Cellular events in tolerance. VII. Decrease in tolerant spleens of PFC precursors stimulated in vitro by specific antigen or mitogen.

Spleen cells from adult mice rendered tolerant to the fluorescein (FL) hapten (as FL-sheep γ-globulin) were analyzed at limiting dilution for the numbers of precursors stimulatable either by specific antigen (FL-polymerized flagellin; FL-POL) or by a polyclonal B-cell activator (E. coli lipopolysaccharide; LPS). As expected, the number of PFC presursors activated by FL-POL was reduced more than fourfold in the spleens of FL-tolerant mice compared to normal controls. In contrast, LPS was able to trigger equivalent numbers of “FL-specific” PFC precursors in both normal and tolerant spleens. However, the clones stimulated by LPS were predominantly the “low-avidity” precursors in FL-tolerant spleens as shown by plaque inhibition studies. In addition, after FL-gelatin enrichment of normal or tolerant spleen cells, which contain equal numbers of antigen-binding cells, we found that purified cells from tolerant mice were in fact reduced in the numbers of clonable precursors upon LPS stimulation. Two other B-cell mitogens, POL and PPD, also failed to activate PFC precursors from FL-gelatin-purified tolerant spleen cells. Our results suggest that some high-avidity clones may be functionally deleted even in adult B-cell tolerance as previously noted for neonatal tolerance.     

Similarity and difference in the mechanisms of neonatally induced tolerance and cyclophosphamide-induced tolerance in mice

The mechanisms of cyclophosphamide (CP)-induced tolerance were investigated by comparing with those of neonatally induced tolerance. When C3H/He Slc (C3H; H-2k, Mls-1b) mice were given i.v. either AKR/J Sea (AKR; H-2k, Mls-1a) or (AKR x C3H)F1 (AKC3F1; H-2k, Mls-1a/b) spleen cells and treated i.p. with CP 2 days later, a long-lasting skin allograft tolerance to AKR was induced in each case without any signs of graft-vs-host disease (GVHD). However, typical signs of GVHD were observed in the C3H mice neonatally tolerized with AKR spleen cells, but not in those tolerized with AKC3F1 spleen cells. The expression of TCR V beta 6, which is strongly correlated with the reactivity to Mls-1a Ag (of donor AKR origin), in the periphery was quite different between the two types of tolerant C3H mice. Namely, in the lymph nodes of the C3H mice tolerized with AKR spleen cells and CP, only CD4(+)-V beta 6+, but not CD8(+)-V beta 6+, T cells selectively disappeared, whereas both of them were abrogated in the lymph nodes of the C3H mice neonatally tolerized of AKR. By contrast, in the thymus of the two types of tolerant C3H mice, both CD4+CD8- and CD4-CD8+ single-positive thymocytes expressing TCR V beta 6 were clonally deleted, suggesting that the thymic involvement was the same in each type of tolerance. These results suggest that the preferential disappearance of the CD4(+)-V beta 6+ T cells (of host origin) and the effector T cells of GVHD (of donor origin) occurred only in the periphery of the C3H mice tolerized with AKR spleen cells plus CP and was attributable to the destruction of Ag-stimulated T cells by the CP treatment. In contrast, the intrathymic clonal deletion of immature V beta 6+ T cells was a common mechanism for both of the tolerance induction systems.     

Transient T and B cell activation after neonatal induction of tolerance to MHC class II or Mls alloantigens.

The neonatal injection of semiallogeneic F1 spleen cells into newborn parental mice results in the induction of tolerance to the corresponding alloantigen (alloAg) and chimerism. In these F1 cell-injected mice, we have previously observed that this state of specific tolerance is associated with the development of a transient lupus-like autoimmune syndrome. In this study, we show that neonatal injection of mice with spleen cells differing from the host at major histocompatibility complex (MHC) class I, class II, class (I + II), or minor lymphocyte stimulating (Mls) alloAg induced a state of specific tolerance characterized by the absence of alloreactive CTL and/or Th cell responses in the spleen and the thymus of 6- to 12-week-old injected mice. However, in mice rendered tolerant to MHC class II or class (I + II) alloAg, the presence of high levels of IgG1 antibodies, of circulating immune complexes, of anti-ssDNA autoantibodies, and of tissue lesions were transiently observed. In these mice, an increased Ia Ag expression on lymphoid spleen cells was also detected at 1 wk. The elevated production of IgG1 and the overexpression of Ia Ag were almost completely prevented by treatment with an anti-IL-4 mAb. Such manifestations of B cell activation and autoimmunity were not observed in mice neonatally injected with F1 cells differing from the host only at MHC class I Ag. In mice neonatally tolerized to Mls Ag, a transient increase in IgG2a production and an overexpression of Ia Ag were detected without features of autoimmunity, and were prevented by anti-INF-gamma mAb treatment. In mice rendered tolerant to MHC class II, class (I + II), or Mls alloAg at birth, the manifestations of B cell activation were associated with the presence of in vivo-activated alloreactive CD4+ T cells in the spleen--but not the thymus--of 1-wk-old injected mice. Together, these results suggest that in mice neonatally injected with semiallogeneic F1 cells, the process of tolerance induction is not efficient during the early postnatal period, and could allow the maturation and peripheralization of some alloreactive CD4+ T cells, leading to transient B cell activation and, depending on the alloAg, to autoimmunity.     

