Soluble factors in tolerance and contact sensitivity to DNFB in mice. VIII. Regulation of T suppressor cell function by autoreactive T helper cells

When cultured with DNP-labeled I-A+ cells, Lyt 2+ T suppressor cells (Ts) from 2,4,-dinitrobenzene sulfonate (DNBS)-tolerized mice are activated to synthesize and release a suppressor factor (SSF) which suppresses the transfer of contact sensitivity to DNFB. The signals required to activate the DNBS-primed Ts to produce SSF were studied in greater detail. As previously observed with fixed DNP-labeled spleen cell stimulators, the supernatants from cultures of DNBS-primed spleen cells and glutaraldehyde-fixed DNP-labeled P388D1 cell monolayers did not contain SSF. When the tolerant cells were harvested from these monolayers and were treated with IL-1, the Ts released the synthesized SSF. Synthesis and release of SSF required Ts recognition of DNP/class I MHC on the hapten-presenting cells followed by interaction with the costimulator IL-1. When the tolerant cells were cultured with fixed DNP-labeled I-A+ or I-A- stimulators to induce SSF synthesis, release was induced by adding either unlabeled or TNP-labeled unprimed spleen cells to the cultures. The release of SSF was blocked when the second stimulators were pretreated with anti-I-A antibody but not with anti-DNP or anti-class I MHC antibodies. These results indicate that the release of SSF by DNBS-primed Lyt 2+ Ts is regulated by the activity of a self-I-A-reactive (i.e., autoreactive) T cell in the tolerant spleen cell population.     

Regulation of contact sensitivity to DNFB in the mouse: effects of adult thymectomy and thymic factor.

The contact sensitivity response to DNFB is decreased after adult thymectomy (ATX). This response decreases to 50% of the control response of normal age-matched mice as soon as 3 weeks after ATX and is not further depressed 9 to 16 weeks after ATX. These results suggest that two T cell subsets of different lifespan are involved in the anti-DNFB response. A circulating thymic factor (FTS) is able to restore the contact sensitivity response to DNFB when injected 3 to 9 weeks after ATX but not 16 weeks later. By contrast, FTS has a depressive effect on the contact sensitivity response to DNFB of normal mice through a cyclophosphamide-sensitive T cell subset. These results suggest that FTS regulates DNFB contact sensitivity by acting on a cyclophosphamide-sensitive T cell subset, still present 9 weeks after ATX but absent after 16 weeks. Thus although the T cell defect, causing a depression of the contact sensitivity reaction to DNFB is quantitatively similar 3 and 16 weeks after ATX, its nature is probably different.     

Tolerance and contact sensitivity to DNFB in mice. VI. Inhibition of afferent sensitivity by suppressor T cells in adoptive tolerance.

Tolerance in contact sensitivity to DNFB can be adoptively transferred to normal mice with lymph node cells from tolerant donors. This tolerance is antigen specific and is mediated by T cells, i.e., "suppressor" T cells. Experiments were carried out to investigate the mechanism(s) by which the suppressor T cells induce tolerance to DNFB contact sensitivity. The suppressor cells were effective only if they were present during the early stages of the afferent limb of sensitization. As measured by DNA synthesis, cell proliferation in the draining lymph nodes of recipients of suppressor cells was found to be significantly less than in control animals indicating that the suppressor cells acted, at least in part, by limiting or inhibiting DNFB-induced cell proliferation. This inhibition was shown to be antigen specific since the DNFB suppressor cells did not inhibit cell proliferation induced by oxazolone, an unrelated contact sensitizer. The ability to DNFB tolerant cells to block afferent sensitization pathways differs from the mechanism of tolerance to picryl chloride, reported by others, where efferent pathways are blocked.     

Tolerance and contact sensitivity to DNFB in mice. V. Induction of tolerance with DNP compounds and with free and membrane-associated DNFB.

