Transmission of hapten-specific tolerance to an unrelated carrier

The principle of linked recognition is well defined in response and suppression. Yet, to our knowledge, it is not explored in the context of tolerance. To investigate, whether the status of tolerance toward a hapten (TNP) can be transferred to a subsequently introduced carrier, animals which were tolerized by a subimmunogenic dose of hapten (TNP) coupled to syngeneic monoclonal anti-TNP IgG, with the rationale of combining the phenomena of low zone tolerance and syngeneic IgG-induced suppression, were challenged with TNP-horse red blood cells (HRBC). Conjugates of high density (40 mM) TNP-syngeneic IgG (TNP40-IgG) were immunogenic and after challenge with TNP-HRBC, animals responded to TNP and to HRBC. Yet, spleen cells (SC) of mice injected with TNP2.5-IgG and challenged with TNP-HRBC were tolerant against TNP as well as the carrier. Limiting dilution (LD) analysis revealed that subimmunogenic doses of TNP coupled to IgG resulted in diminished activation of help, failure to activate contrasuppressor T cells (TCS), and significantly augmented activation of suppressor T cells (TS). On the other hand, after challenge with TNP-HRBC, activation/expansion of carrier-specific helper (TH), suppressor, and contrasuppressor T cells were not affected by previous immunization with subimmunogenic or immunogenic doses of TNP-IgG conjugates, but HRBC-specific TCS could not interact with TNP-specific TS. Hence, to initiate tolerance it was necessary (and sufficient) that an activated and expanded TS population was not counterregulated by TCS. In this situation, an established status of dominance of suppression for the epitope TNP could not be disrupted by an immunogene carrying a multitude of new epitopes; i.e., tolerization by subimmunogenic doses of the individual epitope TNP resulted in unresponsiveness against any immunogen carrying this epitope.     

Phenotypic restriction of antigen-binding specificity on immunized amphibian spleen cells.

Antigen-binding studies have been used to determine the specificity of immunized splenocytes of adult representatives in three groups of Amphibia, the American common newt, Triturus viridescens, the South African clawed toad, Xenopus laevis, and the American leopard frog, Rana pipiens. At question is the basis for organismal response diversity available to lower vertebrates. In vivo immunization with horse erythrocytes (HRBC) followed by trinitrophenylated (TNP) HRBC was effected. Dissociated splenic lymphocytes capable of binding TNP-HRBC and HRBC were counted after preincubation with glycine or TNP-glycine. Since TNP-glycine blocked Triturus immunocytes from binding TNP-HRBC and HRBC equally, while only binding to TNP-HRBC was blocked in the other two species, we have concluded that immunized cells from Triturus, but not Xenopus or Rana, appear to bear surface receptor molecules capable of binding TNP and a determinant of HRBC.     

Ontogeny, Phytogeny, and Cellular Cooperation

The capacity of adult newt (Triturus viridescens) spleen cellsto secrete antibody at 4 C allows simultaneous visualizationand quantification of non-secretory (S^–) and secretory(S⁺) rosette forming cells (RFC). Visualization of mammalianS⁺ RFC requires 37 C, a temperature at which S^– RFC appearto be fragile. RFC can be distinguished as S^– or S⁺ dueto whether one or more layers of erythrocytes adhere to thesurface of sensitized spleen cells. Different doses of horseerythrocytes (HRBC) affect newt S^– RFC and S⁺ RFC differentially.By varying the time between injections of different concentrationsof chicken erythrocytes (CRBC, the "carrier") and a constantdosage of CRBC-TNP (trinitrophenyl, the hapten) it is possibleto measure "helper" activity that correlates with the populationof S^– RFC and is both dose and time dependent. By varyingassay time after "helper" activity has been maximized, one candetermine the cytodynamics of anti-TNP antibody producing cell(APC) activity. For the first time these morphologically separableRFC can be related to their suspected physiologic behavior.A shift from S^– RFC to S⁺ RFC takes place during the immuneresponse. That similar dose-dependent response curves can beshown in adultRana pipiens suggests that the newt responsesrepresent a fundamental vertebrate pattern.     

