Injection Of Tolerogen Research Articles

CTLA-4 regulates expansion and differentiation of Th1 cells following induction of peripheral T cell tolerance.

Intravenous treatment with Ag (peptide)-coupled, ethylene carbodiimide-fixed syngeneic splenocytes (Ag-SP) is a powerful method to induce anergy in vitro and peripheral T cell tolerance in vivo. In this study, we examined the effects of Ag-SP administration on T cell activity ex vivo and in vivo using OVA-specific DO11.10 TCR transgenic T cells. Although treatment with OVA323-339-SP resulted in a strong inhibition of peptide-specific T cell recall responses in vitro, examination of the immediate effects of Ag-SP treatment on T cells in vivo demonstrated that tolerogen injection resulted in rapid T cell activation and proliferation. Although there was an increase in the number of OVA-specific DO11.10 T cells detected in the lymphoid organs, these previously tolerized T cells were strongly inhibited in mounting proliferative or inflammatory responses upon rechallenge in vivo with peptide in CFA. This unresponsiveness was reversible by treatment with anti-CTLA-4 mAb. These results are consistent with the hypothesis that Ag-SP injection induces a state of T cell anergy that is maintained by CTLA-4 engagement.

Induction of hapten-specific immunological tolerance and immunity in B lymphocytes: VII. Correlation between trinitrobenzenesulfonic acid administration, serum trinitrophenyl content, and level of tolerance

The induction of immunological tolerance with trinitrobenzenesulfonic acid (TNBS) was studied by a comparison of the concentration of trinitrophenyl (TNP) in the serum of tolerant mice (TolS) and the degree of unresponsiveness induced as the dose and time of tolerogen injection were varied. The concentration of TNP in TolS was greater with a larger dose of TNBS, as expected, and decreased with time after tolerogen injection in a biphasic manner. The rapid initial decline followed on Day 10 by a more gradual decrease in TNP concentration suggests that there were two classes of TNP conjugates produced by TNBS injection. The serum TNP concentration appeared to correlate to the in vivo response of TNBS-treated mice to thymic-dependent and thymic-independent antigenic challenge while little correlation was evident with the in vitro response.

A spleen-thymus interaction is involved in the tolerization of thymocytes to xenogeneic erythrocytes.

Previous work has shown that thymocytes of rats receiving two i.v. injections, 4 days apart, of large numbers of xenogeneic erythrocytes become tolerant of the injected cells whereas peripheral T cells in the same recipients make a vigorous response to the injected antigen. Results are herein presented of experiments on the effect of splenectomy on the induction of thymocyte tolerance produced in this way. Splenectomy performed either immediately before the first injection of erythrocytes, or between the two injections, completely prevented tolerance induction and tolerance was still incomplete when splenectomy was performed as late as a few hours after the second injection of tolerogen. Sham splenectomy had no effect on the erythrocyte-induced thymocyte tolerance. The results demonstrate an essential role for the spleen in the tolerization of thymocytes in this system. Possible mechanisms and the implications for self tolerance are discussed.

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.

Studies on the resistance to tolerance induction against human IgG in DDD mice: III. Development of the resistance with age and cellular events

Three-week-old DDD mice were easily rendered tolerant to human IgG while 12-week-old mice were tolerized only partially. Mechanisms of the development of the resistance with age were investigated. It was shown by the cell transfer experiments that spleen T cells, purified on a Tetron wool column, from older mice were responsible for the resistance, which was not associated with the loss of suppressor cells with age. To elucidate the possibility of whether tolerogen-sensitive spleen T cells differentiate into resistant ones, cell transfer experiments were carried out in which thymectomized, lethally irradiated mice were reconstituted with spleen cells from 3-week-old mice and then treated with the tolerogen on various days afterward. The results indicated that tolerance was inducible in these hosts to the same degree, irrespective of the timing of the tolerogen injection, while age-matched intact mice gradually acquired the resistance. Then the possibility of whether age of thymus affected tolerance inducibility of the hosts or not was examined. The tolerogen was injected into irradiated, bone-marrow-reconstituted mice which bore either 4- or 7-week-old thymus. It was shown that helper T cells newly generated under younger thymus acquired higher susceptibility to the tolerogen. There was no difference in tolerance inducibility irrespective as to whether bone marrow cells were prepared from younger or older mice. From these observations it was suggested that the resistance to tolerance induction in DDD mice is acquired through the appearance of resistant T cells which are generated from T-cell precursors in bone marrow under the influence of a radioresistant thymic constitution and predominantly located in the spleen.

