Suppressor cells in tolerance to HGG: kinetics and cross-suppression in high dose tolerance--absence in low dose tolerance.

Spleen cells from mice made tolerant with high doses of human gamma-globulin (HGG) specifically suppress the immune response of normal, syngeneic, spleen cells. These suppressor cells were found to be cross-reactive in that they would suppress the immune response of normal spleen cells to bovine gamma-globulin (BGG) as well as to HGG. In contrast, suppressor cells could not be demonstrated in spleens of mice made tolerant with low doses of HGG (i.e., T-cell tolerance), nor could they be found in high dose tolerant mice following a second injection of DHGG at a time when the initial suppressor activity had waned. The role of suppressor cells in the induction, maintenance, and loss of tolerance is discussed.     

Tolerance and contact sensitivity to DNFB in mice. VI. Inhibition of afferent sensitivity by suppressor T cells in adoptive tolerance.

Tolerance in contact sensitivity to DNFB can be adoptively transferred to normal mice with lymph node cells from tolerant donors. This tolerance is antigen specific and is mediated by T cells, i.e., "suppressor" T cells. Experiments were carried out to investigate the mechanism(s) by which the suppressor T cells induce tolerance to DNFB contact sensitivity. The suppressor cells were effective only if they were present during the early stages of the afferent limb of sensitization. As measured by DNA synthesis, cell proliferation in the draining lymph nodes of recipients of suppressor cells was found to be significantly less than in control animals indicating that the suppressor cells acted, at least in part, by limiting or inhibiting DNFB-induced cell proliferation. This inhibition was shown to be antigen specific since the DNFB suppressor cells did not inhibit cell proliferation induced by oxazolone, an unrelated contact sensitizer. The ability to DNFB tolerant cells to block afferent sensitization pathways differs from the mechanism of tolerance to picryl chloride, reported by others, where efferent pathways are blocked.     

Immunologic tolerance to HGG in mice. I. Suppression of the HGG response in normal mice with spleen cells or a spleen cell lysate from tolerant mice.

Adoptive transfer of spleen cells or spleen cell lysates from mice tolerant to human-gamma-globulin (HGG) specifically suppressed the response of normal syngeneic recipients to HGG. The suppressive activity could be transferred for over 100 days after tolerance induction. The suppression induced by both spleen cells and spleen cell lysate was found to be specific as evidenced by a normal response to a challenge with turkey-gamma-globulin or goat erythrocytes. The activity of the suppressive lysate could be removed by passing the material through an HGG immunoadsorbent column but not by passing it through an anti-HGG column or a BSA column. These results indicated that the factor had antigen specificity and was probably not antigen-antibody complexes. That this suppression was not due to a shifting of the kinetics of the antibody response has also been demonstrated. The antigen-specific suppressor factor in the tolerant spleen cell lysates was a protein with a m.w. of approximately 45,000 daltons. The kinetics of the appearance of both suppressor cells and suppressor factor were consistent with a mechanism of active suppression functioning in the maintenance of tolerance to HGG.     

Induction of immunological tolerance against sheep red blood cells in A/J mice by maternal immunization

Female A/J mice were immunized with sheep red blood cells (SRBC) before mating and boosted a few days before delivery. The progeny of these mothers was immunologically tolerant against SRBC at the level of plaque forming cells (PFC). The state of unresponsiveness was antigen specific. Exchange of the newborn mice between control mothers and immunized ones shows clearly that the tolerance is induced by factors present in the milk or the colostrum, respectively. Some others findings suggest that antibodies of the mothers and not small amounts of the injected antigen are responsible for the nearly complete suppression of the immune response of the offspring.     