Neonatal splenic suppressor cells in the chicken. I. In vivo suppression of the immune response to bovine serum albumin by normal and tolerized neonatal spleen cells

The presence of active splenic suppressor cells in neonatal chickens, either normal or tolerant to bovine serum albumin (BSA), was examined by assessment of their effect on both primary and adoptively transferred secondary responses to BSA or sheep red blood cells (SRC). Both normal and BSA tolerized spleen cells were shown to be highly suppressive of secondary anti-BSA responses generated by specifically primed adult spleen cells in inert recipients. Suppression of the secondary anti-BSA response by normal spleen cells was slightly less effective than that seen with BSA tolerant spleen cells. Transfer of BSA tolerant spleen cells into normal recipients, followed by BSA challenge, prevented any significant primary anti-BSA response. In contrast, transfer of normal spleen cells into normal recipients, followed by BSA challenge, failed to show any suppression of the resulting primary response. Neither normal nor BSA tolerant neonatal spleen cells were capable of suppressing either primary or secondary responses to SRC. Thus, chickens tolerized to BSA have suppressor cells specific for the tolerizing antigen. We present evidence that both the tolerance associated suppressors and the suppressors detected in normal neonatal chickens are T cells.     

Loss of carrier-determined tolerance in vitro with loss of receptor blockade.

Experiments were done to determine whether carrier-determined tolerance is reversible and whether the loss of tolerance is accompanied by the loss of receptor blockade. Spleen cells from mice made tolerant with DNP-isologous IgG remained tolerant when transferred to irradiated syngeneic mice. If these same tolerant spleen cells were incubated for 24 hr or more before transfer the tolerance was lost. Autoradiology was done on the tolerant cells with either 125I anti-DNP or 125DNP-KLH, before and after incubation in vitro. When the cells were tolerant the number of DNP ABC was decreased whereas cells having DNP on their surface were increased. When the cells lost tolerance after in vitro incubation, the hapten-bearing cells were no longer present although the number of cells free DNP receptors increased to normal. These data suggest that in carrier determined tolerance the reactivation of tolerant lymphocytes may involve reversible receptor blockade.     

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 of tolerance to serum albumin in adult mice. I. characterization of T suppressor and T helper cells.

The surface markers and size of suppressor cells were determined in adult (BALB/c x C57BL/Ka)F1 mice which were tolerized with a single injection of deaggregated bovine serum albumin (BSA). Suppressor cells from the spleens of tolerazided donors were assayed in a cell transfer system in which graded numbers of cells were injected into irradiated syngeneic mice along with limiting numbers of T cells primed to BSA and an excess of B cells primed to DNP-BSA. Adoptive hosts were challenged with DNP-BSA in saline, and the anti-DNP response was measured. Suppressor cells were antigen specific as shown by the inhibitory activity of BSA-tolerant spleen cells on the response to DNP-BSA, but not to DNP-BGG. Suppressor cells were eliminated by in vitro treatment with anti-Thy 1.2, anti-Ly-2.2, anti-I-J subregion antisera and C, but not with anti-Ly-1 or anti-I-A subregion antisera. Neither unprimed nor primed helper T cells were detected in the spleen of tolerized donors after in vitro treatment with anti-Ly-2.2 antisera. Both helper and suppressor T cells from the spleens of primed or tolerized donors, respectively, showed a rapid sedimentation velocity (S greater than 3.7 mm/hr).     

