Tolerogenic Signals Research Articles

Effects of phosphorylcholine-lipopolysaccharide conjugates on the induction of anti-idiotype-mediated B-cell tolerance

Preincubation of BALB/c spleen cell cultures for 24 hr with phosphorylcholine (PC)-containing antigen together with antibody against the major idiotype (id) of anti-PC antibody renders them irreversibly unresponsive to subsequent stimulation with the antigen alone. In contrast, cultures preincubated for 24 hr with anti-id antibody, either alone or together with lipopolysaccharide (LPS), resulted in an anti-PC response comparable to that induced in control cultures incubated with mock anti-id antibody. After such a 24-hr preincubation with anti-id antibody and various PC-LPS conjugates possessing intact activity for polyclonal B-cell activation, the anti-PC response was inversely proportional to the epitope (PC) density on the LPS conjugates. In addition, similar preincubation of cultures with a non-mitogenic low dose of PC-LPS in the presence of anti-id antibody induced suppression of the anti-PC response as observed with a specific antigen. These results suggest that specific epitope delivers an additional tolerogenic signal during induction of B-cell suppression by anti-id antibody. This epitope effect cannot be replaced by, and is antagonistic to, the mitogenic signal of LPS in the course of B-cell inactivation.

Conversion of a tolerogenic to an immunogenic signal by P388AD.2 cells, a lymphoid dendritic cell-like tumor line.

The lymphoid dendritic cell-like tumor P388AD.2 is capable of converting a tolerogenic signal into an immunogenic one. In the present study, adherent P388AD.2 cells were pulsed with the tolerogen, fluoresceinated (FL) sheep gamma-globulin (SGG), washed, and incubated with normal spleen cells. After 24 hr, the spleen cells were harvested and challenged in secondary cultures with immunogenic doses of FL-thymic independent (TI) antigens. The plaque-forming cell response on day 3 of secondary culture was increased as much as 400% compared with control spleen cultures exposed to untreated P388AD.2 cells. The increased response was specific for the FL-hapten and occurred only when P388AD.2 cells were pulsed with FL-Ig or FL-F(ab')2 fragments, but not with FL-synthetic tolerogens or other FL-antigens. Furthermore, the augmentation required histocompatible T cells in the primary cultures. Additional experiments showed that if cultures were devoid of Lyt-1+ cells but not Lyt-2+ cells, no augmentation occurred. A variety of other macrophage-like tumor cells, some with known antigen presenting properties, were tested for the ability to present tolerogen in an immunogenic fashion. Only an I-A+ J774 clone was able to present tolerogen in an immunogenic fashion. However, we failed to find a correlation between the presence of surface Ia antigen and tolerogen-presenting ability. The results suggest that certain types of cells may play a role in immune regulation by abrogating the tolerogenicity of Ig tolerogens via their presentation in an immunogenic mode.

The effect of T lymphocyte depletion on neonatal tolerance induction, graft-vs.-host disease and cellular chimerism.

Treatment with a monoclonal anti-Thy-1 antibody and complement completely prevented C57BL spleen cells from causing graft-vs.-host disease following their inoculation into newborn CBA mice. The proportion of mice that became tolerant to C57BL antigens, as measured by skin grafting, was significantly less compared with mice given (CBA X C57BL)F1 hybrid cells. This was not due to the elimination of T cells, for antibody-treated F1 cells induced tolerance as readily as complement-treated control F1 cells. To investigate whether the apparent superiority of F1 cells over C57BL cells is attributable to differences in the mechanism inducing and maintaining unresponsiveness, two approaches were followed. First, the level of donor cell chimerism in the spleens of tolerant animals was studied. Though no difference between F1 and C57BL cells was uncovered, the presence of T cells in the donor inocula favored the establishment of chimerism. Second, the involvement of suppressor T cells was examined in adoptive transfer experiments. Splenic suppressor T cells were associated with tolerance regardless of how it was elicited. Preliminary results with F1 cells show that the tolerogenic property is not confined to any one cell type. It is proposed that the greater tolerogenicity of F1 cells is brought about by the presence of host-type (self) antigens, which enable the tolerogenic signals to operate without recourse to antigen processing by host cells.

Analysis of the mechanism of unresponsiveness produced by haptens painted on skin exposed to low dose ultraviolet radiation.

Acute, low dose ultraviolet B radiation of murine body wall skin followed by local application of DNFB produces a state of antigen-specific unresponsiveness. This state is maintained at least in part by an Lyt-1+ T cell that effects unresponsiveness by impairing the induction phase of contact hypersensitivity. The absence of suppressor cells capable of acting at the effector stage of immunity suggests that tolerogenic signals derived from the skin establish suppressor networks that are incomplete and perhaps different from networks that are induced by systemic administration of tolerogens. It is proposed that ultraviolet radiation produces its effects by impairing the antigen-presenting potential of resident Langerhans cells in whose absence hapten-derivatized keratinocytes (or their products) are able to deliver a tolerogenic signal.

