Cellular events in tolerance IX: Maintenance of immunological tolerance in the presence of normal B-cell precursors and in the absence of demonstrable suppression

The cellular events involved in immunological tolerance to fluoresceinated sheep gammaglobulin (FL-SGG) were analyzed at the level of hapten-specific B cells. One single iv injection of FL-SGG induced tolerance as measured by challenge with thymus-dependent (FL-KLH) or thymus-independent (FL-Ficoll) antigens in vivo or thymus-independent (FL-LPS) antigen in vitro. As noted earlier, unresponsiveness was maintained until 6–8 weeks after tolerance induction. Limiting-dilution precursor analysis demonstrated a reduction in B-cell precursors on Day 7 after tolerogen treatment; precursor frequencies returned to control levels by 3–4 weeks. This recovery of precursors in the presence of stable tolerance was not due to suppressor activity. Rather, results show that tolerant hapten-specific B cells are clonally anergic and display a reduced burst size in response to antigen. Hence, unresponsiveness is maintained in the presence of apparently normal precursor levels by an intrinsic defect in antigen-specific B cells.     

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

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

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

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

Reversible Blocking Effect of Anti-mouse Immunoglobulin Serum on the Induction of Immunity and Tolerance <Emphasis Type="Italic">in vitro</Emphasis>

THE induction of blast transformation by incubating lymphocytes with anti-immunoglobulin¹ and anti-allotype² sera has suggested that these cells have immunoglobulin on their surface. This hypothesis was directly verified by the demonstration of immunoglobulin on living mouse lymphoid cells by Raff et al.³. There is much evidence to indicate that immunocompetent cells have surface receptors for antigen. This idea is based on the finding that lymphocytes can bind radioactively labelled antigen to their surface^4,5 and that specific immune unresponsiveness occurs if lymphoid cells are exposed to either highly radioactive antigen⁶ or haptens capable of forming covalent bonds with proteins^7,8. The immunoglobulin nature of these antigen receptors is suggested by recent work showing that the binding of radioactively labelled antigen can be blocked by anti-immunoglobulin sera^5,9. Reports that the adoptive immune response of mouse spleen cells can be inhibited by anti-mouse immunoglobulin sera (AMS)^9,10 suggest that the interaction of antigen with the immunoglobulin receptor sites is a crucial step in the induction of the antibody response. We report here that the inhibitory action of AMS on the immune response is potentially reversible and that the induction of immune tolerance to polymerized flagellin (POL) in vitro may be blocked in the presence of AMS.     

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

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

Membrane dynamics of lymphocyte interaction with antigen and tolerogen.

The principle of hapten-specific carrier-dependent immunologic tolerance was used to study the in vivo and in vitro interaction of lymphocyte membrane receptors with antigen (DNP-KLH) and tolerogen (DNP-MGG). Direct fluorescent techniques were employed to illustrate the binding of tolerogeu and antigen to the same population of lymphoid cells and the subsequent in vivo and in vitro events related to capping and regeneration of membrane receptors.     

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

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

Induction of Immunity and Tolerance to the Dinitrophenyl Determinant <Emphasis Type="Italic">in vitro</Emphasis>

THE immune response in dissociated lymphoid cell cultures offers an opportunity to investigate the interaction of antigen with the surface receptors of immunocompetent cells. Using polymerized flagellin of Salmonella adelaide (POL), evidence was obtained that in vitro processes as different as immunity and tolerance both depend on the direct interaction between antigen and antigen-sensitive cells^1–4. The use of chemically defined determinants in place of natural antigens could simplify the study of the molecular mechanisms underlying immunity and tolerance. Systems used in the past to induce immunity to defined determinants in vitro involved either a particulate antigen⁵ or spleen fragment cultures⁶ and were therefore unsuitable for the detailed study of the interactions occurring on the surface of lymphoid cells. A new system had to be devised. Here I describe the induction of a primary immune response to a hapten–protein conjugate in dissociated spleen cell cultures and the immune tolerance to a chemically defined determinant in vitro.     

The immunoregulatory role of bone marrow. I. Suppression of the induction of antibody responses to T-dependent and T-independent antigens by cells in the bone marrow.

Bone marrow cells (BMC) from normal mice suppressed the in vitro IgM, but not the IgG, antibody (Ab) response of spleen cells. BMC were inhibitory only when added during the first 24 hr of culture, and inhibition was not due to an induced shift in the kinetics of the response. Addition of specifically activated T cells or nonspecific T-cell-replacing factors to normal or T-depleted spleen cell cultures did not abrogate suppression while the response to the T-independent antigen DNP-polymerized flagellin or lipopolysaccharide was also suppressed. BMC did not inhibit background Ab synthesis by normal or primed cells in the absence of antigen and did not inhibit, but stimulated, DNA synthesis in normal spleen cell cultures. In addition, high-avidity Ab synthesis was preferentially suppressed. A possible role for the bone marrow suppressor cell in the induction of B cell tolerance is discussed.     