Mechanism of neonatal idiotype suppression. II. Alterations in the T cell compartment suppress the maturation of B cell precursors

The cellular mechanism in neonatally suppressed BALB/c mice, which maintains the chronic suppressed state of the TEPC-15 idiotype in the antibody response to phosphorylcholine (PC), was investigated. Cells taken from these suppressed mice cannot transfer suppression to adult BALB/c or affect the in vitro response to PC of adult BALB/c spleen cells. However, spleen cells or T cells from neonatally suppressed mice given to neonatal animals induce chronic suppression of the TEPC-15 idiotype in the anti-PC response. Co-transfer of T cells from neonatally suppressed cells with normal T cells prevented the induction of suppression in neonates. Transfer of T cells from normal or keyhole limpet hemocyanin-primed BALB/c increased the expression of TEPC-15 idiotype in chronically suppressed mice, whereas T cells from neonatally suppressed were ineffective. These findings show that T cells in neonatally suppressed mice can affect the development of immature but not mature cells. The restoration of TEPC-15 expression in neonatally suppressed animals by normal T cells and the failure to induce suppression in neonates by co-transfers of T cells from normal and chronically suppressed mice demonstrate the profound role of an altered T cell compartment in sustaining chronic idiotype suppression.     

Modification of the cytotoxic T cell repertoire in neonatal tolerance. Evidence for preferential survival of cells with low avidity for tolerogen

Clonal deletion of developing lymphocytes with potential reactivity for self is thought to play a crucial role in the establishment of self tolerance. One prediction of the clonal deletion hypothesis is that cells bearing receptors with high affinity for self are more likely than cells with low affinity receptors to be deleted from the repertoire. Experimental models of B cell tolerance have provided evidence for the preferential survival of low affinity cells with specificity for tolerogen in tolerant animals, but no comparable evidence exists for T cells. To examine this issue in T cells, cytotoxic T cell lines specific for the Kb mutant class I H-2 molecule, bm1, were generated from C57BL/6 mice rendered neonatally tolerant of bm1 and compared with anti-bm1 lines generated from normal mice. Compared with normal lines, those from tolerant mice differed in five ways: 1) they grew more slowly; 2) they were less efficient at lysing bm1 targets; 3) they showed different patterns of lysis against a panel of third party targets; 4) their cytotoxic activity against bm1 could be increased in the presence of leukoagglutinin, whereas the activity of normal lines was not increased by leukoagglutinin; and 5) their cytotoxic activity was more susceptible to inhibition by anti-Lyt-2 antibody. Taken together, these results demonstrate that the repertoire of the remaining tolerogen-specific cytotoxic T cells in neonatally tolerant mice is different from the normal C57BL/6 anti-bm1 repertoire, and the results are consistent with the idea that the remaining tolerogen-specific cells are low avidity cells that have preferentially escaped the clonal deletion process.     

Depressed Cellular Formation of Antibody to Shigella Antigens In Vitro by Spleen Cell Cultures from Unresponsive Mice

Specific antibody plaque-forming cells (PFC) to Shigella-soluble antigen did not appear in spleen cell cultures from Shigella-tolerant mice, as occurred with similar cultures prepared from normal mice immunized with Shigella antigen prior to sacrifice. Cultures from tolerant mice also failed to form serologically detectable amounts of agglutinins in vitro. Exposure of cell cultures from tolerant mice in vitro to additional antigen had little or no effect on appearance of plaque-forming cells to Shigella. Spleen cells from normal control mice formed readily detectable levels of antibody, as well as specific antibody plaque-forming cells, after similar stimulation with antigen either in vivo or in vitro. The absence of antibody-forming cells in cultures prepared from spleens of tolerant mice was specific since such cultures, as well as those from normal control mice, formed numerous antibody plaques to unsensitized sheep erythrocytes in vitro after in vivo challenge of the mice with sheep erythrocytes. Tolerance to Shigella antigen, as assessed by absence of antibody-forming cells in vitro, persisted for several months. Spleen cell cultures from tolerant mice less than 3 to 4 months of age did not form significant numbers of antibody plaques, even after in vitro exposure to specific antigen. However, spleen cultures prepared from neonatally treated mice, approximately 6 to 8 months old, formed essentially normal numbers of specific PFC in vitro, indicating that the animals had "recovered" from tolerance and that their lymphoid cells were capable of responding to Shigella antigen in vitro. Absence of specific PFC in cell cultures from tolerant animals supports the concept that tolerance is due to a central failure of specific immunocompetent cells and not due to an inhibitory effect caused by either "excess" antigen or humoral antibody.     

