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Adult guinea-pigs injected with a small monovalent conjugate of arsanilic acid, from 2 weeks before to the day of immunization, with hapten- conjugate in adjuvant show a profound depression of hapten-specific delayed sensitivity. The same conjugate given 1–2 weeks after immunization is no longer effective. More than 95 per cent of the intravenously administered monovalent conjugate was found to be excreted in 5 days. Passively administered antibody was without effect on the subsequent development of the hapten-specific delayed sensitivity. These observations are consistent with the hypothesis that the non- immunizing intravenous injection of monovalent conjugate is paralysing to precursor cells if given first, but that once these cells are engaged by adjuvant immunization they are no longer repressible but only transiently inhibitable, as long as high concentrations of monovalent conjugate remain in the circulation. [ABSTRACT FROM AUTHOR]

Passive cutaneous anaphylaxis in the guinea pig has been studied with rabbit antibody to a series of antigens of differing molecular weight. The results indicated that at a given antibody level the weight of antigen needed to elicit a reaction increases with its molecular weight. Previous observations have been confirmed that the amount of antigen needed to elicit a reaction at a high level of antibody is less than that required at a lower level. The results suggest that extremely small amounts of small molecular weight antigens might be sufficient to produce anaphylactic symptoms in highly sensitive individuals. [ABSTRACT FROM AUTHOR]

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.

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.

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.

We have used a fractionation procedure involving bovine serum albumin gradient floatation, adherence to glass, and rosetting with antibody-coated sheep erythrocytes to purify an accessory cell fraction from Lewis rat spleens. The fraction which is of light buoyant density, nonadherent, and FcR- is markedly Ia+, lacks phagocytic ability, is nonspecific esterase negative and under scanning electron microscopy has a heterogeneous morphology with a variety of protuberences described for rat dendritic cells. This putative dendritic cell preparation is very effective at stimulating proliferative responses with concanavalin A and allogeneic cells. When used to reconstitute the reactivity of peritoneal exudate cells of rats immunized with azobenzenearsonate-N-acetyl-L-tyrosine (ABA-Tyr) and subsequently depleted of Ia+ cells, it was shown to be highly effective with ABA conjugates of tyrosine, Ficoll, and polystyrene beads. Thus, despite the apparent absence of phagocytic or degradative abilities, this cell was very efficient at presenting large soluble and insoluble antigens. It is suggested that processing may occur at the cell surface without requiring internalization.

The cellular and molecular basis for the difference in ability of BCG to induce tolerance in BALB/c and DBA/2 mice has been examined by in vitro biofiltration. It was found that incubation with the adherent cells from BALB/c but not DBA/2 spleens could remove the material from BGG which inhibited tolerance induction in BALB/c mice. This material was shown to represent only a trace component in BGG, was present in only certain commercial batches of BGG, and was apparently unrelated to the presence of aggregates or endotoxin.

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.

Immunization of Lewis rats with azobenzenearsonate-N-acetyl-L-tyrosine (ABA-tyr) in complete Freund's adjuvant (CFA), produces a hapten-specific helper T cell response measured by an increase in plaque forming cells (PFC) against a different hapten. The response seen is primarily direct (IgM) PFC unless B cells are primed by injection of trinitrophenylated keyhole limpet hemocyanin (TNP-KLH) prior to immunization with ABA-tyr. The response requires both ABA and TNP to be on the same carrier molecule which can be as diverse as bovine serum albumin (BSA), poly L-glutamine-lysine-tyrosine (L-GLT); however, a D-amino acid polypeptide does not work. The in vitro demonstration of such help was successful only with peritoneal exudate lymphocytes, not spleen or lymph node cells. Repeated pretreatment of rats by intraperitoneal injection of ABA-tyr in incomplete Freund's adjuvant (IFA) induced an unresponsiveness for helper activity to subsequent immunization with the same antigen in CFA. Passive transfer of lymphoid cells from spleens and lymph nodes from rats pretreated with ABA-tyr in IFA followed by boosting with ABA-tyr in CFA induced unresponsiveness to subsequent induction of hapten-specific help.