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Under defined conditions, papain removes IgD from cells while leaving IgM, H-2, Ia, Lyb-2, and complement receptor intact. The effect of such treatment with papain on the induction of tolerance in murine splenic B cells was determined in an in vitro system. Treatment of the cells with papain has no effect on subsequent antibody responsiveness presumably because surface receptors regenerate before and during incubation with immunogen. Removal of increasing amounts of IgD results in increasing susceptibility of thymus-dependent responsive cells to tolerance induction. The tolerance susceptibility of thymus-independent responsive cells, which we have previously suggested are immature cells that bear only IgM, is unaffected by cleavage of IgD. If cells are incubated for 24 h after treatment with papain, cell surface IgD and tolerance resistance return. These results indicate that a surface molecule affects susceptibility of B cells to induction of tolerance and suggest that this molecule may be IgD.
During ontogeny IgD appears later than IgM on splenocytes of neonatal mice (1) and at a time when mice develop a markedly increased immune responsiveness (2). Based on these observations, it was suggested that IgD serves as a "triggering" isotype for induction of immune responses, whereas surface IgM functions as a tolerizing receptor (3). To test this hypothesis, the susceptibility of adult splenocytes (which are predominantly mu(+)delta(+)[4-6]) and neonatal splenocytes (which bear predominantly IgM [mup(+); 1, 4-6]) to tolerance induction were compared. The results indicate that neonatal splenic B cells responsive to thymus dependent (TD) antigens are exquisitely susceptible to tolerance induction compared with those from adult mice (7-9). However, cells from both adult and neonatal mice were highly susceptible to tolerance induction when thymus independent (TI) antigen was used as immunogen (8). These results suggest that the major precursor for the TD response is a mu(+)delta(+)-cell which appears late in ontogeny and is resistant to tolerance induction and that the mup(+)-cell is the major precursor for the TI response and is highly susceptible to tolerance induction. Other differences between responders for TI and TD antigens have been described previously (10-12). To test this concept, adult splenocytes were treated with papain under conditions in which IgD, but not five other surface molecules, was removed (13). Such treated splenocytes were shown to be markedly susceptible to tolerance induction, resembling TD responders from neonatal animals. This experiment was interpreted as indicating that IgD confers resistance to tolerance induction on mu(+)delta(+)-cells. To prove this interpretation, it is necessary to show that specific removal of IgD with anti-delta also results in increased susceptibility to tolerance induction and that treatment with anti-mu does not have a similar effect. In the present studies, we have removed surface IgM or IgD by antibody-induced capping and assessed the tolerance susceptibility of the treated cells. Our results demonstrate that removal of IgD, but no IgM, from TD responders increases their susceptibility to tolerance induction.
Neonatal splenic B cells which are responsive to thymus-dependent antigens (TD) are exquisitely susceptible to induction of tolerance (1,2). This state of tolerance is not mediated by suppressor T cells and is not a result of suboptimal macrophage function (1 and footnote one). In adult mice, induction of B-cell tolerance is not achieved with doses of antigen 1,000-fold higher (1) than those required to produce the same degree of unresponsiveness in neonates. In contrast to these results, studies with T-independent (TI) antigens indicate that neonatal and adult splenic B cells are equally susceptible to tolerance induction (3,4). However, such studies have not ascertained whether the neonate is more resistant to tolerance induction or the adult is hypersusceptible, i.e., does the induction of tolerance in cells responsive to TI antigens resemble that of adult or neonatal cells responsive to TD antigens? The answer is pertinent to determining the relative maturity of the B cells which can be tolerized or respond to TI or TD antigens. We report here the direct comparison of tolerogen sensitivity of adult and neonatal TD and TI responses by inducing tolerance in vitro with trinitophenyl human gamma globulin (TNP(17)HgG) and assaying unresponsiveness with TD and TI forms of the TNP determinant.