The ex vivo toll-like receptor 7 tolerance induction in donor lymphocytes prevents murine acute graft-versus-host disease

Background aims

Acute graft-versus-host disease (aGVHD) remains a major cause of morbidity and mortality after allogeneic hematopoietic stem cell transplantation, mediated by alloreactive donor T cells. Toll-like receptors (TLRs), a family of conserved pattern-recognition receptors (PRRs), represent key players in donors' T-cell activation during aGVHD; however, a regulatory, tolerogenic role for certain TLRs has been recognized in a different context. We investigated whether the ex vivo–induced TLR-2,-4,-7 tolerance in donor cells could prevent alloreactivity in a mismatched transplantation model.

Macrophage-like suppressor cells in rats. I. Inhibition of natural macrophage-like suppressor cells by red blood cells

When trinitrobenzenesulfonic acid (TNBS), the reactive form of trinitrophenyl (TNP) hapten, is injected into a mouse, a brief intrinsic B-cell tolerance to TNP has been shown to result. Yet antigen-binding cells (ABC) with receptors for TNP persist in the TNBS-treated animal.After treatment with Pronase under conditions preserving cell recovery and viability, 80–90% of TNP-ABC failed to bind antigen. After 2 hr in vitro, Pronase-treated 4-day immune TNP-ABC displayed significant recovery of antigen binding, whereas nonimmune TNP-ABC performed the same feat by 18 hr. However, TNP-ABC tested 2 to 11 days after TNBS failed to replace digested receptors by 18 hr in vitro. Thirty days after TNBS, they had recovered this ability. This defective receptor replacement by TNP-ABC was not reversed by colchicine, and was not shared by the sheep-erythrocyte ABC of the same animals, which replaced receptors normally. When challenged with antigen (TNP-sheep erythrocytes) simultaneously with TNBS, recovery by 2 hr was evident on Day 11. When challenged with antigen 4 days after TNBS, receptor regeneration had returned to normal by the next day, and partial recovery of the anti-TNP plaque-forming cell response was evident 4 days later.Thus, the inability to replace receptors and immune unresponsiveness coincides in time, so that a causal relationship between these two defects may be hypothesized. This result contrasts with the membrane locking defect, previously described in the TNP-ABC of TNBS-treated animals, which far outlasted the unresponsive state.     

Tolerance and contact sensitivity to DNFB in mice. VII. Functional demonstration of cell-associated tolerogen in lymph node cell populations containing specific suppressor cells.

Adoptive tolerance to contact sensitivity to DNFB is mediated by suppressor T cells. These cells are induced by iv injection of the hapten DNB-SO₃. Experiments were carried out to investigate the question of simultaneous transfer of tolerogen (DNB-SO₃ or its conjugation product DNP) with the suppressor cells. The results showed that tolerant lymph node cells pretreated in vitro with anti-TNP serum before transfer were unable to induce unresponsiveness to DNFB. Tolerant cells treated with either anti-TNP serum which had been passed over a TNP-affinity column or with polyvalent anti-immunoglobul in serum were not inhibited. These results functionally demonstrate that LN cell populations containing DNFB suppressor cells have accessible hapten (e.g., DNP) associated with their membrane, which is necessary for induction of adoptive tolerance. The hapten (tolerogen) appears to be bound directly to the cell surface rather than as an immune complex.     

The nature of the suppressive effect of interferon and interferon inducers on the in vitro immune response

Viral-induced interferon inhibition of the primary in vitro plaque-formong cell (PFC) response in the mouse (C57B1/6) involves a dynamic relationship between the nature of the antigen, the concentration of interferon added to antigen-stimulated cultures, and the time of addition of interferon relative to antigen addition. The PFC response to a thymus-dependent antigen (sheep red blood cells) was more easily suppressed by viral-induced interferon than was that to a thymus-independent antigen (E. coli 0127 LPS), both in terms of inhibitory concentrations of interferon and the time at which the interferon could be added to cultures after antigen and still inhibit the PFC response. These differential effects of interferon could be related to the difference in cellular requirements (B and T lymphocytes) of the two antigens. Interferon was effective in inhibiting the in vitro PFC response of antigen-primed spleen cells, indicating that it can block the response of memory lymphocytes. By using interferon inducers as inhibitors of the in vitro PFC response, it was possible to show that at least two antigenically distinct interferons may be involved in suppressing the immune response. It is known that one type of interferon is induced by virus and synthetic double-stranded polyribonucleotides. The other type is stimulated by antigen and T cell mitogens. A model is proposed to explain the nature of these interferon inhibitory effects in terms of mediation of immune suppressor cell effects.     

