Nobuo Sakato

The His-probe method: Effects of histidine residues introduced into the complementarity-determining regions of antibodies on antigen-antibody interactions at different pH values

We examined the effects or histidine residues that were artificially introduced into complementarity‐determining regions of antibodies on antigen‐antibody Interactions at different pH values. Using a monoclonal antibody specific ror hen egg‐white lysozyme and three mutant antibodies that contained a histidine residue. we measured binding constants for antibodies and lysozyme at different pH values (PH 5–8). No gross conformational changes were evident over this range of pH vulues, as determined by analysis of the spectra of circular dichroism. Since the charge on a histidine residue is the most likely factor that can vary over this range of pH values, differences on pH‐dependent antigen‐binding patterns observed between the wild‐type and mutant antibodies should be due mainly to the effects of the charges on the histidine residues. The three mutant antibodies showed different and characteristic patterns of pH‐dependent binding to lysozyme, which depended on the location of the artificially introduced histidine residues.

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.

Suppression of the delayed-type hypersensitivity response to hen egg white lysozyme (HEL) by HEL peptides in a genetically high-responder mouse strain: Evidence for requirement of the loop structure for induction of suppressor T cells

The delayed-type hypersensitivity (DTH) response of C3H/HeN mice to hen egg white lysozyme (HEL) can be blocked by a single iv injection of a solution of HEL in buffered saline 7 days before sensitization of animals with HEL in complete Freunds adjuvant (CFA). The minimal structure of HEL required for the suppression was examined by determining the abilities of various HEL-derivative peptides to inhibit HEL-DTH. Treatment of normal mice with Ploop I X II, sequence 57-107 (Cys64-Cys80, Cys76-Cys94), or P17 (sequences 1-27 and 123-129 linked by Cys6-Cys127) 7 days before immunization with HEL resulted in marked suppression of the DTH response. This inhibition of DTH involved generation of suppressor T cells (Ts). The results suggested that two suppressor pathways are involved. These data, together with another recent finding (1) that an entirely different portion of HEL is a suppressor determinant (SD) in A/J mice, indicate that different epitopes act as SDs in different strains of mice. Of the loop region peptides tested, Plc (intact loop I joined to a linear peptide, residues 84-97) was found to be the minimum structure capable of suppressing the HEL-DTH response; loop I or II alone did not cause suppression. Activation of Ts cells by the loop peptide depended on its conformational structure; completely reduced and carboxymethylated Ploop I X II did not cause suppression.

Three Antigenic Regions in p17 of Human Immunodeficiency Virus Type 1 (HIV-1) Revealed by Mouse Monoclonal Antibodies and Human Antibodies in HIV-1 Carrier Sera

We investigated the murine antibody response to recombinant p17 (rp17) of human immunodeficiency virus type 1 (HIV‐1) and the human antibody response directed to p17 in HIV‐1 infection. Three large peptides covering residues 12‐29, 53‐87 and 87‐115 of p17 were synthesized. The cysteine residues 57 and 87 of peptide 53‐87 were reoxidized to form a disulfide bridge. Eighteen out of 19 murine monoclonal anti‐rp17 antibodies had relatively high affinities (KA = 1.9 × 105−1.4 × 108 M−1) with one of the 3 p17 peptides in the liquid phase. Each monoclonal antibody reacted only with one particular peptide and had no reactivity with the other 2 p17 peptides. All the monoclonal antibodies reacted with rp17 in the liquid phase with a reasonable degree of affinity (KA = 2.0 × 105−1.8 × 107 M−1). Four HIV‐1 carrier sera, which were positive in ELISA using rp17 as the antigen, reacted positively in an ELISA using 3 p17 peptides which were used to titrate murine monoclonal antibodies. Murine monoclonal antibodies having specificity for the 3 p17 peptides stained live HIV‐1‐infected cells by means of indirect membrane immunofluorescence, irrespective of their specificity. This suggests that the various portions of p17 (at least 3 regions of p17) were exposed on the surface of live infected cells, probably as short polypeptide chains.

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.

Characterization of the recognition specificity of autoreactive, polyspecific monoclonal antibodies obtained from spleen cells of parasite-infected BALB/c mice

We examined the specificity of four monoclonal antibodies (MoAbs) obtained from Schistosoma japonicum (1D, GAO3), Trypanosoma cruzi (TSLO), and Trichinella spiralis (TSY2) infected BALB/c mice. All four MoAbs reacted not only with autologous parasite antigens but also with various heterologous parasite antigens and normal tissues. The antigen recognition pattern seen on Western blots was almost identical in 1D and GAO3, and in TSLO and TSY2. Furthermore, certain bands were identical among all four MoAbs. The mechanisms responsible for these phenomena are discussed.

Recognition of the Self Idiotype by T Cells: Induction of a Rapid Increase in Cytoplasmic Free Calcium in T Cells Recognizing a Variable L Chain Determinant

To investigate the initial stages of recognition of the self idiotype (Id) by T cells, we examined the early increase in cytoplasmic free calcium ([Ca2+]i) occurring in murine CD4+ T cells specific for a model Id, Id315, following their interaction with the Id. The changes in [Ca2+]i were monitored with stopped‐flow fluorometry bv loading T cells with fura 2, a Ca2+‐binding fluorescent dye. An increase of [Ca2+]i in the Id‐specific T cell line was dependent on the presence of both antigen‐presenting cells (APC) and Id315. When T cells were mixed with APC pulsed with M315 for 90 min at 37 C, a significant increase in T cell [Ca2+]i was observed within one second. A pronounced elevation in [Ca2+]i was also observed in T cells after their interaction with APC which had been pulsed for 90 min with VL‐315 Id‐containing proteins (such as VL‐315, L315, FV‐315 or Fab′‐315 fragments). In contrast, pulsing APC for 5 min with the VL fragment produced little or no change in the [Ca2+]i. These results suggest that VL must be further processed by APC before it can be recognized by T cells. Indeed, a synthetic VL region peptide (positions 91–108, designated as P18) produced an elevation in T cell [Ca2+]i when mixed with APC without pulsing.

Monoclonal Antibodies to O4 Antigen of Vibrio parahaemolyticus

Four monoclonal antibodies were prepared against O4 antigen of Vibrio parahaemolyticus. All the antibodies were shown to be specific for O4 antigen by agglutination with heat‐killed O‐cells of the organism and precipitation with LPS preparations. The inhibition experiments of the precipitations with various sugars and oligosaccharides suggested that the combining sites of these hybridoma antibodies were directed to an antigenic determinant structure containing →3 and →6 linked d‐glucose, d‐galactose, and N‐acetyl‐d‐galactosamine.

VHDJH TO JH JOINING IS NOT BLOCKED IN μ-CHAIN-PRODUCING PRE-B CELLS, IMPLYING THE BREAKDOWN OF ALLELIC EXCLUSION

Intracytoplasmic μ‐positive pre‐B cells (μ+VH315− cells) transformed with Abelson virus continuously produced cells (μ+VH315+ cells) that were double‐stained by anti‐μ and anti‐VH315 antibodies which specifically recognized the immunoglobulin heavy chain variable region identical or closely related to that of MOPC315 myeloma protein. Southern blot analysis indicated that the μ+VH315+ cells were generated from the μ+VH315‐ cells by the VH315·D·JH2 to a germline JH3 joining on the non‐productive VH315·D·JH2 allele, which resulted in the production of μ‐chains with variable region detectable by anti‐VH315 antibodies. Therefore, it is strongly indicated that VHDJH to JH joining is not blocked in μ‐chain‐producing pre‐B cells, and thus is free from allelic exclusion machinery.