Prevention by the MER tubercle bacillus fraction of immunosuppression induced by cancer chemotherapeutic agents

Summary Contact hypersensitivity (CH) to 2,4-dinitro-1-fluorobenzene (DNFB) was induced in guinea pigs and mice by DNFB skin application. Development of CH was suppressed in both species either by cyclophosphamide (CY) treatment after sensitization or by single intravenous injection of dinitrobenzene-sulfonate (DNBS) before sensitization (hapten-induced tolerance). Additional treatment schedules were employed in guinea pigs, with the following results: Suppression of CH by injection of DNBS concomitant with sensitization; abrogation of hapten-induced tolerance by administration of CY before sensitization; and potentiation of CH skin reactivity by administration of CY before sensitization.Pretreatment by two injections of the methanol extraction residue (MER) tubercle bacillus fraction restored significantly the ability of CY treated animals to respond to DNFB sensitization. In contrast, administration of MER either by one injection before sensitization, concomitant with DNFB, or after sensitization did not prevent immunosuppression by CY.MER treatment was not effective in reversing hapten-induced tolerance in mice, and had only an occasional effect on this process in guinea pigs. Abrogation of hapten-induced tolerance and potentiation of DNFB sensitization by CY in guinea pigs were also not influenced by MER treatment.Supported by Contract NO1-CM-12127 from the NCI and by research grants from Concern Foundation, Inc., the Lautenberg Endowment, the National Council for Research and Development, Israel, and the GSF Munich, Germany, and the Leukemia Research Foundation, Inc.     

Specific suppressor T cells in rats active in the afferent phase of contact hypersensitivity

The optimal conditions for the induction of contact hypersensitivity in rats and the characteristics of its suppression were studied using the sensitizing haptens dinitrofluorobenzene (DNFB) and trinitrochlorobenzene (TNCB). The hypersensitivity was shown to be hapten specific in so far as TNCB did not sensitize for DNFB responses but sensitization with DNFB did allow a marginal response in rats challenged with TNCB. Suppression of the sensitization to DNFB and TNCB could be generated by intravenous injection of dinitrobenzenesulphonic acid (DNBS) or trinitrobenzenesulphonic acid (TNBS), respectively, up to 3 weeks before sensitization. This suppression was hapten specific and could be transferred with splenic T cells enriched for lymphocytes carrying the OX8 (Tc/s) cell marker. Only the induction phase of sensitization, however, could be suppressed in that way. No suppression acting upon the effector phase could be detected except for a nonspecific local suppression at the site of a previous challenge with an antigen to which the rat was specifically suppressed. This study shows that suppression of contact hypersensitivity in rats is mediated by specific suppressor T cells of which the activation pathway apparently differs from that postulated for mice.     

Prevention by the MER tubercle bacillus fraction of immunosuppression induced by cancer chemotherapeutic agents

Summary Contact hypersensitivity (CH) to 2,4 dinitro-1-fluorobenzene (DNFB) was induced in BALB/c mice by DNFB skin application. Development of skin CH was suppressed by exposure of the animals after sensitization to the cancer chemotherapeutic drugs cyclophosphamide (CY), sodium methotrexate (MTX), and 5-fluorouracil (5FU). Unresponsiveness to DNFB was also induced in parallel experiments by a single intravenous injection of dinitrobenzenesulfonate (DNBS), either before or concomitant with sensitization. Potentiation of CH skin reactivity was achieved by administration of CY prior to sensitization.Pretreatment by two injections of the methanol extraction residue (MER) tubercle bacillus fraction restored significantly the ability of animals exposed to CY, MTX, or 5FU to respond to DNFB sensitization. The agent did not impair the potentiation of CH skin reactivity that could be effected by administration of CY prior to sensitization.MER treatment was not effective in reversing hapten-induced (DNBS) tolerance in mice.These findings favor the assumption that MER, under the conditions tested, stimulates the function of positively reacting T cells and exerts no enhancing or protective action on suppressor T cells.     