Immune Response against Hamster Erythrocytes in the Low-Responder Mouse Strains

C57BL/6 and AKR mice were treated with hamster erythrocytes (HRBC) in complete Freund's adjuvant (CFA) or incomplete Freund's adjuvant (IFA) and the development of delayed hypersensitivity and antibody production were examined. 1) Delayed hypersensitivity against HRBC antigen, as determined by the peritoneal macrophage disappearance test, was detected in mice sensitized with HRBC in CFA but not in those sensitized with HRBC in IFA. 2) Antibody production against HRBC or hapten TNP after a booster injection of HRBC or trinitrophenylated HRBC (TNP-HRBC) in saline was enhanced by pretreatment with HRBC in CFA or IFA. 3) Delayed hypersensitivity was not detectable after a booster sensitization with HRBC in CFA in mice which had been pretreated with HRBC in IFA 2 weeks earlier. In the mice treated with both HRBC in IFA (day ?21) and in CFA (day ?7), however, an enhanced antibody production against HRBC or TNP was detected after an intravenous injection with HRBC or TNP-HRBC in saline (day 0). These results suggest that sensitized effector lymphocytes in delayed hypersensitivity and helper cells in antibody production may be derived from the same pool of unprimed T cells. The pool of unprimed T cells with a capacity to differentiate into either type of primed T cells may be exhausted after pretreatment with the antigen in IFA, and the primed helper T cells may not be able to differentiate into sensitized lymphocytes even after sensitization with the antigen in CFA, which favors development of delayed hypersensitivity in normal controls.     

Immune Response against Hamster Erythrocytes in the Low-Responder Mouse Strains

The production of anti-hapten antibody after immunization with trinitrophenylated (TNP) hamster erythrocytes (HRBC) or sheep erythrocytes (SRBC) was determined in high- and low-responder mouse strains against HRBC antigen. 1) Anti-TNP antibody was detected in sera of high-responder DDD and CF1 mice after primary immunization with TNP-HRBC, but not in those of low-responder C57BL/6 mice. 2) Anti-TNP antibody was detectable in sera of all the strains after primary immunization with TNP-SRBC. 3) Production of anti-TNP antibody was elicited after a booster injection of TNP-HRBC in low-responder C57BL/6 mice pre-sensitized with HRBC in Freund's complete adjuvant. These results suggest that functions of thymus-derived cells specific for HRBC antigen are deficient in low-responder mice.     

Potentiation of antibody responses by specific IgM: specificity and thymus dependency

Modulation of antibody responses induced by IgM directed against the immunogen was investigated. When IgM directed against ox erythrocytes (ORBC) was given together with trinitrophenyl (TNP)-ORBC, the subsequent antibody response to the carrier, ORBC, as well as the response to the hapten, TNP, was potentiated. In contrast, IgG with carrier specificity inhibited both responses. The hapten-specific potentiation was found in both direct and indirect plaques, and was antigen-dose dependent, i.e., no potentiation was found with the lowest antigen doses. The response to 2,4-dinitrophenyl (DNP)-labeled proteins was potentiated by a monoclonal IgM with specificity for the hapten. The effects were observed both in primary and secondary responses. One strict requirement for IgM potentiation to occur was observed. The determinant against which potentiation was achieved had to be physically linked to the determinant against which the IgM was directed, be it hapten or carrier determinants. Thus, irrelevant IgM-antigen complexes were incapable of potentiating the responses. Similar specificity requirements were found for IgG induced suppression of antibody responses. Experiments with nude mice and their euthymic littermates showed that IgM potentiation of antibody production is T-cell dependent. Furthermore, passive transfer of carrier-primed spleen cells together with antigen challenge suggests that IgM potentiation of secondary antibody responses is dependent on specific carrier-primed immune T cells.     