Immunologic unresponsiveness (tolerance) was induced in a mouse model of contact sensitization to DNFB. The ability to induce tolerance varied with the chemical reactivity of the tolerogen; DNFB was highly tolerogenic, DNBSO3 was moderately tolerogenic, and DNP-lysine was not tolerogenic. Although DNFB is considered a highly reactive compound, tracer studies of injected DNFB showed that it was rapidly excreted. Further studies were therefore done with DNFB attached to mouse erythrocytes. Tolerance to DNFB-RBC was highly specific in vivo; mice tolerant to DNFB showed normal reactivity to TNCB (picryl chloride.) Cells of mice tolerant to DNFB-RBC were also unresponsive to DNBSO3 in vitro. Tolerance to DNFB, DNBSO3, and DNFB-RBC all required time to develop, suggesting that an active process was involved.     

Influence of contact sensitivity to DNFB on the induction of carrier-determined tolerance to DNP.

Contact sensitivity to DNFB, induced by skin painting Balb/c mice with DNFB had no influence on the induction of carrier determined tolerance to DNP. In contrast, contact sensitivity to DNFB, elicited in complete Freund's adjuvant, prevented induction of tolerance to DNP. This effect was not due to the adjuvant alone and was hapten specific since contact sensitivity induced by OCBC in complete adjuvant had no influence on tolerance induction to DNP. In addition, in mice primed with DNFB in complete Freund's adjuvant, the tolerogen becomes immunogenic. It is suggested that the T-cell mediating tolerance to DNP-autologous IgG is different from the T-cell mediating contact sensitivity to DNP autologous carrier. DNFB in complete adjuvant may augment not only contact-sensitized T-cells, which mediate contact sensitivity, but also T-cells which have helper function in the antibody response to DNP.     

Cell-mediated immunity: role of IL-3 and IL-6 in the regulation of contact sensitivity reaction

Delayed type hypersensitivity reaction (DTH) consists of a sequential cascade of steps depending on different types of T cells, as well as mast cells, endothelial cells and macrophages. Recently it has been shown that CD4+ TH1 lymphocytes ("inflammatory type") play a central role in DTH reaction. Activated TH1 cells produce a characteristic pattern of cytokines: IL-2, IL-3, TNF-beta, IFN-gamma. Using the contact sensitivity (CS) reaction on mice as a model system, the role of cytokines in the regulation of DTH is presented, particularly the significance of IL-3 and IL-6. The recent data can be interpreted to show that IL-6 released by activated macrophages (APC cells) in the induction phase of the CS reaction probably stimulate CD8+ T suppressor cells. These in turn inhibit the production of IL-2 and IL-3 by CD4+ TH1 cells followed by a state of unresponsiveness.     

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.     

Regulation of immunity by anti-T-cell antibodies.

Current pharmacologic approaches to immune suppression leave much to be desired. The prevention of allograft rejection and the suppression of autoimmunity generally require treatment with corticosteroids or cytotoxic drugs, or both, which may not be sufficiently effective and which frequently cause serious immediate and long-term complications. With the advent of monoclonal antibody technology, it has become possible to identify and selectively inhibit distinct elements in the immune system that contribute to pathologic immune responses. This achievement has led to new therapeutic strategies that may be safer and more effective than the immunosuppressive therapies currently available. Many of these strategies focus on subsets of T cells because of the critical importance of T cells in immune responses. Monoclonal antibodies directed against CD4 + T cells, T-cell activation antigens, and T-cell receptor families have all shown promise in animal models and, in some cases, in preliminary human trials. The challenge now is to translate this promise into practical new forms of immunosuppressive therapy.     

