal responds to challenge with this antigen. Furthermore, normal lymphocytes fail to mount an adoptive immune response when injected into either type of animal unless the transferred cells have been stimulated with sheep erythrocytes in vitro. Although unirradiated tolerant rats fail to mount an immune response when injected with normal lymphocytes and antigen, preliminary irradiation of the recipients permits an adoptive response (McGregor, McCullagh and Cowans, 1967). This facilitating effect of irradiation is likely to be mediated by damage to suppressor cells in the tolerant host which would otherwise interfere with the ability of transferred normal lymphocytes to mount TABLE 2. Splenic plaque-forming cell responses of neonatal rats irradiated before receiving lymphocytes.
SRBC 10'
Mean PFC response (± S.E.)(xlO-') 25-5±5-8 32±12
10^
18 + 5 31-5 + 4-5 87 ±24
10'
119±11
Six litters of rats received 730 rad whole body irradiation on the day of birth and were injected with 10^ normal syngeneic thoracic duct lymphocytes plus the indicated dose of sheep erythrocytes on the following day. PFC responses were measured 6 days later in all rats and the results represent the mean of the rats in each litter.
an adoptive response (McCullagh, 1974). As the first means of testing the proposition that a radiosensitive suppressor mechanism may be present in neonates, the inffuence of irradiation on the adoptive responses of newborn rats was studied. For simplicity, the effects of different doses of irradiation on responses to the two antigens, sheep and horse erythrocytes, and the consequences of exposure of the neonate to more than one antigen will be considered separately.
RADIOSENSITIVITY OF SUPPRESSOR CELLS
403
(a) Effect of antigen dose on adoptive responses to sheep erythrocytes in irradiated neonates. Six litters of rats were exposed to 730 rad of gamma irradiation on the day of birth and then, approximately 24 h after birth, each rat was injected intravenonsly with 10* normal thoracic duct lymphocytes plus sheep erythrocytes. The mean splenic plaque-forming cell responses to varying doses of sheep erythrocytes as assayed 6 days after challenge are summarized in Table 2. Two features require comment. In the first place, the responses of these 1-day old rats are 50-250 times greater than those observed to follow the transfer of similar inocula to unirradiated neonates (Table 1). Secondly, there is no tendency for higher doses of antigen to suppress responsiveness and no support for the hypothesis that high doses are suppressive and lower doses stimulatory. In this context, it should be noted that the highest dose of antigen used, 10" sheep erythrocytes, was slightly in excess of the intravenous inoculum which invariably suffices for the induction of tolerance in non-irradiated neonates. While these observations fulfil the expectations which a hypothesis based on immunosuppression as the cause of neonatal unreactivity would raise, they are incompatible with an explanation for failure of neonates to support adoptive responses that is based on macrophage immaturity. (b) Effect of exposure to antigen before irradiation on adoptive responses in neonates. The ability of 730 rad irradiation to facilitate adoptive responses could be most readily explained by the presence of a radiosensitive suppressor cell in the neonate. If this interpretation is correct, it is conceivable that exposure of neonates to antigen before irradiation might modify the radiosensitivity of a suppressor cell population. Before testing this possibility, it was necessary to quantitate the radiosensitivity of the mechanism antagonistic to adoptive responses in the neonate. It could be suggested that low doses of irradiation might inactivate suppressor cells while leaving antibody-forming cell precursors intact and, in this situation, irradiation alone might be sufficient to confer responsiveness on the neonate. However, it was found that exposure of neonates to comparatively low doses of irradiation in the absence of any restoration with lymphocytes did not confer responsiveness to challenge with 10* sheep erythrocytes (Table 3, footnote). It was also found that supplying irradiated neonates with an inoculum exclusively of thymus cells after irradiation failed to confer immune responsiveness. Column (a) of Table 3 summarizes the mean responses of rats which received 10* thoracic duct lymphocytes plus 10* sheep erythrocytes after being irradiated on the day of birth and indicates that 350 rad irradiation of the host was as effective in facilitating adoptive responses as 730 rad had been. The influence of previous antigenic exposure on this radiosensitivity was assessed by irradiating a number of litters which had been challenged intravenously with 10"* sheep erythrocytes. These litters were injected with erythrocytes on the day of birth while irradiation was administered either 2 h or 24 h later. It will be observed (Table 3, column (b)) that, if neonates had been exposed to sheep erythrocytes 2 h before irradiation, their resistance to sustaining an adoptive