Augmentation of Suppressed Antibody Responses in Mice During Experimental Chagas'Disease by T Helper Cells Activated in a Time-Dependent Mode of Immunization

ABSTRACT. Mice infected with the protozoan parasite Trypanosoma cruzi , the causative agent of human Chagas'disease, develop immunosuppressed responses to heterologous antigens. Experiments were performed using infected mice in the acute stage of infection to assess immunoregulatory activities during induction of direct plaque-forming cells (DPFC) to sheep erythrocytes (SRBC), hapten-conjugated SRBC (TNP-SRBC), and horse erythrocytes (TNP-HRBC). Studies in vivo demonstrated that anti-SRBC responses were best enhanced when T. cruz -infected mice were injected with primed T cells derived from normal or infected mice immunized four days previously. The presence of enhancing capacities for DPFC responses by T cells from T. cruzi -infected mice were also supported by experiments examining the hapten-carrier effect. Preimmunization of infected mice with SRBC or HRBC four days before injection of hapten-homologous (TNP-SRBC or TNP-HRBC) carrier resulted in markedly augmented anti-hapten antibody responses. These results show that functional help provided by T cells activated during priming and exposed to a challenge dose of antigen (SRBC) in a time-dependent mode can overcome the effect of immunosuppression in T. cruzi -infected mice.     

Phylogenetic origins of immune recognition: Lymphoid heterogeneity and the hapten/carrier effect in the goldfish, Carassius auratus

These studies were designed to determine whether the cellular response of the goldfish, Carassius auratus, to heterologous erythrocytes is comparable in complexity and shares similar recognition mechanisms with those of mammals. Immunocytoadherence has been used to monitor the anti-horse erythrocyte (HRBC) response. The levels and kinetics of antigen-binding activity in the thymus, head nephros, and spleen were established following a single high (25%) or low (0.0025%) HRBC challenge. Rabbit anti-goldfish IgM was found to inhibit antigen binding of thymocytes, as well as other immunocytes. Hapten-carrier immunization was used to explore lymphoid heterogeneity in this species. The anti-hapten (2, 4, 6-trinitrophenyl) response was found to be specifically enhanced by carrier priming. The helper function of carrier-stimulated cells was short lived and greatest when low-dose carrier priming was used. The proportion of low-dose, carrier-stimulated thymus and head nephros antigen-binding cells was substantially enriched by passage through a nylon-wool column. However, previously carrier-primed and hapten-stimulated secretory antigen-binding cells were depleted by comparable column separation. The results of hapten-carrier immunization in combination with column separation are indicative of lymphoid heterogeneity for this species. They suggest that cell-cell collaboration is a general and essential part of the vertebrate immune response. Antigen-specific recognition of both lymphoid subsets seems likely to be mediated by surface immunoglobulin, since anti-IgM inhibits antigen binding of thymus and head nephros immunocytes.     

Effects of BCG (Bacillus Calmette–Guérin) Vaccines on Immune Responses in Mice

The effects of killed and living BCG on antibody production against hamster erythrocytes (HRBC) and the 2, 4, 6-trinitrophenyl (TNP) group were studied in SL mice. Killed and living BCG, each in doses of 0.008 mg, 0.08 mg, 0.8 mg and 8 mg per mouse, were intravenously inoculated 7 days prior to primary immunization with HRBC. Secondary immunization was carried out 28 days later with TNP-HRBC. Anti-HRBC and anti-TNP antibodies were estimated by a hemagglutination test. The results showed that pretreatment with killed or living BCG enhanced the antibody production against both HRBC and TNP. Comparing the effects of these two BCG preparations, it was noted that killed BCG augmented the anti-HRBC antibody production more effectively than living BCG. In regard to the anti-TNP antibody production, living BCG exhibited a greater augmenting effect than killed BCG. This difference in the modes of action of killed and living BCG was remarkable when two groups given 8 mg of killed and living BCG were compared. In addition, it was shown that living BCG at a dose as high as 8 mg was able to augment the anti-TNP antibody production, even in the absence of preceding immunization with HRBC.     

Effects of BCG (Bacillus Calmette-Guérin) vaccines on immune responses in mice. I. Possible effect of BCG on helper T cells.