Abrogation of the capacity of delayed-type hypersensitivity responses to alloantigens by intravenous injection of neuraminidase-treated allogeneic cells

BALB/c or C3H/He mice were inoculated i.v. with allogeneic spleen cells untreated or treated with neuraminidase. Appreciable or potent anti-allo-delayed-type hypersensitivity (DTH) responses were observed when mice were inoculated i.v. with untreated allogeneic cells or inoculated i.v. with those cells followed by s.c. immunization with untreated allogeneic cells. In contrast, i.v. inoculation of neuraminidase-treated allogeneic cells (presensitization) not only failed to induce any significant anti-allo-DTH responses but also abolished the capability of the animals to develop DTH responses after s.c. immunization, indicating the tolerance induction. This tolerance was alloantigen-specific, and rapidly inducible and long lasting. The induction of suppressor cell activity was demonstrated in tolerant mice. However, this activity was associated only with the tolerant state around 4 to 7 days after the i.v. presensitization, but was no longer detected in mice more than 14 days after the presensitization, although these mice exhibited complete tolerant state. When spleen cells from such tolerant mice were transferred i.v. into 600 R x-irradiated syngeneic recipient mice alone or together with normal syngeneic spleen cells, these tolerant spleen cells themselves failed to induce DTH responses but did not exhibit suppressive effect on the generation of DTH responses induced by normal spleen cells co-transferred. These results indicate that i.v. administration of neuraminidase-treated allogeneic cells results in the induction of alloantigen-specific tolerance which is not always associated with the induction of suppressor cell activity but rather with the elimination or functional impairment of alloantigen-specific clones.     

Thymus dependency of neonatal tolerance induction in the absence of detectable suppressor cells

Neonatal thymectomy prevents tolerance induction with bovine serum albumin (BSA) in Wistar Furth (WF) rats whose thymus-derived (T) cell deficit is reconstituted with adult nonadherent peripheral blood lymphocytes (PBL). Sham-thymectomized (STx) rats given PBL become tolerant. To establish whether the adult T cells become tolerant in STx rats, their carrier-reactivity was studied in a cooperative immune response following challenge with methylated BSA (mBSA). The results indicate that carrier-reactive cells, derived from PBL, do become tolerant of BSA in the presence, but not in the absence, of the thymus. To determine whether thymic function during tolerance induction is mediated by suppressor T cells, attempts were made to replace the thymus with various populations of thymocytes or lymphoid cells from neonatal or adult normal rats or neonatal BSA-injected rats. No cell population tried could substitute for the thymus during tolerance induction. In addition, it was found that BSA-tolerant rats with intact thymi do not contain either nonspecific suppressor cells whose activity can be boosted with mBSA or specific suppressor activity demonstrable on transfer to normal rats. Timed thymectomy experiments showed that the thymus is required for more than 2, but less than 5 to 7 days after tolerogen injection for significant tolerance induction. These results imply that the thymus itself is necessary for tolerance induction in a peripheral T-cell population and that its effect is not mediated by suppressor cells. It is suggested that peripheral T helper cells may periodically recirculate through the thymus, at least in young rats, and become tolerant of antigen complexed with Ia antigens in the thymic epithelium. Such a mechanism may be of great importance in the development of self-recognition.     

Reversal of immunological tolerance by aclacinomycin through inhibition of suppressor cell activity

The elimination of suppressor cells by aclacinomycin, which could be the mechanism by which immune responses are enhanced after its administration, was studied in mice in which tolerance had been induced by the injection of high doses of sheep red blood cells (SRBC). We observed that tolerance could not be induced in aclacinomycin-treated mice, and that aclacinomycin inhibited the expression of tolerance to SRBC. This drug also diminished the capacity of spleen cells from SRBC-tolerant mice to inhibit the response of normal animals upon adoptive transfer, indicating that suppressor cells had been eliminated from the tolerant spleen cell population. The efficiency of the elimination of suppressor cells for DTH reactions appears greater than that of suppressor cells for plaque-forming cell responses.     

Tolerance induced by TNP-derivatized syngeneic erythrocytes: evidence for cooperation between hapten-specific T and hapten-specific B lymphocytes in the immune response.

