Regulation of the immune response by antibody. II. The effects of different classes of passive guinea pig antibody on humoral and cell-mediated immunity in rats

The ability of different classes of passively administered guinea pig antibody (γ1, γ2, and IgM) to regulate humoral and cell-mediated immunity to flagellin, polymerized flagellin (POL), and sheep red blood cells (SRBC) was investigated in rats. It was found that at high concentrations, all classes of antibody suppressed the primary antibody responses and usually enhanced the delayed-type hypersensitivity induced by the three antigens. With flagellin and SRBC, the different classes of passive antibody varied in their suppressing and enhancing properties, being in the order: γ2 > γ1 = IgM. At low concentrations, γ1 and IgM enhanced the primary antibody response and suppressed the delayed hypersensitivity induced by flagellin. Such an effect was not observed with either POL or SRBC. Priming for a secondary antibody response was less readily suppressed by all classes of passive antibody. The removal of macrophage cytophilic antibody from γ2 converted this antibody to a preparation (γ2 absorbed) which had effects on humoral and cell-mediated immunity approaching that of γ1 antibody.     

T cell tolerance in the chicken. I. Parameters affecting tolerance induction to human gamma-globulin in agammaglobulinemic and normal chickens.

T cell-mediated delayed hypersensitivity (DH) to human gamma-globulin (HGG) can be induced in chickens by subcutaneous injection of the antigen in complete Freund's adjuvant (CFA). In the present work, it has been demonstrated that specific tolerance of the cells mediating this DH can readily be induced in both normal and bursectomized (BX) FP strain chickens by simple i.v. injection of soluble antigen, regardless of the presence of antibody production to the tolerogen. A significant degree of tolerance at the DH and helper T cell levels could be generated in BX birds by injection of as little as 0.5 mg of HGG; such a dose only induced tolerance in normal birds when it had been previously deagregated by ultracentrifugation. Regular, nondeaggregated antigen could produce tolerance in normal animals, but only at doses of greater than 5 mg. The tolerizing injection induced a primary antibody response in normal birds in all cases, but a secondary response could not be obtained in animals rendered tolerant at the T cell level. Establishment of tolerance appeared to be very rapid, and animals remained refractory to induction of DH for at least 3 weeks after the tolerizing injection. The mode in which the antigen was presented to the animals appeared to be crucial in determining whether tolerance or sensitivity would be established.     

Factors affecting production of contrasensitizing antisera in mice.

Using four different protein antigens, two different strains of mice, and various immunization protocols, we have studied production in mice of immunological enhancement antibodies that specifically suppress induction of delayed hypersensitivity. Primary assay of these antibodies was in vivo, because no in vitro test used detected them dependably. Any antigen priming that favored initiation of humoral antibody responses prepared mice to make these contrasensitizing antibodies vigorously following appropriate boosting. The method of boosting usually was more important than that of priming, high titers regularly developing only when primed mice were boosted with much antigen in a short time and were bled a few days later. The presence or absence of delayed hypersensitivity was immaterial. CAF₁ mice made these antibodies better than CF-1 mice, and antigen effectiveness correlated with propensity to induce humoral antibody formation in mice, decreasing from ovalbumin through human serum albumin and bovine serum albumin to methylated human serum albumin. In certain antigenmouse combinations (e.g., ovalbumin in CAF₁ mice) immunosuppressive antibody production was vigorous and prolonged; in others (e.g., bovine serum albumin in CF-1 mice) it was moderate and brief. From our results one can predict what conditions should induce formation of strongly enhancing/contrasensitizing antisera, and speculate that these conditions also should elicit strong, active immunologic tolerance for averting induction of delayed hypersensitivity.     

Chemical separation of helper cell function and delayed hypersensitivity responses

Studies were performed to investigate the effects of the immunosuppressive chemical TCDD. Fetal and neonatal rats were exposed to TCDD through maternal dosing (5 μg/Kg) at Day 18 of gestation and on Days 0, 7, and 14 of postnatal life. Another group of neonatal rats were exposed to TCDD through maternal dosing on Days 0, 7, and 14 of postnatal life only. Parameters of cell-mediated and humoral immune function were investgiated. TCDD suppressed delayed hypersensitivity responses and responses to the mitogens Con A and PHA without affecting humoral immune function. Suppression of T-cell function was selective in that helper function was not suppressed. Transfer of primed T-lymphocytes from TCDD treated and non-treated animals into neonatally thymectomized animals confirmed this. Results indicate that delayed hypersensitivity function and helper function reside in distinct T-cell subsets.     

