The induction of peripheral T cell unresponsiveness in adult mice by monomeric human gamma-globulin

Monomeric human gamma-globulin (HGG), when injected into adult mice, induces a state of specific immunologic unresponsiveness to further challenge with immunogenic forms of HGG. In this report we have directly determined the role of the thymus in the induction of HGG tolerance and the proliferative responsiveness of T cells from normal and HGG-tolerant mice. Draining lymph node T cells were isolated from HGG-tolerized and -challenged mice, and tested for their proliferative response to HGG in vitro. T cells from untreated but challenged adult CBA/CaJ and A/J mice proliferate in response to HGG, whereas such mice given monomeric HGG before challenge fail to show an HGG-specific proliferative response. APC from tolerant or nontolerant mice were equally effective in the support of Ag-specific proliferation of primed T cells. The influence of the thymus gland on HGG-induced T cell unresponsiveness was assessed by determining whether thymectomized mice could be tolerized to HGG. The results suggest that the generation of T cell tolerance to HGG is independent of thymic function as assayed by both antibody production in vivo and T cell proliferation in vitro. Unresponsiveness of T cells from tolerant mice was not a result of the presence of CD8+ cells since removal of CD8+ cells from lymph node T cells did not alter unresponsiveness to HGG in vitro. Further, mixing tolerant T cells with normal HGG-primed T lymphocytes did not inhibit proliferation of the HGG-primed cells. The results of this investigation suggest that this mouse model of tolerance to HGG represents a thymus-independent unresponsiveness of mature peripheral T cells to a nonself-Ag. Understanding the regulation of tolerance to HGG may give additional insight into the mechanisms required for the maintenance and possibly the induction of tolerance to certain self-Ag in peripheral lymphoid organs.     

Defective B cell tolerance induction in New Zealand black mice. I. Macrophage independence and comparison with other autoimmune strains

B cell unresponsiveness was examined in vitro by using spleen cells from autoimmune NZB, BXSB/Mp male, MRL/Mp-Ipr/Ipr (MRL/l), and control mice, and the tolerogen trinitrophenyl human gamma-globulin (TNP-HGG). The B cell subset responsive to TNP-Brucella abortus in each autoimmune and control strain that was tested was highly susceptible to tolerance induction with the use of high epitope density conjugates (TNP30HGG and TNP32HGG). When a tolerogen with a lower epitope density was used (TNP7HGG), several control strains were all rendered tolerant in a thymic-independent and hapten-specific manner. NZB B cells were resistant to all concentrations of TNP7HGG tested, whereas B cells from BXSB/Mp male and MRL/1 mice were resistant to low concentrations of this tolerogen. NZB mice were resistant in addition to tolerance induction with TNP9HGG, TNP10HGG, and TNP12.7HGG. Experiments were performed to determine whether splenic macrophages played a role in resistance to tolerance in NZB mice. The mixing of NZB and control DBA/2J T cell-depleted splenocytes revealed no modulatory effects by the accessory cells in culture. Moreover, B cells rigorously depleted of macrophages by double Sephadex G-10 column passage exhibited characteristic patterns of resistance or susceptibility in NZB and control strains, respectively. These findings support the conclusion that resistance to tolerance in NZB mice is determined at the B cell level and are consistent with the hypothesis that diverse immunoregulatory disturbances contribute in varying degrees to the development of systemic lupus erythematosus in different inbred strains of mice.     

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.     

Inheritance of tolerance susceptibility to human gamma-globulin in congenic mice.

The genetic control of susceptibility to tolerance induction with human gamma-globulin (HGG) was studied by using H-2 congenic mice. Strains tested that were congenic with C57BL/10Sn were completely tolerized by 1.0 mg deaggregated HGG. In contrast A/Sn mice showed full tolerance whereas A.SW mice were only intermediately tolerant. It was further shown that (B10 X SJL)F1 mice could be rendered tolerant but (B10.S X SJL)F1 mice could not. These data indicate a role for H-2 linked genes in control of tolerance susceptibility. Results obtained with the progeny of (B10.S X SJL)F1 backcrossed to B10.S indicate that two non-H-2 linked genes are involved in control of tolerance induction. Preliminary mapping studies show the H-2 gene located to the left of the IC subregion. These results confirm our previous finding that both H-2 and non-H-2 genes control susceptibility of adult mice to tolerance induction with HGG.     

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.     