PETER McCULLACH
404
immune response was significantly augmented, both 350 and 450 rad proving inadequate to facilitate responses. In contrast, if irradiation was deferred for 24 h after antigen exposure, the capacity to sustain an adoptive response resembled that of animals not exposed to antigen before irradiation (column (c)). These results suggest that exposure to antigen shortly before irradiation may temporarily increase the radioresistance of the suppressor mechanism in the neonate. TABLE 3. Influence of previous exposure to sheep erythrocytes on the plaque-forming cell response to this antigen of irradiated neonates in receipt of lymphocytes. Irradiation (rads)
No. SRBC before irradiation (a)
10'SRBC given 2 h before irradiation (b)
lO** SRBC given 24 h before irradiation (c)
150
3,300 -1- 700 (5)
1,300 ± 500
1,000 ± 500
250
2,300 + 700 (5)
2,000 + 400 (2)
6,500 -1- 4,700 (4)
350 450
37,000 + 11,000* (7)
300* (5)
14,600 + 3,000 (5)
19,400 -1- 4,800t (7)
2,200 + 500t (5)
15,500+ 1,900 (4)
Litters of rats in group (a) were irradiated as indicated on the day of birth and injected with 10* normal thoracic duct lymphocytes plus 10' sheep erythrocytes on the following day. Rats in group (b) were injected with 10' sheep erythrocytes on the day of birth, irradiated 2 h later and injected with 10* lymphocytes plus 10' sheep erythrocytes on the following day. Rats in group (c) were injected with 10* sheep erythrocytes on the day of birth, irradiated 24 h later and injected with 10* lymphocytes plus 10* sheep erythrocytes on the day following irradiation. Splenic plaque-forming cell responses were measured 6 days after the injection of lymphocytes plus antigen in each case and the results are expressed as mean ± S.E. As controls, 13 rats which received 250, 350 or 450 rad on the day of birth were challenged with 10* sheep erythrocytes on the following day. In no case did the plaque-forming cell response exceed 300. Furthermore, the addition of 8 x 10' thymus cells from 5-week old donors to the inoculum of sheep erythrocytes in similar experiments failed to increase these responses. *, t results significantly different.
(c) Effect of preliminary irradiation on adoptive responses to horse erythrocytes in neonates. It will be recalled from Section 2 that adoptive plaqueforming cell responses to horse erythrocytes in non-irradiated neonates, although weak, exceeded those observed against sheep erythrocytes. The magnitude of the effect of irradiation on capacity to sustain adoptive responses also varied between these two antigens. To assess the effect of preliminary irradiation on the capacity of neonates to sustain adoptive responses to horse erythrocytes, a series of rats was subjected to a range of doses of irradiation (50 rad to 730 rad) on the day of birth and were injected with lO** thoracic duct lymphocytes plus 10* horse erythrocytes on the following day (Table 4, column (a)). It is apparent that irradiation facilitated the mounting of adoptive responses although the extent of the amplification achieved by irradiation was less than that observed in the case of sheep erythrocytes.
RADIOSENSITIVITY OF SUPPRESSOR CELLS
405
(d) Effect of challenge tvith two antigens on adoptive responses in irradiated neonates. The preceding experiments have not indicated whether suppression of adoptive responses by neonatal hosts can be antigen specific. To examine this point, the influence of pre-irradiation exposure to sheep erythrocytes on the subsequent ability to sustain adoptive responses against horse erythrocytes was tested. Three experimental designs were employed. In the first of these, rats were injected with 10" sheep erythrocytes on the day of birth and irradiated TABLE 4. Influence of previous exposure to sheep erythrocytes on the immune response to horse erythrocytes in irradiated neonates in receipt of lymphocytes. Pre-irradiation challenge
Nil
SRBC
Post-irradiation challenge
HRBC
HRBC
Irradiation (rads)
(a)
50
Nil
SRBC
HRBC & SRBC
HRBC & SRBC
(b)
(c)
(d)
9,000+1,600 (4)
N.D.