The in vitro induction of T cells which mediate delayed-type hypersensitivity toward horse red blood cells.

Humoral and cell-mediated immunity to the antigen horse red blood cells (HRBC) were induced in vitro. The type of immune response induced, however, was dependent on the concentration of antigen present in the culture. Whereas intermediate concentrations of HRBC induced antibody-forming cells, high and low concentrations of HRBC induced T cells which, on transfer, mediated delayed-type hypersensitivity reactions. The inverse relationship between humoral and cell-mediated immunity often observed in vivo is, therefore, also evident when lymphocytes are stimulated with antigen in vitro.     

Immunoregulation in experimental interstitial nephritis: immunization with renal tubular antigen in incomplete Freund's adjuvant induces major histocompatibility complex-restricted, OX8+ suppressor T cells which are antigen-specific and inhibit the expression of disease

We utilized a model of experimental interstitial nephritis induced by renal tubular antigen in complete Freund's adjuvant to examine a mechanism of immunologic tolerance produced by priming immunization with tubular antigen in incomplete Freund's adjuvant. Brown Norway rats primed with tubular antigen in incomplete adjuvant do not develop significant nephritis after challenge with antigen in complete adjuvant, and this tolerance can be transferred to naive recipients with donor T cells. These T cells also specifically suppress a delayed-type hypersensitivity response to soluble tubular antigen in recipients immunized to produce disease. This suppression is MHC-restricted and is mediated by OX8+ T cells which bind antigen and bear idiotypes cross-reactive with those on antibodies eluted from the tubular basement membrane. Despite the suppression of histologic disease, tolerized animals were able to produce significant titers of antibodies to tubular basement membrane. Our findings demonstrate an additional strategy for altering the natural history of immune-mediated renal disease, and further refine the characterization of the suppressive effect produced by incomplete Freund's adjuvant.     

Characterization and genetic control of the immune response to synthetic polypeptide antigens of defined geometry.

Both humoral and cell-mediated immune responses to the synthetic helical hapten-carrier conjugate poly-Glu-Tyr-Lys(TNP)-(Glu-Tyr-Ala)5 were found to be linked to the major histocompatibility locus in mice and guinea pigs. The responder mouse strains (H-2d haplotype) showed a primary IgM response with an IgG component appearing after the secondary immunization. The antibody response was accompanied by a positive DTH reaction in responder strains. Nonresponder mice (H-2b or H-2k haplotypes) showed neither IgM nor IgG antibodies and the DTH reaction was negative. Administration of the antigen as a complex with an immunogenic carrier was not effective in inducing a response in nonresponder mice. In guinea pig studies, it was found that strain 2 animals were able to mount an antibody response against the TNP-hapten and a DTH response against the polypeptide backbone. Strain 13 animals gave no anti-TNP antibodies at the lower dose levels and DTH activity was entirely negative for all doses of immunizing antigen. Replacement of the TNP hapten by the arsanilazo dipeptide derivative, BOC-gly-ARA-tyrosine, converted the nonresponder strain 13 guinea pigs into complete responders showing antibody and DTH reactions to both the hapten and the polypeptide backbone.     

Studies on antigen-induced arthritis in mice. II. Immunologic correlates of arthritis susceptibility in mice.

Antigen-induced arthritis was developed in mice as a model of human rheumatoid arthritis by using methylated bovine serum albumin (mBSA) as antigen. It was found that most strains were susceptible, whereas CBA mice were resistant. We therefore investigated the humoral and cell-mediated immune responses to mBSA in resistant mice (CBA) and susceptible mice (exemplified by C57BL) to determine whether these were associated with susceptibility to arthritis. The resistant strain (CBA) differed from the suceptible strains in the following respects. First, there was a lower humoral immune response to mBSA as measured by passive hemagglutination, but this could be overcome by a larger immunogenic dose. Secondly, there were differences in response to low doses of DNP-mBSA after mBSA carrier preimmunization. Thirdly, there were striking differences in delayed-type hypersensitivity (DTH) to mBSA as determined by a radioisotopic assay in vivo; the response of CBA mice occurred early, at 5 days, declined quickly, and was weaker, whereas that of C57BL mice developed later and was long sustained. Genetic studies of the DTH response with hybrids and backcrosses showed an oligogenic control of immune responsiveness, with one gene being linked to the H-2b allele of the susceptible C57BL mice, and another being independent of the H-2 complex. Our findings indicate that in mice, susceptibility to antigen-induced arthritis with mBSA correlates with a higher responder state to this antigen, and that T cells are the major if not the only determinant of the high responder state.     