Tolerance to the DNP haptenic determinant was induced with a single i.v. injection of trinitrophenylated syngeneic red blood cells. The tolerant state lasted 1 month and was stable on transfer to irradiated thymectomized syngeneic recipients. Suppressor activity was found soon after injection of tolerogen but was lost before the termination of tolerance. The unresponsive state could be reversed by adding normal thymus cells to tolerant spleen cells but not by normal bone marrow cells. LPS when given with immunogen restored the normal immune response in tolerant mice. Thus the injection of TNP-MRBC induced partial immune unresponsiveness which was characterized by the induction of T cell suppressor activity and by a hapten-specific helper T cells tolerance. Finally, these studies suggest a cooperative interaction between DNP-specific T lymphocytes and DNP-specific B lymphocytes in the immune response to DNP-BGG.     

Specific, transient suppression of the immune response by HGG tolerant spleen cells. II. Effector cells and target cells.

In a previous report, it was shown that spleen cells from mice made tolerant to human gamma-globulin (HGG)5 could specifically inhibit the immune response of normal spleen cells after adoptive transfer to lethally irradiated recipients. However, that report also showed that the suppressive activity was only transiently associated with tolerant spleen cell populations. It was concluded from those experiments that while suppressive activity could be demonstrated in tolerant spleen cells under certain conditions, such activity was not obligatory for the maintainance of the tolerant state. The experiments presented here were performed to determine the nature of the effector cell(s) and the target cell(s) involved in this system of suppression of the immune response. Treatment of cells from tolerant animals with anti-thymocyte serum and complement to remove thymus-derived (T) cells completely abrogated suppresive activity. Removal of adherent cells from tolerant spleen cells by passage over glass wool columns resulted in partial loss of the suppression. The inhibitory activity of the suppressor cells was resistant to 900 R irradiation regardless of whether the tolerant spleen cells were irradiated before or after adoptive transfer. The cellular target(s) for the supprssor cells was examined by using lipopolysaccharide (LPS) as an alternative source of helper activity for the response to HGG. LPS, injected at the time of the initial antigenic challenge of mice that had been reconstituted with tolerant and normal spleen cells, prevented the expression of suppression against bone marrow-derived (B) cells. However, when LPS was presented only at the time of secondary antigenic challenge, it was unable to overcome suppression of the immune response of reconstituted recipients. Thus, LPS could produce a state where the B cells were resistant to suppression, but LPS could not rescue the responsiveness of B cells once the cells in the reconstituted recipient had been suppressed. In addition, the immune response to both the hapten dinitrophenol (DNP) and the carrier (HGG) were suppressed when recipients of tolerant and normal spleen cells were challenged with DNP6HGG. This indicates that T helper cells are also a target for suppression. The results presented in this paper are discussed in relation to a possible mechanism of suppression which proposes that suppressive activity represents the induction of tolerance in immunologically competent cells by HCG which is closely associated with the tolerant spleen cells.     

Studies on the mechanism of transplantation tolerance: I. Do suppressor cells play a role in the induction and maintenance of neonatal transplantation tolerance?

Neonatal transplantation tolerance was induced in CBA (H-2^k) mice to A (H-2^a) mice by injection of (CBA × A)F₁ spleen cells. Animals carrying an A-skin test allograft for more than 4 months without any visible sign of rejection were considered to be permanently tolerant. Permanently tolerant CBA mice were given normal syngeneic spleen cells to abrogate the state of tolerance. Abrogation of tolerance was greatly facilitated by antithymocyte serum (ATS) treatment of tolerant mice prior to the normal syngeneic cell transfer. Survival of A allografts on normal, adult, ATS-treated CBA mice was significantly prolonged (and in many cases “adult” tolerance was achieved) by transfer of spleen cells of syngeneic mice made permanently tolerant at neonatal age. The possible role of the F₁-cell “contamination” in the tolerance-inducing effect of the transferred “tolerant” spleen cells was excluded. The results indicate that ATS-sensitive suppressor cells play a definite role in the induction, maintenance, and transfer of neonatally induced transplantation tolerance.     