N.D.
N.D.
150
14,800 + 2,500 (6)
7,400+1,100 (5)
24,600 + 4,20011 (5)
10,300 + 3,20011 (7)
250
11,700 + 2,200* (7)
9,000+1,500 (3)
30,800 +3,400* S (5)
2,800+1,300 § (4)
350
27,300 +3,900t (6)
16,800 + 3,000 (4)
60,600+7,200t** (5)
3,600+1,200** (5)
450
32,800 + 4,200]: (6)
8,200+1,8001 (9)
33,800+l,300tt (4)
11,800+3,200tt (5)
550
20,400 + 4,400 (5)
N.D.
N.D.
N.D.
650
20,000 + 4,300 (5)
N.D.
N.D.
N.D.
730
15,600+5,900 (8)
N.D.
N.D.
N.D.
Litters of rats were irradiated as indicated on the day of birth. In the experiments summarized in columns (b) and (d), 10' sheep erythrocytes were injected intravenously 2 h before irradiation. All rats received 10^ normal thoracic duct lymphocytes plus 10" horse erythrocytes on the following day, whilst groups (c) and (d) simultaneously received 10' sheep erythrocytes. Splenic plaqueforming cell assays were performed 6 days after lymphocyte injection and the results (horse erythrocytes) are expressed as mean ± S.E. with the number of rats in each experiment being given in brackets. t t significant difference (P
RADIOSENSITIVITY OF SUPPRESSOR CELLS IN NEWBORN RATS by PETER McCULLAGH (From the Department of Immunology, John Curtin School of Medical Research, Australian National University, P.O. Box 334, Canberra City, A.C.T., Australia 2601.) {Accepted for puhUcation November 3, 1975.) Summary. The resistance of neonatal rats to sustaining adoptive immune responses against heterologous erythrocytes following the transfer of normal thoracic duct lymphocytes was examined. Irradiation of the neonatal rat at levels as low as 350 rad was found to be effective in overcoming this resistance, although preliminary exposure to antigen could interfere with facilitation of adoptive responses by irradiation. It is suggested that the failure of the neonate to sustain adoptive immune responses is explicable on the basis of an active suppression and, as a corollary, unresponsiveness resulting either from macrophage immaturity or the transfer of maternal antibody is discounted as a likely explanation for the immunological behaviour of the newborn rat towards the antigens examined.
INTRODUCTION. While the inability of newborn rats and mice to mount immune responses of the magnitude observed in adult animals has been readily accepted as a consequence of the immaturity of their lymphoid system, reports of the failure of lymphoid cells transferred from normal, adult donors to mount adoptive immune responses in neonatal hosts are less readily explained. Impairment of adoptive responses in neonates was first noted by Dixon and Weigle (1957). Antibody was not produced following antigenic challenge of 6-day old rabbits which had received lymph node cells from adult animals unless immune donors were used. Similarly, Nossal (1959) reported that spleen cells from normal adult rats and mice responded poorly to challenge after transfer to intact or irradiated neonates, whereas cells from immune donors could respond, provided they had been incubated with antigen before transfer. These poor adoptive responses following the transfer of cells from immunocompetent adults to neonates have most commonly been ascribed to immaturity of the phagocytic cells of the host. Thus, Argyris (1968) was able to augment the immune response of newborn mice to sheep erythrocytes by transferring peritoneal cells, provided that the cell dosage and time of administration were appropriate. She surmised
400
PETER McCULLAGH
that, during development of the neonate, an initial period of deficiency in both immunocompetent cells and macrophages was succeeded by a phase when macrophages alone were inadequate. A similar conclusion was drawn by Bendinelh, Senesi and Falcone (1971), whilst Hardy, Globerson and Danon (1973) found that peritoneal exudate cells from neonatal mice were markedly inferior to similar cells from adults in reconstituting the reactivity of irradiated adult mice to antigen. The aim of this and the succeeding paper (McCullagh, 1975) was to examine the immunological reactivity of neonatal rats to heterologous erythrocytes in the light of the present understanding of the nature of immunological tolerance of such antigens. Accordingly, evidence is presented for the existence of a dominant