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The significance of immunological immaturity for the induction of tolerance
~. Theoret. Biol. (1962) 3, 503-5 °8
The Significance of Immunological Immaturity for the Induction of Tolerancet A. LENGEROV~
Institute of Experimental Biology and Genetics, Czechoslovak Academy of Sciences, Prague, Czechoslovakia (Received 2 February 1962 and in revised form 23 May 1962 ) The mechanism underlying the induction of immunological tolerance in adult cell populations is discussed, and especially the question of whether this state of tolerance, established in different experimental systems, is based on the suppression of reactivity in functionally mature lymphoid cells or on their gradual replacement by a tolerant population derived from immature cells which acquired tolerance while differentiating in the presence of chronic antigenic stimulation.
In its original concept (BiUingham, Brent & Medawar, 1956), immunological tolerance was defined as a state of specific non-reactivity to a certain antigen or antigens displayed by an otherwise normally reactive individual, which is due to prior exposure to the same antigen before the maturation of the faculty of immune response. According to this concept, the tolerance responsive phase of development of an individual is confined fairly strictly to a period coinciding roughly with its foetal life, whereas immunity is the regular reaction to be expected if the individual is confronted with the same antigenic stimulus for the first time while already immunologically mature. This duality of reaction to antigen as manifested at the level of the organism might obviously reflect two qualitative phases in the functional development of its lymphoid cells, on which antigen acts first as an inhibitor and later as an inducer of specific immune response. Further analysis casts some doubt on this simple scheme. For example, the question arises as to whether immunological maturity detected at the level of the whole organism fully expresses the stage of development of all its lymphoid cells or only that of the majority. Immunological maturation of a lymphoid cell might be defined by its progression from sensitivity to reactivity (Lederberg, 1959). The immature cell being rather sensitive, exposure to antigen could prevent it from developing further; this might be more or less drastic interference based on destruction of the cell (Burnet, 1959) or on some sort of functional block (Szilard, 196o ). t This investigation was supported by the International Atomic Energy Agency. 503
5o4
A.
LENGEROV/~
Immunological maturation of an individual might thus be visualized by a set of distribution curves expressing the relative frequency of successive types of differentiating lymphoid cells (Fig. i). In an immunologicaUy immature individual all the lymphoid cells might evidently be in the tolerance responsive phase of their development (curves (a)a, (b)a); this would account for the relative ease with which tolerance can be induced in such Co)
(b)
0
0 Q
i
" 0 0
C
b
Successive types of differentiating lymphoid cells
FIO. I. Hypothetical distributions of lymphoid cells in an immunologicallymaturing individual, a: prenatal period, b: postnatal period, c: adult age. 0 Tolerance-responsivecells (or cells with low requirementsfor tolerance-inducingdose of antigen). • Immunity responsive cells (or cells with extremely high requirements for toleranceinducing dose of antigen whose reaction to lower amount of antigen is immunity). (~ Transient type. organisms. If the whole cell population then matured simultaneously ((a), curves b, c) the mature individual would contain only mature lymphoid cells (c) reacting uniformly when stimulated with a given dose of antigen. If, however, new mutant ctones arose (Burnet, 1959) or a stem cell reserve were present (Loutit, 1956; Lederberg, 1958 ) from which reactive cells could differentiate throughout the animal's life, then in later developmental stages of an individual the population of lymphoid cells would be heterogeneous ((b), curves b, c) containing in different proportions all the successive cell types. To maintain tolerance would then virtually mean inducing it in newly arising or differentiating cells. This would explain the part taken by chronic antigenic stimulation in the maintenance of tolerance and its disappearance after the dissipation of non-replicating antigen (Smith & Bridges, 1958; Mitchison, 1959). Under this condition and that of the limited life-span of mature lymphoid cells, the tolerance responsive phase would not be confined to the perinatal period. It should therefore be possible to confer tolerance on adults on the same principle as on newborn
TOLERANCE AND I M M U N O L O G I C A L M A T U R I T Y
505
