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.     

In vivo assessment of tumor-induced nonspecific suppression of contact sensitivity

Summary Normal BALB/c mice were assessed for 2,4-dinitrofluorobenzene (DNFB)-induced contact sensitivity following adoptive transfer of macrophages (Mo). T cells, or their derived products, from normal or tumor-bearing hosts (TBH). Contact sensitivity (CS) was measured by a quantitative radioisotopic ear assay, a total in vivo system based on localization of IP-injected iodinated human serum albumin ([¹²⁵I]HSA) in the DNFB-challenged ear. Adoptive transfer of low or high doses of TBH T cells or their derived supernatants into normal recipients suppresed their responsiveness, while Mo supernatants enhanced it. Moreover, in all cases adoptive transfer of TBH cells or supernatants resulted in a lower CS response than did their normal counterparts. These results further corroborate our previous in vitro data indicating that T cells, or Mo and T cell soluble products, possess immunoregulatory capabilities in vivo.Abbreviations DNFB 2,4-dinitrofluorobenzene - TBH tumor-bearing host - Mo macrophages - ¹²⁵I-HSA iodinated-human serum albumin - CMI cell-mediated immunity - CS contact sensitivity; Ig, immunoglobulin - C complement     

Prethymic T cell precursors express receptors for antigen

An anti-idiotype serum raised in BALB/c mice against syngeneic lymph node T cells from 2,4-dinitrofluorobenzene (DNFB)-sensitized mice was used to study the early expression of antigen receptors on developing T cells. Normal BALB/c bone marrow cells were treated with either anti-Thy-1.2 plus complement or anti-Thy-1.2 and anti-idiotype plus complement before use in the reconstitution of lethally irradiated syngeneic mice. Five weeks after reconstitution, recipient mice were assayed for both contact sensitivity (CS) and in vitro proliferative responses to DNFB. Mice reconstituted with bone marrow cells treated with both anti-Thy and anti-idiotype sera showed a significant decrease in reactivity to DNFB in both assay systems when compared with mice reconstituted with marrow treated with anti-Thy only. CS response to the noncross-reacting hapten oxazolone was identical in both recipient groups. Bone marrow mixing experiments showed no evidence of anti-idiotype-induced suppressor cells in these experiments. These data provide strong evidence that at least some T cell precursors express receptors for antigen prethymically.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

Suppressor T cell mechanisms in contact sensitivity. II. Afferent blockade by alloinduced suppressor T cells.

We investigated the mechanism(s) by which MHC-restricted suppressor T cells (Ts) induced by i.v. injection of allogeneic DNP-modified lymphoid cells (alloinduced Ts) suppress the DNFB contact sensitivity response. It was shown that alloinduced Ts acted only during the early phases (afferent limb) of sensitization. They were incapable of suppressing previously sensitized recipients or of inhibiting the expression of DNFB-immune LN cells when co-transferred into normal recipients. The target of alloinduced Ts seems to be cell proliferation, i.e., inhibition of antigen-induced cell proliferation (DNA synthesis) in Ts recipient mice. The failure of recipients of alloinduced Ts to generate DNFB-immune LN cells capable of transferring contact sensitivity to normal recipients also suggests that these Ts act by preventing the development of an expanded clone of mature immune T cells. The suppressive effects of alloinduced Ts also were inhibited by prior in vitro treatment with anti-TNP serum. The data are discussed in terms of current models of suppression, and are compared to mechanisms of suppression in other contact sensitivity models.     

Dermal dendritic cells, and not Langerhans cells, play an essential role in inducing an immune response

Langerhans cells (LCs) serve as epidermal sentinels of the adaptive immune system. Conventional wisdom suggests that LCs encounter Ag in the skin and then migrate to the draining lymph nodes, where the Ag is presented to T cells, thus initiating an immune response. Platelet-activating factor (PAF) is a phospholipid mediator with potent biological effects. During inflammation, PAF mediates recruitment of leukocytes to inflammatory sites. We herein tested a hypothesis that PAF induces LC migration. Applying 2,4-dinitro-1-fluorobenzene (DNFB) to wild-type mice activated LC migration. In contrast, applying DNFB to PAF receptor-deficient mice or mice injected with PAF receptor antagonists failed to induce LC migration. Moreover, after FITC application the appearance of hapten-laden LCs (FITC+, CD11c+, Langerin+) in the lymph nodes of PAF receptor-deficient mice was significantly depressed compared with that found in wild-type mice. LC chimerism indicates that the PAF receptor on keratinocytes but not LCs is responsible for LC migration. Contrary to the diminution of LC migration in PAF receptor-deficient mice, we did not observe any difference in the migration of hapten-laden dermal dendritic cells (FITC+, CD11c+, Langerin-) into the lymph nodes of PAF receptor-deficient mice. Additionally, the contact hypersensitivity response generated in wild-type or PAF receptor-deficient mice was identical. Finally, dermal dendritic cells, but not LCs isolated from the draining lymph nodes after hapten application, activated T cell proliferation. These findings suggest that LC migration may not be responsible for the generation of contact hypersensitivity and that dermal dendritic cells may play a more important role.     