Early endotoxin tolerance is associated with alterations in bone marrow-derived macrophage precursor pools

Early endotoxin tolerance has been defined as the transient period after an initial sublethal exposure to LPS during which a normally responsive individual is rendered hyporesponsive. Little is known about the cellular mechanisms that underlie this phenomenon. In this study, an early tolerance system was established by the injection of mice with 25 micrograms of E. coli K235 LPS. Maximal hyporesponsiveness in response to a challenge injection was observed 3 to 4 days after the initial injection, and normal responsiveness returned by 8 days after the initial exposure to LPS. Further experiments described herein demonstrate that the acquisition and maintenance of the tolerant state coincides temporally with an increase in the number of macrophage progenitor cells in the bone marrow. Cell-sizing profiles of the bone marrow cells from tolerized mice indicate an enrichment for a population of cells that are significantly larger than in bone marrow preparations from control mice. By density gradient sedimentation, it was shown that the denser population of cells from tolerized mice contained the increased numbers of progenitor cells, which, by cytology, were immature monocytic cell types. The increased numbers of macrophage progenitors was sustained after a second (challenge) injection of LPS. These results indicate that early endotoxin tolerance is associated with an increase in a population of bone marrow cells that is enriched for macrophage progenitors and suggests the possibility that the lack of responsiveness observed during the hyporesponsive period is related to a failure of these immature cell types to respond to LPS.     

Neonatally induced tolerance to soluble protein antigens. II. A role for the thymus in prolonging tolerance

Mice were rendered tolerant to bovine serum albumin (BSA) or fowl γ-globulin (FGG) by neonatal injection. Spleen and thymus cells from tolerant mice were able to suppress responsiveness of normal adult spleen cells, but only if tolerant donor mice were between the ages of 6 weeks and the age at which mice were no longer tolerant (10 weeks for BSA tolerance and 20 weeks for FGG tolerance). To determine whether T-cell-dependent suppression was obligatory for the maintenance of tolerance, neonatal nude and euthymic littermate mice were injected with tolerizing doses of FGG. FGG-specific B-cell tolerance in nude mice lasted until the mice were 8 weeks of age. In sharp contrast, B-cell tolerance in euthymic littermates lasted until 22 weeks of age. These results are consistent with a “fail-safe” role of T-cell-dependent immune suppression in the maintenance of tolerance.     

Early loss of precursors of CTL and IL 2-producing cells in the development of neonatal tolerance to alloantigens

Bulk culture and limiting dilution analysis (LDA) were used to follow the ontogeny of the tolerant state in CBA/ HT6T6 mice neonatally tolerized to allogeneic histocompatibility antigens. Advantage was taken of the fact that the lymph nodes (LN) of young mice show immunocompetence before spleen cells do, allowing analysis of actual reactivity as early as 1 wk of age. At 1 wk, the LN cells of mice tolerized i.v. showed a loss of CTL reactivity in bulk culture specific for the tolerizing antigens; a corresponding specific decrease was seen in the frequency of CTL precursors (CTLp). At the same age, however, proliferative responses and interleukin 2 (IL 2) production in MLC were nonspecifically depressed in the tolerized animals. LDA of IL 2 producer precursor frequency (IL- 2Tp ) showed that there was a nonspecific loss of 50% of functional alloreactive IL- 2Tp , accompanied by a larger specific decrease of 90% in the frequency of IL- 2Tp responding to the injected alloantigens. These characteristics of the tolerant state persisted through at least 4 wk of age. Neither the proliferative nor CTL response deficiencies could be overcome by the addition of Con A supernatant containing IL 2. Mixing experiments failed to show evidence of suppressor cell involvement in the loss of the proliferative response. Our results indicate that the specific loss of alloreactivity after tolerization is due to clonal inactivation or deletion of both CTLp and IL- 2Tp , which is obvious as early as 7 days of age. In addition, the differences in the specificity of the clonal inactivation between CTLp and IL- 2Tp suggest the existence of independent mechanisms for tolerization.     