Antibody-mediated regulation of T cell responses. I. Characterization of a monoclonal antibody which specifically regulates contact hypersensitivity to DNFB in BALB/c mice

Contact hypersensitivity (CS) to 2,4-dinitrofluorobenzene (DNFB) in BALB/c mice is regulated by autoanti-idiotypic antibody. This report describes the preparation and characterization of a monoclonal antibody, 2-16.1, which has characteristics previously described for the serum anti-idiotypic antibodies. Monoclonal 2-16.1 was prepared by fusing lymph node (LN) cells from optimally sensitized BALB/c mice to the P3X myeloma. The monoclonal product of the cloned hybridoma is an IgM (K) immunoglobulin which does not bind to DNP-protein but which does bind to other immunoglobulins with anti-DNP specificity, primarily of the IgM class. Functionally, 2-16.1 inhibits the efferent limb of the CS reaction as measured by passive transfer of immunity. This inhibition is antigen-specific and appears to require the presence of a subset of Ia+ T cells in the DNFB-immune LN cell population. Suppression of transfer of immunity is strain-specific. Finally, suppression occurs only in the absence of complement, indicating that a lytic mechanism is not involved and that 2-16.1 does not recognize determinants expressed on the effector T cells of the CS reaction. Collectively, these results indicate that 2-16.1 is a monoclonal anti-idiotypic antibody, and that the hybridoma CSDNP 2-16.1 represents a clone of B cells which is stimulated during the primary CS response to DNFB and whose antibody product is involved in the endogenous, active regulation of this T cell-mediated response.     

Desensitization of contact allergy to DNFB in mice. I. Description of a model system

We investigated the down-regulation of contact sensitivity (desensitization) in mice sensitized to DNFB. Mice were sensitized with DNFB, desensitized with antigen 2 wk later, and resensitized 2 wk after desensitization. Large doses of antigen (DNFB or DNBSO3) produced about 50% inhibition of the anamnestic response as measured by ear swelling after challenge with DNFB. Desensitization was antigen specific and long lasting. Lymph node cells from desensitized mice showed diminished antigen-induced proliferation in vitro. Although the anamnestic response can be inhibited by afferent- or efferent-acting suppressor cells, such suppressor cells were not demonstrated in desensitized animals. The most likely explanation is that antigen desensitizes by inactivating effector cells for contact sensitivity, although suppressor mechanisms have not been completely excluded.     

Desensitization of contact allergy to DNFB in mice. III. Characteristics of immediate desensitization induced by haptenated spleen cells

The immediate effects and mechanisms of desensitization of contact sensitivity to dinitrofluorobenzene (DNFB) were investigated. Intravenous injection of dinitrophenol (DNP)-labeled syngeneic spleen cells caused significant antigen-specific inhibition (greater than 40%) of the contact response within 24 hr in mice that had been sensitized 2 weeks prior to desensitization. With low concentrations of the hapten used for labeling, allogeneic spleen cells were found to be more efficient than syngeneic ones in inducing the down-regulation of the contact response. The most efficient desensitization was produced by DNP-cells that differed from the recipient at the MLS locus. Haptenated spleen cells induced the production of suppressor mechanisms, as spleen cells from animals desensitized with DNP-cells were able to down-regulate recipients in adoptive transfer and could block the passive transfer of contact sensitivity. Procedures that interfere with the development of suppressor cells, e.g., cyclophosphamide and adult thymectomy, interfered with desensitization by DNP-cells. These results are in contrast to desensitization with soluble dinitrobenzenesulfonic acid (DNBS), where suppressor mechanisms have not been shown. The mechanisms for desensitization depend upon the form of the desensitizing antigen.     

Soluble factors in tolerance and contact sensitivity to DNFB in mice. X. IL-2 is the activation signal mediating release of synthesized suppressor factor

