Tsunehisa Amano

Regulation of delayed-type hypersensitivity (DTH) response to hen egg-white lysozyme (HEL).

Delayed-type hypersensitivity (DTH) can be demonstrated in A/J mice immunized with hen egg-white lysozyme (HEL) in complete Freunds adjuvant (CFA) by challenging primed animals in the ear with aqueous HEL. Normal A/J mice receiving soluble HEL derivative peptide, P-Ib, sequence 29-54 and 109-123 linked by a single S-S bond, 7 days prior to immunization with HEL showed much lower DTH response specific to the protein. The inhibition of DTH reactivity is due to active suppression and involves the generation of suppressor T-cells (Ts). Thus, the suppression induced with a single i.v. injection of P-Ib solution was able to be transferred into syngeneic recipients by the spleen cells from mice pretreated with P-Ib. These suppressor cells are T-cells, since their ability to suppress DTH is completely abrogated by treatment wit anti-Thy 1.2 and complement. Amongst HEL derivative peptides tested in the present study, only P-Ib could induce the tolerance.

Induction of idiotype-specific suppressor T-cells with soluble Fv fragment

Abstract Delayed-type hypersensitivity (DTH) responses to the idiotype (Id) of the isologous immunoglobulins (Igs) could be demonstrated in Balb/c mice immunized with M315 or its Fv fragment (Fv-315) in complete Freunds adjuvant (CFA) by challenging primed animals in the ear with aqueous M315. Normal Balb/c mice receiving soluble M315 or Fv-315 seven days prior to sensitization with M315 showed marked suppression of the DTH specific to the Id but not to the other Id. The suppression has been shown to be attributed to active suppression and involves the generation of suppressor T-cells, Thus, the suppression induced with a single i.v. injection of free Fv-315 solution was transferable with both spleen cells and thymocytes from mice pretreated with Fv-315. These suppressor cells are T-cells since their ability to suppress DTH is completely abrogated by treatment with anti-Thy 1.2 serum and complement.

Idiotypic Analysis of Antibodies to Hen Egg-White Lysozyme (HEL): III. Further Studies on the Idiotypic Cross-Reactivity among Anti-HEL Antibodies in Mice

An isolated antibody preparation directed to the native hen egg‐white lysozyme (HEL) from a single A/J mouse (#a‐11), termed IdHELa‐11, was inoculated into rabbits to produce anti‐idiotypic sera (anti‐Id). The antisera were extensively absorbed with normal A/J Ig to render them idiotype specific. Radioimmunoassay utilizing 125IdHELa‐11 and the anti‐Id sera (R103 and R104) was performed to examine the idiotypic cross‐reactivity of the humoral immune response to HEL in various mouse strains and other animal species. Idiotypes shared by IdHELa‐11 were detected in the sera of five mouse strains tested, but not in any of the examined sera of other animal species such as rats, goats, guinea pigs, or sheep, indicating the occurrence of species‐specific cross‐reactive idiotypes (CRI) of antibodies to HEL. Our experiments also suggested the presence of intrastrain CRI. The present experiments, along with our earlier results (15), suggest that idiotypic cross‐reactivity among murine antibodies to HEL appears to be weak. Thus when R103 and R104 were the anti‐Id sera used, the frequency of occurrence of CRI shared by IdHELa‐11 in 5 μg of anti‐HEL antibody in strain A mice was 74 ng and 111 ng; in other strains it was 25–44 ng and 60–98 ng, respectively.

A local antigenic determinant distribution in a continuous antigenic region at residues 38-54 of hen egg white lysozyme.

The precise location of the antigenic determinants in a continuous antigenic region at residues 38–54 of hen egg white lysozyme (lysozyme) was investigated using the inhibition test of binding of Nα-[14C]acetyl fragment 38–54 with goat (three individuals) and sheep (four individuals) anti-lysozyme antisera by various synthetic and proteolytic fragments of lysozyme. From these inhibition studies, we found that in this region there were three independent antigenic determinants, consisting of residues 38–45, 40–48, and 44–54, respectively. The existence and the specificity of the antibodies directed to these determinants were further examined with isolating the specific antibodies by affinity chromatography on columns to which the fragment 38–45, 44–48, and 46–54 were bound. The results indicated that these determinants partially overlapped one another in amino acid sequence, but the antibodies directed to them could recognize only each corresponding determinant. These antibodies were also shown to be reactive with native lysozyme as well as a reduced and S-carboxymethylated derivative of lysozyme, and to be found in goat and sheep anti-lysozyme antibodies. The amounts of these antibodies calculated from the binding capacities were in the range from 0 to 48 μg/ml of antisera. These values corresponded to a small fraction of the total precipitable anti-lysozyme antibodies and were as high as 0.8% of the total. The ratios of the amounts of these antibodies differ in individuals or in different species of animals. The binding affinities of Nα-[14C]acetyl fragment 38–54 with these antibodies were in the range from 1.3 × 107 to 2.6 × 108m−1. The double-reciprocal plots of the antigen binding with these antibodies drew almost a straight line compared with those of a mixture of several antibody populations, that is, whole antisera.

Suppression of Hen Egg-White Lysozyme (HEL)-Specific Delayed-Type Hypersensitivity Responses in Mice by Suppressor T Cells after Neonatal Administration of Anti-Idiotypic Antibodies

Anti‐idiotypic rabbit antiserum (anti‐Id) directed to the idiotypes of anti‐hen egg‐white lysozyme (HEL) antibody from a single C3H mouse (No. 2) was shown to be capable of recognizing only a fraction of the anti‐HEL antibody populations produced by other C3H mice. Experiments were performed to examine the effect of this particular anti‐Id on the delayed‐type hypersensitivity (DTH) response specific for the same protein antigen. A group of 60‐day‐old C3H mice which had been administered anti‐Id within 24 hr after birth were tested for HEL‐DTH response. The results indicated that the DTH response was completely suppressed by the anti‐Id treatment. The inhibition of DTH reactivity is due to active suppression and involves the generation of suppressor T cells. Thus, the suppression induced with a single injection of anti‐Id was transferable with both spleen cells and thymocytes from mice that received anti‐Id. These suppressor cells are T cells since their ability to suppress DTH is completely abrogated by treatment in vitro with anti‐Thy 1.2 serum and complement.