Induction of tolerance via skin depleted of Langerhans cells by a chemical carcinogen

Since treatment of mouse skin with the chemical carcinogen 7,12-dimethylbenz[alpha]anthracene (DMBA) substantially decreases the density of cutaneous Langerhans cells (LC), the immune status of mice sensitized to 2,4-dinitrofluorobenzene (DNFB) through DMBA-treated skin was investigated. Mice did not develop contact sensitivity to DNFB when applied to DMBA-treated dorsal trunk skin, whereas sensitization resulted when DNFB was applied to untreated abdominal wall skin. Mice immunized with DNFB via DMBA-treated skin did not respond to subsequent immunization through untreated dorsal trunk skin, demonstrating the generation of suppressor cells which could inhibit the activation of effector lymphocytes. Adoptively transferred spleen cells from mice immunized with DNFB through DMBA-treated skin inhibited the response of sensitized hosts, indicating the presence of efferently acting suppressor cells which could inhibit the function of effector lymphocytes. This study has demonstrated that sensitization via skin depleted of LC by chemical carcinogen treatment induces an active state of tolerance rather than immunity.     

Suppressor cells for the afferent phase of contact sensitivity to picryl chloride: inhibition of DNA synthesis induced by T cells from mice injected with picryl sulfonic acid.

Previous reports have shown that picryl sulfonic acid (PSA) induces suppressor T cells that inhibit the effector phase of contact sensitivity, whereeas its DNP counterpart, dinitrobenzenesulfonate (DNBS) induces cells that inhibit the afferent phase of sensitization. Accordingly, cells from mice injected with DNBS, but not PSA, could be shown to inhibit the DNA synthesis in the lymph nodes that occurs during sensitization. It is now shown that PSA does induce T cells that suppress DNA synthesis but this can only be detected with enriched T cells or by using a regimen of PSA injection different frm previously used to induce suppressor cells for the effector phase. The T cells did not affect responses to oxazolone or dinitrofluorobenzene (DNFB) and were distinguishable from suppressors of the efferent phase in that they could be produced in adult thymectomized but not cyclophosphamide-treated mice. T cells from mice injected with DNBS that inhibited DNA synthesis to DNFB had the same properties.     

Immunomodulating activities of staphylococcal enterotoxins. I. Effects on in vivo antibody responses and contact sensitivity reaction

The immunomodulating effects of staphylococcal enterotoxins on in vivo immune responses in C57BL/6 mice were examined. Of the five serological types A (SEA), B, C, D, and E (SEE), only SEA and SEE markedly suppressed the antibody response to sheep red blood cells (SRBC) when injected 1 day before or on the day of immunization with SRBC. Further study of SEA revealed that it did not affect the antibody response to a thymus-independent antigen, salmonella flagella, but did affect the T-cell-mediated immune response. Contact sensitivity to dinitrofluorobenzene (DNFB) was suppressed when SEA was injected before sensitization or before challenge with DNFB, indicating that SEA affected both the afferent and efferent phases of DNFB contact sensitivity. As the suppression of DNFB contact sensitivity could be transferred by anti-Thy-1.2 antibody-sensitive spleen cells of SEA injected donors into normal or DNFB-sensitized recipients, the suppression was thought to be an active one. However, SEA could augment the DNFB contact sensitivity when injected on the third day after sensitization with DNFB. These results indicate that the immunomodulating effects of SEA can be mediated by the T-cell function.     

Immune suppression with supraoptimal doses of antigen in contact sensitivity. I. Demonstration of suppressor cells and their sensitivity to cyclophosphamide.

Immunologic suppression was induced in a mouse model of contact sensitization to DNFB by using supraoptimal doses of antigen. In these studies, in vivo measurement of ear swelling as an indication of immunologic responsiveness correlated well with measurement of in vitro antigen-induced cell proliferation. This unresponsiveness was specific, since supraoptimal doses of DNFB did not interfere with the development of contact sensitivity to another contactant, oxazolone. The decrease in responsiveness is a form of active suppression, as lymphoid cells from supraoptimally sensitized donors transferred suppression to normal recipients. Furthermore, pretreatment with cyclophosphamide (Cy) reversed the suppression seen in supraoptimally sensitized animals but had no effect on the optimal sensitization regimen. These results indicate that supraoptimal doses of contactants can activate suppressor cells and that precursors of these cells are sensitive to Cy. Such suppressors regenerate within 7 to 14 days after Cy treatment. The ability of Cy pretreatment to affect supraoptimal sensitization without affecting optimal sensitization confirms other reports indicating that the observed results of Cy treatment depend critically upon the dose of antigen used.     