Antigen-specific suppressor cells in hapten-specific carrier-determined tolerance.

Tolerance to the hapten 2,4-dinitrophenyl (DNP) induced by the injection of DNP coupled to isologous IgG (carrier-determined tolerance) is associated with a receptor blockade of antigen-binding lymphocytes. In the present study, hapten-specific suppressor cells were detected in the spleens of mice made tolerant by intravenous injection of 20 microgram DNP-IgG. When spleen cells from mice rendered tolerant to DNP were co-cultured with normal spleen cells in Marbrook cultures, the response to DNP-Ficoll was suppressed, while the response to sheep red blood cells was not altered. Depletion of T cells from these spleens restored the normal anti-DNP response. The suppressor cells were not detectable in the spleen lymphocyte population of mice in the early stages of tolerance but were present on day 7 after injection of tolerogen, and disappeared by day 14. Mice injected with larger doses of 1 mg or four weekly doses of 200 microgram DNP-IgG did not have detectable suppressor cells. Thus, it appears that a short-lived suppressor T cell is generated in carrier-determined tolerance. This cell most likely plays a minor role in the mechanism of carrier-determined tolerance and may be associated with the receptor blockade which is seen early in tolerance.

Studies on the resistance to tolerance induction against human IgG in DDD mice: I. Organ differences of tolerogen susceptibility and cellular sites responsible for the resistance

Cellular sites of the tolerogen resistance in DDD mice against human IgG (HGG) were examined by reconstitution experiments in which cells of various lymphoid organs from tolerized mice were transferred into lethally irradiated syngeneic recipients with or without the supplement of an excess number of untreated T or B cells. It was shown that T cells but not B cells in the spleen and bone marrow-locating B cells were tolerogen resistant. Kinetic profiles of tolerance induction were compared among thymus, lymph node, and spleen T cells. Thymus cells fall into unresponsive state as early as 2 days after the tolerogen (tHGG) injection when only partial tolerance was observed in lymph node T cells. By 1 week of tolerogen treatment, the tolerant state was completed in both thymus cells and lymph node T cells, while spleen T cells showed marked resistance. Tolerance induced in thymus cells and spleen T cells was of relatively short duration and responsiveness was completely recovered by 5 weeks after the injection of tHGG. At this time lymph node T cells still showed hyporesponsiveness. The differences in tolerance inducibility were also shown among different lymphoid organs in tolerogen dose response. Lymph node T cells were very sensitive to tolerance induction, giving no response even by the injection of 0.01 mg of tHGG. Thymus cells were much less sensitive with the gradual loss of responsiveness by increasing the amount of tHGG. In contrast, spleen T cells showed gradual resistance with increasing amount of tHGG, indicating that some positive response was evoked in spleen T cells by a relatively high dose of tHGG. These results seem to suggest that the tolerogen resistance of spleen T cells may be due to their capability of showing positive response against the tolerogenic material. This was also suggested by the fact that the treatment with cyclophosphamide following the tolerogen injection diminished completely the responsiveness against the subsequent challenge immunization.

Tolerance Induced by TNP-Derivatized Syngeneic Erythrocytes: Evidence for Cooperation between Hapten-Specific T and Hapten-Specific B Lymphocytes in the Immune Rensponse

Abstract 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 cell 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.

Mechanisms of tolerance to HGG induced in neonatal and adult mice.