Alteration of Gene Expression during the Induction of Freezing Tolerance in Brassica napus Suspension Cultures

Brassica napus suspension-cultured cells can be hardened to a lethal temperature for 50% of the sample of −20°C in eight days at room temperature with abscisic acid. During the induction of freezing tolerance, changes were observed in the electrophoretic pattern of [³⁵S]methionine labeled polypeptides. In hardening cells, a 20 kilodalton polypeptide was induced on day 2 and its level increased during hardening. The induction of freezing tolerance with nonmaximal hardening regimens also resulted in increases in the 20 kilodalton polypeptide. The 20 kilodalton polypeptide was associated with a membrane fraction enriched in endoplasmic reticulum and was resolved as a single spot by two-dimensional electrophoresis. In vitro translation of mRNA indicate alteration of gene expression during abscisic acid induction of freezing tolerance. The new mRNA encodes a 20 kilodalton polypeptide associated with increased freezing tolerance induced by either abscisic acid or high sucrose. A 20 kilodalton polypeptide was also translated by mRNA isolated from cold-hardened B. napus plants.     

Lack of requirement for cell cooperation in the antibody response to DNP conjugated to levan.

The cellular basis of the antibody response to dinitrophenylated levan (DNP-LE) was investigated using a combination of in vivo and in vitro techniques. The results indicated that the induction of the primary response to DNP-LE is independent of the function of macrophages or T cells. However, these cells may regulate the response to DNP-LE (as for other T-independent antigens), as suggested by the prolongation of the response by adjuvants. No evidence of collaboration between carrier (LE)-reactive and hapten (DNP)-reactive B cells in the induction of the response to DNP-LE was obtained (either by inducing tolerance to LE (in vivo or in vitro) or culturing in the presence of excess LE). Thus DNP-LE, like other thymus-independent antigens (e.g., DNP-polymerized flagellin, DNP-Ficoll) may trigger B cells directly.     

Tolerance induction to a thymus-dependent antigen in vitro: treatment of nonadherent cells with tolerogen biologically filtered in vitro

Highly tolerogenic bovine gamma globulin (BGG), a thymus-dependent antigen, was prepared by biologic filtraration in vitro. It readily induced tolerance in vivo in BALB/c mice and also rendered their nonadherent lymph node cells tolerant after in vitro incubation. Biologic filtration in vitro was carried out by incubating 2.5 × 10⁷ lymph node cells with 10 mg of nontolerogenic BGG in 10 ml of Eagle's medium containing 2% normal mouse serum at 37 °C for 6 hr. The BGG-containing medium was clarified by centrifugation and was used without further dilution.For tolerance induction in vitro, lymph node cells were separated into adherent and nonadherent populations on Falcon plastic. These cells were incubated for 0–18 hr at 37 °C with biologically filtered BGG (bBGG). After incubation, the cells were washed three times and (2–2.5) × 10⁷ nonadherent or 4 × 10⁶ adherent cells were injected iv with their untreated counterpart into lethally irradiated mice which had received 10⁶ bone marrow cells. The recipients were then challenged with 300 μg of aggregated BGG, and tolerance was assayed by the elimination of labeled BGG, rosette formation, and passive hemagglutination. Spleen cells were similarly treated for comparison. Our findings show that tolerance was not induced in vitro in adherent lymph node cells. However, in the nonadherent populations, those from the lymph node but not the spleen were rendered tolerant. The acquisition of tolerance in vitro was gradual. It was dependent upon the length of exposure to bBGG and required at least 6 hr.     

Functional evidence for abnormal maturation within a B-cell subpopulation of NZB mice

NZB mice develop a systemic autoimmune disease and have a subpopulation of B lymphocytes that spontaneously produce excessive amounts of IgM. These abnormal B cells reside within a specific B-cell subset that is affected by the CBA/N defect. In normal mice, this B-cell subset acquires in vitro responsiveness to certain thymus-independent antigens (TI-2) relatively late in ontogeny. We compared the functional development of neonatal B cells from NZB mice to that of normal mice of the same H-2 type. The acquisition of in vitro responsiveness to the TI-1 antigen, TNP-LPS and the TI-2 antigens, TNP-Dextran, TNP-Ficoll, and FITC-Ficoll was examined. TNP-LPS could elicit a response from both normal and NZB neonates. In contrast, responses to the TI-2 antigens were elicited early in life (<1 week) only from or at a higher level from NZB neonates. However, an accelerated appearance of B-cell differentiation antigens was not detected in NZB neonates compared to normal strains. We conclude, therefore, that a maturation or triggering defect occurs in a small B-cell subpopulation of NZB mice very early in life.     

