Transplant. Rev. (1975), Vol. 26 Published by Munksgaard, Copenhagen, Denmark No part may be reproduced by any process without written permission from the aulhor(s)
The Role of Specific Suppressor T Cells in Immune Tolerance D. NACHTIGAL, I. ZAN-BAR & M. FELDMAN
BACKGROUND The mechanism of immune tolerance is both one of the most studied and one of the most controversial fields of immunological research. The first attempt at proposing a coherent explanation for this phenomenon is to be credited to Bumet (1959), who formulated his hypothesis as an offshoot of the clonal selection theory of acquired immunity, claiming that selective elimination of antigen-oriented clones may explain all types of specific unresponsiveness. Burnet's hypothesis provides a very elegant and relatively simple explanation for the phenomenon of immune tolerance, and since it was formulated during the heyday of molecular genetics, its basically genetical orientation captured the imagination of immunologists at that time. However, his interpretation has so far found no direct experimental support, and up till now it remains mainly of a speculative nature. Evidence has been accumulating in the meantime, which indicates that at least some models of unresponsiveness do not fit into Burnet's suggested pattern of specific deletion. This followed from the finding that, in some cases of induced tolerance, the capacity to respond to the tolerizing antigen could be restored rapidly in lymphoid cells of tolerant donor animals, in most instances by manipulating the respective lymphocytes in vitro (Hemphill et al. 1966, McGregor et al. 1967, Intini et al. 1971, Sjoberg 1972). Moreover, this seems also to be true for natural 'self' tolerance (Cohen et al. 1971, 1972). Thus, an altemative explanation was suggested, namely that tolerance may result from an inherently reversible specific block of potentially immunocompetent cells. Interesting insight into a possible suppressive mechanism operating in tolDepartment of Cell Biology, Tbe Weizmann Institute of Science, Rebovot, Israel. Supported by a grant from tbe Thyssen Foundation.
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D. NACHTIGAL, I. ZAN-BAR & M. FELDMAN
erance was provided by McCulIagh (1970a, b) who studied rats made tolerant to sheep red blood cells (SRBC). He confirmed the well-documented observation that induced tolerance cannot usually be reversed by supplementing the tolerant animals with normal lymphoid cells from non-tolerant donors, unless the recipients are sub-lethally irradiated before grafting. This limitation used to be interpreted as a requirement to create a 'physiological space' for the grafted cells, by means of reducing the recipients' lymphoid cell population. However, McCulIagh demonstrated that grafted normal thoracic duct lymphocytes (TDL) did 'take' in tolerant rats without previous irradiation and could be shown to be specifically competent on sub-transfer from tolerant to normal lethally irradiated rats. McCulIagh concluded that the reason for the failure to restore tolerant rats with normal syngeneic cells lies in an 'inimical factor' to responsiveness, which operates in the tolerant hosts. The existence of a specifically suppressive mechanism in tolerance, as well as the evidence of its potential reversibility, suggested that viable 'tolerant' cells may actually exist. Since such suppressive mechanisms, tmlike mechanisms of clonal deletion, lend themselves relatively easily to experimental analysis, we considered this approach as an auspicious point of attack for the study of immune tolerance. The following review sums up the available evidence concerning the role of suppressor T cells in specific unresponsiveness within the frame of our own observations in the field. SUPPRESSED MEMORY IN TOLERANT MICE It has been widely reported (Glenny & Hopkins 1924, Brent & Gowland 1962, Siskind & Howard 1966, Nossal & Austin 1966) that induction of tolerance by antigen stimulation in the 'high zone' is accompanied by a transient, low-level, immune response. When we induced tolerance to human serum albumin (HSA) in a strain of adult C3H/ebw mice, we foimd that the animals developed allergic sensitivity to the tolerizing antigen, and had to be desensitized before each antigen injection to protect them from lethal shock (Zan-Bar et al. 1974). By injecting a parallel sample group of mice without desensitizing them first, the kinetics of both transient antibody foraiation and deaths from allergic shock could be followed during tolerization (Figure 1). The data demonstrate a transitory rise in antibody titer, which reaches its peak on week 5 of the induction process, while deaths from shock reach 100 % on week 4. The allergic sensitivity decreases rapidly to zero on week 6, while the antibody response drops practically to zero level at the end of eight weeks. Thus, it was evident that a distinct stimulation of immune processes preceded, in this case, the establishment of unresponsiveness. This presented a
89
T SUPPRESSORS IN TOLERANCE
problem: what was the eventual fate of the immunocompetent cells which had been primed before tolerance supervened? Speculating along the lines of the clonal elimination hypothesis, one would assume that the respective cells died out because of over-stimulation as a result of which the specific clone was eradicated (Jensen & Simonsen 1963). Alternatively, tolerance in the model described might represent a situation in which potentially-competent cells remain in a specifically suppressed state. The possibility would then exist that the primed cells might survive as memory cells in a mode of suppression. Since tolerant mice can be restored by reconstitution with normal, syngeneic spleen cells after sub-lethal gamma irradiation, the response of such reconstituted mice was compared with that of normal non-tolerant controls identically treated. It was postulated that the primed cells of tolerant animals, if they survived, might be relatively radio-resistant, since they would represent an expanded clone (Katz & Benacerraf 1972). Indeed, experiments employing differential gamma irradiation revealed that tolerant mice, when given low irradiation doses and reconstituted with normal spleen, were hypo-responsive in comparison with non-tolerant controls up to a total-body irradiation dose of about 600 R (Figure 2). When, however, the dose of irradiation was in-
