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.     

Immune response to chemically modified flagellin. IV. Further studies on the relationship between humoral and cell-mediated immunity

Acetoacetylation converts flagellin from an antigen which preferentially induces humoral antibodies to an antigen which exclusively provokes cell-mediated immunity and, under certain circumstances, induces antibody tolerance. Studies reported in this paper revealed that the acetoacetylated flagellins expressed similar immunological properties in flagellin primed rats as in normal rats. Thus, on the one hand, acetoacetylation destroyed the capacity of flagellin to trigger a secondary antibody response, but on the other hand, the acetoacetyl-flagellins very effectively induced delayed-type hypersensitivity reactions in flagellin primed animals. It was concluded from these results that humoral and cell-mediated immunity may be opposing immunological processes in both unprimed and primed animals.Acetoacetylated flagellin induced antibody tolerance in both strain W (low responder) and J (high responder) Wistar rats. Maximum tolerance was induced 12 hr after injection of antigen, but in strain J animals the tolerance had disappeared by 48 hr, whereas in strain W rats tolerance persisted for >28 days. The potential to recover from tolerance in strain J rats appeared to coincide with the level of delayed hypersensitivity at the time of challenge. However, this delayed hypersensitivity disappeared when breaking of tolerance occurred. These results suggest that the T cells which participate in delayed hypersensitivity reactions may also act as “helper” cells in antibody responses. On the other hand, it was found that priming for a secondary antibody response by flagellin appeared to coincide with development of primary antibodies rather than with induction of delayed-type hypersensitivity. The relative importance of specific T and B cells in these phenomena is discussed.     

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.     

Cellular aspects of tolerance. II. Unresponsiveness of B cells

The responsiveness of bone marrow cells from tolerant donors was examined by reconstitution of lethally irradiated tolerogen-free recipients. In these animals, stem cells from tolerant donors gave rise to immunologically competent antigen sensitive B cells. The antibody produced by these cells could be detected by a sensitive plaque assay in liquid and by antigen elimination. The antibody was not demonstrable by an assay which only detected plaque forming antibody which was highly avid or was formed in large quantity per cell. In lethally irradiated animals, partially purified B cells from a tolerant animal could not cooperate with T cells from normal donors to reconstitute immunological responsiveness to immunogenic doses of the tolerance inducing antigen. We concluded that antigen sensitive B cells in the bone marrow become unresponsive following administration of tolerogenic forms of antigen. Responsiveness of the reconstituted recipient animals was due to the differentiation of donor stem cells and subsequent antibody production by their descendants. Earlier contradictory findings could be unified in terms of these observations and conclusions.     

Immunity to carcinogen-induced transplantable fibrosarcoma in B2B2 chickens: V. Relationship to tumor cell-specific delayed hypersensitivity and serum antibody

Lasting immunity to the chemically induced (DMBA) fibrosarcomas (CHCT-NYU1, 2, and 4) of SC chickens ($\text{B2}\text{B2}$) can be obtained by injection of Corynebacterium parvum (CP)-primed chickens with tumor cells and CP in one wing and tumor cells alone in the other wing. The local delayed hypersensitivity (DH) reaction to CP in the wing inhibits local growth completely, whereas the tumor on the contralateral side shows transient growth. In the present studies, the development of tumor immunity was studied in detail by monitoring DH and antibody formation to the tumor cells and adoptive immunity with spleen cells in Winn tests. Injection of NYU1 cells alone in normal or CP-treated animals induced transient immunity in Winn tests in 50% of the animals, weak DH reactivity, and antibody detectable by immunofluorescence within the first 2 weeks. Chickens receiving both NYU1 cells and CP in one wing and NYU1 cells alone on the other side developed stronger DH reactions to the tumor cells and a higher incidence of immunity in Winn tests which was sustained throughout the period of observation. Antibody levels were similar to those of animals receiving tumor cells alone. In contrast, injection of CP and tumor cells on one side without a tumor challenge on the contralateral side did not induce detectable immunity in CP-primed chickens. Chickens immunized to NYU2 and 4 cells were also tested for DH reactivity and antibody formation. Studies on the cross-reactivity between the tumor lines showed that there was cross-reactivity at the humoral level while at the cellular level this was not apparent. However, animals immune to one tumor line rejected transplants of another tumor line. Observations on the antibody specificity(s) suggested that it was not directed against minor histocompatibility or avian sarcoma viral antigens. SC embryo fibroblasts could induce DH, and serum antibody induced by tumor cells usually reacted also with such embryo cells.     