Allo-I-J determinants participate in maintenance of neonatal H-2 tolerance

To evaluate the role of IJ antigens in maintenance of the tolerant state in adult H-2 tolerant mice, we have attempted to abolish tolerance by injecting monoclonal antibodies (mab) specific for host, donor, or third party IJ antigens into adult H-2 tolerant mice. Abolition of tolerance was evidenced by the rejection of fresh test skin grafts bearing the tolerated antigens. Whole H-2 tolerant mice treated with anti-IJ mab specific for donor (allo) IJ antigens rejected their test skin grafts, indicating that tolerance had been abolished. When two other types of tolerant mice were tested, we found that mice tolerant of class II antigens alone, but not mice tolerant of an IJ thru D disparity, were susceptible to the anti-donor IJ mab treatment. In addition, adult tolerant mice were unaffected by treatment with either anti-host or anti-third party IJ mab. When tested in vitro, lymphoid cells from tolerant mice, the tolerance of which was abolished by anti-IJ mab, remained unresponsive to the tolerogen, just as untreated (control) tolerant mice, in several in vitro assays (e.g., mixed lymphocyte reaction, cytotoxic T cell precursor frequency and bulk cell-mediated lysis without growth factor). Mice treated with antidonor IJ mab, however, unlike mice treated with anti-host or third party IJ mab, were capable of generating tolerogen-specific T cells in the absence of exogenous growth factor. Thus in the strain combinations we used, adult mice tolerant of either the entire H-2 region or of the class II major histocompatibility complex region alone are susceptible to abolition of the tolerant state by treatment with anti-donor IJ mab. Coincidentally, lymphoid cells from these mice generate sufficient endogenous T helper activity to activate the tolerogen-specific cytotoxic T cells. We suspect that these latter cells may be responsible for rejection of grafts bearing the tolerated antigens.     

Tolerance mechanism in experimental ovarian and gastric autoimmune diseases.

Neonatal splenocytes, neonatal thymocytes, or phenotypically mature adult thymocytes, transferred from normal BALB/c mice to syngeneic athymic nu/nu (or SCID) mice, led to autoimmune oophoritis and autoimmune gastritis, with corresponding serum autoantibodies, in the recipients. The overall disease incidence was 73%; the pathology ranged from mild to severe, with complete loss of ovarian follicles and gastric parietal cells. CD4+ neonatal spleen cells and CD4+ CD8- adult thymocytes were required for autoimmune disease induction. Adult spleen cells did not elicit disease, but they prevented disease when co-transferred with neonatal spleen cells. However, in confirmation of an earlier report by Sakaguchi et al., (J. Exp. Med. 161:72, 1985), a subset of adult splenic T cells expressing a low level of CD5 molecules elicited similar autoimmune diseases. Thus, self-reactive T cells responsible for autoimmune disease of the stomach and ovary are not effectively deleted in the thymus, and they exist in the peripheral lymphoid organs of normal mice. We conclude that the functional expression of the self-reactive T cells is ontogenetically regulated; whereas T cells in the neonatal mice readily elicited autoimmune diseases in nu/nu recipients, regulatory cells may render self-reactive T cells in the normal adults unresponsive.     

Break of neonatal Th1 tolerance and exacerbation of experimental allergic encephalomyelitis by interference with B7 costimulation

Ig-PLP1 is an Ig chimera expressing proteolipid protein-1 (PLP1) peptide corresponding to aa residues 139-151 of PLP. Newborn mice given Ig-PLP1 in saline on the day of birth and challenged 7 wk later with PLP1 peptide in CFA develop an organ-specific neonatal immunity that confers resistance against experimental allergic encephalomyelitis. The T cell responses in these animals comprise Th2 cells in the lymph node and anergic Th1 lymphocytes in the spleen. Intriguingly, the anergic splenic T cells, although nonproliferative and unable to produce IFN-gamma or IL-4, secrete significant amounts of IL-2. In this work, studies were performed to determine whether costimulation through B7 molecules plays any role in the unusual form of splenic Th1 anergy. The results show that engagement of either B7.1 or B7.2 with anti-B7 Abs during induction of EAE in adult mice that were neonatally tolerized with Ig-PLP1 restores and exacerbates disease severity. At the cellular level, the anergic splenic T cells regain the ability to proliferate and produce IFN-gamma when stimulated with Ag in the presence of either anti-B7.1 or anti-B7.2 Ab. However, such restoration was abolished when both B7.1 and B7.2 molecules were engaged simultaneously, indicating that costimulation is necessary for reactivation. Surprisingly, both anti-B7.1 and anti-B7.2 Abs triggered splenic dendritic cells to produce IL-12, a key cytokine required for restoration of the anergic T cells. Thus, recovery from neonatally induced T cell anergy requires B7 molecules to serve double functions, namely, costimulation and induction of cytokine production by APCs.     

Antigen recognition and IL-2 receptor gene expression as evidence against clonal deletion in mice with neonatally induced transplantation tolerance.