Polyvalent epitope inhibits but added with anti-idiotype antibody enhances idiotype suppression.

When BALB/c spleen cell cultures were preincubated with polyvalent PC-antigens, regardless of their immunogenicity, cultures became resistant to id suppression induced by subsequent treatment with anti-id antibody and immunogenic PC-antigen. In addition, incubation with both anti-id and any of these antigens assisted the induction of rapid, irreversible B cell tolerance. These results suggest that B cells bearing receptors for an epitope undergo an initial triggering in response to polyvalent epitope regardless of the T dependence and/or immunogenicity of the antigen. An additional signal must then facilitate the differentiation of the partially activated B cells to produce antibody. Therefore, a nonimmunogenic polyvalent epitope-bearing molecule can deliver either an initial triggering signal or a tolerogenic signal, depending on the presence of anti-id antibody, and it is as efficient as a complete immunogen.

Interruption of tolerance toward a haptenic initiator of delayed-type hypersensitivity.

We have found that an immunomodulator, dextran, prevents initiation of tolerance for an unrelated haptenic stimulus of contact hypersensitivity, picryl chloride. To prevent tolerance optimally, 1 to 10 mg of dextran per 25g mouse was required to be injected i.v. 2 hr before administering tolerogen. The immunomodulator was seen to be effective in mice with diverse major histocompatibility backgrounds. Furthermore, prevention of tolerance was seen to be independent of the condition of the hapten used to initiate unresponsiveness, since tolerance was abrogated when attempted with uncoupled hapten or with hapten attached to allogeneic or syngeneic spleen cells. In addition, administering dextran before tolerogen did not convert the tolerogenic signal into an immunogenic one so that hapten-specific DTH could be detected. While the precise mechanism of dextran's interruption of immunologic unresponsiveness for DTH has yet to be elucidated, the principle that tolerance can be readily and easily interrupted with this immunomodulating agent has been firmly established.

Immunoregulatory circuits which modulate responsiveness to suppressor cell signals: contrasuppressor cells can convert an in vivo tolerogenic signal into an immunogenic one.

The intravenous injection of 2,4,6-trinitrophenyl (TNP)-labeled peritoneal exudate cells (TNP-PEC) into CBA mice fails to produce a state of hypersensitivity; rather, it renders recipient mice incapable of mounting a contact hypersensitivity response when they are subsequently immunized with a reactive form of the specific hapten. However, if precultured neonatal spleen cells are injected along with the cells that induce tolerance (TNP-PEC), not only is the development of tolerance inhibited but sensitization to TNP develops. The neonatal spleen cell responsible for turning the tolerogenic signal into an immunogenic one is I-J+ and adheres to the Vicia villosa lectin. Thus, it expresses markers that distinguish contrasuppressor effector cells from helper cells (D. R. Green et al., Eur. J. Immunol. 1981. 11:973), indicating that activated contrasuppressor cells can act as potent, helpful regulatory cells in vivo.

Role of IgD in the immune response and tolerance. I. Anti-delta pretreatment facilitates tolerance induction in adult B cells in vitro.

Adult spleen cells from C57BL.Ige mice, which generally are resistant to in vitro tolerance induction in the B-cell compartment, became hyporesponsive (tolerant) when cultured with antigen in the presence of an anti-allotype serum. Both antigen and anti-delta had to be present for this effect, which was hapten-specific and did not occur in C57BL/L mice, which lack the Ig5-1 allotype of the delta-chain detected in this system. Preculture with anti-mu serum plus antigen, in contrast, did not cause tolerance induction in adult spleen B cells of either strain. These results suggest that the surface IgD may act as a failsafe receptor to prevent tolerance induction in adult B cells. Tolerance studies with spleen cells from mice with markedly reduced numbers of IgD+ve cells, because of regimen of repeated injections of anti-delta serum beginning at birth (delta-suppressed mice), confirmed the importance of membrane IgD in preventing tolerance, because such delta-suppressed mice were hypersusceptible to tolerance by antigen alone. Inasmuch as immature B cells lack IgD on their surface, these studies suggest that acquisition of IgD is an important maturational step in the ability of murine B cells to discriminate tolerogenic and immunogenic signals.

Spleen cells from animals tolerant to a thymus-dependent antigen can be activated by lipopolysaccharide to synthesize antibodies against the tolerogen.