Studies on delayed hypersensitivity to protein antigen. Induction of delayed hypersensitivity by chemically modified antigen.

it was shown in our previous paper that mice primed with chemically modified bacterial alpha-amylase (BaA), which was neither cross-reactive with anti-BaA antibody nor able to induce a humoral anti-BaA response, developed enhanced responses to a subsequent challenge with native BaA and that the magnitude of the immunological memory was closely related to the priming dose of modified BaA. This paper describes the experimental conditions for induction of delayed hypersensitivity (DH) by modified BaA in relation to the development of immunological memory for antibody response to native BaA. Mice primed with either an intraperitoneal (i.p.) or subcutaneous (s.c.) injection of modified BaA in complete Freunds adjuvant (CFA) developed enhanced anti-BaA as the immunogen and modified BaA as the eliciting antigen, the relationship of anti-BaA responses to a subsequent challenge with BaA. In contrast, when mice were immunized with an s.c. injection of the modified BaA only, a significant level of DH to native BaA could be induced, as measured by the footpad reaction (FPR). The highest degree of DH was observed in mice given 50 micrograms of modified BaA. DH was detectable within 5 days and persisted for 25 days after immunization. In the reciprocal combination of native BaA as the immunogen and modified BaA as the eliciting antigen, the relationship of anti-BaA responses to DH was examined. The primary anti-BaA responses induced by an i.p. injection of large doses of BaA was markedly higher than those induced by an s.c. injection, while DH was exhibited only in mice given s.c. injection of BaA in CFA. With respect to DH to native BaA induced by the modified BaA, it was shown that C3H/He mice were high and C57BL/6 mice were low responders.     

Studies on the in vivo induction and suppression of direct and lectin-dependent, cell-mediated cytotoxicity

Alloimmunization (C57BL/6, anti-P815) can result in the development of cytolytic effector cells capable of mediating direct antigen-specific, cell-mediated cytotoxicity (DCMC) and nonspecific lectin-dependent, cell-mediated cytotoxicity (LDCC). The induction of DCMC appeared to require challenge with large numbers (10⁸) of viable replicating P815 cells, whereas LDCC reactivity was obtained following challenge with high or low (10⁴) numbers of replicating or mitomycin C-treated P815 cells. Other alloantigen preparations, e.g., soluble antigen or membrane preparations, failed to induce DCMC or LDCC. An examination of the effects of high- and low-dose challenge using viable P815 cells demonstrated that high-dose challenge resulted in strong DCMC, LDCC, a readily detectable humoral response, and some delayed-type hypersensitivity, whereas low-dose challenge yielded LDCC, strong delayed-type hypersensitivity, and suppressor cell activity. The development of DCMC was severely suppressed in animals primed with a low-dose P815 challenge and subsequently rechallenged with 10⁸ P815. Further development of LDCC was similarly suppressed. It appears that although both DCMC and LDCC effector cells are susceptible to previously activated suppression, during a primary in vivo response a portion of the LDCC effector cells develop beyond a critical stage before suppression is expressed.     

Type II collagen-induced arthritis in mice. III. Suppression of arthritis by using monoclonal and polyclonal anti-Ia antisera

Pretreatment of mice genetically susceptible to type II collagen-induced arthritis (CIA) with monoclonal or polyclonal antisera specific for I region gene products (Ia antigens) suppressed or delayed the onset of CIA, whereas pretreatment with anti-Ia to an irrelevant haplotype was without effect. The humoral response to type II collagen was transiently depressed 14 days after immunization but antibody levels did not differ significantly after 28 days. The peak delayed-type hypersensitivity to type II collagen was unaffected by anti-Ia treatment. Monoclonal antibody of one anti-Ia specificity enhanced both the antibody response and the arthritis incidence in one mouse strain.     