Direct demonstration of specific suppressor T cells in the mice tolerant to type III pneumococcal polysaccharide: two-step requirement for development of detectable suppressor cells

Spleen cells from CAF1 mice made tolerant to type III pneumococcal polysaccharide (S3) with S3 coupled to syngeneic spleen cells (S3-SC) develop S3-specific suppressor T cells (Ts). These Ts could be demonstrated consistently only when spleen cells from tolerant mice were cultured in vitro with the specific antigen and the specific tolerogen. Spleen cells from normal mice cultured under the same conditions did not suppress the antibody response to S3. When different numbers of Ts were transferred to normal CAF1 mice, an unusual dose-effect pattern was observed. Maximal suppression of the S3 response occurred when relatively low numbers of Ts, 3 to 30 x 10(5) per recipient, were transferred, whereas larger numbers of cells, 150 x 10(5) per recipient, were not suppressive. These results indicate that a presumably T-independent antigen, S3, can activate antigen-specific Ts. These Ts exhibit unusual dose effects upon transfer and require both an in vivo induction period and in vitro activation for development of maximal activity. These latter observations suggest that S3 may activate a different population of T cells with suppressor function than do conventional T-dependent antigens. The loss of suppression observed when greater than optimal numbers of cells were transferred suggests that a second type of T cell, which has the ability to 'neutralize' the activity of S3-specific Ts, is also induced in the same spleen cell population.     

Induction of immunologic tolerance in nursing neonates by absorption of tolerogen from colostrum.

Deaggregatedhuman gamma-globulin (DHGG) injected into female mice within 24 hr after delivery of a litter enters the colostrum and is absorbed intact through the intestine by nursing neonates. This absorbed HGG was present in the neonatal circulation at concentrations of 0.3 to 0.6 mg/ml of serum under the experimental conditions used. This absorption of HGG by the nursing neonate resulted in a complete, specific, tolerant state to HGG. This tolerant state was stable upon adoptive cell transfer and could not be abrogated by transfer of normal syngeneic spleen cells.     

Supressor activity of spleen cells during medically induced immunologic tolerance]

SRBC tolerance was induced in mice (CBA X C57BL/6) F1 by single intraperitoneal injection of 6 X 10(9) SRBC and of cyclophosphamide (100-200 mg/kg) in 44-46 hours. Spleen cells of tolerant mice obtained at various periods after the tolerance induction (in 12-26 days) failed to decrease their immune response to SRBC after administration to intact syngeneic recipients. Contrary to intact mice, tolerant animals were incapable of producing suppressor cells after a single SRBC immunization. Only when 3 additional injections of high SRBC doses (6 X 10(9)) were given to tolerant mice the spleen cells in them acquired the capacity to inhibit the immune response after administration to normal mice. It is supposed that the absence of suppressor cells in induction of the immunological tolerance by means of cyclophosphane was caused by the processes of clone elimination. Suppressor cells can originate in tolerant animals under the effect of intensive antigenic stimulation, this leading to enhancement of the tolerance state as a result of additional SRBC injections.     

Immunologic tolerance to dinitrophenylated human gamma globulin induced via colostrum

Immunologic unresponsiveness or tolerance was induced in neonatal mice via colostrum by injection of dinitrophenylated human gamma globulin (DNP-HGG) into the mother on the day of birth. Unresponsiveness persisted in the neonates for at least 21 weeks. This longlasting tolerance appeared to be the result of an unresponsiveness to the carrier determinants. Hapten-specific B-cell tolerance was assessed in mice receiving high- or low-epitope-density tolerogen and it was observed that the low-epitope-density tolerogen (DNP₁HGG) resulted in carrier-specific tolerance only. Although mice tolerized with the high-epitope-density conjugates were found to be slightly hyporesponsive in their in vivo B-cell responses, their in vitro hapten-specific responses were normal. This tolerant state induced via colostrum was compared to tolerance induced in utero. This earlier contact with tolerogen resulted in more profound alterations in hapten-specific B-cell responses. An additional interesting finding was that the colostrally induced tolerant state was transmitted to the next generation.     