The effect of aging on the induction of humoral and cellular immunity and tolerance in two long-lived mouse strains.

Aging is a complex process that adversely affects most if not all components of the immune system. In this report, two long-lived mouse strains have been compared in ability to generate both antigen-specific immunity and tolerance. Although CBA/CaJ mice produced high levels of antibody following injection of aqueous preparations of aggregated human gamma-globulin (AHGG), C57BL/6 mice made only meager antibody responses to such preparations. Age dramatically affects the humoral anti-HGG response to aqueous AHGG in both strains, but the meager response of young C57BL/6 mice was at insignificant levels in aged C57BL/6 mice. Conversely, both mouse strains generated good responses following injection of HGG in complete Freund's adjuvant at both the T and B cell level as evidenced by in vitro antigen-specific T cell proliferation and anti-HGG antibody production. Aged mice of both strains showed a marked decrease in the production of serum anti-HGG antibody in comparison to young mice. Although the antigen-specific T cell proliferative response was significantly decreased in aged CBA/CaJ mice, such proliferation was not affected in aged mice of the C57BL/6 strain. Removal of CD8+ cells from lymph node T cells of either young or aged C57BL/6 mice did not increase the antigen-specific proliferative response, suggesting that loss of CD8+ suppressors during the aging process is not responsible for the high level of antigen-specific T cell proliferation in aged C57BL/6 mice. Tolerance to HGG was readily induced in both young and aged C57BL/6 and CBA/CaJ mice although aged mice demonstrate a modest resistance to tolerance induction when compared to their young counterparts. This resistance was observed in both antibody production and antigen-specific T cell proliferation.     

The role of hapten-reactive T lymphocytes in the induction of autoimmunity in mice. I. Establishment of the experimental conditions for the termination of immunological tolerance to human gamma globulin.

In order to study the role of hapten-reactive helper T cells in the induction of autoimmunity in mice, an attempt was made to establish an experimental model for the development of hapten-reactive helper T cells and the termination of immunological tolerance against heterologous proteins. Spleen cells taken from mice which were immunized with hapten-isologous protein conjugates (PAB-MGG) demonstrated helper activity for the anti-DNP antibody response of DNP-primed B cells responding to DNP and PAB-conjugated protein, but spleen cells from hapten-heterologous protein conjugate (PAB-HGG)-primed mice could not respond to PAB-determinant. Thus, hapten-reactive helper T cells can develop in mice by the immunization with hapten-isologous protein conjugate, but not with hapten-heterologous protein conjugate. However, spleen cells from mice which had been rendered tolerant by treatment with 2.5 or 0.2 mg of DHGG and then immunized with PAB-HGG could demonstrate helper activity responding to PAB-determinant. This helper activity was PAB-specific, because these spleen cells did not demonstrate helper activity if PAB-determinant was omitted in the primary and the secondary antigen. This helper activity was abrogated by the treatment of spleen cells with anti-θ serum and complement. Thus, hapten-reactive helper T cells were successfully induced by the challenge with hapten-heterologous protein conjugate in carrier-protein tolerant mice. When mice were treated with 2.5 or 0.2 mg of DHGG, no anti-HGG antibody response was induced by the challenge with HGG or PAB-HGG. However, the termination of HGG-tolerance was demonstrated only when the mice were preimmunized with PAB-MGG to raise PAB-rcactive helper T cells, treated with 0.2 mg of DHGG, and then challenged with PAB-HGG. This termination of immunological tolerance was not observed when the mice were preimmunized with PAB-BαA to raise PAB-specific B cells and anti-PAB antibody, or when the mice were treated with 2.5 mg of DHGG. Thus, if HGG-specific B cells remain intact in mice such as treated with low dose of DHGG, these B cells can be activated by some bypass mechanisms in the presence of PAB-reactive helper T cells through the PAB-determinant even in the absence of HGG-reactive helper T cells. These data clearly showed the role of hapten-reactive helper T cells in the termination of immunological tolerance and provide experimental supports to the hypothesis on the termination mechanism proposed by Weigle. The cellular mechanism for the development of hapten-reactive helper T cells in tolerant animals and the cellular mechanism of autoantibody production were discussed on the basis of T-B cell collaboration.     

Influence of antigen dose on antibody production of intact and splenectomised Xenopus laevis.