Nonantibody-mediated suppression of delayed hypersensitivity to human gamma-globulin in mice.

Development of delayed hypersensitivity (DHS) to human γ-globulin (HIgG) in mice was documented by histological analysis, by the kinetics of footpad swelling in animals exhibiting humoral or delayed responses, and by the failure of sera to transfer delayed reactions to normal, syngeneic recipients. Since cyclophosphamide (CY) treatment resulted in diminished humoral and augmented delayed reactions, we used this as a tool to explore the nature of the regulatory mechanisms which affect expression of this type of cell-mediated immunity. In order to evaluate the effect which the presence or absence of antigen-specific cells might exert on expression of DHS, we subjected mice to experimental regimes which would result in lymphocyte proliferation or depletion, respectively (see Bachvaroff, R., and Rapaport, F. T., Cell. Immunol. 15, 336, 1975). Cell proliferation was induced by injection of 80 μg of aqueous antigen on Day ?4; this was followed by sensitization with HIgG-CFA (Freund's adjuvant) on Day 0, and footpad challenge on Day 13. These mice exhibited strong humoral reactivity; three of six died of anaphylaxis following footpad challenge, and the remaining three showed a diminished delayed response. Similarly treated mice that, in addition, received 6 mg of CY 3 days after injection of aqueous antigen and, therefore, would have antigen-specific cells present showed greatly diminished humoral reactivity, due to B-cell depletion. However, they also exhibited a marked diminution in delayed responsiveness. The data clearly demonstrate that a nonantibody-mediated, possibly cell-directed, regulatory influence is exerted on DHS where cell proliferation has occurred. We next examined the impact which the depletion of proliferating cells would exert on the expression of DHS. Cell depletion was attempted by giving one injection of aqueous antigen (Day 0) early in a regime of chronic CY administration (Days ?1 through +3) ; antigen-induced proliferating cells would be susceptible to CY and, therefore, depleted under these conditions. The results show that mice receiving both aqueous antigen and CY have depressed humoral and markedly diminished delayed reactivity compared to animals that were injected with CY alone. Thus, the augmenting effect which CY exerts on DHS is abrogated by stimulation with aqueous antigen. One interpretation is that CY removes a regulatory cell population in the normal animal, thereby allowing enhanced expression of delayed responsiveness. Clearly, regulatory function cannot be attributed solely to bumoral antibody production.     

Induction of delayed-type hypersensitivity with proteins made insoluble by polymerization

Protein antigens, made particulate by polymerization with ethyl chloroformate, were incorporated in Freund's complete adjuvant and used for footpad immunization of rats and guinea pigs. A comparison was made with animals similarly immunized with the native, soluble protein. Two to three weeks after immunization of rats with polymerized bovine serum albumin (Pol-BSA) and up to 8 weeks after immunization of guinea pigs with polymerized diphtheria toxoid, in vivo and in vitro evidence of delayed-type hypersensitivity (DTH) was found without measurable serum antibodies. Ten times more polymerized than soluble BSA was needed to induce comparable levels of DTH. This was not, however, true in the case of serum antibodies, since soluble BSA induced higher titers than the 1000 times larger amount of Pol-BSA. In addition, the titers in polymer-immunized rats were consistently low or under detectable level when followed up to 5 months after priming. These findings encourage the belief that insolubilization of antigens by polymerization guides the immune response toward cell-mediated immunity, whereas antibody formation becomes weaker. However, boosting of polymer-primed animals with soluble antigen resulted in the production of high levels of antibody.     

IL-4 from Th2-type cells suppresses induction of delayed-type hypersensitivity elicited shortly after immunization