An in vitro model for induction of immunologic unresponsiveness to turkey gamma-globulin in primed mouse spleen cells, II. Antigen-specific suppressor cells.

Unresponsiveness induced to turkey γ-globulin (TGG) in cultures of TGG-primed spleen cells by incubation with high concentrations of soluble TGG (sTGG) was shown to involve a state of active suppression. Upon transfer to secondary cultures of primed spleen cells stimulated with an optimal dose of TGG-conjugated erythrocytes, such tolerant spleen cells were able to actively inhibit a secondary plaque-forming cell response to TGG in these cultures. Almost complete inhibition was observed with a tolerant cell to primed cell ratio of as low as 0.1. The suppression was antigen specific in that tolerant spleen cells which were suppressive for the secondary TGG response were unable to inhibit a primary response to sheep erythrocytes. T cells were shown to be required for the suppressor effect, in that (i) suppressor activity could be removed by complement-mediated lysis with an anti-Thy 1.2 antiserum and (ii) suppressor activity was retained in the effluent fraction after passage of suppressor spleen cells over a nylon wool column. Induction of the T-cell suppressor activity was found to be associated with a loss of T-cell helper activity within the TGG-pulsed cell population. The presence of adherent cells was not required for induction of suppressor activity. Furthermore, the suppressor effect was found to be resistant to 1000 R of γ irradiation.     

Suppressor cells in contact sensitivity. Effect on T cells helping antibody production to picryl chloride.

T cells from mice injected with picryl sulfonic acid have previously been shown to suppress the effector and possibly other phases of contact hypersensitivity reactions to picryl chloride. In this report we examine their effect on T cells helping the early direct anti-TNP plaque-forming cell response of mice painted with picryl chloride. They did not directly inhibit the activity of the helper cells but did inhibit the ability of mice to generate helper cells after skin painting. The suppressor cells were T cells as tested by passage through nylon wool columns and sensitivity to anti-θ serum. Viable syngeneic cells were required for suppression and their effect was specific. The suppressor cells could not be generated in adult thymectomized mice but could be produced in mice treated with high doses (200 mg/kg) of cyclophosphamide. These properties are distinct from those of suppressor T cells produced following immunization with picryl chloride but are the same as those of other suppressor cells induced by PSA which inhibit contact hypersensitivity.     

Induction of tolerance to syngeneic IgE in neonatal mice

We have previously shown that adult A/J mice are tolerant to syngeneic IgE at the level of T cells, but not B cells. T cells of mice are responsive until the age of 2 to 3 wk, which correlates with the time of appearance of serum IgE. Tolerance can be induced earlier by neonatal administration of IgE in saline. We report here that purified nonimmune adult B cells, but not T cells, can transfer the state of tolerance to neonatal mice. As few as 2 x 10(6) B cells are effective. If IgE-bearing or IgE-secreting cells prove to be responsible, the amount of cell-bound IgE that can induce tolerance must be very small. The results also indicate that suppressor T cells do not have a major role in maintenance of self-tolerance to IgE.     