Antigen persistence and serum antibody production in intact Xenopus were monitored using human gamma globulin (HGG), in adjuvant, in various immunisation schedules. Retention of HGG in spleen and serum was directly related to the quantity injected. However, antibody responses to a dose range between 1 mu-g-6 mg antigen were similar in intensity. These were detected in the serum two weeks after injection and at this stage were exclusively mercapto-ethanol (ME) sensitive; ME-resistant antibodies had appeared by four weeks. No antibodies were detected below a dose of 100 ng HGG. The effect of splenectomy on antibody levels was tested using HGG/adjuvant or sheep erythrocytes (SRBC) in saline. Splenectomised toads showed impairment of antibody responses only to threshold doses of HGG (100 ng) but to a wider range of SRBC doses.     

The induction of immunological unresponsiveness in previously immunized mice.

HGG unresponsiveness can be induced in primed A/J mice; however, such induction is difficult and requires multiple injections of large doses of soluble HGG (SHGG). Although single injections (1.5–25 mg) of deaggregated HGG (DHGG) did not result in a significant secondary immune response, an unresponsive state to a subsequent injection of aggregated HGG (AHGG) was not induced. When the dose of DHGG was even smaller (0.5 mg), a normal secondary response was obtained similar to that observed following injection of AHGG. Evidence is presented here which suggests that the difficulty encountered in inducing unresponsiveness may be in part due to partial aggregation of DHGG by persisting antibody in the circulation. The PFC to HGG produced after injection of either AHGG or DHGG or during induction of unresponsiveness to SHGG apparently involved cell division, since all three responses were inhibited by vinblastine. The reduction in PFC in primed mice injected with DHGG or SHGG was not due to selective inhibition of PFC secreting certain classes or subclasses of immunoglobulins.     

A reexamination of humoral tolerance in chickens congenitally infected with an avian leukosis virus.

In a reexamination of congenital infection of chickens with ALV, one of the classical models of immunologic tolerance, we were unable to demonstrate that there was any evidence suggesting humoral immune reactivity to the infecting virus. In ALV-F42 congenitally infected birds that have a persistent viremia and no neutralizing antibody detectable by conventional means, we could find no evidence of host IgG deposits in the kidneys, nor any suggestion of renal pathology, or damage. In addition, attempts to precipitate any putative circulating infections virus-antibody complexes by treatment with a highly specific anti-globulin were negative nor were there titer differences in the viremic plasmas of bursectomized birds compared to nonbursectomized birds, indicating that the chickens were not making either a masked neutralizing or a nonneutralizing antibody response to the infecting virus. The evidence presented indicates that congenital infection of chickens with ALV does, at least at the humoral level, result in a state of immunologic tolerance to the infecting agent.     

Macrophage handling of a tolerogen and the role of IL 1 in tolerance induction in a helper T cell clone in vitro

The human gamma-globulin (HGG)-specific helper T cell clone AB.7.D7 can reconstitute the plaque-forming cell response of HGG-primed B cells. Tolerance induction at the level of T cell help results from exposure of the AB.7.D7 cells to 10 micrograms monomeric HGG. The monokine IL 1 was found to interfere with tolerance induction in AB.7.D7 cells in a dose-dependent manner. Furthermore, interference with tolerance induction was dependent upon the T cells being presented with IL 1 at the same time as monomeric HGG, the tolerogen. IL 1 and monomeric HGG could not be demonstrated to interact to make nontolerogenic soluble aggregates, however. It was found that monomeric HGG was unable to stimulate the production of either membrane or secreted IL 1 by splenic macrophages and in addition was not degraded by peritoneal exudate cells. Heat-aggregated HGG, which is highly antigenic and nontolerogenic, is a good stimulus for IL 1 production and is processed by macrophages into peptides of varying sizes. These data are consistent with the suggestion that a tolerogenic signal results from T cell recognition of a nondegraded antigen in the absence of a signal from IL 1. It is possible, however, that small amounts of processed antigen, undetectable by us, are involved.     

Insulin-specific tolerance induction. II. Tolerant helper T cells can be rescued by insulin complexed to Brucella abortus

Murine antibody responses to heterologous insulins are under H-2-linked immune response (Ir) gene control. We previously demonstrated that the immune response to insulin in Freund's complete adjuvant (CFA) can be specifically inhibited by prior injection of soluble insulin i.v. Unresponsiveness requires at least 4 days after i.v. injection to develop, and once induced, it lasts 4 wk or more. Unresponsiveness is caused by T cell, but not B cell, tolerance; furthermore, we have been unable to demonstrate any role for suppressor T cells in this unresponsiveness. The following experiments examine the nature of the T cell tolerance induced by i.v. injection of insulin, and the data suggest that helper T cells were not clonally deleted by this procedure. The functional activity of the tolerized T cells can be rescued by stimulation with insulin covalently complexed to the type 1 T-independent (TI-1) antigen, Brucella abortus. This observation suggests that tolerance induced by soluble insulin is due to clonal anergy rather than clonal deletion of helper T cells; thus, this system could provide a model for determining the cellular events involved in tolerance induction and reversal in helper T cells.     