The pure delayed-type hypersensitivity reaction obtained in 4-day ovalbumin-sensitized mice after antigen challenge in the footpad was abrogated by transfer of in vitro expanded, antigen-specific lymphoblasts derived from ovalbumin-hyperimmunized donors (high antibody producers), 12 h before immunization. This effect was specific inasmuch as Trypanosoma cruzi-specific blasts derived from Tc-Ag-hyperimmunized mice did not inhibit delayed-type hypersensitivity in ovalbumin-immunized recipients. The ovalbumin-specific blasts displayed a Th2 cytokine profile, secreting IL-4 and IL-10 upon restimulation in vitro with ovalbumin, but not IFN-gamma or IL-2. In addition, recipients of such cells produced much more IgG1 and IgE antibodies. When the frequency of T-cell blasts was enriched among these cells, transfer of four million cells was enough to prevent the induction of delayed-type hypersensitivity. Neutralization of IL-4 alone just before cell transfer not only restored the delayed-type hyper-sensitivity reaction, but also maintained it in a plateau for at least 72 h after challenge. Recipients treated in this way also showed a shift back towards a Th1 phenotype, indicated by the increase in IL-2, IFN-gamma and IL-12 synthesis. No synergistic action was observed when IL-4 and IL-10 were concomitantly neutralized. These results indicate that activation of Ag-specific Th2 cells early in the course of the immune response to a protein antigen provides an immunological environment rich in IL-4, thus leading to the inhibition of cell-mediated immunity.     

Modulation of delayed-type hypersensitivity and acquired cellular resistance by orally administered viable indigenous lactobacilli in Listeria monocytogenes infected Wistar rats

Aims: Various probiotic lactobacilli have been reported to modulate immunity. In this study we investigate the effects of viable indigenous Lactobacillus strains Utr-1, Utr-2 and Utr-3, on T cell-mediated immunological memory responses. METHODS AND RESULTS: In Listeria monocytogenes infected rats it was demonstrated that short-term daily ingestion of Lactobacillus strain Utr-3 significantly decreased delayed-type hypersensitivity (DTH) expression, whereas long-term, daily oral administration of Lactobacillus strain Utr-3 and Lactobacillus strain Utr-2 significantly enhanced acquired cellular resistance (ACR) towards Listeria re-infection. CONCLUSIONS: Our findings demonstrate that certain indigenous Lactobacillus strains are capable of modulating T cell-mediated immunity. SIGNIFICANCE AND IMPACT OF THE STUDY: Our results support the importance of indigenous microflora analysis in probiotic lactobacilli studies.     

Evanescent delayed-type hypersensitivity: mediation by effector cells with a short life span.

Four days after i.v. immunization of mice with optimal low doses of heterologous erythrocytes (2 x 10(5) RBC), strong delayed-type hypersensitivity (DTH) responses can be elicited in the footpad. At later intervals after immunization, DTH responsiveness is progressively diminished and replaced by 4-hr antibody-dependent reactions. These evanescent T cell-mediated DTH responses, which are progressively replaced by antibody-dependent reactions, resemble Jones-Mote type delayed hypersensitivity responses of humans and guinea pigs. Since higher doses of immunizing antigen activate suppressor mechanisms that inhibit DTH responses, we examined the possibility that the evanescence of DTH in mice immunized with an optimal low dose of antigen might also be due to suppression. Using techniques that could clearly demonstrate the suppression produced by high antigen doses, we failed to find evidence for either humoral or cellular suppression in optimally immunized mice with declining of DTH responses. Thus, it appears that the evanescence of produced by optimal low dose immunization with RBC may be due to an intrinsic short life span of the effector cells rather than to the activation of an identifiable shut-off mechanism.     

Ocular immune responses. I. Priming of A/J mice in the anterior chamber with azobenzenearsonate-derivatized cells induces second-order-like suppressor T cells

We studied the cellular immune responses to ocular anterior chamber (AC) priming of mice. A/J mice primed subcutaneously with azobenzenearsonate-coupled spleen cells (ABA-SC) manifested delayed-type hypersensitivity (DTH) in the form of footpad swelling when challenged 5 days later with the diazonium salt of ABA. Mice inoculated with ABA-SC in the anterior chamber at the time of subcutaneous priming, however, were tolerant to ABA. Subconjunctival inoculation with ABA-SC did not tolerize; rather it primed for DTH. Antibodies against ABA were not detectable in significant amounts in mice made tolerant by AC inoculation. The AC-induced tolerance was shown to result from hapten-specific T cell-mediated suppression. Suppressor T cells (Ts) arising from AC priming suppressed the efferent limb of the immune response and did not bear detectable cross-reactive idiotype (CRI) surface receptors. In these phenotypic and functional respects, AC-induced Ts differed from first-order Ts (Ts1) that result from i.v. priming. The results are discussed with respect to immune privilege and the anterior chamber of the eye.     