Effects of early antigen exposure through lactation on later specific antibody responses in mice

This study characterized totally the effects of early Ag exposure by the suckling route on later specific antibody responses. When mother mice of BALB/c or C57BL/6 strains were injected with deaggregated human gamma-globulin (HGG) immediately after delivery, total amounts of HGG in sera of offspring increased until 2 wk of age. The catabolism of transferred HGG was extremely slow and the half-life was about 3 wk in both strains. Hence, small amounts of Ag in mothers, 0.5 micrograms in C57BL/6 and 50 micrograms in BALB/c, could tolerize their offspring effectively. As these were minimum tolerogenic doses, the strain difference in ease of tolerance induction is apparent already during suckling. The study on timing dependent effects of HGG-specific antiserum on tolerance induction by mothers given 50 micrograms HGG demonstrated that the tolerance is achieved within the 1st wk of lactation in C57BL/6 offspring, but not in BALB/c offspring, and the restoration from the tolerance needs more than 6 wk under circumstances, supposedly, without free Ag. Whereas the tolerance was induced in a dose-dependent manner in each class of antibody, the dissociation of tolerant states between IgM, IgG, and IgE antibody classes was found in C57BL/6 offspring. It is interesting that C57BL/6 offspring were sensitized weakly, but significantly, by mothers given subtolerogenic doses. However, this was not apparent in BALB/c. Thus, the Ag dose and the animal strain are related closely to the consequences of this Ag exposure. The aging of suckling mice within the first 2 wk of life or immunomodulators administered early in life did not seriously affect the consequences. Studies on a cellular basis showed that the tolerance is caused by the selective defect in helper T cell function and the suppressor cell activity is not associated with the mechanisms. This contrasts with other models of oral tolerance.     

Neonatally induced tolerance to HGG: duration in B cells and absence of specific suppressor cells.

A specific, long lasting, tolerant state to human gamma-globulin (HCG) was established in neonatal A/J mice. These suckling mice received the tolerogen in the colostrum of their mother who had been injected with DHGG. The tolerant state could not be accounted for by "factors" other than HGG in the colostrum. The duration of this tolerance in the intact animal and in the B cell population was 16 to 18 weeks. Naturally occuring nonspecific suppressor cells were evident but specific suppressor cells could not be demonstrated. These results are discussed in relation to possible mechanisms of the induction of tolerance to self.     

Induction of tolerance in athymic mice with an antigen which is highly immunogenic in euthymic mice.

Adult congenitally athymic (nu/nu) mice were found to be unable to respond to aggregated human γ-globulin (AHGG), the normally immunogenic form of HGG, unless first reconstituted with specific T cells. However, pretreatment of nude mice with AHGG prior to T-cell reconstitution resulted in the induction of unresponsiveness. This state of tolerance was specific since pretreated animals responded normally to the noncross-reacting antigens turkey γ-globulin or DNP-Ficoll. Transfer of spleen cells from nude mice pretreated with AHGG into normal littermates did not significantly affect a subsequent anti-HGG response of the recipients. Conversely, nude mice pretreated with AHGG and reconstituted with normal littermate spleen cells were hyporesponsive to challenge with AHGG. The results of these experiments are discussed in reference to various models for the induction of B-cell unresponsiveness.     

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.     

Strain differences in tolerance induction to human gamma-globulin subclasses: dependence on macrophages.

BALB/c and DBA/2 mice differ with respect to ease of tolerance induction with HGG, BALB/c mice being the resistant strain. When tested for susceptibility to tolerance induction with individual IgG subclasses, both strains were easily rendered unresponsive with IgG₁ and IgG₂ and less so with IgG₄. A strain difference appeared with IgG₃, where only BALB/c mice showed complete resistance to tolerance induction. Mixtures of the IgG subclasses Showed that IgG₁ and IgG₂ accounted for most of the tolerance to whole HGG seen in both strains, while addition of IgG₃ to the mixture made DBA/2 completely tolerant but reversed the trend toward tolerance in the BALB/c mice. By rosette assay it was found that BALB/c macrophages had receptors for IgG₃ (and to a lesser extent for IgG₄). These results are consistent with the hypothesis that resistance to tolerance induction with HGG is dependent on the presence of a receptor on the macrophage for a minor IgG subclass.     

The effect of allogeneic spleen cells on the induction of immunologic tolerance

It is shown, in this study, that the transfer of parental immunocompetent spleen cells to F₁ hybrid mice interferes with the induction of tolerance to human gamma globulin (HGG) in the F₁ recipients. The transfer of syngeneic spleen cells did not cause this interference, but the transfer of HGG-tolerant parental spleen cells was as effective as that of their normal counterparts. Hence, the interference with tolerance induction was attributed to the histoincompatibility of donor and host cells and, more specifically, to the graft-vs-host reaction. A mechanism of interference with the expression of tolerance by an antigen-specific T-cell bypass is discussed.     