Maturation of the lymphoid system. I. Induction of tolerance in neonates with a T-dependent antigen that is an obligate immunogen in adults.

A/J mice displayed a striking ontogenetic difference in the capacity to respond to DNP-Ficoll, a T-independent antigen, and to aggregated human gamma-globulin (AHGG), a T-dependent antigen. Thus, whereas responses to DNP-Ficoll of 4-day-old mice were similar in magnitude to those of adult animals, responses to AHGG did not become pronounced until mice were some 30 to 40 days of age. The inability of young animals to respond to AHGG was reflective of a negative consequence of lymphocyte/antigen interaction, since such mice became specifically unresponsive to subsequent challenges with AHGG. Unresponsiveness induced by neonatal injection of AHGG lasted 50 to 60 days, in contrast to that induced by deaggregated HGG, which persisted some 100 days longer. The unresponsive state induced by injection of neonates with AHGG maintained itself upon adoptive transfer and did not appear to be linked to suppressive factors associated with either serum or lymphoid cells for its maintenance. Finally, AHGG was also shown to be capable of inducing unresponsiveness in neonatal, athymic mice. These results demonstrate that AHGG, the normally immunogenic form of HGG in adult mice, can serve as an effective tolerogen when administered into a neonatal environment.     

Avian antigen-binding cells. I. Characterization of antigen-binding T cells from bursectiomized chickens by autoradiography.

Antigen-binding cells (ABC) from spleens of HGG-immunized, bursectomized agammaglobulinemic (Bx) chickens were detected by direct autoradiography with 125I-HGG and by sandwich autoradiography with HGG plus 125I-goat-anti-HGG. The specificity of antigen binding was demonstrated by 1) inhibition of binding of 125I-HGG by preincubation with unlabeled HGG and 2) a specific increase in ABC after immunization. The ABC from Bx chickens were not B cells, as shown by the virtual absence of immunoglobulin-bearing cells in this population and by the lack of inhibition of antigen binding by anti-immunoglobulin sera. The ABC were not macrophages and did not bind HGG via Fc receptors because their frequency was unchanged after passage over nylon wool or incubation with antigen-antibody complexes. The temperature dependence and azide stabilization of the ABC were characteristic of antigen-binding T cells. Therefore, T cells capable of binding soluble antigen were demonstrated in Bx chicken spleen, which is free of contamination by B cells and passively adsorbed antibody.     

Hapten-specific carrier-dependent tolerance induction in man in vitro

We sought to determine whether hapten-specific tolerance can be induced in cultured human lymphocytes in vitro. Unfractionated as well as T and B cells from peripheral blood lymphocytes of healthy human volunteers were cultured with different hapten-carrier conjugates before in vitro challenge with dinitrophenyl (DNP) linked to keyhole limpet hemocyanin. Hapten-specific antibody was detected in the supernatant by solid-phase radioimmunoassay. Both hapten specificity and carrier dependence in addition to the cellular basis of tolerance induction were examined. The results show that hapten-specific tolerance of antibody production was induced by human gamma-globulin (HGG) conjugated to DNP but not by other conjugates of DNP nonhuman gamma-globulin, as well as human serum albumin. Moreover, both T and B cells are involved in tolerance induction to DNP-HGG in vitro. The significance of tolerance in human in vitro for the specific therapy of autoimmune disease is discussed.     

Tolerance induction in antigen-specific helper T cell clones and lines in vitro

The induction of T cell tolerance in vitro was investigated by using HGG-specific murine helper T cell (Th) clones and cell lines. It was found that exposure of Th to monomeric HGG (tolerogen) for 18 hr rendered the Th unable to reconstitute the PFC response of HGG-primed B cells. The tolerant state was not a result of Th cell death, as up to 100% of Th could be recovered after exposure to the monomer, and in addition, the recovered cells proliferated in response to IL2. B cells were shown not to be significantly affected by the presence of monomeric HGG in amounts calculated to be carried over from the tolerization cultures into the assay cultures. Consequently, it was concluded that interaction between Th and monomeric HGG induced unresponsiveness at the T cell level. A comparison of the tolerogenic potential of monomeric, soluble, and aggregated HGG revealed that only the monomer could induce tolerance in Th. Monomeric HGG was also shown to induce tolerance in an antigen-specific manner. Th reactive to HGG could be tolerized by monomeric HGG, but not Th reactive to FGG or OVA. Helper function of Th was also shown to be antigen specific in that HGG-reactive Th helped only HGG-primed B cells. Certain HGG-specific Th clones were found to be refractory to tolerization with monomeric HGG, whereas other clones derived from the same uncloned cell line were tolerizable.     

