Two signals are required for accessory cells to induce B cell unresponsiveness. Tolerogenic Ig and prostaglandin

We previously demonstrated that a lymphoid dendritic cell-like tumor line (P388AD.2) presented a normally tolerogenic signal, fluoresceinated sheep gamma-globulin (FL-SGG), as an immunogenic one. In contrast, macrophages derived from the peritoneal cavity potentiated the ability of FL-SGG to induce B cell unresponsiveness. In this paper we examined whether two different Ia+ splenic accessory cells differentially presented tolerogen to spleen cells or fluorescein (FL)-binding B cells. Interestingly, lymphoid dendritic cells presented FL-SGG to spleen cells and elicited augmented anti-FL antibody responses, whereas splenic macrophages presented this same moiety and elicited hapten-specific B cell unresponsiveness. The mechanism of splenic macrophage-elicited B cell negative signaling was investigated, and it was found that B cell unresponsiveness was abrogated in the presence of the cyclooxygenase inhibitor indomethacin. This observation suggested a crucial role for PG in B cell negative signaling. The addition of 10 nM PGE2 restored unresponsiveness in cultures treated with indomethacin and tolerogen-pulsed macrophages, even though this dose of PG had no effect on the ability of B cells to be triggered by an immunogenic signal. A role for T cells was excluded, inasmuch as purified hapten-specific B cells were specifically tolerized by FL-SGG-pulsed macrophages. Lymphoid dendritic cells pulsed with FL-SGG did not deliver a tolerogenic or immunogenic signal to FL-specific B cells. However, when PGE2 was supplied, B cell unresponsiveness was induced. Finally, we tested whether "non-tolerogenic" doses of FL-SGG could render hapten-specific B cells unresponsive in the presence of PGE2, but in the absence of accessory cells. Interestingly, the combination of non-tolerogenic amounts (10 to 1000 pg/ml) of FL-SGG in conjunction with PGE2 induced unresponsiveness, whereas neither moiety alone was effective. These results suggest that splenic macrophages and lymphoid dendritic cells exert opposing effects on the immune system as evidenced by the induction of negative or positive B cell signaling. Our observations suggest that one of the key factors in controlling whether an accessory cell delivers a tolerogenic signal is the ability to secrete PG.     

A novel role for macrophages: the ability of macrophages to tolerize B cells

We investigated the ability of macrophages (M phi) to present the tolerogen fluoresceinated sheep gamma-globulin (FL-SGG) to B cells. M phi pulsed with FL-SGG or murine FL-IgG2 were able to tolerize normal spleen B cells specifically as assessed by the plaque-forming cell (PFC) response to the antigens FL-Brucella abortus (FL-BrA) and FL-polymerized flagellin (FL-POL). Tolerance was not induced when M phi were pulsed with a variety of other FL antigens or the synthetic tolerogen FL-D-glutamic acid-D-lysine (FL-D[G,L]). The ability of M phi to tolerize B cells was not T cell-dependent, because populations of both T-depleted B cells and hapten-specific B cells were tolerizable. M phi-induced B cell tolerance did not exhibit genetic restriction with regard to the H-2 haplotype of the presenting M phi or the responding B cells. A variety of different types of peritoneal M phi, including normal resident M phi and those elicited by thioglycollate or concanavalin A (the latter are predominantly la+), could tolerize B cells after being pulsed with FL-SGG. We compared tolerogen-pulsed M phi to soluble tolerogen for the ability to tolerize B cells and found that tolerogen bound to M phi was more than 10 times as potent as an equivalent amount of soluble tolerogen. In contrast to the ability of M phi to present FL-SGG in a tolerogenic fashion to B cells, the P388AD lymphoid dendritic cell-like tumor line presented FL-SGG in an immunogenic mode to B cells. Tolerogen bound to P388AD cells could specifically augment a PFC response to both FL-BrA and FL-POL. We suggest that certain types of M phi may play a role in unresponsiveness by enhancing the tolerogenicity of soluble antigen, whereas other accessory cells may present the same moieties in an immunogenic fashion.     

Role of self carriers in the immune response and tolerance. III. B cell tolerance induced by hapten-modified-self involves both active T cell-mediated suppression and direct blockade.