Two signals are required for the in vitro activation of Lyt2+ T suppressor cells (Ts) from mice tolerized with 2,4-dinitrobenzene sulfonate (DNBS) to produce soluble suppressor factors (SSF) which suppress the transfer of contact sensitivity to dinitrofluorobenzene (DNFB). Recognition of DNP/class I MHC (signal one) stimulates the Ts to synthesize SSF. Release of SSF requires a soluble mediator (signal two) produced by the interaction of L3T4+ T cells from tolerant mice with I-A on metabolically functional cells in the DNP-presenting cell population. The purpose of this study was to examine the nature of this second Ts activation signal. Coculture of tolerant spleen cells and glutaraldehyde-fixed (Glu-) DNP-labeled spleen cells (DNP-SC) resulted in the synthesis but not release of SSF. Addition of either IL-1 or IL-2 to these cultures induced SSF release. Treatment of such cultured cells with the anti-murine IL-2 receptor antibody PC 61.5.3 blocked the IL-2- and IL-1-stimulated release of SSF. Release of SSF was also blocked when tolerant cells were cultured with (unfixed) DNP-SC in the presence of a monoclonal anti-IL-2 antibody. IL-2 but not IL-1 was able to stimulate the Ts to release synthesized SSF in the absence of L3T4+ TH activity. First, addition of IL-2 to cocultures of tolerant cells and DNP-presenting I-A- cells induced release of the synthesized SSF, whereas addition of IL-1 did not. Second, IL-2 also stimulated SSF release in cocultures of L3T4+ T cell-depleted tolerant cells and Glu-DNP-SC, whereas IL-1 did not. Tolerant cells pretreated with IL-2 and then washed were able to synthesize and release SSF upon culture with Glu-DNP-SC. Pretreatment of tolerant cells with IL-1 did not stimulate SSF release upon subsequent culture with Glu-DNP-SC. These results indicate that the Lyt2+ Ts from DNBS-tolerant mice express IL-2 receptors and IL-2 is the lymphokine which induces the Ts to release synthesized SSF. Thus, IL-2 provides a differentiative signal during the functional activation of these regulatory T cells.     

Soluble factors in tolerance and contact sensitivity to DNFB in mice. VI. Cellular and lymphokine requirements for stimulating suppressor factor production in vitro

Coculture of spleen cells from mice tolerized with 2,4-dinitrobenzenesulfonate (DNBS) and DNP-labeled spleen cells (DNP-SC) activates Lyt-2+ T suppressor cells (Ts) to synthesize and release a suppressor factor (SSF) into the supernatant, which suppresses the transfer of contact sensitivity to DNFB. The purpose of the present study was to examine in greater detail the signals required to activate DNBS-primed Ts to produce SSF. The supernatant from cultures of tolerant cells and glutaraldehyde-fixed DNP-SC did not have SSF. In contrast, the soluble cell lysate from these cultures did contain the suppressive activity. Pretreatment of glutaraldehyde-fixed DNP-SC with either anti-DNP or anti-class I, but not anti-class II MHC, antibodies blocked SSF synthesis. The addition of IL 1 to cultures of DNBS-tolerant cells and glutaraldehyde fixed DNP-SC restored the ability of the Ts to release the synthesized factor. These results indicate that Ts recognition of the hapten/class I MHC determinant stimulates the synthesis of SSF, and a costimulator is required to induce the release of the factor. The supernatants from cultures of either L3T4-depleted tolerant cells and DNP-SC or tolerant cells and anti-I-A antibody-treated DNP-SC had no SSF activity. The addition of a costimulator (IL 1) also restored the ability of the Ts to release the synthesized factor in cultures of L3T4-depleted tolerant cells and DNP-SC. These results suggest that an L3T4 cell in the DNBS-primed cell population interacts with I-A determinants on a cell in the DNP-stimulator population to initiate the generation of the mediator required for SSF release. This further suggests that the Ts is unable to induce the costimulator from the hapten-presenting cell during interaction with the DNP/class I MHC ligand. Therefore, the production of SSF is regulated not only by the presentation of the appropriate hapten/MHC determinant but also by the interactions of cells that function in generating the costimulator needed to induce release of the suppressor factor.     

Requirements for induction of T cell tolerance to DNFB: efficiency of membrane-associated DNFB.