In 1973 we repeated some of the experiments initially carried out by Chiller, Weigle, and their colleagues (Chiller et al., 1974) by attempting to terminate the tolerant state to human γ-globulin (HGG) induced in adult A/J mice by reconstitution with normal or immune lymphocytes or by the injection of crossreacting γ-globulin. We found the same results, i.e. that it was nor possible to terminate this tolerant state by either method. One would have thought that if clonal deletion was the sole mechanism responsible for this tolerance, that by providing the missing specific lymphocyte, these mice could be reconstituted to a fully normal status. Both of these methods were highly successful in terminating the tolerance to bovine serum albumin (BSA) induced in neonatal rabbits (Benjamin and Weigle, 1970; Benjamin, 1974). The results obtained in the attempts to terminate this HGG tolerance with heterologous γ-glob-ulins made us suspect that there was much more involved than the simple deletion of HGG-specific clones. The data shown in Table I illustrate this point quite well. Adult A/J male mice that had been injected with 2.5 mg deaggregated HGG (DHGG) do not respond to subsequent injections of aggregated HGG (AHGG) (Group 2), nor is this tolerant state terminated by the injection of aggregated bovine γ-globulin (ABGG) (Group 3). In fact the response of such HGG tolerant mice to the specific determinants on BGG is much reduced (compare the BGG response of Groups 3 and 4 — Table I). The reverse is also true, i.e. mice tolerant to BGG do not respond well to HGG specific determinants. Ruben et al. (1973) attributed this lack of response to non-crossreacting determinants to the loss of crossreactive T-helper cells in these HGG tolerant mice. However, it seemed to us that it could also have been due to the presence of crossreactive suppressor cells induced by the injection of tolerogen.

Adjuvanticity (immunity-inducing property) of cord factor in mice and rats.

Cord factor, a glycolipid in mycobacteria, was found to make a tolerogenic protein antigen immunogenic when injected 0 to 2 days before tolerogen injection in mice and rats. Cord factor was also found to increase phagocytic function of the reticuloendothelial system of mice, and the relationship between the immunity-inducing capacity and the phagocytic function is discussed.

Hapten-bearing cell in carrier-determined tolerance.

AbstractThe relationship between tolerance to the 2,4‐dinitrophenyl (DNP) hapten and cells bearing this hapten on their surface, was investigated using autoradiography. Animals injected with nontolerogenic conjugates of DNP‐ε‐lysine‐IgG, DNP‐azo‐IgG, and DNP‐mustard‐IgG (J. Immunol. 1975. 115: 1409) did not have splenic lymphocytes bearing DNP on their surface 5 days after their last injection, while those injected with tolerogenic conjugates of DNP‐ε‐lysine‐IgG did have splenic hapten‐bearing cells. Control studies showed that I25I‐labeled anti‐DNP used to reveal hapten‐bearing cells did react with the nontolerogenic conjugates on the cell surface. There was no difference in the numbers of hapten‐bearing cells in animals receiving either 100 μg or 1000 μg of tolerogenic DNP‐IgG 5 days after the injection. Animals injected with 1000μg of tolerogen remained tolerant for 6 months. These tolerant animals still had hapten‐bearing cells in their spleens 6 weeks after injection. In contrast, animals which had lost tolerance 3 months after 200 μg of tolerogen injection did not have hapten‐bearing cells.These experiments confirm that the hapten DNP remains on the surface of hapten‐binding cells in tolerance only. It is unlikely that the carrier alone, either nonspecifically or specifically via Fc binding, accounts for the presence of the tolerogen on lymphoid cells because it would be observed with nontolerogenic as well as tolerogenic DNP‐IgG conjugates. A direct relationship between tolerance and hapten‐bearing cells exists. The significance of this correlation to the mechanism of tolerance is discussed.

In vitro tolerance induction to a thymus-dependent antigen with BALB/c and C57BL/6 lymph node cells: effect of tolerogen concentration and duration of exposure.

Further studies on tolerance induction in vitro to bovine gamma globulin (BGG) in nonadherent BALB/c lymph node cells showed that it was dependent upon both the dose of tolerogen and the time of exposure. Nearly complete tolerance was achieved at a dose of 1.0 mg/ml and an incubation time of 12-18 hours. When C57BL/6 strain was used for comparison with the BALB/c strain because of its relative ease to become tolerant after in vivo injection of tolerogen, the rate of tolerance induction of its nonadherent lymph node cells was not different from that of BALB/c cells. Thus, in the absence of other host factors, the acquisition of tolerance by lymphocytes is gradual and may reflect the time required for a cell to reach a susceptible phase of the cell cycle and/or the activation of suppressor cells.