The induction of B cell unresponsiveness with hapten-modified syngeneic murine lymphoid cells (hapten-modified self, HMS) can be achieved in vivo and in vitro. Tolerance in vivo in mice required a latent period of 3 to 4 days. Moreover, B cell unresponsiveness could not be induced by HMS in athymic nude mice, although their nu/+ littermates were rendered hyporesponsive by HMS. Pretreatment of normal mice with cyclophosphamide (cyclo) prevented their susceptibility to tolerance induction by haptenated lymphoid cells. Nude mice became sensitive to HMS-induced suppression if they were first reconstituted with spleen cells from normal (but not cyclo-treated) donors.Interestingly, labeling of H-2 antigens was not necessary for tolerance induction by HMS since haptenated teratoma cells (lacking H-2) were tolerogenic in normal recipients.In contrast, suppression of the in vitro response to haptenated flagellin occurred equally well with nude, nu/+ and anti-Ly 2 + C-treated spleen cells. These data suggest that cyclo-sensitive modified self-reactive (T) cells may regulate the immune response and mediate tolerance to HMS in vivo. However, the in vitro “blockade” of B cell reactivity may be directly mediated on hapten-specific PFC precursors.     

Induction of B lymphocyte tolerance by an oligovalent antigen. I. Influence of the read-out system.

A single intravenous injection of deaggregated preparations of lightly substituted dinitrophenylated human gamma globulin (DNP-HGG) induced DNP-specific tolerance in adult CBA mice, as judged by their failure to mount an IgM anti-DNP antibody-forming cell (AFC) response following challenge with the thymus-independent antigen, polymerized flagellin substituted with DNP (DNP-POL). Tolerance was also readily achieved in nude mice. Experiments using bovine serum albumin as the DNP carrier in both strains suggested that this was a less effective carrier for tolerance induction. The spleen cells from mice injected with DNP-HGG failed to respond to challenge with DNP-POL in vitro, but marked recovery of responsiveness occurred when the cells were challenged after adoptive transfer.These observations indicate that tolerance among antibody-forming cell precursors may selectively affect subpopulations. They further show that the choice of a read-out system used to analyze tolerance in B cells may critically influence the results.     

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.     

In vitro studies of the genetically determined unresponsiveness to thymus-independent antigens in CBA/N mice.

The X-chromosome-linked B lymphocyte defect of CBA/N mice has been studied in vitro by comparing the ability of (CBA/N X DBA/2)F1 (X-/X- X X+/Y) male (X-/Y) and female (X-/X+) spleen cells to respond to the thymus-independent antigen DNP (or TNP)-AECM-Ficoll. (CBA/N X DBA/2)F1 male spleen cells failed to generate significant in vitro anti-TNP antibody responses to DNP- or TNP-AECM-Ficoll, in contrast to spleen cells from F1 female (X-/X+) mice which responded normally to these T-independent antigens. Spleen cells from male F1 mice responded almost as well as F1 female cells to the thymus-dependent antigen, TNP-sheep red blood cells (TNP-SRBC) in vitro. Adding F1 male cells to F1 female cells failed to reduce the response of the latter to DNP-AECM-Ficoll, suggesting that the inability of F1 male cells to respond was not due to active suppression. The response of F1 male spleen cells to TNP-SRBC was not impaired by adding high concentrations of TNP-AECM-Ficoll indicating that the mechanism of unresponsiveness was not tolerance induction in all TNP-specific precursors. Lymphocytes from F1 male mice were capable of forming anti-TNP antibody after stimulation with lipopolysaccharide (LPS) in high concentrations; DNP-AECM-Ficoll had no effect on this polyclonal response. B lymphocytes from mice bearing only the X-chromosome of the CBA/N strain thus display a profound defect in B cell activation. This functional defect may represent either an inability of the defective B cells to be activated by thymus-independent antigens or the absence of a sub-class of B cells which respond to thymus-independent antigens.     

Receptor-ligand interactions and tolerance induction in mature virgin B cells

The membrane events and the nature of the receptors involved in the induction of thymus-independent high-zone tolerance were investigated. Tolerance was induced in vitro by incubating cells for the 4 hr with DNP-polysaccharide conjugate. The degree of tolerance was estimated by determining the subsequent cellular response to antigenic challenge in vitro. Treatment with agents that inhibited energy metabolism, membrane fluidity, and movement of membrane receptors all inhibited the induction of tolerance. Agents which affect the integrity of cytoskeletal elements also interfered with tolerance induction. Taken together these results indicate that the induction of high-zone thymus-independent tolerance is an active process involving at least some aspects of antigen induced receptor modulation. The specific receptor involved appears to be IgM since tolerance could be induced by exposing cells first to subtolerogenic doses of antigen and then to antibodies specific for the IgM receptor.     

