Biomedical subjects

A Nisonoff

Publications and source records attributed to A Nisonoff.

At least 37 records · Page 2Linked to original sources

PubMed1989

Three-dimensional structure of Fab R19.9, a monoclonal murine antibody specific for the p-azobenzenearsonate group.

The crystal structure of Fab R19.9, derived from an anti-p-azobenzenearsonate monoclonal antibody, has been determined and refined to 2.8-A resolution by x-ray crystallographic techniques. Monoclonal antibody R19.9 (IgG2b kappa) shares some idiotopes with a major idiotype (CRIA) associated with A/J anti-p-azobenzenearsonate antibodies. The amino acid sequences of the variable (V) parts of the heavy (VH) and light (VL) polypeptide chains of monoclonal antibody R19.9 were determined through nucleotide sequencing of their mRNAs. The VL region is very similar to that of CRIA-positive anti-p-azobenzenearsonate antibodies as is VH, except for its third complementarity-determining region, which is three amino acids longer; it makes a loop, unique to R19.9, that protrudes into the solvent. A large number of tyrosine residues in the complementarity-determining region of VH and VL, with their side chains pointing towards the solvent, may have an important function in antigen binding.

Amino Acid Sequence↗

PubMed1989

Isotype switching by a microinjected mu immunoglobulin heavy chain gene in transgenic mice.

Immunization of transgenic mice carrying an immunoglobulin mu heavy chain resulted in a response dominated by expression of the transgene variable region. Unexpectedly, in a large proportion of the antibody produced by immunized mice, the transgene variable region was associated with IgG rather than IgM. This demonstrates that the transgene can undergo an isotype switch. Four transgenic founder lines all exhibited transgene isotype switching despite the likelihood of random chromosomal integration of the transgene. In addition one of the lines was analyzed by breeding studies and the transgene was found to be genetically unlinked to the immunoglobulin heavy chain (Igh) locus. These results indicate that a precise chromosomal location is not required for isotype switching and suggest the possibility that the isotype switching process can occur interchromosomally.

Animals↗

PubMed1988

Naturally occurring cytotoxic T lymphocyte precursors with specificity for an Ig idiotype.

The humoral response to the p-azobenzenearsonate hapten in the A/J mouse includes the major cross-reactive idiotype associated with anti-p-azobenzenearsonate (CRIA) found in all immunized mice. Limiting dilution cultures of non-immunized spleen cells of A/J mice with irradiated B hybridoma cells bearing the Ig idiotype, CRIA, in the presence of T cell growth factors developed cytotoxic activity against the CRIA-bearing hybridoma; in some wells this activity was completely abrogated by an anti-idiotype mAb specific for CRIA or by a univalent hapten antigen, tyrosine-p-azobenzenearsonate, indicating the existence of cytotoxic T cell precursors (CTL-P) specific for one or more idiotopes of CRIA in normal spleen cells. The CTL clones lysed targets in a H-2D-restricted manner and were cytotoxic for CRIA-bearing hybridoma lines, but not for CRIA-non-bearing, IgG1k-bearing hybridoma lines. These CTL-P were detected at a high frequency (1/4,500 to 1/10,000) in a spleen cell population of non-immunized, relatively aged A/J mice (16 to 30 wk of age), and at a lower frequency in spleen cells of younger A/J mice (8 wk of age). However, they were not detected in normal spleen cells of B10.A (CRIA-non-producer) mice at any age (less than 1/6 x 10(5)). Normal Ighd-congenic C.AL-20 mice (16 wk of age), that are CRIA producers had as a high frequency of the CTL-P as did A/J mice, whereas normal Ighb-congenic C.B-20 mice (CRIA-non-producers) had none. In the spleen cells of the CRIA-producers, cytotoxicity of the CTL-P developed only in cultures with small numbers of seeding cells. They were completely absent in cultures with greater numbers of cells; this may be due to the presence of suppressor cells of lower frequency but greater potency. In lymph node cells or PBL of relatively aged A/J mice, the CTL-P were also detected, but only in cultures containing higher cell numbers, and at low frequency (between 1/5 x 10(5) to 1/2 x 10(6)). In thymocytes of 8-wk-old A/J mice, they were occasionally detected at very low frequency (less than or equal to 1/1 x 10(6)), but were not present in the bone marrow cells at any age. These results demonstrate the high incidence of the generation of CTL-P specific for an autologous Ag, and indicate that CRIA on B cells may induce CTL specific for CRIA. However, the development of CTL-P may be inhibited by co-existent suppressor cells under normal conditions.

Animals↗

PubMed1988

Immunological responsiveness of neonatal A/J mice to isotypic determinants of syngeneic IgE.

We have previously shown that adult A/J mice produce high titers of anti-IgE with isotypic or idiotypic specificities in response to challenge with a conjugate of KLH with syngeneic monoclonal IgE. Thus, B cells that can synthesize anti-IgE are present in the mice. Adult mice are unresponsive to unconjugated IgE in CFA, suggesting that tolerance exists at the level of T cells. The present study shows that neonatal mice produce anti-IgE antibodies in response to unconjugated IgE in CFA, but that this capacity is lost after the age of 2-3 wk. The loss of responsiveness corresponds closely with the appearance of detectable IgE in serum, suggesting that the IgE may induce tolerance. The affinities of anti-IgE antibodies produced by neonatal mice fall in the range of values obtained with KLH-IgE in adult mice. Tolerance to unconjugated IgE in CFA can be induced in neonatal mice by administration of IgE in saline. In addition, the tolerant state can be induced by adoptive transfer of spleen cells from adult mice. The time-dependent acquisition of tolerance provides a useful model for studying mechanisms of tolerance and autoimmunity.