Tolerance to contact sensitization with DNFB, a T cell-dependent phenomenon, was induced in mice by preparations of DNFB coupled to mouse RBC or spleen cells. Such tolerance is dose related, wanes with time, and can be transferred to normal animals with lymphoid cells (presumably containing suppressors). Tolerance to DNFB-RBC can be produced by whole DNFB-RBC, by ghosts of these cells, by sonicates of the ghosts, and by detergent-treated DNFB-RBC ghosts. Tolerance cannot be produced by larger amounts of DNFB-RBC components not associated with membrane. The ability of various DNP compounds to stimulate DNA synthesis in DNFB-sensitized cells also correlates with their ability to bind to protein components; i.e., DNFB is a far more efficient stimulator than DNBSO, whereas DNPlysine does not stimulate at all. Thus, the ability to sensitize or to tolerize with DNFB congeners is related to their ability to couple to proteins. It appears that the active induction of T cell tolerance requires that tolerogen be coupled to cell membranes. Since both T cell sensitization and tolerance to DNFB are best produced by DNFB-membrane, the actual occurrence of one state or the other must depend on the molecular method of "presentation" of DNFB-membrane.     

The role of the macrophage as the stimulator cell in contact sensitivity.

In this study we examined the requirement for the type of stimulator cell for thymus-derived (T) lymphocyte activation to simple chemical haptens. T cells from picryl chloride-immune guinea pigs were challenged in vitro with various trinitrophenyl (TNP)-conjugated syngeneic stimulator cells and the extent of activation was determined by an increase in DNA synthesis. Hapten-specific T cell activation occurred with TNP-conjugated peritoneal exudate cells (PEC) and purified macrophages but not with TNP-conjugated erythrocytes, thymocytes, or nonadherent lymph node cells or PEC. In addition, T cell activation also occurred with TNP-conjugated guinea pig leukemia cells, but only in the presence of macrophages. Furthermore, it was shown that macrophages were required to process and/or present TNP-conjugated leukemia cell antigens rather than simply providing a growth-promoting function. These results suggest that a macrophage-like stimulator cell is required for hapten-specific T cell activation and that this particular stimulator cell may be important in contact sensitivity.     

Soluble factors in tolerance and contact sensitivity to DNFB in mice. VII. Characterization of a monoclonal, efferent-acting suppressor factor with specificity for DNP/H-2Kd

To study further soluble factors which regulate contact sensitivity (CS) to 2,4-dinitrofluorobenzene (DNFB), hapten-primed spleen cells from BALB/c mice were used to make T-cell hybridomas. A hybrid constitutively producing a suppressor factor was identified and cloned (clone 3-10). Incubation of BALB/c DNFB immune lymph node cells (LNC) in the 3-10 supernatant suppressed the ability of the immune cells to transfer CS to DNFB. The passive transfer of CS to oxazalone or to 2,4,6-trinitrochlorobenzene (TNCB) was not suppressed by the 3-10 factor. The hapten specificity of the 3-10 factor further was demonstrated by the ability of DNFB immune LNC but not LNC from unsensitized or from TNCB-sensitized mice to absorb the factor. The 3-10 factor also was adsorbed by DNFB-immune LNC from mice that were syngeneic with BALB/c mice at the K locus of the MHC (e.g., B10.D2 and D2.GD). Pretreatment of DNFB-immune LNC with monoclonal anti-Kd antibody or with anti-DNP antibodies blocked the ability to adsorb the factor. These results indicated that the 3-10 suppressor factor binds to DNP/H-2Kd complexes on immune LNC. Nylon wool-purified T cells (83% Thy-1.2+) from DNFB-immune LNC were able to adsorb the factor as well as unseparated immune LNC. Furthermore, treatment of immune LNC with anti-Thy-1.2 plus C' abrogated the ability of the cells to adsorb the factor, indicating that the cellular target of the 3-10 factor is a T cell. In addition, treatment of the immune LNC with an autoantiidiotypic antiserum (CS 231) plus C', which depletes DNP-specific delayed-type hypersensitivity effector T (TDH) cells, also abrogated the ability of the cells to adsorb the factor. Finally, the suppressor factor was adsorbed and eluted from DNP affinity columns but was not adsorbed by TNP affinity columns. Collectively, these results indicate that although the monoclonal 3-10 suppressor factor has affinity for DNP, focusing of the factor on the TDH cells requires recognition of DNP in the context of the appropriate MHC determinant, Kd.