individuals except for the time and amount of antigen required. The antigen used to induce tolerance would always get partly lost owing to immune elimination by the reactive cells and its level would have to be maintained by repeated administration. However, the stock of mature cells would gradually be depleted and replaced by a tolerant population of cells differentiating under the effect of antigen. As tolerance is not detectable as something positive but only as a lack of reactivity it should obviously take some time before it could be revealed at the level of the organism, where it would be masked by immunity as long as non-tolerant cells survived. The rather long times taken for gain and loss of tolerance in recent experiments by Mitchison (1962) are interpreted by this author as an objection to the possibility of mere accumulation and degradation of antigen by a homogeneous cell population and in favour of cell turnover as a mechanism involved in the induction of tolerance in adult cells. Simonsen (I96Z), however, found that immunologically competent cells derived from adult mouse spleen can be rendered tolerant to antigens of their newborn hosts in as short a time as twenty-four hours; cell turnover and population replacement can, of course, scarcely be held responsible for this immediate effect. A cell population which becomes non-reactive at such a rate apparently still contains the formerly reactive cells in a state of functional block or exhaustion. If this were the whole explanation it would be pointless to assume the existence of a stem-cell reserve in an adult organism to account for the occurrence of tolerance in Simonsen's system. In that case, adult mouse spleen might contain only mature lymphoid cells as expressed by curve (a)c; the question arises, of course, whether the mechanism of non-reactivity established here in formerly reactive cells is identical with that operating in immature lymphoid cells or whether there is another mechanism involved. If the capacity of being rendered tolerant were retained throughout the life of every lymphoid cell, then not the time of introduction of antigen but rather the quantity of antigen might be the decisive factor between tolerance and immunity. The quantitative requirements of the reactive cells might be much higher than those of immature cells but in spite of it the accumulation of large amounts of antigen (if available at once) they might be accomplished rather rapidly. In that case, the establishment of tolerance in adult cells would not necessarily be a longterm process. If, however, lymphoid cells undergo qualitative changes during differentiation and their tolerance responsive phase is limited, then Simonsen's finding of immediate loss of reactivity in adult cells might be a less general phenomenon due possibly to something specific for his experimental system. One factor to be considered here might be the quantitative relationship between the antigen (represented by the whole organism) and the reactive site (represented by a relatively small inoculum),
506
A. LENGEROV,~
which is hard to achieve in any other system. The removal of reactive cells which would otherwise die out slowly might be here accelerated by the excess of antigen on a different principle (Gorer & Boyse, 1959; Simonsen, 196o). In some graft-to-host systems, however, the reactivity of immunologically competent ceils could be maintained for three to five transfers (Simonsen, 1957) or was shown to persist several days in the first recipient (Russell, 1962); thus the immediate loss of reactivity is not a general phenomenon even here. At any rate, the immediate inhibition of the reactive ceils, for example, by the excess of antigen in certain immunogenetic combinations, does not exclude their gradual replacement by a tolerant population recruited from the immature proportion of transferred spleen ceils. It might be of some interest to discuss in this connection a few data obtained in a third experimental system which from the quantitative point of view lies just between the two above extreme cases where the reactive site and antigen were represented by host and graft or vice versa. In this third system) the interactions were followed of two replicating mesenchymal cell grafts in the environment of a non-reactive host. (Lethally irradiated mice were restored by a pooled inoculum of haematopoietic cells--adult bone marrow or embryonic liver--from two donor strains differing antigenicatly from each other.) The idea was to discover whether mutual graftto-graft immunological tolerance which could be established in the given immunogenetic system when both the grafts were from embryonic donors could also be attained when one of them was derived from adult donors. Since in both cases one of the grafts contained a chromosome marker (T6), the establishment of tolerance could be tested by examining mitotic cells in haematopoietic and lymphoid tissues of the hosts whose own ceils had been destroyed by irradiation (Ford & Hamerton, i956 ). Co-existence of both types of cells (T6-1- and T 6 - - ) was taken as proof of mutual graft-tograft tolerance, whereas absence of one of them was attributed to the failure of the persisting graft to acquire tolerance of the other and to immune elimination of the missing cells. Full details of these experiments are given elsewhere (Lengerov~, Micklem & Dent, 1961; Clarke, Lengerov~i & Micklem, 196z ). The summarized results are presented in Table I. The considerable contrast between experiment x and z seems to indicate that in the given immunogenetic system it is hardly, if at all, possible to induce tolerance in immunologically mature ceil populations by exposing them to a single massive dose of replicating antigen; this applies both to the immediate and the gradual establishment of tolerance. Since the cell content of both grafts was similar and their close contact was secured by their shared homing instinct to the depleted spaces of host's myeloid and lymphoid tissues, the conditions for antigenic overloading of the reactive
Experiment
embryonic
adult 787
755]"
T6-
i + i ?
