The role of immunogenicity in the induction of tolerance with conjugates of arsanilic acid.

A number of azobenzenearsonate (ABA) conjugates have been prepared and tested for ability to react with antibody, to sensitize for hapten-specific delayed hypersensitivity and to induce hapten-specific unresponsiveness. All conjugates tested by in vitro or in vivo methods show a capacity to react with preformed antibody. Conjugates of L-amino acid polymers are immunogenic and induce tolerance. Conjugates of D-amino acid polymers or conjugates with high density of ABA groups are nonimmunogenic and fail to induce tolerance. Since paired D- and L-polymer conjugates react comparably with preformed antibody but differ markedly in tolerance induction, it is argued that combination with an antibody-like receptor molecule on the surface of an immune-competent cell is not a sufficient condition for tolerance. The lack of effectiveness of sterically crowded conjugates as well as D-polymer conjugates argues for a preliminary biologic "processing" of antigen necessary for induction of immunity or tolerance. Such a processing event might well involve enzymatic attack on the antigen.

Genetic control of cutaneous basophil hypersensitivity.

CBH to DNP-GL could be elicited only in responder guinea pigs which possess the genetic ability to develop classic delayed type hypersensitivity to DNP-PLL, the response to which is governed by the same gene. Since the defect in nonresponder animals seems to reside at the level of their T cells and not B cells, these results lend support to the contention that CBH, as well as DH, is dependent on and probably mediated by T cells.

Cutaneous basophil hypersensitivity. I. A new look at the Jones-Mote reaction, general characteristics.

Jones-Mote reactivity, defined as a delayed-type skin reaction, occurs transiently early in the course of immunization with protein antigens or hapten conjugates with or without the adjuvant effect of tubercle bacilli. The skin reaction is typically a flat, well-circumscribed erythema with little induration beginning at about 6 hr, reaching a peak at 18-24 hr, and fading or gone at 48 hr. Immunogenic carrier requirements for hapten-specific Jones-Mote hypersitivity resemble those of antibody production rather than of classic delayed hypersensitivity. Skin test antigen requirements indicate that the Jones-Mote reaction involves an active stimulatory response rather than combination with preformed antibody, since ABA conjugates of nonimmunogenic D-polymers do not work. Studies with ALS and carrageenan suggest that the lymphocyte is an important contributor to the reaction, but the macrophage is not. Because the reactions studied here are operationally different from those described by Jones and Mote and because they have a characteristic histology, the term "cutaneous basophil hypersensitivity" is proposed.

Immunochemical study of antigenic specificity in delayed hypersensitivity. V. Immunization with monovalent low molecular weight conjugates.

Hapten-specific delayed hypersensitivity was produced by immunization of guinea pigs with arsanilic acid conjugated to N-acetyltyrosine or other small aromatic molecules. Such hapten-specific delayed sensitivity could be passively transferred by peritoneal exudate cells. While a conjugate made from a polymer of D-amino acids was ineffective in producing sensitization, the conjugate made with D-tyrosine was effective, suggesting that the inability of D-amino acid polymers to be broken down by enzymes might be bypassed by use of the monomer. The effectiveness of such monomers in producing delayed sensitivity, but not antibody production, is consistent with a hypothesis that different types of antigenic determinants are involved in the production of each.

Cutaneous basophil hypersensitivity. II. A light and electron microscopic description.