The Induction of Hapten-Specific T Cell Tolerance by Using Hapten-Modified Lymphoid Cells

Abstract BALB/c mice were made tolerant to the T cell-dependent phenomenon of contact sensitivity to DNFB by i.v. injection of syngeneic lymphoid cells which had been previously modified with DNFB in vitro. The highly efficient unresponsiveness, as measured by ear challenge and in vitro antigen-induced cell proliferation, was shown to follow dose-response kinetics both in vivo and in vitro and to be exquisitely specific for the DNP moiety. The kinetics of tolerance induction were shown to be very rapid and had been previously shown to be long lasting. Unresponsiveness was more efficient when the hapten-modified cells were introduced by the i.v. route and tolerance could be increased by repeated injection of tolerogen. The tolerance could be transferred to normal syngeneic recipients by spleen and/or lymph node cells from tolerant donors. A wide variety of hapten-modified lymphoid cells, including mixed cell populations and enriched populations of T cells, B cells, and macrophages, were capable of inducing tolerance. The unresponsiveness was dependent merely on the association of DNP to the lymphoid membrane proteins and not upon the viability of the hapten-modified cells. These experiments support the hypothesis that in hapten-specific T cell sensitivity, as exemplified by contact sensitivity to DNFB, specific T cell tolerance in actively induced by hapten on self-membrane. In other hapten-specific antibody-forming systems, tolerance appears to be most readily induced by hapten on soluble self protein or on a nonimmunogenic carrier.

Cellular events in tolerance: V. Detection, isolation and fate of lymphoid cells which bind fluoresceinated 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 10 5 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.

Quantitative and qualitative analyses of the antibody response of adult mice tolerized with deaggregated bovine gamma-globulin. I. At the serum antibody level.

The antibody response of mice to bovine γ-globulin(BGG) was suppressed either specifically by an intravenous injection of deaggregated soluble BGG (sBGG) or nonspecifically by X-irradiation. Immunization with the subcutaneous injection of BGG in Freund's incomplete adjuvant was given to mice either various days after sBGG injection or immediately after X-irradiation. Antigen-elimination (AE) test and passive hemagglutination(PHA) test were employed for estimating the immune status. The AE test indicated that the induction of tolerance was accomplished in the first 2 days after sBGG injection and that the tolerant state was stable at least for about 30 days thereafter. The degree of suppression by 1000 μg of sBGG corresponded to that obtained by X-irradiation at the dose of 400 R or more, and 100 μg of sBGG was equivalent to 300 R X-irradiation. The PHA test indicated, however, that such a correspondence as mentioned above between the dose of tolerogen and that of X-irradiation was not so stable as was seen by the AE test. Thus, the PHA titers of tolerized animals tended to recover up to the level of untolerized animals during the period of time from 10 days to 20 days after the tolerogen injection. Such discrepancies between the features in the AE test and those in the PHA test seemed attributable to a low avidity antibody formation in the tolerized animals, as judged by the hemagglutination-dissociation test. Hemagglutination by means of the sera from tolerized animals was seen to be reversed by the addition of free antigen more easily than the hemagglutination achieved by the sera of control animals or X-irradiated animals. The relationship between PHA titers and AE capacities of antibodies was investigated by the passive immunization of normal mice previously given the antigen. The result showed that the PHA titer did not always correlate with the AE capacity.

Quantitative and qualitative analyses of the antibody response of adult mice tolerized with deaggregated bovine gamma-globulin. II. At the cellular level.

The antibody response of mice to bovine γ-globulin(BGG) was suppressed either specifically by an intravenous injection of deaggregated soluble BGG (sBGG) or nonspecifically by X-irradiation. Immunization with the subcutaneous injection of BGG in Freund's incomplete adjuvant was given to mice either various days after sBGG injection or immediately after X-irradiation. Antigen-elimination (AE) test and passive hemagglutination(PHA) test were employed for estimating the immune status. The AE test indicated that the induction of tolerance was accomplished in the first 2 days after sBGG injection and that the tolerant state was stable at least for about 30 days thereafter. The degree of suppression by 1000 μg of sBGG corresponded to that obtained by X-irradiation at the dose of 400 R or more, and 100 μg of sBGG was equivalent to 300 R X-irradiation. The PHA test indicated, however, that such a correspondence as mentioned above between the dose of tolerogen and that of X-irradiation was not so stable as was seen by the AE test. Thus, the PHA titers of tolerized animals tended to recover up to the level of untolerized animals during the period of time from 10 days to 20 days after the tolerogen injection. Such discrepancies between the features in the AE test and those in the PHA test seemed attributable to a low avidity antibody formation in the tolerized animals, as judged by the hemagglutination-dissociation test. Hemagglutination by means of the sera from tolerized animals was seen to be reversed by the addition of free antigen more easily than the hemagglutination achieved by the sera of control animals or X-irradiated animals. The relationship between PHA titers and AE capacities of antibodies was investigated by the passive immunization of normal mice previously given the antigen. The result showed that the PHA titer did not always correlate with the AE capacity.