state responsible for suppressing immune responses in neonatal rats. The first paper is concerned with the facilitation of adoptive immune responses in neonates by preliminary irradiation of the host and the infiuence of earlier exposure to antigen on the effects of irradiation. The second paper (McCullagh, 1975) delineates the origin and some of the properties of the cells which enable the neonate to suppress adoptive responses and establishes the presence of antibody-forming cell precursors in the neonate. MATERIALS AND METHODS. Rats used in the preliminary experiments were from an outbred albino colony. The remaining experiments utilized (DA x PvG/c)F^ hybrid rats. Thoracic duct lymph was collected from each donor in 5 ml of medium 199 containing 100 units of heparin at room temperature, cannulation of the thoracic duct having been performed by the method of Gowans (1959). In the preliminary group of experiments peritoneal cells which were collected from previously unstimulated peritoneal cavities injected with sheep erythrocytes shortly before irrigation were incubated with thoracic duct lymphocytes as previously described (McGullagh, 1970). Injections of both lymphocytes and sheep erythrocytes were invariably administered by the intravenous route unless an alternative is indicated. The volume injected was generally 0-15 ml. Spleens were disrupted on stainless steel wire grids to produce single cell suspensions and direct plaque-forming cell estimations were performed by means of Gunningham and Szenberg's (1968) modification of the technique of Jerne, Nordin and Henry (1963). Haemolysin titres of sera were measured as described in a previous paper (McGullagh, 1970). Rats received whole body irradiation in rotating drums placed 25 cm from a 100 curie ••^Go source. PRELIMINARY E X P E R I M E N T S .
The results of an unreported group of experiments performed 7 years previously, being relevant to the experiments described in this paper, are briefly summarized at this point. The aim of the earlier investigation, undertaken in outbred albino rats, had been to detemiine whether exposure to an antigenic challenge in vitro could equip lymphocytes to mount an adoptive response in a neonatal host. To elucidate this point, 3 groups of rats each received 2-5 X 108 leucocytes (thoracic duct lymphocytes with or without peritoneal macrophages) via the intraperitoneai route within 72 h of birth and were exsanguinated 7 days later to provide sera for haemolysin determination. The first group, which, received thoracic duct lymphocytes plus 10^ sheep erythrocytes, attained a mean titre (logo) of 4-3 ± 0-2 (71 rats). A second group injected with freshly mixed antigen-primed peritoneal cells and lymphocytes had a mean titre of 4-7 ± 0-5 (30 rats). In contrast, the third group of
RADIOSENSITIVITY OF SUPPRESSOR CELLS
401
9 rats, which received a cellular mixture similar in composition to that given to the second group with the difference that the component populations had been incubated together for 8 h prior to transfer, developed a mean haemolysin titre of 7-4 ± 1-0 (P < 0-01). It has been demonstrated previously that lymphocytes submitted to incubation in this manner are thereby stimulated for antibody formation (McGullagh, 1970).
RESULTS. Splenic plaque-forming cell response of neonates to heterologous erythrocytes. The mean total splenie plaque-forming cell response on the 4th to the 7th days after challenge of 32 2-day old rats with 10* sheep erythrocytes failed to exeeed 250 on any day. A similar response was observed in 18 rats challenged with 10^ horse erythrocytes. TABLE 1. Splenic plaque-forming cell responses of young rats in receipt of thoracic duct lymphocytes. Recipient's age (days)
Total response (mean ± S.E.) 5 days
6 days
7 days
3
270±120(10)*
42O±I8O(12)
320 ±70 (9)
5
t t t
8 II
270 ±40
(6)
1800 ±600 (6) 2400±1800(12)
t t t
Each recipient was injected with 10" riormal syngeneic thoracic duct lymphocytes plus 10' sheep erythrocytes. This injection was administered to the 3-day old rats intravenously and to older animals by the intraperitoneal route. * Numbers in parentheses refer to the number of rats examined in each group. t Assays were not performed at these times.