Delayed onset erythematous skin reactions elicited in guinea pigs early in the course of sensitization with azobenzenearsonate-protein conjugates or with protein antigens in incomplete Freund's adjuvant or in saline were found to have a characteristic morphology which sets them apart from delayed hypersensitivity and the classic antibody mediated reactions. The principle feature was massive dermal infiltration with basophilic leukocytes. Mononuclear cells of several types including activated and small lymphocytes, monocytes, macrophages, and blast cells were also present. Such reactions have in the past been designated Jones-Mote hypersensitivity, but we prefer the descriptive term cutaneous basophil hypersensitivity (CBH) for the reasons given. Occasional basophils extruded their granules, and individual granules, retaining their characteristic ultrastructure, were commonly seen in the interstitium. However, intercellular junctions between endothelial cells were closed except during cell emigration and there was no morphologic evidence of an histamine-like effect. The majority of basophils, moreover, did not degranulate but underwent nuclear pyknosis and cytoplasmic degeneration and were phagocytosed by macrophages. Phagocytosed basophil granules retained their ultrastructure. Skin tests performed at late intervals after sensitization had a different time course and morphology. Animals sensitized with protein antigens in complete Freund's adjuvant developed delayed hypersensitivity; however, reactions elicited in such animals at early (but not late) intervals after sensitization contained a prominent basophil component. We interpret such reactions to be a mixture of delayed hypersensitivity and cutaneous basophil hypersensitivity. The function of the basophil in CBH and its relation to the mononuclear cells which accompany it are unknown, and various possibilities are discussed. We conclude that cutaneous basophil hypersensitivity is a distinct immunologic and morphologic entity, occurring early in the course of sensitization with protein antigens incorporated in any of several vehicles. The mechanism of the reaction is presently unknown, and a general hypothesis to explain its pathogenesis has been proposed.

Mechanism of delayed reactions.

Arsanilic acid conjugates of polymers of L-typrosine, glutamic acid, and alanine are immunogenic and can elicit hapten-specific, delayed-hypersensitivity reactions in sensitized guinea pigs. Conjugates of the D-amino acid polymers are neither immunogenic nor capable of eliciting delayed reactions. Mixtures of small amounts of conjugates capable of eliciting a delayed reaction with larger amounts of D-amino acid polymer conjugates produce only small delayed reactions. I suggest that the delayed reaction is an active response requiring the continued participation of immunogenic material in sensitized animals; it is not the reaction of preformed antibody-like material with the antigenic determinant.

Structural requirements for T-cell recognition of tyrosine-azobenzene-arsonate in the rat and mouse.

The ability of various analogues of tyrosine-azobenzenearsonate (ABA-tyr) to elicit responses in CBA mice and Lewis rats was studied in order to determine the essential features for association with Ia molecules on accessory cells and T-cell recognition. Exquisite specificity for the AsO3H2 group was found in the rat while substantial cross-reactivity was seen in the mouse when other acidic but not neutral groups were substituted. The azo linkage was found to be essential for specificity as was the phenolic ring of tyrosine. Reaction was found to be less specific for changes in the COOH group than the NH2 group of the tyrosine moiety indicating the associations of this end with Ia molecules was dependant on both charge and hydrophilic interactions and differed also between rat and mouse cells. It was concluded that an antigen's reaction with T-cells is affected by an epitope, determining specificity, an agretope, determining association with an appropriate Ia molecule, and an ability to be processed down to these minimal essential structures.

The presentation of L-tyrosine-azobenzenearsonate by different mouse Ia molecules uses a common agretope.

The T cell response to L-tyrosine-azobenzenearsonate (ABA-tyr) has been studied using T cell lines and clones derived from three different mouse strains, B10.BR, B10.A (5R) and C57B1/6. In all cases, the arsonate group in conjunction with the amino group of tyrosine formed the functional T cell epitope. Molecules without any one or both of these groups are non-stimulatory. The hydrophobic moiety consisting of the azo-linked benzene rings forms the agretope of the molecule, as is evident from competitive inhibition of T cell stimulation by non-stimulatory analogues lacking the epitope. Substitutions on the benzene ring at ortho or meta positions resulted in decreases in ability to compete, indicating the likelihood of steric inhibition of binding of the agretope with the Ia molecule. This pattern was observed for clones and lines restricted by IAk, IAb and IEb/k MHC class II molecules. Peptides from lambda repressor protein, P84-98 and P73-88, showed haplotype specificity in their ability to inhibit ABA-tyr-induced proliferation of T cell clones, BRTC-4 and B6TC, respectively. The binding constants of ABA-tyr analogues were considered to be comparable to those of lambda repressor peptides because equimolar concentrations resulted in similar levels of competition. A cluster of aromatic amino acids on the floor of most MHC class II molecule binding sites might provide strong hydrophobic interaction with azo-linked benzene rings of ABA-tyr, thus accounting for its immunogenicity in all strains of mice studied.