The effects of killed and living BCG on antibody production against hamster erythrocytes (HRBC) and the 2, 4, 6-trinitrophenyl (TNP) group were studied in SL mice. Killed and living BCG, each in doses of 0.008 mg, 0.08 mg, 0.8 mg and 8 mg per mouse, were intravenously inoculated 7 days prior to primary immunization with HRBC. Secondary immunization was carried out 28 days later with TNP-HRBC. Anti-HRBC and anti-TNP antibodies were estimated by a hemagglutination test. The results showed that pretreatment with killed or living BCG enhanced the antibody production against both HRBC and TNP. Comparing the effects of these two BCG preparations, it was noted that killed BCG augmented the anti-HRBC antibody production more effectively than living BCG. In regard to the anti-TNP antibody production, living BCG exhibited a greater augmenting effect than killed BCG. This difference in the modes of action of killed and living BCG was remarkable when two groups given 8 mg of killed and living BCG were compared. In addition, it was shown that living BCG at a dose as high as 8 mg was able to augment the anti-TNP antibody production, even in the absence of preceding immunization with HRBC.     

Hapten-IgG-induced suppression of the in vitro antibody response: role of hapten density and of antigenic challenge.

We have studied the suppression of the in vitro antibody response of mouse spleen cells to the trinitrophenyl (TNP) hapten by conjugates of TNP-human IgG (TNP-HGG). Normal mouse spleen cells were incubated with conjugates of HGG and of HGG fragments, with different hapten densities. They were then challenged with either a T-dependent or a T-independent antigen. Highly substituted (12 mol of TNP/mol of HGG) conjugates induced a dose-dependent suppression, apparent after short-term incubation at 4 °C, of both T-dependent and T-independent responses. Conjugates of Fab′₂ and Fab′ were as suppressive as conjugates of IgG, whereas conjugates of albumin, aggregated IgG, and β2 microglobulin lacked suppressive activity. In contrast, the lightly substituted conjugates TNP₈-HGG and TNP₂-Fab′₂ induced a time-dependent suppression, affecting only the T-dependent response. This suggests that the suppressive effect of hapten-IgG conjugates is mediated by two different mechanisms according to the density of hapten on the IgG carrier. When these conjugates are used as tolerogens in the in vivo situation, both mechanisms would operate to a variable extent, and this could account for the remarkable tolerogenic properties of hapten-IgG conjugates.     

Augmentation of suppressed antibody responses in mice during experimental Chagas' disease by T helper cells activated in a time-dependent mode of immunization

Mice infected with the protozoan parasite Trypanosoma cruzi, the causative agent of human Chagas' disease, develop immunosuppressed responses to heterologous antigens. Experiments were performed using infected mice in the acute stage of infection to assess immunoregulatory activities during induction of direct plaque-forming cells (DPFC) to sheep erythrocytes (SRBC), hapten-conjugated SRBC (TNP-SRBC), and horse erythrocytes (TNP-HRBC). Studies in vivo demonstrated that anti-SRBC responses were best enhanced when T. cruzi-infected mice were injected with primed T cells derived from normal or infected mice immunized four days previously. The presence of enhancing capacities for DPFC responses by T cells from T. cruzi-infected mice were also supported by experiments examining the hapten-carrier effect. Preimmunization of infected mice with SRBC or HRBC four days before injection of hapten-homologous (TNP-SRBC or TNP-HRBC) carrier resulted in markedly augmented anti-hapten antibody responses. These results show that functional help provided by T cells activated during priming and exposed to a challenge dose of antigen (SRBC) in a time-dependent mode can overcome the effect of immunosuppression in T. cruzi-infected mice.     