Splenic plaque-forming cell response of neonates injected toith thoracic duct lymphocytes and heterologous erythrocytes. The consequence of transferring inocula of 10* normal thoracic duct lymphocytes plus 10* sheep erythrocytes to young rats is summarized in Table 1. The 5-, 8- and 11-day old recipients in this experiment were injected intraperitoneally. The very low response in the first week of life is apparent. As subsequent experiments in this paper utilize rats younger than 3 days, the responses at this time should be eontrasted with the results of those experiments. That the transfer of inadequate numbers of cells was not a major faetor in determining these poor responses was demonstrated by inereasing the number of normal lymphocytes injected. Thus, the transfer of 4 x 10* lymphocytes to 1-day old rats (the largest dose that eould be administered intravenously) resulted in a total spleen response of 2,900 ± 1,000 plaque-forming cells (mean of 12 reeipients). While this response was appreciably larger than those observed with smaller inocula of lymphocytes, it will be seen to be very small in eomparison with those obtained in later experiments.
402
PETER
MCCULLAGH
The capacity of neonatal rats to sustain adoptive responses to horse erythrocytes was superior to that observed in the case of sheep erythrocytes. One-dayold recipients of 10^ thoracic duct lymphocytes plus 10* horse erythrocytes gave mean total splenic plaque-forming cell responses of 1,770 ± 500 (16 rats), 2,760 ± 500 (31 rats) and 1,190 ± 600 (12 rats) on the 5th, 6th and 7th days respectively after challenge. Variation in the challenging dose of antigen did not significantly alter these results. Administration of 10^ horse erythrocytes with 10^ lymphocytes was followed by a peak response of 1,100 ± 200 plaque-forming cells per spleen on the 7th day while substitution of 10" horse erythrocytes produced a peak of 2,500 ± 1,600 on the 6th day. Splenic plaque-forming cell response of irradiated neonates injected with lijmphocytes. Neonatal rats resemble adult rats tolerant of sheep erythrocytes in that neither type of animal responds to challenge with this antigen. Furthermore, normal lymphocytes fail to mount an adoptive immune response when injected into either type of animal unless the transferred cells have been stimulated with sheep erythrocytes in vitro. Although unirradiated tolerant rats fail to mount an immune response when injected with normal lymphocytes and antigen, preliminary irradiation of the recipients permits an adoptive response (McGregor, McCullagh and Cowans, 1967). This facilitating effect of irradiation is likely to be mediated by damage to suppressor cells in the tolerant host which would otherwise interfere with the ability of transferred normal lymphocytes to mount TABLE 2. Splenic plaque-forming cell responses of neonatal rats irradiated before receiving lymphocytes.
SRBC 10'
Mean PFC response (± S.E.)(xlO-') 25-5±5-8 32±12
10^
18 + 5 31-5 + 4-5 87 ±24
10'
119±11
Six litters of rats received 730 rad whole body irradiation on the day of birth and were injected with 10^ normal syngeneic thoracic duct lymphocytes plus the indicated dose of sheep erythrocytes on the following day. PFC responses were measured 6 days later in all rats and the results represent the mean of the rats in each litter.
an adoptive response (McCullagh, 1974). As the first means of testing the proposition that a radiosensitive suppressor mechanism may be present in neonates, the inffuence of irradiation on the adoptive responses of newborn rats was studied. For simplicity, the effects of different doses of irradiation on responses to the two antigens, sheep and horse erythrocytes, and the consequences of exposure of the neonate to more than one antigen will be considered separately.