Competition among class II major histocompatibility molecules for presentation of tyrosine-azobenzenearsonate occurs in vivo and in vitro.

We have compared by functional assays the relative preference among Ia molecules for their ability to present tyrosine-azobenzenearsonate (ABA-Tyr) to T cells. Immunization of B10.BR mice (IAk, IEk) with ABA-Tyr resulted in the induction of IAk-restricted T cells only. Immunization of B10.A(5R) mice (IAb, IEb/k) gave only IEb/k-restricted T cell clones even though IAb-restricted responses could be induced in C57BL/6 mice (IAb). These results indicated that IAk was preferred over IEk and IEb/k was preferred over IAb for presentation of ABA-Tyr. A comparison between IAk and IEb/k made by immunizing [B10.BR x B10.A(5R)]F1 mice (IAk, IEk, IAb, IEb/k), showed that IEb/k was favored over IAk. No IAb- or IEk-restricted response was seen. Further attempts were made to compare Ia preference for ABA-Tyr presentation by competitive inhibition assays. It could be shown that the presence of IEb/k molecules on an accessory cell interfered with the ability of IAb molecules on the same cell to present ABA-Tyr to an IAb-restricted T cell clone by direct competition. Such a competition was not observed between IAk and IEk. Finally, it could be shown that addition of ABA-Tyr inhibited the presentation of moth cytochrome-c peptide (81-103) by IEb/k but did not influence its presentation by IEk. From these functional studies we suggest that the binding affinity of ABA-Tyr with the Ia molecules will fall in the order: IEb/k greater than IAk greater than IAb greater than IEk.

Hapten-specific T cell response to azobenzenearsonate-N-acetyl-L-tyrosine in the Lewis rat. I. Induction and suppression of delayed-type hypersensitivity and in vitro proliferative responses.

Pretreatment of Lewis rats with a single i.p. injection of ABA-N-acetyl-tyrosine in incomplete Freund's adjuvant induced an unresponsiveness for delayed-type hypersensitivity to subsequent immunization with the same antigen in complete Freund's adjuvant. Complete suppression of in vitro antigen-induced proliferative responses required repeated pretreatment. Passive transfer of lymphoid cells from spleen and lymph nodes but not sera from suppressed rats induced unresponsiveness of hapten-specific T cell functions. Nylon wool-nonadherent cells and cells panned on F(ab')2 of rabbit anti-Lewis rat Ig plates suppressed the induction of DTH and in vitro antigen-stimulated proliferation. Adult thymectomy increased DTH and failed to abolish the induction of suppression.

The azobenzenearsonate conjugate of poly-glutamic-lysine-tyrosine will induce tyrosine-azobenzenearsonate-specific T-cell responses and clones in nonresponder mice.

Mice of the H-2b haplotype are low responders to ABA-tyr. However, when they were immunized with ABA coupled to poly-GLT15 for which they are nonresponders, they developed strong proliferative responses to ABA-tyr in draining lymph node cells. Clones derived from these cells were highly reactive to ABA-tyr although the original mice were not. No evidence was found to indicate that suppression played a role in the failure to respond to ABA-tyr. Characterization of two clones showed an absolute specificity for the arsonic acid group and the Azo linkage. Alterations in the terminal amino acid residues produced varying changes in reactivity which could not be ascribed unequivocally to an effect on epitope or agretope.

Immune response in guinea pigs to two different lipid conjugates of bovine serum albumin.

A lipid conjugate of BSA was made by coupling dodecylamine to the COOH groups of BSA (DA-BSA). Studies on the immunogenicity of this material and one made by coupling dodecanoic anhydride to NH2 groups (D-BSA) demonstrated that cell-mediated immunity could preferentially be generated by lipid conjugation to an antigen as measured by delayed skin reaction, invitro blast transformation, and antibody formation. DA-BSA was found superior by both in vivo and in vitro tests. Both the conjugates retained the ability to suppress delayed type hypersensitivity, but only DA-BSA retained the ability to elicit optimum skin reaction in a sensitized animal and to precipitate anti-BSA antibody.