Trinitrobenzene sulfonic acid effects in two amphibian model systems

The induction of hapten-specific tolerance was investigated in two amphibia, Notophthalmus viridescens and Xenopus laevis. Responses to trinitrophenylated (TNP)-Ficoll and TNP-lipopolysaccharide (LPS), as well as to horse erythrocytes (HRBC) were examined in both species, following an intraperitoneal injection of 2,4,6-trinitrobenzenesulfonic acid (TNBS). The less evolutionarily advanced newt, Notophthalmus, failed to respond to all three immunogens after TNBS administration. While Xenopus became completely tolerant upon challenge with TNP-Ficoll and partially tolerant with TNP-LPS, full capacity to respond to HRBC was retained. Therefore, specific tolerance was induced in Xenopus, but not in Notophthalmus. The tolerance with TNP-Ficoll in the toad, Xenopus, was short lived and return to responsiveness appeared to be related inversely to levels of TNP protein in the sera of TNBS-treated animals. The thymic dependence of this tolerance could not be determined, because adult thymectomy (ATx) abrogated the response to TNP-Ficoll in control nontolerized animals. Responses to TNP-LPS and HRBC were unaffected by ATx. These data, in conjunction with TNBS-induced differential tolerance to the TNP moiety, suggest carrier-dependent hapten-specific B-cell heterogeneity in the toad which differs in certain ways from that recently described for murine systems.     

ABA-specific helper T cells in A/J mice bear the major cross-reactive idiotype

Attempts were made to induce azobenzenearsonate (ABA)-specific helper cell responses in A/J mice. These were measured by an increase in TNP plaque-forming cells following administration of the double hapten conjugate ABA-bovine serum albumin-TNP. Immunization with ABA coupled homologous immunoglobulin or spleen cells produced ABA-specific help only when the same carrier was used to boost. Hapten-specific help was achieved by two injections of ABA-N-acetyltyrosine in complete Freund's adjuvant 5 weeks apart. This help was passively transferable by T cells as shown by its elimination with anti-Thy 1.2 serum and complement treatment. The presence of the major ABA cross-reactive idiotype (CRI) on these T helper cells could be similarly shown by the elimination of help when the cells were treated with rabbit anti-CRI antibody and complement prior to passive transfer. The same treatment did not effect ABA-specific helper activity of CBA/J mice.     

Production of auto-anti-idiotypic antibody during the normal immune response. XII. Enhanced auto-anti-idiotypic antibody production as a mechanism for apparent B-cell tolerance in rabbits after feeding antigen

Rabbits fed trinitrophenylated bovine serum albumin (TNP-BSA) generated fewer anti-TNP plaque-forming cells but greater numbers of hapten (TNP)-augmentable IgM and IgG PFC following immunization with TNP-Ficoll or TNP-Brucella abortus than did animals not previously fed antigen. Spleen and mesenteric and bronchial lymph nodes were similarly affected. In addition more auto-anti-idiotype (Id) antibody (anti-anti-TNP) was eluted by hapten from spleen cells of antigen-fed rabbits than from spleen cells of control rabbits not prefed antigen. Gel filtration studies ruled out the possibility that the Id binding activity in the eluates was due to immune complexes. The isotype of the anti-Id was IgG except in one rabbit where it was IgM. The results are consistent with the interpretation that the production of auto-anti-Id antibody is one of the factors responsible for the specific depression of the IgM and IgG immune responses which follows antigen feeding. In contrast the antigen feeding resulted in priming for an IgA anti-TNP response without detectable hapten-augmentable IgA PFC.     

Allergic contact dermatitis induced by intratracheal administration of hapten

It is known that the skin is the natural route for induction of allergic contact dermatitis (ACD). Because the lung is another potential portal of entry for sensitizing chemicals, studies were performed to evaluate immune responses to haptens deposited in the lung. As a result, a new method for inducing ACD was developed. Intratracheal (IT) inoculation of 2,4,6-trinitrobenzene sulfonic acid (TNBS) led to maximal ear swelling 24 hr after challenge on the ear with 2,4,6-trinitrochlorobenzene (TNCB) in carrier. This response was specific for immunizing hapten. Furthermore, it was equally possible to induce ACD by intratracheal inoculation of trinitrophenyl- (TNP) modified bronchoalveolar cells (BAC) or haptenated spleen cells. Adoptive transfer studies demonstrated that the hypersensitivity that resulted from IT inoculation of TNBS or TNP BAC could be transferred with cells. With a monoclonal anti-hamster Ia reagent, it was demonstrated that, like Langerhans cells, 80 to 90% of cells in the bronchial lavage fluid expressed Ia determinants on their membrane. These cells were morphologically indistinguishable from macrophages. Because an individual is capable of being sensitized when hapten is introduced by the pulmonary or epicutaneous routes, the possibility is raised that the alveolar macrophages in the lung possess a similar capability for antigen presentation of hapten in the induction of ACD as does the Langerhans cell.     