Anaphylactic properties of mouse monoclonal IgG2a antibodies

Mouse monoclonal antibodies (10 hybridoma antibodies specific for soluble antigens, 8 hybridoma antibodies specific for H-2 $\text{K}\text{D}$ antigens, and 9 myeloma immunoglobulins, among which 5 had a known specificity) of the IgG1, IgG2a, IgG2b, IgG3, IgA, and IgM isotypes were studied for their ability to induce mouse mast cell degranulation in vitro, in the presence of specific antigen or after heat aggregation. Monoclonal IgG1 antibodies, as well as IgG2b, IgG3, IgA, and IgM behaved as polyclonal antibodies of corresponding classes: all IgG1 induced mast cell degranulation with typical characteristics of IgG-mediated anaphylactic reactions, whereas IgG2b, IgG3, IgA, and IgM did not. By contrast, 2 hybridoma IgG2a and 3 myeloma IgG2a induced intense mast cell degranulation that could not be explained by a contamination with IgG1 or IgG1-IgG2a hybrid molecules. IgG2a-mediated reactions were observed in four different situations: soluble antigen-hybridoma IgG2a complexes, specific H-2 antigen-bearing mast cells challenged with hybridoma IgG2a anti-H-2, heat-aggregated myeloma IgG2a, and soluble antigen-myeloma IgG2a complexes. The conclusion was reached that mouse mast cells could be activated by mouse monoclonal IgG2a antibodies through a noncytotoxic, complement-independent mechanism involving mast cell Fcγ receptors.     

Neuropilin-1 Expression Is Induced on Tolerant Self-Reactive CD8+ T Cells but Is Dispensable for the Tolerant Phenotype

Establishing peripheral CD8⁺ T cell tolerance is vital to avoid immune mediated destruction of healthy self-tissues. However, it also poses a major impediment to tumor immunity since tumors are derived from self-tissue and often induce T cell tolerance and dysfunction. Thus, understanding the mechanisms that regulate T cell tolerance versus immunity has important implications for human health. Signals received from the tissue environment largely dictate whether responding T cells become activated or tolerant. For example, induced expression and subsequent ligation of negative regulatory receptors on the surface of self-reactive CD8⁺ T cells are integral in the induction of tolerance. We utilized a murine model of T cell tolerance to more completely define the molecules involved in this process. We discovered that, in addition to other known regulatory receptors, tolerant self-reactive CD8⁺ T cells distinctly expressed the surface receptor neuropilin-1 (Nrp1). Nrp1 was highly induced in response to self-antigen, but only modestly when the same antigen was encountered under immune conditions, suggesting a possible mechanistic link to T cell tolerance. We also observed a similar Nrp1 expression profile on human tumor infiltrating CD4⁺ and CD8⁺ T cells. Despite high expression on tolerant CD8⁺ T cells, our studies revealed that Nrp1 had no detectable role in the tolerant phenotype. Specifically, Nrp1-deficient T cells displayed the same functional defects as wild-type self-reactive T cells, lacking in vivo cytolytic potential, IFNγ production, and antitumor responses. While reporting mostly negative data, our findings have therapeutic implications, as Nrp1 is now being targeted for human cancer therapy in clinical trials, but the precise molecular pathways and immune cells being engaged during treatment remain incompletely defined.     

Characterization of the human blood lymphocytes that produce a histamine-induced suppressor factor (HSF)

The cells which elaborate a soluble suppressor factor in vitro in response to histamine (histamine-induced suppressor factor or HSF) were partially characterized in the present studies. Human blood T- and B-cell populations were purified by affinity chromatography with rabbit anti-human F(Ab′)₂ and examined for their ability to make HSF. Highly purified populations of T cells, but not B cells, produced HSF in response to varying concentrations of histamine (10^?4 to 10^?4M). The HSF-producing cells were characterized further by means of affinity chromatography with columns containing conjugates of insolubilized histamine as well as by rosette formation with IgG (Tγ)- or IgM (Tμ)-coated ox red blood cells. These studies revealed the following: (a) Cells that synthesize HSF are retained on histamine (but not control) columns; (b) cells with histamine receptors comprise approximately 50% of the Tγ subpopulation but are not found in the Tμ subpopulation; (c) cells not retained by histamine columns have a reduced capacity to develop into suppressor cells following stimulation by concanavalin A or specific antigen (compared to unfractionated or control column passed cells). In addition, it was shown that cells synthesizing HSF predominantly express histamine type 2 receptors: (d)4-Methyl histamine (H₂ agonist), but not 2-methyl histamine (H₁ agonist), was capable of inducing HSF production; (e) cimetidine (H₂ antagonist) inhibited HSF production but chlorpheniramine (H₁ antagonist) did not. Taken together, these experiments suggest that T lymphocytes capable of expressing suppressor function following activation by histamine, specific antigen, concanavalin A, or perhaps through their Fc receptors may either be heterogeneous within the same subpopulation or more likely be the same cell with the complement of receptors described above.