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.

The inhibition of cutaneous basophil hypersensitivity reactions by a heterologous anti-guinea pig T cell serum.

Systemic treatment with a heterologous anti-T cell serum of guinea pigs immunized with EA in IFA markedly suppressed CBH reactivity to specific antigen and T cell mitogens, as judged by gross reactivity, histology, and skin histamine. The antiserum produced a marked drop in circulating lymphocytes, mainly at the expense of T cells, as indicated by the ability of surviving lymphocytes to rosette with rabbit RBC. It was postulated that the suppression of CBH reactivity is due to the depletion of T cells, which would have released a factor chemotactic for basophils. The data therefore provide further evidence that cutaneous reactions rich in basophils are primarily dependent on a population of T cells.

An intracellular pathway is required for ABA-tyrosine presentation to T cells.

Although tyrosine-azobenzenearsonate (ABA-Tyr) is not degraded by proteolytic enzymes, its presentation by accessory cells is inhibited by lysosomotropic agents such as chloroquine. Presentation of ABA-poly-L-glutamic, alanine, tyrosine (ABA-GAT) is similarly inhibited by chloroquine, but in contrast to ABA-Tyr it is also inhibited by leupeptin. Finally formaldehyde fixation of accessory cells after pulsing with ABA-Tyr but not before permits successful stimulation of ABA-specific hybridoma cells. These results suggest that a lysosomal pathway but not digestion is necessary for the association of ABA-Tyr and la molecules for presentation.

Hapten-specific T cell response to azobenzenearsonate-N-acetyl-L-tyrosine in the Lewis rat. III. Effects of peptide-spacer structure on eliciting ABA-specific helper activity with TNP-haptened ABA-peptide-Ficoll.

A series of antigens was synthesized in which peptide spacers were inserted between the ABA group and TNP-Ficoll. When these antigens were used to assess helper activity in Lewis rats immunized with ABA-tyr, it was found that an increase in the ABA-epitope density and an increase in the peptide spacer size both increased the efficacy of the antigen in eliciting ABA-specific help, manifest by an enhancement of anti-TNP PFC. Substitution of D- for L-amino acids progressively decreased the ability of these conjugates to elicit help until D-tyr-D-ala-D-ala, which when used for coupling ABA to the TNP-Ficoll resulted in a nonimmunogenic molecule. When these same Ficoll conjugates were used to study ABA-specific in vitro proliferative responses, it was found that introduction of even a single D-amino acid into the spacer greatly reduced reactivity. By contrast the ABA-peptides, free of the Ficoll backbone, were all equivalent on a molar basis in their ability to elicit ABA-specific in vitro proliferation, regardless of their content of D-amino acids. These results suggest some form of digestion is required in the processing of these antigens before they can elicit helper activity. This processing can occur only if one or more L-amino acid residues are present. If the ABA-peptides are free of the Ficoll backbone, they are all capable of stimulating T cell proliferation without apparent further processing.

ABA-specific responses are I region restricted by the carriers used for immunization.

Immunization of mice with ABA coupled to carriers to which they are nonresponders gives rise to ABA-specific proliferative responses in lymph node cells. When C3H/HeN and CBA/J nonresponder mice are immunized with ABA on (T,G)-A-L (an I-A-restricted carrier in responder mice), the responses to ABA-tyr and ABA coupled to a variety of unrelated carriers are solely I-A restricted as determined by inhibition with anti-IA and anti-I-E sera. When ABA on GLT (an I-E-restricted carrier in responder mice) is used for immunization, the responses are both I-A and I-E restricted. Thus, ABA-specific responses in nonresponder mice appear in part to be restricted by the carrier used for immunization. B10.S mice, lacking functional I-E molecules, channel their ABA-specific responses entirely through I-A when immunized with ABA-GLT. These results support the hypothesis that the failure in nonresponders lies in a functional deficit in the T cell repertoire rather than an inability of accessory cells to present antigen.