Mechanisms of B cell tolerance. I. Tolerance to dextran B1355 induced with the oxidized dextran.

The bacterial dextran B1355, which is normally a potent thymus-independent immunogen, was made tolerogenic by oxidation. The injection of the oxidized dextran into BALB/c mice before, at the same time, or up to 4 days after the injection of the immunogenic form of the dextran resulted in a marked immunologically specific suppression of the number of anti-dextran antibody-forming cells found in the spleen. This suppression resulted from a direct inactivation of antibody-forming cell precursors rather than from either inhibition of antibody secretion or the exhaustive utilization of precursor B cells that have been observed in other tolerance systems. A substantial degree of tolerance was achieved after only a 1-hr in vivo exposure of the spleen cells to the tolerogen. At a dose of 1 mg of oxidized dextran per mouse, tolerance persised for at least 3 weeks. A complete recovery was apparent by 10 weeks. The stability of the tolerance was demonstrated by transferring tolerant spleen cells to irradiated recipients. The response in the recipient animals to an immunogenic dextran challenge remained suppressed. It appears that the tolerogenicity of the oxidized dextran is due to its ability to couple covalently with free amino groups in or near the receptor site of the cell membrane via the reactive dialdehyde groups of the dextran.     

Interaction of gangliosides with B cells in splenic fragment cultures

The effect of gangliosides upon murine adult B cells at the single precursor cell level was examined using the splenic focus assay. Adult B cells were stimulated in in vitro organ fragment culture by a hapten-modified carrier protein in the presence of an excess of carrier-primed T cells. The addition of a potential tolerogen in the form of antigen coupled to a carrier not previously presented to the T cells resulted in a temporary unresponsiveness of the onset of antibody production in adult B cells, but not a permanent state of tolerance. This delay could be eliminated by the addition of purified murine gangliosides during the presentation of the hapten coupled to the unrecognized carrier. The ganglioside preparation was fractionated by ion-exchange chromatography and the active fraction was found to be a disialoganglioside. These results suggest that the ganglioside may interfere with membrane receptor-related events occurring during or after antigen binding or cross-linking to responding B cells.     

Influence of cortisone on immunologic tolerance in vivo: Cortisone prolongs the duration of unresponsiveness but fails to affect its induction

The influence of cortisone administration on either the induction or the duration of immunologic tolerance was examined in vivo. Tolerance induced by isologous IgG coupled to fluorescein was chosen because the hapten-bearing cell can be directly visualized and the hapten-specific immune response to either a TD antigen or a TI₂ antigen can be tested. It was found that cortisone facilitates the maintenance of tolerance, but fails to affect its induction to either class of antigen. Fluorescein-IgG-bearing cells are cortisone resistant. They are seen for a longer period of time in animals treated with cortisone and tolerogen than in animals treated with tolerogen, and fluorescent cells are either T or B cells. We propose that cortisone facilitates the maintenance of tolerance by maintaining a receptor blockade in vivo. This finding might have clinical implications for the treatment of autoimmunity.     

Expression of IgG memory response in vitro to thymus-dependent and thymus-independent antigens

A model is described in which expression of IgG secondary antihapten responses of large magnitude can be initiated in vitro without resorting to in vivo boosting prior to culture. The number of IgG plaque-forming cells (PFC) is frequently as much as 100-fold greater than that of IgM PFC. Spleen cells from mice primed with trinitrophenylated keyhole limpet hemocyanin (TNP-KLH) several months earlier are stimulated in vitro to produce an anti-TNP plaque-forming cell response 7–10 days later. The in vitro IgG response can be elicited with either a thymus-dependent antigen (TNP-KLH) or thymus-independent antigens (TNP-T₄ bacteriophage or DNP-dextran). The kinetics of the responses to these two forms of antigen differ in that the thymus-independent response peaks two days earlier. The IgG response to both forms of antigen requires the presence of 2-mercaptoethanol (2-ME) even though macrophages are not depleted prior to culture. In the absence of the reducing agent both thymus-dependent and thymus-independent IgG responses were diminished ≥90%. The magnitude of the response to thymus-independent antigens emphasizes the ability of these materials to elicit IgG expression in memory B cells provided optimal conditions for memory development and in vitro expression exist.     