_ E
IMMUNE RESPONSE DEATHS FROM SHOCK
3.0
I"
100
O CD
The Role of Specific Suppressor T Cells in Immune Tolerance D. NACHTIGAL, I. ZAN-BAR & M. FELDMAN
BACKGROUND The mechanism of immune tolerance is both one of the most studied and one of the most controversial fields of immunological research. The first attempt at proposing a coherent explanation for this phenomenon is to be credited to Bumet (1959), who formulated his hypothesis as an offshoot of the clonal selection theory of acquired immunity, claiming that selective elimination of antigen-oriented clones may explain all types of specific unresponsiveness. Burnet's hypothesis provides a very elegant and relatively simple explanation for the phenomenon of immune tolerance, and since it was formulated during the heyday of molecular genetics, its basically genetical orientation captured the imagination of immunologists at that time. However, his interpretation has so far found no direct experimental support, and up till now it remains mainly of a speculative nature. Evidence has been accumulating in the meantime, which indicates that at least some models of unresponsiveness do not fit into Burnet's suggested pattern of specific deletion. This followed from the finding that, in some cases of induced tolerance, the capacity to respond to the tolerizing antigen could be restored rapidly in lymphoid cells of tolerant donor animals, in most instances by manipulating the respective lymphocytes in vitro (Hemphill et al. 1966, McGregor et al. 1967, Intini et al. 1971, Sjoberg 1972). Moreover, this seems also to be true for natural 'self' tolerance (Cohen et al. 1971, 1972). Thus, an altemative explanation was suggested, namely that tolerance may result from an inherently reversible specific block of potentially immunocompetent cells. Interesting insight into a possible suppressive mechanism operating in tolDepartment of Cell Biology, Tbe Weizmann Institute of Science, Rebovot, Israel. Supported by a grant from tbe Thyssen Foundation.
88
D. NACHTIGAL, I. ZAN-BAR & M. FELDMAN
erance was provided by McCulIagh (1970a, b) who studied rats made tolerant to sheep red blood cells (SRBC). He confirmed the well-documented observation that induced tolerance cannot usually be reversed by supplementing the tolerant animals with normal lymphoid cells from non-tolerant donors, unless the recipients are sub-lethally irradiated before grafting. This limitation used to be interpreted as a requirement to create a 'physiological space' for the grafted cells, by means of reducing the recipients' lymphoid cell population. However, McCulIagh demonstrated that grafted normal thoracic duct lymphocytes (TDL) did 'take' in tolerant rats without previous irradiation and could be shown to be specifically competent on sub-transfer from tolerant to normal lethally irradiated rats. McCulIagh concluded that the reason for the failure to restore tolerant rats with normal syngeneic cells lies in an 'inimical factor' to responsiveness, which operates in the tolerant hosts. The existence of a specifically suppressive mechanism in tolerance, as well as the evidence of its potential reversibility, suggested that viable 'tolerant' cells may actually exist. Since such suppressive mechanisms, tmlike mechanisms of clonal deletion, lend themselves relatively easily to experimental analysis, we considered this approach as an auspicious point of attack for the study of immune tolerance. The following review sums up the available evidence concerning the role of suppressor T cells in specific unresponsiveness within the frame of our own observations in the field. SUPPRESSED MEMORY IN TOLERANT MICE It has been widely reported (Glenny & Hopkins 1924, Brent & Gowland 1962, Siskind & Howard 1966, Nossal & Austin 1966) that induction of tolerance by antigen stimulation in the 'high zone' is accompanied by a transient, low-level, immune response. When we induced tolerance to human serum albumin (HSA) in a strain of adult C3H/ebw mice, we foimd that the animals developed allergic sensitivity to the tolerizing antigen, and had to be desensitized before each antigen injection to protect them from lethal shock (Zan-Bar et al. 1974). By injecting a parallel sample group of mice without desensitizing them first, the kinetics of both transient antibody foraiation and deaths from allergic shock could be followed during tolerization (Figure 1). The data demonstrate a transitory rise in antibody titer, which reaches its peak on week 5 of the induction process, while deaths from shock reach 100 % on week 4. The allergic sensitivity decreases rapidly to zero on week 6, while the antibody response drops practically to zero level at the end of eight weeks. Thus, it was evident that a distinct stimulation of immune processes preceded, in this case, the establishment of unresponsiveness. This presented a
89
T SUPPRESSORS IN TOLERANCE
problem: what was the eventual fate of the immunocompetent cells which had been primed before tolerance supervened? Speculating along the lines of the clonal elimination hypothesis, one would assume that the respective cells died out because of over-stimulation as a result of which the specific clone was eradicated (Jensen & Simonsen 1963). Alternatively, tolerance in the model described might represent a situation in which potentially-competent cells remain in a specifically suppressed state. The possibility would then exist that the primed cells might survive as memory cells in a mode of suppression. Since tolerant mice can be restored by reconstitution with normal, syngeneic spleen cells after sub-lethal gamma irradiation, the response of such reconstituted mice was compared with that of normal non-tolerant controls identically treated. It was postulated that the primed cells of tolerant animals, if they survived, might be relatively radio-resistant, since they would represent an expanded clone (Katz & Benacerraf 1972). Indeed, experiments employing differential gamma irradiation revealed that tolerant mice, when given low irradiation doses and reconstituted with normal spleen, were hypo-responsive in comparison with non-tolerant controls up to a total-body irradiation dose of about 600 R (Figure 2). When, however, the dose of irradiation was in-
_ E
IMMUNE RESPONSE DEATHS FROM SHOCK
3.0
I"
100
O CD