Thymus-dependent protein antigens such as fowl gamma globulin (FGG) and dinitrophenylated-human gamma globulin (DNP-HGG), readily induced tolerance of the B cell in the absence of T cells even when these antigens were not deaggregated. However, when the same doses of antigen were given in the presence of T cells, the B-cell population was shown to be protected from tolerance induction, especially when the antigen was not in a deaggregated form. In this case, there was in fact evidence of a priming effect, manifest in both the B-cell and T-cell populations. The priming effect on the B-cell population was demonstrated by an increased response of mice pretreated with DNP-HGG, upon challenge with DNP conjugated to a heterologous carrier. The priming effect on the T-cell population was evident in a helper effect demonstrated in vitro. However, when euthymic mice which had been pretreated with large doses of FGG or DNP-HGG were challenged with the homologous carrier, the results were different. In this case, there was a profound suppression of the response against the carrier or the hapten on that carrier. Suppressor activity was also demonstrated in vitro and was shown to be sensitive to treatment with anti-theta-serum plus complement. Additionally it was shown that the effector phase of the suppression had a definite nonantigen-specific component. Thus, in pretreated euthymic mice, provided the homologous carrier was present, the response to a heterologous carrier was also suppressed. To account for the observation that nondeaggregated antigens can induce B-cell tolerance in athymic mice, but B-cell priming and T-cell-mediated suppression in euthymic mice, it is proposed that B-cell tolerance occurs when antigen at some critical dose interacts with the B cell in the absence of some second signal. This second signal is normally provided by the macrophage, probably with the assistance of the T cell, and its effect is to divert the result of the interaction of the B cell with antigen towards immunization and away from tolerance induction. When a large dose of an antigen that tends to form aggregates is given to an animal possessing functional T cells, both T-dependent helper and T-dependent suppressor activities are generated, thus accounting for a situation where the B-cell population is immunized, but B-cell activation is suppressed in the presence of the original carrier.
The numbers and fate of antigen-binding cells (ABC) in neonatal and adult mice rendered tolerant to fluorescein (FL)-labeled heterologous gamma-globulins were studied. Similar numbers of FL-ABC were observed 1 day after tolerogen in both adult and neonatal mouse spleens: by 7 days after tolerization, no FL-ABC were observed in either case. Reinjection with FL-tolerogen at 7 days led to the detection of normal numbers of ABC in adult mice but significantly reduced numbers in neonates. This suggests that neonatal ABC either have been deleted or have failed to resynthesize surface receptors. Two weeks after tolerance induction, spleen cells from these tolerant mice were cultured with Escherichia coli lipopolysaccharide (LPS), a polyclonal B cell mitogen, or with specific antigen. Tolerant adult spleen cells made an equivalent anti-FL response to that of the uninjected controls when stimulated with LPS, but were unresponsive to specific antigenic triggering. In contrast, spleen cells from neonatally tolerized mice were unresponsive to either specific or nonspecific (LPS) stimulation. Thus, these neonatally tolerized spleen cells lose sensitivity to polyclonal-stimulating agents (along with their receptors), or more simply, are deleted.
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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.
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.
Hapten [2,4-dinitrophenyl (DNP)]-specific tolerance was induced in nonimmune or DNP-hemocyanin (DNP-KLH) primed mice by administering hapten-conjugated type 3 pneumococcal polysaccharide (DNP-lys-S3). The dose of DNP-lys2.5-S3 required to suppress the primary anti-DNP antibody responses was approximately ten times higher than that required to suppress the secondary response. Large doses of lightly substituted antigen (DNP-lys0.6-S3) had no effect on primary antibody responses, while small doses of this conjugate suppressed 90-95% of the secondary response. The conclusion from this (presumably B cell) tolerance model is that B lymphocytes "mature" in their susceptibility to tolerization following primary contact with immunogen, since primed cells are inactivated by lower doses of tolerogen, and by tolerogen with lower epitope density, than nonimmune B cells. These and other data suggest that the tolerance threshold of B lymphocytes is related to their state of differentiation, and especially to their antigen-binding characteristics.
The relative susceptibility of neonatal and adult murine splenocytes to induction of B-cell tolerance was studied in vitro. Adult cells required approximately 1,000-fold more trinitrophenyl-human gamma globulin to be rendered tolerant than did cells from 9- to 12-day-old neonates. The potential effects of suppressor T cells were excluded by pretreating the cultured B cells with anti-Thy-1 and C' and the helper T cells with anti-Ly-2.2 and C'. The possible role of cell surface immunoglobulin isotypes in contributing to this observed difference is discussed.
BDF1 mice were made tolerant by a single i.v. injection of 1 mg of DNAP-gamma1 or by weekly i.v. injections of 0.2 mg of DNP-gamma1 given for a month. In both instances, spleen cells of tolerant animals were fractionated to obtain pure populations of T cells (nonimmunoglobulin-bearing cells), referred to as tolerant T cells, and B cells (immunoglobulin-bearing cells) referred to as tolerant B cells (immunoglobulin-bearing cells) referred to as tolerant B cells. The control cells were similarly fractionated to obtain normal T and B cells. Mixtures of tolerant T cells and normal B cells, or conversely, normal T cells and tolerant B cells were used to repopulate lethally irradiated recipients. These recipients were then immunized with dinitrophenyl-keyhole limpet haemocyanin and in certain instances with other antigen horse red blood cells. The immune response to both antigens was measured using the direct hemolytic plaque assay. It was found that both T and B cells were tolerant and that tolerance was hapten specific at both T- and B-cell levels. While B-cell tolerance was demonstrated at a 1/1 T/B ratio, a 4/1 T/B ratio was necessary to show T-cell tolerance. Thus, the hapten-specific carrier-determined tolerance involves not only B cells but also T cells. The implication of this finding for the cellular mechanism of tolerance in an experimental model closely related to self tolerance is discussed.