Induction and persistence of suppression of contact hypersensitivity against bystander haptens and alloantigens in rats

The shift of suppression from a tolerizing hapten to a so-called bystander antigen was investigated in this study using contact hypersensitivity to trinitrochlorobenzene (TNCB) and dinitrofluorobenzene (DNFB) and delayed type hypersensitivity (DTH) to alloantigens in the rats as experimental models. Primary suppression of contact hypersensitivity was induced by intravenous injection of the water-soluble forms of TNCB and DNFB. A shift of the suppression to the bystander hapten was found if the tolerizing and bystander hapten were mixed and applied to the same area of skin during the sensitization procedure, but not if they were applied to separate areas of skin. With alloantigens, bystander suppression developed only when the sensitizing allogeneic cells were mixed with hapten-modified syngeneic cells. It was not induced by hapten-modified allogeneic cells. Once induced, such bystander suppression of the response to haptens persisted independently of the primarily tolerizing hapten, and it could be adoptively transferred with spleen cells. These results favour the concept that the bystander suppression is mediated by the non-specific action of suppressor cells generated specifically during the mixed sensitization rather than by an antigen bridge.     

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.     

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.     

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.     

Phenotypic and functional characterization of ultraviolet radiation-induced regulatory T cells

Sensitization through UV-exposed skin induces regulatory T cells (Treg). In contrast to the classical CD4+CD25+ Treg that act contact dependent, UV-induced Treg (UV-Treg) suppress via IL-10, indicating a distinct subtype that requires further characterization. Depletion studies revealed that UV-Treg express the glucocorticoid-induced TNF family-related receptor (GITR) and the surface molecule neuropilin-1. The injection of T cells from UV-tolerized mice after depletion of UV-Treg into naive recipients enabled a contact hypersensitivity response, indicating that tolerization also induces T effector cells. Adoptive transfer experiments using IL-10-deficient mice indicated that the IL-10 required for suppression is derived from UV-Treg and not from host-derived cells. Activation of UV-Treg is Ag specific, however, once activated suppression is nonspecific (bystander suppression). Hence, speculations exist about the therapeutic potential of Treg generated in response to Ag that are not necessarily the precise Ag driving the pathogenic process. Thus, we studied the consequences of multiple injections of 2,4-dintrofluorobenzene (DNFB)-specific Treg into ears of naive mice followed by multiple DNFB challenges. DNFB-specific Treg were injected once weekly into the left ears of naive mice and DNFB challenge was performed always 24 h later. After three injections, a challenging dose of DNFB was applied on the right ear. This resulted in pronounced ear swelling, indicating that the subsequent boosting of DNFB-specific Treg had caused sensitization of the naive mice against DNFB. These data demonstrate that UV-Treg express GITR and neuropilin-1 and act via bystander suppression. However, constant boosting of Treg with Ag doses in the challenging range results in final sensitization that might limit their therapeutic potential.     

Immunopotentiating effects of amphotericin B. I. Enhanced contact sensitivity in mice.

Contact sensitivity responses to dinitrofluorobenzene (DNFB) or oxazolone were enhanced by amphotericin B (AmB) administration. This adjuvant effect of AmB was documented in mice by ear thickness measurements, ear histology, and the 5-iodo-2'-deoxyuridine-125I ear assay. The optimum immunopotentiating effect of AmB required its simultaneous administration at the time of skin sensitization. AmB-induced adjuvant effects were also observed in adoptive transfer experiments in which syngeneic recipients of lymph node cells from animals sensitized with DNFB plus AmB gave stronger contact sensitivity responses than recipients of cells from mice sensitized with DNFB alone. AmB also interfered with tolerance induction by i.v. dinitrobenzene sulfonic acid, suggesting that its adjuvant effects involve inhibition of suppressor cells or their precursors.     

IL-12 breaks dinitrothiocyanobenzene (DNTB)-mediated tolerance and converts the tolerogen DNTB into an immunogen