Specific neonatally induced tolerance to Mls locus determinants

Neonatal injection of CBA/HT6T6 (H-2k, Mlsb) mice with adult, Mls-incompatible (CBA/J [H-2k, Mlsd] X CBA/HT6T6)F1 spleen cells results in the abrogation of cell proliferation and interleukin 2 (IL 2) production in bulk mixed lymphocyte cultures, when spleen cells from the inoculated mice are tested at 6 to 8 wk of age with stimulator cells expressing the Mlsd of the tolerizing inoculum. In limiting dilution assays, this tolerant state was manifested in a 25- to 550-fold (280-fold average) decrease in the frequency of precursors of Mlsd-responsive IL 2-producing T cells. Tolerance was specific in that the frequencies of precursors of IL 2-producing cells responding to Con A, allogeneic H-2d, and self-Ia were not affected. The observed low frequency of Mls-responsive cells was due neither to extensive chimerism resulting in the dilution of Mlsd-responsive cells by the nonresponsive F1 cells of the inoculum, nor to the action of suppressor cells. These findings indicate that neonatal injection of Mls-incompatible spleen cells produces a state of specific tolerance by a clonal deletion or inactivation mechanism. This specific tolerance supports the view that 1) the Mls locus encodes or regulates the expression of defined alloantigenic determinants and 2) Mls-incompatible responder mice have specific receptors for Mls determinants on clonally distributed IL 2-producing responder T cells.     

Suppression of cytotoxic response to histoincompatible cells. II. Analysis of the role of two independent T suppressor pools in maintenance of neonatally induced allograft tolerance in mice.

The kinetics of appearance of the precursors of SuppA cells (capable of inhibiting CTLp leads to CTL) or SuppB cells (capable of inhibiting (stem cells leads to CTLp) in neonatal mice, as well as the appearance of SuppA/SuppB cells in mice given neonatal innoculations of semiallogeneic spleen cells has been investigated. The data obtained are consistent with the idea that SuppA cells have a natural role to play in the induction of neonatal tolerance, whereas SuppB cells may be more important for the maintenance of the tolerant state. Unlike the level of SuppB cells, the level of SuppA cells in tolerant mice seems to be modulated by the presence of the tolerizing determinants. Data are provided to show that SuppB cells, once induced in tolerant mice, can adoptively transfer specific allograft unresponsiveness to newborn syngeneic mice in the absence of added tolerizing antigen, whereas SuppA cells are not able to do so. These data fit the notion that SuppB cells may be responsible for the phenotype of clonal deletion.     

Cyclosporine therapy of rat heart allograft recipients and release of interleukins (IL 1, IL 2, IL 3): a role for IL 3 in graft tolerance?

LEW rat recipients of (LEW X BN)F1 strain heterotopic cardiac transplants treated with cyclosporine A (CsA) (15 mg/kg/day intramuscularly, 7 days) retain grafts indefinitely despite drug withdrawal. Donor-specific suppressor T cells that are active in passive transfer experiments have been harvested from long-term CsA-treated hosts. Although CsA is known to inhibit in vitro cytokine release, the in vivo effects of the CsA on the lymphokine cascade are not known. We investigated the action of the drug upon spontaneous and mitogen-induced interleukin 1 (IL 1), interleukin 2 (IL 2), and interleukin 3 (IL 3) release by spleen cells obtained from the following groups of rats: 1) normal, i.e., untreated and ungrafted; 2) grafted, acutely rejecting; 3) grafted, actively treated; and 4) under CsA-induced state of "tolerance." The results demonstrate that in vivo CsA therapy inhibits monocyte (IL 1 release) as well as lymphocyte function (IL 2 and IL 3 release) only during the inductive phase (the 7 days of treatment). During the "tolerant" phase, mitogen (Con A and LPS)-induced release of interleukins was quantitatively similar to that noted in normal animals. In contrast, a remarkable increase in the spontaneous production of IL 3 was observed in the "tolerant" group. Because cytokine release is not inhibited in the "tolerant" state, our data strongly support the concept that maintenance of the state of unresponsiveness is governed by the emergence of suppressor cells. The correlation of increased spontaneous production of IL 3 during this period leads us to postulate that this interleukin may be implicated in the activation or clonal expansion of suppressor cells, and hence may play a role in graft tolerance.