RADIOSENSITIVITY OF SUPPRESSOR CELLS
403
(a) Effect of antigen dose on adoptive responses to sheep erythrocytes in irradiated neonates. Six litters of rats were exposed to 730 rad of gamma irradiation on the day of birth and then, approximately 24 h after birth, each rat was injected intravenonsly with 10* normal thoracic duct lymphocytes plus sheep erythrocytes. The mean splenic plaque-forming cell responses to varying doses of sheep erythrocytes as assayed 6 days after challenge are summarized in Table 2. Two features require comment. In the first place, the responses of these 1-day old rats are 50-250 times greater than those observed to follow the transfer of similar inocula to unirradiated neonates (Table 1). Secondly, there is no tendency for higher doses of antigen to suppress responsiveness and no support for the hypothesis that high doses are suppressive and lower doses stimulatory. In this context, it should be noted that the highest dose of antigen used, 10" sheep erythrocytes, was slightly in excess of the intravenous inoculum which invariably suffices for the induction of tolerance in non-irradiated neonates. While these observations fulfil the expectations which a hypothesis based on immunosuppression as the cause of neonatal unreactivity would raise, they are incompatible with an explanation for failure of neonates to support adoptive responses that is based on macrophage immaturity. (b) Effect of exposure to antigen before irradiation on adoptive responses in neonates. The ability of 730 rad irradiation to facilitate adoptive responses could be most readily explained by the presence of a radiosensitive suppressor cell in the neonate. If this interpretation is correct, it is conceivable that exposure of neonates to antigen before irradiation might modify the radiosensitivity of a suppressor cell population. Before testing this possibility, it was necessary to quantitate the radiosensitivity of the mechanism antagonistic to adoptive responses in the neonate. It could be suggested that low doses of irradiation might inactivate suppressor cells while leaving antibody-forming cell precursors intact and, in this situation, irradiation alone might be sufficient to confer responsiveness on the neonate. However, it was found that exposure of neonates to comparatively low doses of irradiation in the absence of any restoration with lymphocytes did not confer responsiveness to challenge with 10* sheep erythrocytes (Table 3, footnote). It was also found that supplying irradiated neonates with an inoculum exclusively of thymus cells after irradiation failed to confer immune responsiveness. Column (a) of Table 3 summarizes the mean responses of rats which received 10* thoracic duct lymphocytes plus 10* sheep erythrocytes after being irradiated on the day of birth and indicates that 350 rad irradiation of the host was as effective in facilitating adoptive responses as 730 rad had been. The influence of previous antigenic exposure on this radiosensitivity was assessed by irradiating a number of litters which had been challenged intravenously with 10"* sheep erythrocytes. These litters were injected with erythrocytes on the day of birth while irradiation was administered either 2 h or 24 h later. It will be observed (Table 3, column (b)) that, if neonates had been exposed to sheep erythrocytes 2 h before irradiation, their resistance to sustaining an adoptive
PETER McCULLACH
404
immune response was significantly augmented, both 350 and 450 rad proving inadequate to facilitate responses. In contrast, if irradiation was deferred for 24 h after antigen exposure, the capacity to sustain an adoptive response resembled that of animals not exposed to antigen before irradiation (column (c)). These results suggest that exposure to antigen shortly before irradiation may temporarily increase the radioresistance of the suppressor mechanism in the neonate. TABLE 3. Influence of previous exposure to sheep erythrocytes on the plaque-forming cell response to this antigen of irradiated neonates in receipt of lymphocytes. Irradiation (rads)
No. SRBC before irradiation (a)
10'SRBC given 2 h before irradiation (b)
lO** SRBC given 24 h before irradiation (c)
150
3,300 -1- 700 (5)
1,300 ± 500
1,000 ± 500
250
2,300 + 700 (5)
2,000 + 400 (2)
6,500 -1- 4,700 (4)
350 450
37,000 + 11,000* (7)
300* (5)
14,600 + 3,000 (5)
19,400 -1- 4,800t (7)
2,200 + 500t (5)
15,500+ 1,900 (4)
Litters of rats in group (a) were irradiated as indicated on the day of birth and injected with 10* normal thoracic duct lymphocytes plus 10' sheep erythrocytes on the following day. Rats in group (b) were injected with 10' sheep erythrocytes on the day of birth, irradiated 2 h later and injected with 10* lymphocytes plus 10' sheep erythrocytes on the following day. Rats in group (c) were injected with 10* sheep erythrocytes on the day of birth, irradiated 24 h later and injected with 10* lymphocytes plus 10* sheep erythrocytes on the day following irradiation. Splenic plaque-forming cell responses were measured 6 days after the injection of lymphocytes plus antigen in each case and the results are expressed as mean ± S.E. As controls, 13 rats which received 250, 350 or 450 rad on the day of birth were challenged with 10* sheep erythrocytes on the following day. In no case did the plaque-forming cell response exceed 300. Furthermore, the addition of 8 x 10' thymus cells from 5-week old donors to the inoculum of sheep erythrocytes in similar experiments failed to increase these responses. *, t results significantly different.