Epicutaneous application of dinitrothiocyanobenzene (DNTB) induces tolerance against its related compound dinitrofluorobenzene (DNFB), because DNTB-pretreated mice cannot be sensitized against the potent hapten DNFB. This tolerance is hapten-specific and transferable. In this study, we demonstrate that IL-12 can break DNTB-mediated tolerance. Furthermore, naive mice treated with IL-12 before DNTB application responded to DNFB challenge with a pronounced ear swelling response without previous sensitization to DNFB, showing that IL-12 can convert the tolerogen DNTB into an immunogen. No differences in numbers or regulatory activity were observed between CD4+CD25+ regulatory T cells isolated from mice treated with DNFB, DNTB, or IL-12 followed by DNTB. However, the number of CD207+ Langerhans cells in regional lymph nodes of DNTB-treated mice was significantly lower than in animals treated with DNFB or IL-12 plus DNTB. Additionally, CD11c+ dendritic cells (DC) isolated from regional lymph nodes of DNTB-treated mice had a significantly lower ability to stimulate T cell proliferation and produced reduced amounts of inflammatory cytokines. Application of both DNFB and DNTB induced apoptotic cell death of DC in the epidermis and the regional lymph nodes. However, the number of apoptotic DC in regional lymph nodes was significantly higher in DNTB-treated animals compared with mice treated with DNFB or IL-12 plus DNTB. Therefore, we conclude that DNTB-mediated tolerance is secondary to inefficient Ag presentation as a result of apoptotic cell death of DC and that IL-12 converts the tolerogen DNTB into an immunogen by preventing DNTB-induced apoptosis of DC.     

Immunopotentiating effects of amphotericin B. II. Enhanced in vitro proliferative responses of murine lymphocytes.

Enhanced in vitro proliferative responses to DNBSO3 were seen in lymph node cells and spleen cells after in vivo sensitization of mice with DNFB plus AmB compared with mice primed with DNFB alone. The T cell proliferation in the nylon column nonadherent fraction for both groups was highly similar, and the enhanced lymph node cell proliferation with AmB was demonstrated to be in the nylon adherent population consisting of both T and B cells. These and earlier studies of immunopotentiation by AmB are consistent with a mechanism that depends on selective interaction of the polyene with a subset of T cells and a resultant impairment of the normally induced suppressor regulation that limits the magnitude and duration of immune responses.     

CD4+ and CD8+ T cell priming for contact hypersensitivity occurs independently of CD40-CD154 interactions

The primary effector cells of contact hypersensitivity (CHS) responses to dintrofluorobenzene (DNFB) are IFN-gamma-producing CD8(+) T cells, whereas CD4(+) T cells regulate the magnitude and duration of the response. The requirement for CD40-CD154 engagement during CD8(+) and CD4(+) T cell priming by hapten-presenting Langerhans cells (hpLC) is undefined and was tested in the current study. Similar CHS responses to DNFB were elicited in wild-type and CD154(-/-) animals. DNFB sensitization of CD154(-/-) mice primed IFN-gamma-producing CD8(+) T cells and IL-4-producing CD4(+) T cells. However, anti-CD154 mAb MR1 given during hapten sensitization inhibited hapten-specific CD8(+), but not CD4(+), T cell development and the CHS response to challenge. F(ab')(2) of MR1 failed to inhibit CD8(+) T cell development and the CHS response suggesting that the mechanism of inhibition is distinct from that of CD40-CD154 blockade. Furthermore, anti-CD154 mAb did not inhibit CD8(+) T cell development and CHS responses in mice depleted of CD4(+) T cells or in CD4(-/-) mice. During in vitro proliferation assays, hpLC from mice treated with anti-CD154 mAb during DNFB sensitization were less stimulatory for hapten-primed T cells than hpLC from either control mice or mice depleted of CD4(+) T cells before anti-CD154 mAb administration. These results demonstrate that development of IFN-gamma-producing CD8(+) T cells and the CHS response are not dependent on CD40-CD154 interactions. This study proposes a novel mechanism of anti-CD154 mAb-mediated inhibition of CD8(+) T cell development where anti-CD154 mAb acts indirectly through CD4(+) T cells to impair the ability of hpLC to prime CD8(+) T cells.     

Effect of methanol extraction residue tubercle bacillus fraction treatment in vivo and in vitro on the release and activity of suppressor factor inhibiting the efferent phase of contact sensitivity in mice

BALB/c mice subjected to injection of dinitrobenzenesulfonate (DNBSO3) and skin painting with dinitrofluorobenzene (DNFB) produce splenic T-suppressor (Ts) cells that, when transferred to DNFB-sensitized recipients, suppress the efferent (eff) phase of contact sensitivity (CS). Splenocyte populations containing such Ts-eff cells release a specific soluble suppressor factor (SSF) in vitro that similarly suppresses CS in sensitized recipient mice. Treatment with the methanol extraction residue (MER) tubercle bacillus fraction, a known immunomodulating agent, of animals exposed to DNBSO3 and DNFB ("DNBSO3-DNFB" donors) prevented release of SSF by their spleen cells at in vitro incubation. Incubation of DNBSO3-DNFB donor splenocytes with MER in vitro also abolished SSF release, and treatment with MER of test animals prior to DNFB sensitization prevented the suppressive action of subsequently administered SSF. The observations are discussed with regard to the potentiating capacities of MER on cellular immunity and states of resistance.     