Immunological tolerance was induced in adult mice by the injection of 5 mg of deaggregated hapten-protein conjugate. The tolerant state was confirmed 4-19 days later by the failure of such animals to mount an immune response against an aggregated form of the same thymus-dependent hapten-protein conjugate as well as by the inability of spleen cells from tolerant animals to respond to a thymus-independent hapten-carrier conjugate. Even though the animals were fully tolerant, their spleen cells were activated by lipopolysaccharide (LPS) in vitro to produce normal numbers of plaque-forming cells against the hapten. The finding that spleen cells from tolerant animals could be activated by LPS into synthesis of antibodies against the tolerogen indicates that tolerance to thymus-dependent antigens does not affect B cells, but presumably only T cells. It is suggested that the only stringent test for the existence of B-cell tolerance is the inability of polyclonal B-cell activators to activate antibody synthesis against the tolerogen. The findings make it unlikely that B-cell tolerance to autologous thymus-dependent antigens exists and further indicate that such antigens cannot deliver activating or tolerogenic signals to B cells, although they are competent to combine with and block the Ig receptors.

Antigen recognition. IV. Discrimination by antigen-binding immunocompetent B cells between immunity and tolerance is determined by adherent cells.

Mouse spleen cells capable of specifically binding intrinsically tritium-labeled polymerized flagellin (POL) (labeling by biosynthesis of flagellar protein) via IgM receptors were found to comprise a distinct population of about 20-50 cells per 10(6) lymphocytes. Evidence is presented that the majority of mouse spleen cells binding tritium-labeled POL undergoes blastogenesis after antigen capping, antigen shedding, and receptor reformation. Under conditions of tolerance induction in vitro, however, loss of antigen from the cell surface was inhibited. Such inhibition of antigen redistribution and shedding was reversed by a short pulse of colchicine and new antigen receptors were formed. In spite of this, colchicine had no effect on the tolerant state. However, tolerance could be broken, regardless of presence or absence of the alkaloid, with radioresistant theta-negative accessory (A) cells (adherent cells) from normal but not from tolerant spleen cell populations. "Tolerant" A cells, although they were incapable of cooperating in a response to POL, were capable of participating in a response to a second unrelated antigen. It is concluded that tolerance to POL in vitro is induced by mechanisms other than the physical blocking of bone marrow-derived (B) cell receptors by antigen. Most likely, the discrimination by the B cell between a tolerogenic and immunogenic signal is mediated by A cells.

Preventive Effect of Hapten-Reactive Thymus-Derived Helper Lymphocytes on the Tolerance Induction in Hapten-Specific Precursors of Antibody-Forming Cells

The role(s) of helper T lymphocytes in preventing or altering tolerance induction in DNP-specific B lymphocytes was studied. As DNP-reactive helper T cells were reactive against the DNP-portion of the DNP-D-GL molecule, we could probe definitively the physiological role of helper T cells in preventing tolerance induction in B lymphocytes by DNP-D-GL. The results demonstrated that the induction of DNP-specific B cell tolerance by DNP-D-GL can be completely prevented by the presence of DNP-reactive helper T cells, and provide evidence that one critical role of helper T cell participation in humoral responses to antigens is to circumvent the development of a tolerogenic signal that, in the absence of such T cell function, might otherwise ensue after binding of the antigenic determinants by specific B lymphocytes.

Cellular events in tolerance. IV. The effect of a graft-versus-host reaction and endotoxin on hapten- and carrier-specific tolerance.

AbstractThe induction of hapten‐specific and carrier‐specific unresponsiveness has been used as a model of B and T cell unresponsiveness, respectively. Endotoxin (lipopolysaccharide (LPS)) and a graft‐versus‐host (GVH) reaction were then evaluated for their ability to interfere with the induction of B and T cell tolerance in vivo. It was found that LPS, but not a GVH reaction had adjuvant activity in that LPS could convert a B cell tolerogenic signal into an immunogenic one. However, both treatments could partially prevent B cell tolerance induction with trinitrophenyl‐isologous IgG. Under conditions in which the GVH‐induced immunosuppression was minimal, carrier (T cell) tolerance was also reduced by the GVH reaction. LPS did not directly interfere with carrier unresponsiveness. We conclude that LPS can block the induction of unresponsiveness in B cells, whereas a GVH reaction can partially affect both T and B cell tolerance induction.

IMMUNOLOGICAL TOLERANCE IN BONE MARROW-DERIVED LYMPHOCYTES

The present studies were designed to probe the role(s) of T cells in preventing or altering tolerance induction in hapten-specific B cells. This was accomplished by using hapten conjugates of normally immunogenic heterologous carriers to selectively inhibit 2,4-dinitrophenyl (DNP)-primed B cells in adoptive transfer experiments in vivo. The data provide strong indications that one critical role of T-cell participation in humoral responses to antigens is to circumvent the development of a tolerogenic signal that, in the absence of such T-cell function, might otherwise ensue after binding of the antigenic determinants by specific precursor B lymphocytes.