Hapten-specific tolerance induced by hapten conjugates of D-glutamic acid, D-lysine (D-Gl) or isologous gamma-globulin: evidence for central B cell tolerance in the presence of carrier-primed helper T cells.

A comparison has been made of the well known hapten-specific tolerance systems induced, respectively, by hapten-D-GL or hapten-isologous gamma-globulin conjugates. The principal question addressed in this study concerned the comparative maintenance of B cell tolerance, induced by one or the other method, after adoptive transfer into carrier-primed, irradiated recipient animals and, in addition, what role, if any, might be played by T lymphocytes in the tolerant donor cell population in maintaining such tolerance. The results clearly show that insofar as the hapten-specific B cell is concerned, no obvious difference exists in the capacity to maintain tolerance adoptive transfer between the hapten-D-GL and hapten-isologous gamma-globulin systems; such cells remained tolerant even in the presence of excess helper T cell activity. Moreover, under the conditions employed, depletion of T lymphocytes from the tolerant donor cell population did not affect the maintenance of hapten-specific B cell tolerance after adoptive transfer to irradiated recipients.     

The use of haptenated-immunoglobulin molecules to induce tolerance in B cells from neonatal mice

The role of the Fc region of trinitrophenylated (TNP)-immunoglobulins (Ig) in their ability to induce tolerance in immature B cells was examined. With the use of B cells from neonatal mice, tolerogens that could or could not bind to Fc receptors were assessed for their ability to induce tolerance. This was accomplished by tolerizing spleen cells in bulk culture and assessing the degree of tolerance by challenging the cells with the thymus-independent antigen TNP-Brucella abortus (TNP-BA) in limiting dilution cultures. It was found that by using tolerogens containing 10 to 11 haptens per Ig molecule, immature B cells were very susceptible to tolerance induction. Mature B cells were not as susceptible. This increased susceptibility was independent of the Fc portion of the tolerogen, because TNP11-HGG and a TNP10-F(ab')2 induced equivalent degrees of unresponsiveness. When the TNP density was lowered to approximately five haptens per Ig molecule, those Ig molecules that contained Fc portions were superior tolerogens with the use of B cells from 6-day-old mice. Thus, a TNP4-HGG, TNP7-mouse IgG1, and TNP6-mouse IgG2a were more effective tolerogens than either TNP5-F(ab')2 or TNP6-mouse IgG3. These results confirm previous findings that immature B cells are inherently more susceptible to tolerance induction than mature B cells. They also suggest that very lightly haptenated Ig molecules may depend on Fc receptor-binding for effective tolerance induction. Finally, by means of a cytofluorograph, the surface IgD (sIgD) and sIgM phenotypes of splenic B cells from neonates of increasing age were determined. When comparing the phenotype of maturing cells with their tolerance susceptibilities, a correlation between the appearance of sIgD and the acquisition of resistance to tolerance was observed.     

B cell tolerance. I. Analysis of hapten-specific unresponsiveness induced in vitro in adult and neonatal murine spleen cell populations.

The cellular mechanisms and tolerogen dose requirements of hapten-specific unresponsiveness induced in vitro by using 2,4,6-trinitrophenyl human gamma-globulin (TNP17HgG) were analyzed in adult and neonatal murine splenocytes. Tolerance induction in both cell populations was found to be independent of non-B cell effects including BAtheta-positive cells, Ly 2.2-positive cells, adding or reducing the number of macrophages, and large excesses of HgG. The tolerance induced was specific and not "infectious", further excluding a role for suppressor T cells. Neonatal splenic B cells were rendered tolerant by doses of TNP17HgG 1000-fold less than those required to produce similar tolerance in splenic B cells from adults. These findings support the concept of functional clonal abortion as a mechanism for producing tolerance to self antigens.     

Functional heterogeneity of virgin and memory B murine lymphocytes revealed by the utilization of cyclosporin A: an overview

The effects of cyclosporin A on the generation and revelation of B memory cells by thymus-independent (TI) antigens was investigated. A class 1 (TNP-LPS) and a class 2 (TNP-Ficoll) TI antigens were used for priming an elicitation. Evidence is presented that cyclosporin A does not interfere with the generation of hapten-specific (TNP) B memory cells by TNP-LPS or DNP-Ficoll. Cyclosporin A does not affect the revelation of B memory cells by TNP-LPS, but inhibits their revelation by TNP-Ficoll. These findings are discussed in terms of two distinct B cell lineages leading to antibody-forming cells and memory cells precursors, and in terms of heterogeneity of B memory cells.     