198]-
T6+
Relative frequency of dividing ceils in bone marrow, spleen and lymph nodes scored 5-zz weeks after inoculation
]" T 6 + cells were administered i - x o days after T 6 - - .
embryonic
embryonic
(CBA x A)F1 (T6--)
(C57B1 × T6T6) or (A × T6T6)F~ (T6+)
Origin of the grafts
Immunity of adult T 6 - against embryonic T6 + cells
Mutual tolerance betax'een embryonic T 6 - - and T6-b cells
Interpretation
Immunological interactions of two cellular grafts in the environment of lethally irradiated CBA mice
TABLE I
o
o o
o
508
A. L E N G E R O V A
cells were more favourable than in the host-to-graft system. They were, of course, far less favourable than in the graft-to-host system, but experimental evidence is missing of whether the absence of immediate loss of graft-to-graft reactivity is due to the quantitative or rather to the immunogenetic relationships involved. Two different circumstances might account for the fact that tolerance was not established in the adult graft even by gradual recruitment of a tolerant population from the stem cells maturing in the presence of antigen. First, this failure might be due to absence of potentially tolerant stem cells in adult bone marrow from which the adult graft was derived. Second, the reproducing capacity might not by itself be sufficient to allow the antigen (represented by the embryonic graft) to persist long enough to induce tolerance in newly differentiating stem cells from the adult graft. Since the mature cells from the latter obviously did not lose their immune reactivity at the first contact with the antigen, they would presumably eliminate the cells of the embryonic graft, which, being tolerant to the adult, would be unable to retaliate. It is quite possible that repeated administration might permit the cells of the embryonic graft to survive beside those originating from the adult. At any rate, immunological reactivity on the part of adult grafts need not generally be a favourable condition for its secondary loss as suggested by Simonsen (196o) for the graft-to-host system; it may rather interfere with or at least delay the process of establishment of tolerance in stem cells possibly only by competing for the antigen in question. REFERENCES BILLINGHAM,R. E., BRENT,L. & MEDAWAR,P. B. (I956). Phil. Trans. Roy. Soc. B239, 357. BURNET,F. M. (x 959). "The Clonal Selection Theory of Acquired Immunity". Cambridge University Press. CLARKE, C. M., LENGEROVA,A. & MICKLEM, H. S. (I962). Folia biol. (Praha) 8, in press. FORD, C. E. & HAMERTON,J. L. (i956). Stain Technol. 31, 247. GORER, P. A. & BOYSE, E. A. (1959)- Immunology 2, I82. LEDERBERO, J. (1958). ft. cell. comp. Physiol. 52, Suppl. I, 383. LEDERBERG, J. (1959). Science 129, 1649. LENGEROVA,A., MICKLEM, H. S. & DENT, T. (x96z). Folia biol. (Praha) 7, 3o9 • LOOTIT, J. F. (I956). In Lectures on the Scientific Basis of Medicine. Athlone Press, London 5, 439. MITCHISON, N. A. (1959). In "Biological Problems of Grafting". Blackwell, Oxford, p. 239. MITCHISON, N. A. (1962). In "Mechanisms of Immunological Tolerance". Publishing House of the Czech. Acad. Sci., Prague, in press. RUSSELL, P. S. (I962). In "Mechanisms of Immunological Tolerance". Publishing House of the Czech. Acad. Sci., Prague, in press. SIMONSEN, M. (1957). Acta Path. Microbiol. Scand. 4o, 48o. SIMONSEN, M. (x96o). Ann. N . Y . Acad. Sci. 73, 834. SIMONSEN, M. (I962). In "Mechanisms of Immunological Tolerance". Publishing House of the Czech. Acad. Sci., Prague, in press. SMITH, R. T. & BRIDGES,R. A. (I958). J. exp. 21¢ed. Io8, 227. SZILARD,L. (1960). Proc. nat. Acad. Sei., Wash. 46, 293.