Neonatal transplantation tolerance was induced in B10.A mice by the injection of spleen and bone marrow cells from semiallogeneic [C57BL/10(B10) x B10.A] F1 donors. The neonatally treated mice accepted skin grafts from B10 donors. Spleen cells from tolerant animals did not respond by proliferation to tolerated B10 antigens in vitro. However, spleen cells from tolerant mice recognized specific (B10) antigens and synthesized mRNA for the inducible 55-kDa interleukin-2 receptor (IL-2R) as did cells from normal animals. Maintenance of this early phase of cell activation upon contact with tolerated antigens is direct evidence against clonal deletion as a mechanism, in this particular model of neonatally induced transplantation tolerance.     

Studies of tolerance to DNA in NZB/NZW F1 mice.

Administration of sDNA-poly-D-lysine (DNA-PDL) to newborn NZB/NZW F1 mice (B/W) was previously shown to prolonge survival and to decrease nephritis and DNA antibodies. In this study, B/W mice treated from birth with DNA-PDL were found to be tolerant to immunization with sDNA on PDL or mBSA carriers in adjuvants. Tolerance to sDNA was present and was hapten-specific carrier-dependent. IgG and IgM anti-nDNA circulating antibodies were suppressed. Continuous tolerization was necessary to maintain tolerance. Tolerance to sDNA could be transferred by spleen cells, by tolerized thymus cells, and by tolerized bone marrow cells, suggesting that both T and B cells participated in this phenomenon.     

Tolerance induction during ontogeny. III. Carrier recognition by the immature and adult immune system determines tolerogenicity of hapten-carrier conjugates

Hapten-specific B-cell tolerance may be induced by nonimmunogenic hapten derivatives of carboxylmethylcellulose or methylcellulose (MC) in adult, neonatal, or irradiated fetal liver reconstituted mice. Such tolerance was shown to occur independent of T cells, and a receptor blockade has been ruled out as a causative mechanism. Oxidation and subsequent reduction of the vicinal hydroxyl groups of both carriers significantly reduces their tolerogenic potential in adult mice, yet their hapten derivatives remain nonimmunogenic. Such chemical modification of the carrier does not affect the molecular weight and not only does not reduce the binding avidity but increases it for either free antibody- or antigen-binding cells. We have examined the ability of the immature immune system to functionally discriminate between the nominal and the chemically modified hapten conjugate. Like adult mice, the immunologically immature animals were invariably capable of distinguishing between the tolerogenic and the nontolerogenic carrier. Mice treated during ontogenic development with 2,4-dinitrophenyl (DNP)-MC were found to be hapten specifically tolerant when challenged at 4 weeks of age with the TI-2 antigen DNP-Ficoll (F) but not when challenged with the polyclonal activator lipopolysaccharide (LPS) or the TI-1 antigen DNP-Brucella. Moreover, neonatal mice treated for 8 weeks with 2,4,6-trinitrophenyl-ovalbumin (TNP-OVA) were hapten specifically tolerant when challenged with TNP-OVA or the TI-1 antigen TNP-LPS but responded to a challenge with the TI-2 antigen TNP-F. These data suggest that B-cell tolerance in adult as well as in immunologically immature mice is not only carrier dependent but, in addition, that the carrier selects the B subpopulation to be rendered unresponsive. The most popular version of the clonal abortion hypothesis puts no constraints upon the nature of the antigen as long as the B cell is ontogenically "predisposed" toward being rendered unresponsive upon contact with a ligand of sufficiently high binding avidity. Our data are at variance with this prediction.     

Absence of suppression in natural and induced tolerance to F antigen

The role of suppression in natural and induced tolerance to F antigen was investigated in two sets of experiments. In the first, CBA mice were submitted to pretreatments which decrease suppression and the antibody response to self- or allo-F type was investigated. The second set of experiments involved the transfer of spleen cells from tolerized or from naturally tolerant mice into normal mice which were then primed with allo-F, as well as the co-transfer of tolerant and primed lymphocytes into normal mice, to test whether tolerant lymphocytes present suppressor cells. The results indicate that the immune response against allo-F antigen is normally kept in a low level by a suppressive mechanism, and that F-specific suppressor T cells are absent from tolerant mice.Abbreviations used in this paper ATx adult thymectomy - BSS buffered salt solution - CFA Freund's complete adjuvant - CY cyclophosphamide - F.1 type-1 F antigen - F.2 type-2 F antigen - PBS phosphate-buffered saline - RIA radioimmunoassay - Th T helper cell - Ts T suppressor cell