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.     

Oral administration of hapten inhibits in vivo induction of specific cytotoxic CD8+ T cells mediating tissue inflammation: a role for regulatory CD4+ T cells

We investigated whether oral tolerance could block the development of an inflammatory response mediated by CD8+ T cells, using a mouse model of oral tolerance of contact sensitivity (CS) to the hapten 2, 4-dinitrofluorobenzene (DNFB). In this system, the skin inflammatory response is initiated by hapten-specific class I-restricted cytotoxic CD8+ T (CTL) cells, independently of CD4 help. Oral delivery of DNFB before skin sensitization blocked the CS response by impairing the development of DNFB-specific CD8+ effector T cells in secondary lymphoid organs. This was shown by complete inhibition of DNFB-specific CTL and proliferative responses of CD8+ T cells, lack of specific IFN-gamma-producing CD8+ T cells, and inability of CD8+ T cells to transfer CS in RAG20/0 mice. RT-PCR and immunohistochemical analysis confirmed that recruitment of CD8+ effectors of CS in the skin at the site of hapten challenge was impaired in orally tolerized mice. Sequential anti-CD4 Ab treatment showed that only depletion of CD4+ T cells during the afferent phase of CS abrogated oral tolerance induction by restoring high numbers of specific CD8+ effectors in lymphoid organs, whereas CD4 depletion during the efferent phase of CS did not affect oral tolerance. These data demonstrate that a single intragastric administration of hapten can block in vivo induction of DNFB-specific CD8+ CTL responsible for tissue inflammation and that a subset of regulatory CD4+ T cells mediate oral tolerance by inhibiting expansion of specific CD8+ effectors in lymph nodes.     

Age-related changes in cell-mediated immunity in BALB/C mice.

The effect of increasing age on various tests of cell-mediated immunity was investigated in BALB/c mice both in vitro and in vivo with four different assay systems. The following results were obtained. 1) In contact sensitivity to DNFB, old mice (age 60 to 80 weeks) showed no differences in sensitization when compared to young adult mice (age 8 to 12 weeks). (In contrast, old NZB/W mice showed impaired contact sensitization when compared with young NZB/W MICE.)2) Unlike the reaction in contact sensitivity, cells from old BALB/c mice were defective in eliciting a graft-vs-host reaction. This was true also when a partially purified population of T cells was transferred. 3) In the mixed lymphocyte reaction, cells from old mice were as efficient or better than cells from young adult BALB/c mice in responding to or stimulating allogeneic cells. 4) Responses to PHA and Con A (Both T cell mitogens) were greatly reduced when old cells were cultured as compared with cells from young adult mice. Thus, we have found that within the same batch of mice, increasing age was associated with increased capabilities in some measures of cell-mediated immunologic function and decreased capabilities in other measures of the same.     

Desensitization of contract allergy to 2,4-dinitro-1-fluorobenzene in mice. II. Characteristics of "immediate" desensitization

The immediate effects and mechanisms of desensitization of contact sensitivity to 2,4-dinitro-1-fluorobenzene (DNFB) were investigated. Mice were sensitized with DNFB, desensitized with antigen 2 weeks later, and challenged 1 day after desensitization. Significant inhibition (approximately 50%) of contact sensitivity was observed after iv injections of large doses of dinitrobenzene sulfonic acid (DNBS) or dinitropenol (DNP)-labeled spleen cells. Haptenated red blood cells (RBC) did not induce any significant immediate desensitization but produced significant inhibition of an anamnestic response 2 weeks later. The immediate desensitization induced by DNBS was antigen nonspecific. Although the contact sensitivity response itself could be inhibited by afferent- or efferent-acting suppressor cells, such cells were not demonstrated in desensitized animals. DNBS appears to desensitize by inactivating effector cells for contact sensitivity, although it appears that suppressor mechanisms could be activated by other physiochemical forms of the desensitizing antigen.     

Circulating thymic hormone levels in zinc deficiency

The effect of zinc deficiency (Zn^?) on the circulating thymic hormone (FTS) levels in A/J mice was studied. After 3 weeks of feeding the mice a Zn^? diet, FTS levels were markedly reduced and after 17 weeks, FTS was undetectable. By contrast, the zinc-supplemented (Zn⁺) group seemed to maintain FTS levels better than the normal diet group with aging. On the other hand, spleen spontaneous rosette-forming cells (sRFC) were studied for their azathioprine (AZ) sensitivity in A/J mice on different diets. The Zn^? mice had fewer sRFC than did the normally fed or Zn⁺ mice. The role of zinc in controlling levels of FTS and thus thymic function is discussed.     