The regulatory effects of cytokines on the induction of a peripheral immunologic tolerance in mice.

Previous reports have demonstrated that injection of rIL-1 alpha into mice abrogates the ability of deaggregated human gamma-globulin (HGG) to induce a state of antigen specific immunologic tolerance in vivo. Our results demonstrate that human rIL-1 beta and a bioactive nonapeptide of human IL-1 beta inhibit the induction of tolerance to HGG suggesting that IL-1 affects tolerance induction through a noninflammatory mechanism of action because the immunoactive nonapeptide possesses only immunomodulatory properties. Further, TNF-alpha but not IL-6, cytokines with many bioactivities in common with IL-1, was found to inhibit the induction of tolerance. Therefore, it appears unlikely that IL-6 plays a role in the pathway by which either IL-1 or TNF-alpha interferes with tolerance induction. Although IL-2, IL-4, and IFN-gamma were incapable of directly affecting the induction of tolerance to HGG, it was determined that IL-4 and IFN-gamma were capable of inhibiting the ability of IL-1 to abrogate tolerance induction. It has been suggested that IL-1 induces the generation of endogenous IL-1 in vivo. Further, it has been demonstrated that IFN-gamma as well as IL-4 inhibits the synthesis of IL-1. Inasmuch as IL-4 and IFN-gamma inhibit the ability of IL-1 to abrogate tolerance induction, it appears that it is the endogenously generated IL-1 that interferes with tolerance induction. It was also determined that neither IL-4 nor IFN-gamma inhibits the activity of IL-1 which is consistent with results reported by others. Thus, results presented here suggest that the inhibition of tolerance induction to HGG by IL-1 may involve the stimulation of endogenous IL-1 synthesis.     

Selective improvement of thymus and some T cell dysfunctions in NZB mice by in utero thymulin treatment

NZB mice were treated during gestation with thymulin, a thymus-secreted, zinc-associated nonapeptide. Control pregnant NZB mice received either zinc alone or saline alone. Offspring from all three groups of NZB mothers, and age-matched DBA/2 mice, were tested for the following immunologic parameters: thymulin serum levels at 2 and 5 wk of age; splenic anti-sheep red blood cell (anti-SRBC) plaque-forming cell (PFC) numbers after immunization at birth or at 2 wk of age; anti-human gamma-globulin (anti-HGG) antibody titers after immunization at 2 wk of age, with or without prior tolerance induction at birth with deaggregated HGG; spontaneous IgM serum levels at 2 and 5 wk of age; spontaneous splenic anti-trinitrophenyl (anti-TNP) PFC numbers at 2 wk of age. As compared with DBA/2 mice, young NZB mice exhibited low circulating thymulin titers, high antibody responses to SRBC and to HGG, resistance to tolerance induction by deaggregated HGG, increased spontaneous IgM serum levels, and increased spontaneous anti-TNP PFC numbers. However, marked reductions in anti-SRBC and anti-HGG antibody production, both thymus-dependent responses, were observed in the young NZB offspring of thymulin-treated mothers as compared with NZB controls born from zinc- or saline-treated mothers. A delay in the postnatal decrease of serum thymulin levels was also noted in the offspring of thymulin-treated mothers. Interestingly, these effects of in utero thymulin treatment tended to become more pronounced with advancing age during the postnatal period. Conversely, IgM serum levels, spontaneous anti-TNP PFC and sensitivity to tolerance induction were not affected by thymulin treatment during fetal life. Taken together, the data suggest that in utero exposure to pharmacologic concentrations of thymulin induces a persistent and selective improvement of some thymus and T cell dysfunctions but has no effect on intrinsic B cell abnormalities of NZB mice.     

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.     

Interference with tolerance induction in vivo by inhibitors of prostaglandin synthesis

Prostaglandins (PG) have been implicated as modulators of both humoral and cellular immune responses. In order to evaluate a possible role for PG in tolerance, the effect of inhibitors of prostaglandin synthesis on tolerance induction and circumvention has been investigated. Injection of deaggregated human gamma-globulin (DHGG) into A/J mice leads to unresponsiveness to a subsequent challenge with immunogenic aggregated human gamma-globulin (AHGG). Administration of indomethacin (IM) or acetylsalicylic acid (ASA) shortly before and after DHGG injection prevents tolerance induction. PGE2 reverses the tolerance overriding effect provided by IM. IM is not able to overcome unresponsiveness when given 10 and 20 days after tolerance induction, at a time point when both T and B lymphocytes are tolerant. As previously shown, lipopolysaccharide (LPS) both inhibits the induction of tolerance to HGG and circumvents tolerant T helper cells late in tolerance when competent B cells are present. In contrast, IM is unable to circumvent T-helper cell tolerance when given at Day 60 after tolerogen, when B cells (but not T cells) are responsive. Furthermore, LPS acts as an adjuvant, B-cell mitogens, inducer of polyclonal Ig secretion, and primes mice when given with tolerogen, while IM has none of these properties. These results indicate a difference between the effects of IM and LPS on tolerance and a possible role of PG in DHGG-mediated tolerance induction.     