Purified goat antibodies against mouse mu-chains and rabbit antibodies against mouse Ig determinants, and their Fab fragments, inhibited the development of IgM-bearing B cells in explant cultures of 14-day mouse fetal liver, and caused the disappearance of cell surface IgM in explant and dissociated cell cultures of more developed lymphoid tissues. While treatment of cultures of fetal or newborn liver, or adult bone marrow, with low concentrations (less than or equal to 10 mug/ml) of anti-Ig for less than or equal to 24 h caused the complete, but reversible, disappearance (modulation) of cell surface IgM, treatment for greater than or less than 48 h produced irreversible IgM suppression. In contrast, anti-Ig-induced suppression of cell surface IgM in cultures of adult spleen or lymph nodes required much higher concentrations of antibody (greater than or equal to 100 mug/ml) and was always reversible. These differences between immature and mature IgM-bearing cells could not be related to differences in the amount of surface IgM on the cells. The remarkable sensitivity of newly formed B cells to IgM modulation and irreversible IgM suppression when ligands bind to their Ig receptors, may have important implications for B-cell tolerance to self antigens.
Previous studies indicated that T cells are required for tolerance induction by hapten-modified syngeneic spleen cells (TNP-SC) in vivo. The role of T cells in the maintenance of this unresponsive state has been examined herein. By three criteria--limiting dilution precursor analysis, removal of T cells by anti-Thy-1 + C, and direct mixing experiments--we show that T cells are required for the continued suppression of the B cell response to the T-independent antigen, TNP-POL. Suppressor cells can also be induced by TNP-teratoma cells, which lack detectable H-2 antigens. Both anti-Ly-1 + C and anti-Ly-2 + C treatment reversed suppression induced by TNP-SC. These results demonstrate that normal B cell reactivity is present in the spleens of mice rendered tolerant by haptenated self, but that Ly-1,2,3 or Ly-1 + Ly-2,3 suppressor T cells prevent their responsiveness.
Exposure of 2,4-dinitrophenyl (DNP)-hemocyanin-primed spleen cells to DNP-conjugated pneumococcal polysaccharide (DNP-S3) in vivo or in vitro renders such cells unresponsive to DNP-hemocyanin challenge following adoptive transfer. The unresponsiveness is hapten-specific, independent of the presence of T cells and adherent cells, and not due to either toxic effect of S3 or carry-over of tolerogenic amounts of cell-bound DNP-S3, and thus presumably represents a model of B cell tolerance. The degree of suppression induced depends upon the dose of the tolerogen and most strikingly on the duration of exposure of cells to the tolerogen. Thus 2 to 6 h exposure to DNP-S3 has an insignificant effect on anti-DNP responses, while 24 and 48 h exposures in vitro and in vivo are highly suppressive. Such a dynamic process of tolerance induction suggests a "multiple hit" phenomenon, implying the generation of suppressive signals by repeated cycles of tolerogen-receptor interactions. The process can be interrupted by removal of extracellular DNP-S3, although this does not reverse the unresponsiveness that has already been induced.
Injection of mice already making antibodies to 2,4-dinitrophenylated (DNP) Ficoll with tolerizing doses of DNP-pneumococcal polysaccharide (DNP-lys-S3) markedly inhibits the secretion of anti-DNP antibodies by IgM antibody-forming cells. The present study shows that the degree of inhibition depends not only on the dose of DNP-lys-S3 but also on the duration of exposure to antigen. DNP-lys-S3 was detectable on the surface of antibody-forming cells at a time when their rate of secretion was unimpaired, thus suggesting that the inhibition involves intracellular events subsequent to the binding of antigen to the cell membrane. The inhibition was reversible if antibody-forming cells were exposed to antigen for 24 h, and then cultured for 18 h in its absence, but became irreversible if the treatment period was extended to 48 h. The relevance of this model of inhibition of lymphocyte function by antigen to possible mechanisms of B cell tolerance is discussed.
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