Dim light at night increases immune function in Nile grass rats, a diurnal rodent

With the widespread adoption of electrical lighting during the 20th century, human and nonhuman animals became exposed to high levels of light at night for the first time in evolutionary history. This divergence from the natural environment may have significant implications for certain ecological niches because of the important influence light exerts on the circadian system. For example, circadian disruption and nighttime light exposure are linked to changes in immune function. The majority of studies investigating the effects of light exposure and circadian disruption on the immune system use nocturnal rodents. In diurnal species, many hormones and immune parameters vary with secretion patterns 180° out of phase to those of nocturnal rodents. Thus, the authors investigated the effects of nighttime light exposure on immunocompetence in diurnal Nile grass rats (Arvicanthis niloticus). Rats were housed in either standard 14-h light (L):10-h dark (D) cycles with L ～150 lux and D 0 lux or dim light at night (dLAN) cycles of LD 14:10 with L ～150 lux and D 5 lux for 3 wks, then tested for plasma bactericidal capacity, as well as humoral and cell-mediated immune responses. Rats exposed to dLAN showed increased delayed-type hypersensitivity pinna swelling, which is consistent with enhanced cell-mediated immune function. dLAN rats similarly showed increased antibody production following inoculation with keyhole lymphocyte hemocyanin (KLH) and increased bactericidal capacity. Daytime corticosterone concentrations were elevated in grass rats exposed to nighttime dim light, which may have influenced immunological measures. Overall, these results indicate nighttime light affects immune parameters in a diurnal rodent.     

Mechanisms of suppressed cell-mediated immunity and impaired antigen-induced interleukin 2 production in granuloma-bearing mice

Pulmonary granulomas were induced in BALB/c mice immunized with methylated bovine serum albumin in complete Freund's adjuvant by the intratracheal injection of plain agarose beads or beads conjugated to specific antigen. Large hypersensitivity granulomas developed around antigen-coupled beads in immunized animals. Smaller but still prominent granulomatous reactions developed around plain beads in immunized mice. In nonimmunized animals, both plain and antigen conjugated beads produced very small granulomas. Granuloma formation in sensitized animals was associated with suppressed delayed-type hypersensitivity reactions induced by the footpad injection of specific and nonspecific antigens. Lymph node cells from sensitized granuloma-bearing mice with cutaneous anergy showed suppressed specific and nonspecific antigen-induced proliferative responses in vitro. These cells also showed suppressed interleukin 2 production in response to specific antigen. Although no soluble suppressive factor was detected in granuloma extracts, suppressor cells were found in lymph nodes of granuloma-bearing mice, which could inhibit antigen-induced production of interleukin 2 by lymph node cells from immunized mice. Antigen-specific immunoglobulin G antibody production was not suppressed in immunized granuloma-bearing mice. Previous studies from our laboratory have demonstrated migration inhibition factor and interleukin 1 activities in aqueous extracts prepared from granuloma-bearing lungs of immunized mice. These results and the findings reported here indicate that granuloma formation and the associated anergy observed in this system are primarily expressions of cell-mediated immunity; selective suppression of in vivo and in vitro expressions of cell-mediated immunity in granuloma-bearing mice may be due to impaired antigen-induced interleukin 2 production; and such impairment is caused by suppressor cells.     

Antigen-specific CD8+ T cells switch the immune response induced by antigen from an IgG to a cell-mediated mode.

We have developed an in vivo/in vitro immunization procedure with xenogeneic RBC as Ag that results in the generation of a lymphoid population that expresses potent delayed-type hypersensitivity (DTH) and produces little, if any, antibody. This lymphoid population contains Ag-specific CD8+ T cells that can inhibit the induction of a strong IgG response. These CD8+ T cells are shown to not only inhibit the antibody response in an Ag-specific manner but allow the Ag to induce cells of the target population to express DTH. Furthermore, the Ag-specific inhibition of the antibody response and the Ag-specific enhancement of the induction of DTH appear to be coordinately regulated, as the same number of CD8+ T cells cells is required to achieve both effects. Thus these CD8+ T cells are shown to switch the response induced by Ag from a humoral to a cell-mediated mode. These regulatory characteristics are consistent with a physiologic role for these cells of ensuring the absence of antibody production during a strong, cell-mediated response.