Augmentation of specific immune response against a syngeneic SV40-induced sarcoma in mice by depletion of suppressor T cells with cyclophosphamide.

When cyclophosphamide was administered to mice before immunization with syngeneic SV40-transformed cells, the specific immune response elicited, as was measured by the tumor cell neutralization assay with a syngeneic SV40-induced sarcoma, was stronger and lasted longer as compared to the response generated in non-cyclophosphamide-treated mice. The augmentation effect of the drug was dependent on cyclophosphamide concentration, being optimal at 100 mg/kg, and on the time of drug administration in relation to antigen immunization, being optimal at 2–4 days before antigen administration. Transfer of T cells from normal syngeneic mice to drug-treated animals abolished the cyclophosphamide-induced augmentation of immune response. These results implied that cyclophosphamide-sensitive T cells suppressed the in vivo generation of specific effector T cells against SV40-induced sarcoma.     

The role of the thymus in the maintenance of natural killer cells in vivo

This report describes a model for investigating the role of the thymus in regulating natural killer (NK) cell activity in vivo. Evidence is presented that the thymus can regulate NK cells, and that at least some NK cells can develop without thymic help. Marrow from thymectomized rats depleted of circulating T cells by thoracic duct cannulation was transplanted into rats without a thymus (1 degree ATX.BM). These 1 degree ATX.BM rats had NK cell levels above controls 3 months after reconstitution but markedly depressed NK cell levels by 9 months. When 1 degree ATX.BM marrow was used to reconstitute rats with or without a thymus, those without a thymus (2 degrees ATX.BM) exhibited low NK cell levels after 3 months, and a similar result was obtained when 2 degrees ATX.BM marrow was used to reconstitute 3 degrees ATX.BM rats. The low NK cell levels in 2 degrees and 3 degrees ATX.BM rats were due to a deficiency in spontaneously cytotoxic NK cells, as they had normal numbers of interferon-responsive pre-NK cells. Spleen cells from 2 degrees and 3 degrees ATX.BM rats produced less interferon than control spleen cells when cultured with P815 tumor cells in vitro. However, 2 degrees and 3 degrees ATX.BM rats had higher numbers of large granular lymphocytes than controls despite their low NK cell levels. In marked contrast to 2 degrees and 3 degrees ATX.BM rats, spleen cells from 4 degrees ATX.BM rats had higher levels of cytotoxicity and a higher frequency of both spontaneously cytotoxic and pre-NK cells than controls. The 4 degrees ATX.BM rats also had the highest frequency of large granular lymphocytes in the spleen.     

Contact hypersensitivity reactions to dinitrofluorobenzene mediated by monoclonal IgE anti-DNP antibodies

Several studies have suggested a possible role for IgE antibodies in the pathogenesis of cutaneous hypersensitivity reactions that reach maximum intensity 24 to 48 hr after antigen challenge. The recent availability of murine monoclonal IgE anti-hapten antibodies has made possible the direct examination of the range of cutaneous inflammatory reactions that can be mediated by such antibodies. We have examined the effects of passively sensitizing BALB/c mice with monoclonal IgE anti-dinitrophenyl (DNP) antibody 48 hr before antigen challenge. Inflammatory responses were assessed by measuring ear swelling in mice challenged on the ears with the reactive hapten 2,4-dinitrofluorobenzene (DNFB). Compared with unsensitized controls, the ears of mice passively sensitized with IgE anti-DNP displayed a biphasic pattern of ear swelling after DNFB challenge. An early, transient response (present within 15 to 30 min of challenge and returning to control levels within 4 to 9 hr) was followed by a second, more persistent increase in ear swelling that peaked 24 to 48 hr after challenge. This biphasic pattern of ear swelling seen in IgE-sensitized mice was temporally indistinguishable from that observed in mice conventionally sensitized for allergic contact dermatitis reactions by epicutaneous application of DNFB 5 days before DNFB ear challenge. Antigen specificity of the IgE-mediated contact hypersensitivity reactions was demonstrated by the failure of mice passively sensitized with IgE anti-DNP to display early or delayed ear swelling greater than unsensitized controls when challenged with either of two noncross-reacting haptens, fluorescein isothiocyanate or oxazolone. Mice passively sensitized with a monoclonal IgA anti-DNP antibody (MOPC 315) 48 hr before DNFB challenge failed to display early or delayed ear swelling greater than unsensitized controls. Heat inactivation of the IgE anti-DNP ascitic fluid at 56 degrees C for 30 min completely abolished its capacity to passively sensitize mice for contact hypersensitivity reactions after DNFB challenge. These results document the existence of an antigen-specific, IgE-mediated, delayed-in-time cutaneous hypersensitivity response that can be elicited by epicutaneous challenge (contract) with a reactive hapten.