Clonal abortion of bone marrow T cell precursors: T cells acquire specific antigen reactivity prethymically

Lethally irradiated (900 R) mice were reconstituted with bone marrow cells from syngeneic donors that had been tolerized 2 to 3 wk earlier to either DNP or TNP compounds. Five weeks after reconstitution, these animals were tested for their ability to mount a delayed hypersensitivity (DH) response to the tolerizing haptens. Recipient mice were specifically tolerant to the hapten that was used to induce tolerance in the marrow donor. Mixing experiments in which mice were reconstituted with marrow from DNP-tolerant and TNP-tolerant donors showed no indication of active suppression or effective antigen carry-over in this system. This observation held true even in experiments in which mice were reconstituted with a mixture of marrow from tolerant and normal donors at a ratio of 5:1. Thus the reduced responsiveness in recipient mice seemed to be due to the functional elimination of hapten-responsive T cell precursor (pre-T) clones. Recipient unresponsiveness was also shown to be MHC restricted. Maintenance of unresponsiveness appeared to be due to the restricted access of regenerating pre-T cell clones to the maturational influence of the recipient's thymus.     

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.     

Transfer of agammaglobulinemia in the chicken. I. Generation of suppressor activity by injection of bursa cells

Spleen cells from adult agammaglobulinemic (bursectomized) chickens taken 1 to 3 weeks after an injection of histocompatible bursa cells can inhibit the adoptive antibody response to B. abortus of normal spleen or bursa cells in irradiated recipients. Spleen cells from Aγ chickens not injected with bursa cells generally do not. Moreover, bursectomized chickens which have been reconstituted with spleen cells within the first week after hatching do not respond with suppressor cell formation upon bursa cell injection. This apparent “autoimmunization” with bursa cells induces suppressor T cells which are only minimally sensitive to treatment with mitomycin C or to 5000 R γ irradiation. The suppressor activity is neither induced nor potentiated by concanavalin A in vivo. It is much stronger in spleen than in thymus cells and appears to be macrophage independent and to require intact cells. The cell component which stimulates the suppressor activity is more pronounced on bursa than on spleen cells, and is at most present to a very limited extent on bone marrow, thymus, or peritoneal exudate cells. It is better represented in comparable cell numbers of Day 17 than of Day 14 embryonic bursa. The inducing cell component is present in the membrane fraction of disrupted bursa cells. Immunization with bursa cells from B locus histoincompatible chickens leads to suppressor activity against histocompatible bursa cells. Although the removal of Ig-bearing cells from bursa greatly diminishes its immunizing capacity, injection of serum IgM and IgG does not induce suppressor cells. It is suggested that tolerance to a B-cell antigen is lacking in adult Aγ chickens, resulting in an autoimmune response upon exposure to B cells. The B-cell antigen may be a cell surface-specific form of Ig, a complex of Ig and a membrane component, or a differentiation antigen which appears simultaneously with Ig during ontogeny.     

The induction of tolerance to thyroglobulin significantly reduces the severity of thyroiditis in obese strain chickens

Obese strain chickens develop severe spontaneous autoimmune thyroiditis several weeks after hatching, characterized by mononuclear cell infiltration and antibodies to thyroglobulin (Tg). The presence of antibodies to Tg suggests that Tg is an important antigen in this disease, but it does not provide definitive evidence. To clarify this point, Obese strain chicks were tolerized at hatching with Tg and then examined up to 6 wk later for antibodies to Tg, thyroid pathology, and function. Various tolerance regimens were tested. The optimal conditions were i.v. injection of Tg within 24 hr of hatching, and injection of at least 1 mg. Tg isolated from normal thyroid glands was satisfactory, and it did not have to be deaggregated. Tolerance induced by the above procedure significantly retarded all parameters of autoimmunity, although by 6 wk of age some of the tolerized chicks had severe thyroiditis. Multiple weekly injections of Tg were no more effective than a single injection at hatching. Interestingly, a single injection at hatching was very effective, yet it was cleared from the circulation within 24 hr. In summary, tolerance induced with Tg had a profound effect on the disease and thus provides good evidence for the role of Tg in this disease.