The Significance of Immunological Immaturity for the Induction of Tolerancet A. LENGEROV~
Institute of Experimental Biology and Genetics, Czechoslovak Academy of Sciences, Prague, Czechoslovakia (Received 2 February 1962 and in revised form 23 May 1962 ) The mechanism underlying the induction of immunological tolerance in adult cell populations is discussed, and especially the question of whether this state of tolerance, established in different experimental systems, is based on the suppression of reactivity in functionally mature lymphoid cells or on their gradual replacement by a tolerant population derived from immature cells which acquired tolerance while differentiating in the presence of chronic antigenic stimulation.
In its original concept (BiUingham, Brent & Medawar, 1956), immunological tolerance was defined as a state of specific non-reactivity to a certain antigen or antigens displayed by an otherwise normally reactive individual, which is due to prior exposure to the same antigen before the maturation of the faculty of immune response. According to this concept, the tolerance responsive phase of development of an individual is confined fairly strictly to a period coinciding roughly with its foetal life, whereas immunity is the regular reaction to be expected if the individual is confronted with the same antigenic stimulus for the first time while already immunologically mature. This duality of reaction to antigen as manifested at the level of the organism might obviously reflect two qualitative phases in the functional development of its lymphoid cells, on which antigen acts first as an inhibitor and later as an inducer of specific immune response. Further analysis casts some doubt on this simple scheme. For example, the question arises as to whether immunological maturity detected at the level of the whole organism fully expresses the stage of development of all its lymphoid cells or only that of the majority. Immunological maturation of a lymphoid cell might be defined by its progression from sensitivity to reactivity (Lederberg, 1959). The immature cell being rather sensitive, exposure to antigen could prevent it from developing further; this might be more or less drastic interference based on destruction of the cell (Burnet, 1959) or on some sort of functional block (Szilard, 196o ). t This investigation was supported by the International Atomic Energy Agency. 503
5o4
A.
LENGEROV/~
Immunological maturation of an individual might thus be visualized by a set of distribution curves expressing the relative frequency of successive types of differentiating lymphoid cells (Fig. i). In an immunologicaUy immature individual all the lymphoid cells might evidently be in the tolerance responsive phase of their development (curves (a)a, (b)a); this would account for the relative ease with which tolerance can be induced in such Co)
(b)
0
0 Q
i
" 0 0
C
b
Successive types of differentiating lymphoid cells
FIO. I. Hypothetical distributions of lymphoid cells in an immunologicallymaturing individual, a: prenatal period, b: postnatal period, c: adult age. 0 Tolerance-responsivecells (or cells with low requirementsfor tolerance-inducingdose of antigen). • Immunity responsive cells (or cells with extremely high requirements for toleranceinducing dose of antigen whose reaction to lower amount of antigen is immunity). (~ Transient type. organisms. If the whole cell population then matured simultaneously ((a), curves b, c) the mature individual would contain only mature lymphoid cells (c) reacting uniformly when stimulated with a given dose of antigen. If, however, new mutant ctones arose (Burnet, 1959) or a stem cell reserve were present (Loutit, 1956; Lederberg, 1958 ) from which reactive cells could differentiate throughout the animal's life, then in later developmental stages of an individual the population of lymphoid cells would be heterogeneous ((b), curves b, c) containing in different proportions all the successive cell types. To maintain tolerance would then virtually mean inducing it in newly arising or differentiating cells. This would explain the part taken by chronic antigenic stimulation in the maintenance of tolerance and its disappearance after the dissipation of non-replicating antigen (Smith & Bridges, 1958; Mitchison, 1959). Under this condition and that of the limited life-span of mature lymphoid cells, the tolerance responsive phase would not be confined to the perinatal period. It should therefore be possible to confer tolerance on adults on the same principle as on newborn
TOLERANCE AND I M M U N O L O G I C A L M A T U R I T Y
505