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.     

Antigen recognition in delayed-type hypersensitivity.

It is still uncertain if cell-mediated immune reactions are more or less specific than antibody-mediated reactions. Accordingly, hapten and carrier specificity were examined in delayed hypersensitivity in guinea pigs. Hapten specificity was demonstrated with 2,4-dinitrophenyl (DNP)-guinea pig albumin (GPA), 2,6-DNP-GPA, 2,4,6-trinitrophenyl (TNP)-GPA, and dansyl (DNS)-GPA. Guinea pigs immunized with each of these conjugates were tested 7 days later with the immunogen and the other conjugates. Strong delayed skin responses were highly specific for the immunogen; there were some weak cross-reactions among the nitrophenyl conjugates, no crossre-actions between the DNS and nitrophenyl conjugates, and no responses to unconjugated GPA. Conjugates carrying different numbers (1–45) of 2,4-DNP groups per molecule were all able to elicit specific responses to 2,4-DNP.Carrier specificity in delayed hypersensitivity was confirmed by immunizing with 2,4-DNP-GPA, and challenging with the immunogen, with 2,4-DNP coupled to bovine albumin (BSA), rabbit IgG, ovalbumin, and hemocyanin. Strong responses were seen to the immunogen, a weak response to 2,4-DNP-BSA, and no response to the other conjugates. Specific immune recognition of both hapten and carrier determinants is therefore required for expression of delayed hypersensitivity. These cell-mediated reactions thus appear to be more specific than those of antibody-mediated reactions in solution.     

Epitope-specific regulation. IV. In vitro studies with suppressor T cells induced by carrier/hapten-carrier immunization

Sequential immunization with a carrier molecule and a new epitope (hapten) conjugated to the carrier (carrier/hapten-carrier immunization) induces specific suppression for IgG antibody production to the new epitope (hapten) on the carrier. Once induced, this "epitope-specific" suppression persists and specifically suppresses subsequent in vivo IgG antibody responses to the hapten presented on the same or on an unrelated carrier molecule. In vitro studies presented here characterize the surface markers and specificity of suppressor T cells generated in carrier/hapten-carrier-immunized animals. Thus we show (1) that spleen cells from these donors suppress in vitro IgG anti-hapten antibody production by cocultured hapten-primed spleen cells; (2) that some but not all of the suppressor cells carry surface Lyt-2; (3) that at least some of the suppressor cells have receptors for the inducing hapten (DNP); and (4) that, unlike the suppression obtained in vivo, the in vitro suppression extends to IgG responses to unrelated carrier protein epitopes presented in association with the inducing hapten.     

Defective helper T-cell activity in LPS-unresponsive C3H/HeJ mice

C3H/HeJ mice, unresponsive to LPS, exhibit a defective ability to mount antibody responses to T-dependent immunogens. The anti-TNP antibody response to TNP-HRBC, a T-dependent immunogen, was found to be lower in these mice as compared to LPS-responsive C3H/HeN mice, whereas the anti-TNP antibody response to TNP-Ficoll, a T-independent immunogen, was of the same magnitude in C3H/HeJ and C3H/HeN mice. An impaired helper activity of C3H/HeJ HRBC-primed spleen cells was demonstrated in a titration assay in which graded numbers of C3H/HeJ or C3H/HeN HRBC-primed spleen cells were added to cultures containing a constant number of unprimed spleen cells from either C3H/HeJ or C3H/HeN mice and the immunogen TNP-HRBC. The reduced helper T-cell activity of C3H/HeJ HRBC-primed spleen cells appears to be independent of macrophage defects, since C3H/HeJ and C3H/HeN macrophages were found equally effective in antigen presentation as evaluated by an in vitro antigen-specific T-cell proliferation assay. The difference in helper T-cell activity between these two substrains probably reflects a lower number and/or proliferation rate of antigen-responsive T cells in C3H/HeJ mice.