Cellular events in tolerance. V. Detection, isolation and fate of lymphoid cells which bind fluoresccinated antigen in vivo.

The binding of tolerogen to specific receptors of lymphocytes and the subsequent fate of such cells was directly studied in Lewis rats injected with fluorescein-labeled sheep gamma globulin (F-SGG). This tolerogen produced unresponsiveness both in SGG-specific T cells (carrier tolerance) and F-specific antibody-forming cell precursors. The former (T-cell tolerance) was still significant more than 60 days after tolerogen whereas tolerance in the latter (B-cell tolerance) had waned by that time.Cells which have bound the tolerogen (antigen-binding cells, ABC) in vivo were detectable by direct immunofluorescence of washed spleen cell suspensions from rats injected with F-SGG up to 7 days previously. These cells were isolated using antifluorescein affinity columns, and shown to contain immunocompetent precursors for F- and SGG specific responses.The frequency of such ABC was between 30 and 80 per 10⁵ spleen, lymph node or bone marrow cells; no ABC were detected in the thymus. Both Ig positive and Ig negative cells were found to be ABC; Ig negative ABC usually showed a “capped” fluorescent pattern whereas Ig positive ABC generally were “spotted.”By 10 days after injection, ABC were not detectable in the spleen, lymph nodes, thymus or bone marrow of tolerant rats. Furthermore, reinjection of F-SGG after this time did not label any cells. This suggests that antigen-binding cells are not present at this time or that such cells, if available, lack receptors. In contrast, rats previously injected with a lower non-tolerogenic dose of F-SGG or an immunogenic form (F-SGG on bentonite) possessed cells at these later times which could be labeled with F-SGG. Thus, ABC remain detectable following immunogen or a subtolerogeic dose of F-SGG, but disappear in tolerant rats.By approximately 40 days after initial high dose tolerogen injection (when B cell tolerance has started to wane), cells capable of binding a second dose of F-SGG again became detectable. It is suggested that high doses of F-SGG are bound by specific lymphocytes (identifiable as ABC) and that these cells either fail to regenerate new receptors or die. As tolerance begins to wane, either new receptors or new cells are generated.     

Cellular basis for a hapten-specific state of tolerance induced in vitro.

A hapten-specific unresponsive state was induced in vitro by the incubation of normal murine spleen cells with highly conjugated dinitrophenylated bovine gamma-globulin (DNP-BGG) or a dinitrophenylated copolymer of D-glutamic acid and D-lysine (DNP-D-GL) for 24 hr. After this incubation period spleen cells were washed and cultured for 4 days with the thymic-independent antigen dinitrophenylated polyacrylamide beads (DNP-PAA) or the thymic-dependent antigen trinitrophenylated burro the erythrocytes (TNP-BRBC). Preincubation with either DNP-BGG or DNP-D-GL led to a specific depression of the in vitro anti-hapten plaque-forming cell response. The degree of depression was dependent upon the concentration of the tolerogen and the duration of preincubation. The response to DNP-PAA or TNP-PAA beads was depressed to a greater degree than was the response to TNP-BRBC. The cellular basis of the immunologic unresponsiveness induced by DNP-BGG was attributable to an inhibition of B cell function whereas the unresponsive state induced with DNP-D-GL was due to both a specific inhibition of B cell function and the activation of antigen-specific suppressor T cells.     

The induction of cell-mediated immunity and tolerance to insulin with insulin coupled to syngeneic lymphoid cells

The induction of delayed type hypersensitivity (DTH) and tolerance to DTH against bovine insulin in mice were explored. DTH was induced with insulin in complete Freund's adjuvant (CFA) and was assessed by ear swelling in vivo and by antigen-driven cell proliferation in vitro. Using the concept that thymus cell unresponsiveness is most easily accomplished via antigen on syngeneic membranes, tolerance was induced by iv injection of syngeneic lymphoid cells which had been coupled to insulin with carbodiimide. Mice tolerized with insulin-coupled cells and then sensitized with insulin-CFA had diminished ear swelling in vivo and decreased insulin-driven cell proliferation in vitro. This unresponsiveness was antigen specific but was also inconstant in degree with regard to suppression of ear swelling, most likely because of variability in coupling of insulin to cells. Proliferative responses were more uniformly suppressed, suggesting the possibility that two target cells were being tolerized. Thus, as with other proteins, the biologically active insulin can be used to induce tolerance.     