individuals except for the time and amount of antigen required. The antigen used to induce tolerance would always get partly lost owing to immune elimination by the reactive cells and its level would have to be maintained by repeated administration. However, the stock of mature cells would gradually be depleted and replaced by a tolerant population of cells differentiating under the effect of antigen. As tolerance is not detectable as something positive but only as a lack of reactivity it should obviously take some time before it could be revealed at the level of the organism, where it would be masked by immunity as long as non-tolerant cells survived. The rather long times taken for gain and loss of tolerance in recent experiments by Mitchison (1962) are interpreted by this author as an objection to the possibility of mere accumulation and degradation of antigen by a homogeneous cell population and in favour of cell turnover as a mechanism involved in the induction of tolerance in adult cells. Simonsen (I96Z), however, found that immunologically competent cells derived from adult mouse spleen can be rendered tolerant to antigens of their newborn hosts in as short a time as twenty-four hours; cell turnover and population replacement can, of course, scarcely be held responsible for this immediate effect. A cell population which becomes non-reactive at such a rate apparently still contains the formerly reactive cells in a state of functional block or exhaustion. If this were the whole explanation it would be pointless to assume the existence of a stem-cell reserve in an adult organism to account for the occurrence of tolerance in Simonsen's system. In that case, adult mouse spleen might contain only mature lymphoid cells as expressed by curve (a)c; the question arises, of course, whether the mechanism of non-reactivity established here in formerly reactive cells is identical with that operating in immature lymphoid cells or whether there is another mechanism involved. If the capacity of being rendered tolerant were retained throughout the life of every lymphoid cell, then not the time of introduction of antigen but rather the quantity of antigen might be the decisive factor between tolerance and immunity. The quantitative requirements of the reactive cells might be much higher than those of immature cells but in spite of it the accumulation of large amounts of antigen (if available at once) they might be accomplished rather rapidly. In that case, the establishment of tolerance in adult cells would not necessarily be a longterm process. If, however, lymphoid cells undergo qualitative changes during differentiation and their tolerance responsive phase is limited, then Simonsen's finding of immediate loss of reactivity in adult cells might be a less general phenomenon due possibly to something specific for his experimental system. One factor to be considered here might be the quantitative relationship between the antigen (represented by the whole organism) and the reactive site (represented by a relatively small inoculum),
506
A. LENGEROV,~
which is hard to achieve in any other system. The removal of reactive cells which would otherwise die out slowly might be here accelerated by the excess of antigen on a different principle (Gorer & Boyse, 1959; Simonsen, 196o). In some graft-to-host systems, however, the reactivity of immunologically competent ceils could be maintained for three to five transfers (Simonsen, 1957) or was shown to persist several days in the first recipient (Russell, 1962); thus the immediate loss of reactivity is not a general phenomenon even here. At any rate, the immediate inhibition of the reactive ceils, for example, by the excess of antigen in certain immunogenetic combinations, does not exclude their gradual replacement by a tolerant population recruited from the immature proportion of transferred spleen ceils. It might be of some interest to discuss in this connection a few data obtained in a third experimental system which from the quantitative point of view lies just between the two above extreme cases where the reactive site and antigen were represented by host and graft or vice versa. In this third system) the interactions were followed of two replicating mesenchymal cell grafts in the environment of a non-reactive host. (Lethally irradiated mice were restored by a pooled inoculum of haematopoietic cells--adult bone marrow or embryonic liver--from two donor strains differing antigenicatly from each other.) The idea was to discover whether mutual graftto-graft immunological tolerance which could be established in the given immunogenetic system when both the grafts were from embryonic donors could also be attained when one of them was derived from adult donors. Since in both cases one of the grafts contained a chromosome marker (T6), the establishment of tolerance could be tested by examining mitotic cells in haematopoietic and lymphoid tissues of the hosts whose own ceils had been destroyed by irradiation (Ford & Hamerton, i956 ). Co-existence of both types of cells (T6-1- and T 6 - - ) was taken as proof of mutual graft-tograft tolerance, whereas absence of one of them was attributed to the failure of the persisting graft to acquire tolerance of the other and to immune elimination of the missing cells. Full details of these experiments are given elsewhere (Lengerov~, Micklem & Dent, 1961; Clarke, Lengerov~i & Micklem, 196z ). The summarized results are presented in Table I. The considerable contrast between experiment x and z seems to indicate that in the given immunogenetic system it is hardly, if at all, possible to induce tolerance in immunologically mature ceil populations by exposing them to a single massive dose of replicating antigen; this applies both to the immediate and the gradual establishment of tolerance. Since the cell content of both grafts was similar and their close contact was secured by their shared homing instinct to the depleted spaces of host's myeloid and lymphoid tissues, the conditions for antigenic overloading of the reactive
Experiment
embryonic
adult 787
755]"
T6-
i + i ?