(c) Effect of preliminary irradiation on adoptive responses to horse erythrocytes in neonates. It will be recalled from Section 2 that adoptive plaqueforming cell responses to horse erythrocytes in non-irradiated neonates, although weak, exceeded those observed against sheep erythrocytes. The magnitude of the effect of irradiation on capacity to sustain adoptive responses also varied between these two antigens. To assess the effect of preliminary irradiation on the capacity of neonates to sustain adoptive responses to horse erythrocytes, a series of rats was subjected to a range of doses of irradiation (50 rad to 730 rad) on the day of birth and were injected with lO** thoracic duct lymphocytes plus 10* horse erythrocytes on the following day (Table 4, column (a)). It is apparent that irradiation facilitated the mounting of adoptive responses although the extent of the amplification achieved by irradiation was less than that observed in the case of sheep erythrocytes.
RADIOSENSITIVITY OF SUPPRESSOR CELLS
405
(d) Effect of challenge tvith two antigens on adoptive responses in irradiated neonates. The preceding experiments have not indicated whether suppression of adoptive responses by neonatal hosts can be antigen specific. To examine this point, the influence of pre-irradiation exposure to sheep erythrocytes on the subsequent ability to sustain adoptive responses against horse erythrocytes was tested. Three experimental designs were employed. In the first of these, rats were injected with 10" sheep erythrocytes on the day of birth and irradiated TABLE 4. Influence of previous exposure to sheep erythrocytes on the immune response to horse erythrocytes in irradiated neonates in receipt of lymphocytes. Pre-irradiation challenge
Nil
SRBC
Post-irradiation challenge
HRBC
HRBC
Irradiation (rads)
(a)
50
Nil
SRBC
HRBC & SRBC
HRBC & SRBC
(b)
(c)
(d)
9,000+1,600 (4)
N.D.
N.D.
N.D.
150
14,800 + 2,500 (6)
7,400+1,100 (5)
24,600 + 4,20011 (5)
10,300 + 3,20011 (7)
250
11,700 + 2,200* (7)
9,000+1,500 (3)
30,800 +3,400* S (5)
2,800+1,300 § (4)
350
27,300 +3,900t (6)
16,800 + 3,000 (4)
60,600+7,200t** (5)
3,600+1,200** (5)
450
32,800 + 4,200]: (6)
8,200+1,8001 (9)
33,800+l,300tt (4)
11,800+3,200tt (5)
550
20,400 + 4,400 (5)
N.D.
N.D.
N.D.
650
20,000 + 4,300 (5)
N.D.
N.D.
N.D.
730
15,600+5,900 (8)
N.D.
N.D.
N.D.
Litters of rats were irradiated as indicated on the day of birth. In the experiments summarized in columns (b) and (d), 10' sheep erythrocytes were injected intravenously 2 h before irradiation. All rats received 10^ normal thoracic duct lymphocytes plus 10" horse erythrocytes on the following day, whilst groups (c) and (d) simultaneously received 10' sheep erythrocytes. Splenic plaqueforming cell assays were performed 6 days after lymphocyte injection and the results (horse erythrocytes) are expressed as mean ± S.E. with the number of rats in each experiment being given in brackets. t t significant difference (P