The induction of hapten-specific immunological tolerance and immunity in B lymphocytes. V. Evidence for b-cell deletion in vitro with serum from tolerant mice.

The serum from mice that had been rendered specifically tolerant (TolS) to the trinitrophenyl (TNP) hapten by the injection of trinitrobenzenesulfonic acid (TNBS) is effective in the in vitro induction of immunological unresponsiveness in murine spleen cells. This tolerance system was investigated with particular emphasis upon the mode of induction. The observed inhibition by TolS of responses to the thymic-independent (TI) antigen TNP-lipopolysaccharide (TNP-LPS) was stable following adoptive transfer to lethally irradiated recipients and was due neither to the delay of in vitro responsiveness nor to effector cell blockade at the level of the antibody-forming cell. Neither suppressor cells nor cell-bound tolerogen carry-over were responsible for the tolerance induced by TolS. TNP-LPS doses, including a wide range of polyclonal activating concentrations, were ineffective in reversing the unresponsive state induced by cocultivation with TolS. Additionally, unconjugated LPS in either fetal calf serum (FCS)-containing or FCS-free cultures did not break tolerance. This failure of polyclonal activating substances to reverse the unresponsive state suggests that blockade of TNP-specific receptors is not the mechanism of tolerogenesis, since such compounds trigger cells polyclonally through nonimmunoglobulin receptors. Tolerance induced by incubation of spleen cells with TolS for 24 hr followed by extensive washing was stable whether the immunogenic stimulus was the TI antigen TNP-LPS or the thymic-dependent (TD) form of the hapten, TNP-sheep erythrocytes (TNP-SRC). Washing spleen cells at elevated temperatures after preculturing with TolS to avoid possible reassociation of surface Ig (sIg)-bound TNP conjugates did not lead to escape from tolerance. Antigen-free incubation for 24 hr following cultivation with TolS was equally unsuccessful in reversing the unresponsive state. Thus, extensive washing following tolerance induction and antigen-free cultivation where unblocking or turnover and resynthesis of sIg receptors should have taken place provided no support for receptor blockade as the mode of in vitro induction and maintenance of tolerance by TolS. Treatment with the proteolytic enzyme pronase with the intention of removing potential tolerogen from the cell surface revealed a stable tolerant state. Incubation with anti-Ig or anti-TNP antisera under conditions designed to allow capping and removal of sIg-bound tolerogen or surface-bound TNP conjugates also failed to reverse the tolerance induced by incubation with TolS. The results presented here and previously lend no support to active or passive suppression or blockade of reactive cells as the mechanism of tolerance induction in vitro by TolS. The data are consistent with the hypothesis that TolS-induced unresponsiveness is due to a functional deletion of TNP-specific B lymphocytes. Furthermore, the similarities observed between the induction of tolerance by TNBS injection and TolS-induced unresponsiveness are consistent with the suggestion that TNBS-induced tolerance in vivo is mediated by a component of TolS which is active as a tolerogen in vitro.     

The effects of carrier-specific helper T-cell tolerance on antibody avidity in the anti-hapten response.

Rats rendered tolerant to ultracentrifuged sheep γ-globulin (SGG) have been shown to make a poor anti-trinitrophenyl (TNP)-specific antibody response upon challenge with TNP-SGG in complete Freund's adjuvant (CFA). We have been able to use this carrier-tolerance system in studying specific helper T-cell unresponsiveness to IgG and IgM antibody responses. By using the plaque inhibition technique to measure antibody avidity, we found that there appears to be no difference in the avidity of antibody responses to TNP between the SGG-tolerant and control groups when both are challenged with TNP-SGG in CFA. This was found to be true in both the 19 and 7S antibody responses in vivo as well as in an adoptive transfer model. In addition, studies on the maturation of 19 and 7S antibody responses showed no differences in antibody avidity between carrier-tolerant and control groups. These findings imply that carrier-specific helper T cells do not play a controlling role in determining whether high- or low-avidity hapten-specific B-cell precursors will proliferate in response to challenge with a hapten-carrier conjugate.