198]-
T6+
Relative frequency of dividing ceils in bone marrow, spleen and lymph nodes scored 5-zz weeks after inoculation
]" T 6 + cells were administered i - x o days after T 6 - - .
embryonic
embryonic
(CBA x A)F1 (T6--)
(C57B1 × T6T6) or (A × T6T6)F~ (T6+)
Origin of the grafts
Immunity of adult T 6 - against embryonic T6 + cells
Mutual tolerance betax'een embryonic T 6 - - and T6-b cells
Interpretation
Immunological interactions of two cellular grafts in the environment of lethally irradiated CBA mice
TABLE I
o
o o
o
508
A. L E N G E R O V A
cells were more favourable than in the host-to-graft system. They were, of course, far less favourable than in the graft-to-host system, but experimental evidence is missing of whether the absence of immediate loss of graft-to-graft reactivity is due to the quantitative or rather to the immunogenetic relationships involved. Two different circumstances might account for the fact that tolerance was not established in the adult graft even by gradual recruitment of a tolerant population from the stem cells maturing in the presence of antigen. First, this failure might be due to absence of potentially tolerant stem cells in adult bone marrow from which the adult graft was derived. Second, the reproducing capacity might not by itself be sufficient to allow the antigen (represented by the embryonic graft) to persist long enough to induce tolerance in newly differentiating stem cells from the adult graft. Since the mature cells from the latter obviously did not lose their immune reactivity at the first contact with the antigen, they would presumably eliminate the cells of the embryonic graft, which, being tolerant to the adult, would be unable to retaliate. It is quite possible that repeated administration might permit the cells of the embryonic graft to survive beside those originating from the adult. At any rate, immunological reactivity on the part of adult grafts need not generally be a favourable condition for its secondary loss as suggested by Simonsen (196o) for the graft-to-host system; it may rather interfere with or at least delay the process of establishment of tolerance in stem cells possibly only by competing for the antigen in question. REFERENCES BILLINGHAM,R. E., BRENT,L. & MEDAWAR,P. B. (I956). Phil. Trans. Roy. Soc. B239, 357. BURNET,F. M. (x 959). "The Clonal Selection Theory of Acquired Immunity". Cambridge University Press. CLARKE, C. M., LENGEROVA,A. & MICKLEM, H. S. (I962). Folia biol. (Praha) 8, in press. FORD, C. E. & HAMERTON,J. L. (i956). Stain Technol. 31, 247. GORER, P. A. & BOYSE, E. A. (1959)- Immunology 2, I82. LEDERBERO, J. (1958). ft. cell. comp. Physiol. 52, Suppl. I, 383. LEDERBERG, J. (1959). Science 129, 1649. LENGEROVA,A., MICKLEM, H. S. & DENT, T. (x96z). Folia biol. (Praha) 7, 3o9 • LOOTIT, J. F. (I956). In Lectures on the Scientific Basis of Medicine. Athlone Press, London 5, 439. MITCHISON, N. A. (1959). In "Biological Problems of Grafting". Blackwell, Oxford, p. 239. MITCHISON, N. A. (1962). In "Mechanisms of Immunological Tolerance". Publishing House of the Czech. Acad. Sci., Prague, in press. RUSSELL, P. S. (I962). In "Mechanisms of Immunological Tolerance". Publishing House of the Czech. Acad. Sci., Prague, in press. SIMONSEN, M. (1957). Acta Path. Microbiol. Scand. 4o, 48o. SIMONSEN, M. (x96o). Ann. N . Y . Acad. Sci. 73, 834. SIMONSEN, M. (I962). In "Mechanisms of Immunological Tolerance". Publishing House of the Czech. Acad. Sci., Prague, in press. SMITH, R. T. & BRIDGES,R. A. (I958). J. exp. 21¢ed. Io8, 227. SZILARD,L. (1960). Proc. nat. Acad. Sei., Wash. 46, 293.