Immune responses in mice infected with lactic dehydrogenase virus. II. Contact sensitization to DNFB and characterization of lymphoid cells during acute LDV infection.

The effect of acute infection with lactic dehydrogenase virus (LDV) on the development of contact sensitivity to DNFB was studied in Balb/c mice. LDV infection inhibited contact sensitivity to DNFB. The extent of inhibition observed depended on the timing of LDV infection relative to the first (sensitization) and last (challenge) antigenic stimulation. The possibility that the observed inhibition of contact sensitivity to DNFB could be due to a depletion of the T-dependent areas of lymphoid tissues during acute LDV infection was considered. Lymph nodes, spleen and thymus from normal animals, and from mice infected with LDV 1 or 2 days previously, were examined histologically. Also, the proportion of T cells to Ig positive cells in cell suspensions of these tissues was determined. Results did not suggest a T cell depletion during acute infection with LDV.     

IL-1 receptor signaling is required at multiple stages of sensitization and elicitation of the contact hypersensitivity response

Contact hypersensitivity (CHS) is a CD8 T cell-mediated response to hapten skin sensitization and challenge. The points at which IL-1R signaling is required during this complex, multistep immune response have not been clearly delineated. The role of IL-1R signaling during 2, 4 dinitro-1-fluorobenezene (DNFB) sensitization to induce hapten-specific CD8 effector T cells and in the trafficking of the effector T cells to the DNFB challenge site to elicit the response were investigated using IL-1R deficient mice. DNFB-sensitized IL-1R(-/-) mice had low CHS responses to hapten challenge that were caused in part by marked decreases in hapten-specific CD8 T cell development to IL-17- and IFN-γ-producing cells during sensitization. Hapten-primed wild type CD8 T cell transfer to naive IL-1R(-/-) mice did not result in T cell activation in response to hapten challenge, indicating a need for IL-1R signaling for the localization or activation, or both, of the CD8 T cells at the challenge site. Decreased CD8 T cell priming in sensitized IL-1R(-/-) mice was associated with marked decreases in hapten-presenting dendritic cell migration from the sensitized skin to draining lymph nodes. Transfer of hapten-presenting dendritic cells from wild type donors to naive IL-1R(-/-) mice resulted in decreased numbers of the dendritic cells in the draining lymph nodes and decreased priming of hapten-specific CD8 T cells compared with dendritic cell transfer to naive wild type recipients. These results indicate that IL-1R signaling is required at multiple steps during the course of sensitization and challenge to elicit CHS.     

Key role for mast cells in nonatopic asthma

The mechanisms involved in nonatopic asthma are poorly defined. In particular, the importance of mast cells in the development of nonatopic asthma is not clear. In the mouse, pulmonary hypersensitivity reactions induced by skin sensitization with the low-m.w. compound dinitrofluorobenzene (DNFB) followed by an intra-airway application of the hapten have been featured as a model for nonatopic asthma. In present study, we used this model to examine the role of mast cells in the pathogenesis of nonatopic asthma. First, the effect of DNFB sensitization and intra-airway challenge with dinitrobenzene sulfonic acid (DNS) on mast cell activation was monitored during the early phase of the response in BALB/c mice. Second, mast cell-deficient W/W(v) and Sl/Sl(d) mice and their respective normal (+/+) littermate mice and mast cell-reconstituted W/W(v) mice (bone marrow-derived mast cells-->W/W(v)) were used. Early phase mast cell activation was found, which was maximal 30 min after DNS challenge in DNFB-sensitized BALB/c, +/+ mice but not in mast cell-deficient mice. An acute bronchoconstriction and increase in vascular permeability accompanied the early phase mast cell activation. BALB/c, +/+ and bone marrow-derived mast cell-->W/W(v) mice sensitized with DNFB and DNS-challenged exhibited tracheal hyperreactivity 24 and 48 h after the challenge when compared with vehicle-treated mice. Mucosal exudation and infiltration of neutrophils in bronchoalveolar lavage fluid associated the late phase response. Both mast cell-deficient strains failed to show any features of this hypersensitivity response. Our findings show that mast cells play a key role in the regulation of pulmonary hypersensitivity responses in this murine model for nonatopic asthma.