The ability of bacterial lipopolysaccharide to modulate the induction of unresponsiveness to a state of immunity. Cellular parameters.

Studies were performed to define the cellular parameters involved in the interference with the induction of immunologic unresponsiveness to human gamma globulin (HGG) by bacterial lipopolysaccharide (LPS). Mice which were injected with deaggregated HGG (tolerogen) and with LPS did not become tolerant to that antigen, but rather became primed to a subsequent challenge with immunogen. The ability to prime with tolerogen and LPS was also demonstrated in an adoptive transfer system. The temporal relationship between the injection of tolerogen and that of LPS was critical for priming to occur. The injection of tolerogen and LPS not only primed mice to HGG, but also resulted in a primary antibody response to HGG. The capacity of LPS to interfere with the induction of tolerance was restricted to B cells and did not affect the ability to induce unresponsiveness in T cells. The secondary response to HGG in mice primed by tolerogen and LPS was found to be T-cell independent. These observations are interpreted and discussed from the standpoint of the ability of LPS to circumvent required T-cell cooperation and to modulate to tolerogenic stimulus into an immunogenic signal.

Fate of antigen-binding cells in unresponsive and immune mice.

Antigen-binding cells (ABC) to the antigen human gamma globulin (HGG) were quantitated in lymphoid tissues of A/J mice at various times after the injection of deaggregated HGG (tolerogen), aggregated HGG (immunogen), or saline. The reaction of lymphoid cells with highly labeled HGG was specific to that antigen since binding could be inhibited by excess unlabeled HGG, but not by unrelated non-cross-reacting proteins. Compared with normal mice, there was a marked decrease in the numbers of ABC in the spleens of unresponsive animals evident as early as 12 h after the injection of tolerogen. A marked increase in ABC was observed in the spleens of immunogen-injected mice, beginning at 24 h and reaching a peak at 3 days. In bone marrow, no difference in the number of ABC was found among the three experimental groups until day 20, when a reduction in ABC was observed only in tolerogen-injected mice. No quantitative difference in the thymuses in the experimental groups could be determined because of the paucity of ABC displayed by normal thymus cells.

Studies on M and G antibody response of mice to bovine serum albumin. II. Induction of tolerance.

Tolerogenic effect of soluble deaggregated bovine serum albumin (sBSA) on the γM and γG antibody responses was studied. Normal and tolerogen-treated animals were immunized with the intravenous injection of alum-precipitated BSA (net 0.1 mg) and a bacterial endotoxin (0.01 mg), since this immunizing procedure was the most adequate to elicit the strong responses of both types of antibodies. Both γM and γG responses were decreased evenly by a single injection of small dose (0.01 or 0.1 mg) of sBSA. Larger doses (1 mg or 5 mg) caused the marked suppression of γM response, but the γG response was reduced only slightly. The tolerant state was facilitated by weekly injections of 0.1 mg of sBSA, both γM and γG responses being abrogated profoundly by more than three injections. The induction of such a complete tolerance was not ascribed solely to either the total dose or the period of time between the first tolerogen injection and the challenge immunization, but to the retention of antigen more than a certain threshold amount in the body for some duration.

Cellular Events during Induction of Immunologic Unresponsiveness in Adult Mice

Abstract A specific and completely unresponsive state in adult mice was induced at the cellular level with ultracentrifuged human γ globulin (HGG). The cellular kinetics of induction of unresponsiveness were quantitated using a cell transfer system and the hemolytic plaque assay to detect plaque-forming cells to HGG. The results demonstrated that although 100% of the cells were not unresponsive until the 4th or 5th day after injection of tolerogen, 75% of the cells were unresponsive 6 hr following tolerogen. The kinetic curve did not reflect sequential induction to determinants located on the Fc and Fab fragments of HGG. Furthermore, the process of inducing unresponsiveness to HGG did not demonstrate antibody-forming cells detectable by the hemolytic plaque assay in the spleens of tolerogen-treated mice.