Th1 and Th2 clones differ in their response to a tolerogenic signal.

Th1 and Th2 clones specific for human gamma globulin (HGG) were compared and shown to differ in terms of the effects of tolerance induction on Ag-induced proliferation and helper activity. In developing a method to induce tolerance, splenic APC that had been pulsed with HGG and then fixed with 0.15% paraformaldehyde (HGG-FAPC) were used as a means to present Ag to the Th clones in the absence of costimulatory signals. Both Th1 and Th2 clones recognized HGG-FAPC as evidenced by their ability to proliferate to HGG-FAPC. Unlike Th2, Th1 proliferated to HGG-FAPC only in the presence of T cell-depleted allogeneic spleen cells as a source of accessory cell signals. The inability of Th1 cells to proliferate in the absence of costimulatory signals was due to Ag-specific inactivation: Th1 clones preincubated with HGG-FAPC were unable to proliferate when recultured with HGG and irradiated APC. In contrast to Th1 clones, Th2 clones showed no decrease in their Ag-induced proliferative capacity after exposure to any concentration of HGG-FAPC. However, when examined by using a second assay system, that of providing help for anti-HGG antibody production by primed B cells, Th2 preincubated with HGG-FAPC were markedly inhibited (up to 90%) in their ability to provide help. Preincubation with HGG-FAPC also inhibited the helper activity of the one Th1 clone that was found to induce a significant secondary antibody response. Taken together, the results suggest that exposure of Th1 to tolerogen in the form of HGG-pulsed fixed APC inactivates Th1 proliferative capacity, and possibly Th1 helper activity as well. Exposure of Th2 cells to a tolerogen suppresses the mechanism by which the Th2 cells provide Ag-induced B cell help, but does not inhibit the mechanism by which they proliferate to HGG. Furthermore, the results define a model that incorporates Ag processing as well as Ag presentation in the induction of tolerance in vitro.     

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.     

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.     

Adjuvant effect on maintenance and escape from tolerence.

When mice are injected with deaggregated human gamma globulin (HGG), and HGG-tolerant state ordinarily is produced and persists despite subsequent challenges with an immunizing dose of HGG in saline or with an immunizing dose of HGG in Mycobacterium adjuvant. Subsequent administration of an immune elimination dose of radiolabeled HGG, at 27 days and 47 days does not break the tolerant state. Of special interest is the observation that when complete adjuvant containing increasing amounts of mycobacterial components was administered in conjunction with antigen very early in the tolerance induction phase 5 days after TID, it appears to prevent tolerance production. Mice challenged 5 and 17 days after the tolerance-inducing inoculation exhibit a statistically significant increase in circumvention of tolerance when compared with individuals challenged on the 7 and 17 day schedule. This increased circumvention of tolerance, as evidenced by 5 day challenge mice, seems to be related both to the mycobacterial content of the adjuvant and murine strain.     

Strain Differences in the Immunogenicity of Aggregated Human IgG and the Adjuvant Action of Lipopolysaccharide on the Low-Responder Strain of Mice

Strain differences in the antibody response to human IgG (HGG) were observed when aggregated HGG was injected intravenously. Lipopolysaccharide (LPS) administered subsequently markedly enhanced the antibody response to HGG in low responder C57BL/6 mice as compared with that in high responder DDD, C3H/He or (C57BL/6 × DDD)F₁ mice. Aggregate-free preparation of HGG at a dose of 0.5 mg induced immunological tolerance in all strains of mice tested. LPS injected subsequently converted tolerogenic, aggregate-free HGG into immunogen in DDD mice but not in C57BL/6 mice. To determine the correlation between adjuvanticity and mitogenicity of LPS, spleen cells from normal mice were cultured in the presence of LPS and ³H-thymidine uptake was measured. Spleen cells of DDD mice incorporated three times as much ³H-thymidine as those of C57BL/6 mice. There seems no strong correlation between both activities of LPS. The data obtained are discussed in terms of strain differences in the macrophage function for processing the antigen.