Aging↗

PubMed1988

Induction and properties of syngeneic murine anti-immunoglobulin D.

High titers of autoantibodies directed to isotypic determinants of IgD were produced by inoculation of syngeneic monoclonal IgD, conjugated covalently to keyhole limpet hemocyanin, into adult or neonatal inbred mice. Anti-idiotypic antibodies were induced at the same time. The average affinity of the mouse antibodies (Ka approximately equal to 10(7) M-1) is similar to that of rabbit anti-IgD and of syngeneic anti-IgE induced by the same procedure. Results indicate that B cells of the mice are not tolerant to serum IgD and that tolerance is maintained at the level of T cells. Direct interaction of the syngeneic anti-IgD with cell-surface IgD was minimal, and there was no convincing evidence that cell-surface IgD was down-regulated in the anti-IgD-producing mice. Further studies, preferably employing monoclonal anti-IgD, are required to determine whether epitopes on cell-surface IgD can be recognized by syngeneic anti-IgD. The ability to generate in vivo high titers of anti-IgD should facilitate the production of such monoclonal antibodies.

Animals↗

PubMed1987

Production of syngeneic autoreactive monoclonal antibodies specific for isotypic determinants of IgE.

Methods are described for the production of syngeneic mouse anti-IgE monoclonal antibodies (mAb). Hybridomas were prepared by using spleen cells from mice immunized with a conjugate of keyhole limpet hemocyanin with monoclonal IgE. Serum titers varied from approximately 40 to 1000 micrograms/ml. The anti-IgE mAb were isolated by affinity chromatography on columns containing immobilized monoclonal IgE. The mAb are specific for isotypic determinants of IgE and do not react with other immunoglobulin isotypes. One of the mAb, which has a high affinity for IgE (Ka = 4.7 X 10(8) M-1), should be useful for studies of regulation of IgE. The applicability of the mAb to quantitative assays for IgE was demonstrated.

Animals↗

PubMed1987

Characteristics of two idiotypic subfamilies of murine anti-p-azobenzenearsonate antibodies.

A family of antibodies bearing a common or cross-reactive idiotype, termed CRIC, predominates in the response of most BALB/c mice to the p-azobenzenearsonate (Ar) hapten, but represents a minor component of the anti-Ar response of most A/J mice. Previous results have suggested that the VH region of CRIC is encoded by two different germ-line genes in both strains. We have determined extensive mRNA sequences for VH and VL, developed specific idiotypic reagents and measured affinities for two subfamilies of CRIC, designated CRIC1 and CRIC2. Both were found to be minor components of A/J anti-Ar antibodies, and CRIC1, but not CRIC2, is a major component of the BALB/c response. The two subfamilies utilize different VH germ-line genes but the same, or nearly identical V kappa genes. The VH nucleotide sequences of CRIC1 and CRIC2 exhibit approximately 90% homology. The D regions of both families are short (one or two amino acid residues) and some can be accounted for on the basis of known JH sequences alone. Affinity differences may account for the dominance of CRIA over CRIC1 and CRIC2 in A/J mice, but results obtained with allotype-congenic mice indicate that background (non-V region) genes are also important in controlling levels of expression of the CRIC1 idiotype. Our data suggest that the A/J germline VH gene that gives rise to the CRIC2 family of antibodies may be identical with a previously sequenced BALB/c germ-line VH gene. On the basis of these and earlier data it is suggested that extensive differences between inbred strains of mice in their complements of VH genes do not result from the accumulation of many mutations in these genes. An alternative possibility is that the differences arise from deletions and/or duplications of VH genes.

Amino Acid Sequence↗

PubMed1987

Induction of high titers of anti-IgE by immunization of inbred mice with syngeneic IgE.

We have generated high titers (up to 1 mg/ml) of antibodies to isotypic determinants of IgE by immunization of A/J mice with syngeneic monoclonal IgE conjugated to keyhole limpet hemocyanin. As much as 3 mg of anti-idiotypic antibodies per ml was induced at the same time. In contrast to conventional rheumatoid factors, the anti-isotypic antibodies are of moderately high affinity (10(7)-10(8) M-1). Assays of the anti-IgE antisera indicated the presence of IgE, both free and in the form of immune complexes; the latter values are minimum estimates owing to masking of isotypic determinants. Regulatory effects of these high titers of anti-IgE can now be investigated. Such studies will be facilitated by the availability of monoclonal, syngeneic anti-IgE antibodies.

Animals↗

PubMed1987

Identity of the V kappa 10-Ars-A gene segments of the A/J and BALB/c strains.

To characterize the light chain gene segments involved in the murine immune response to keyhole limpet hemocyanin p-azophenylarsonate (Ars), we have determined the amino acid and/or nucleotide sequences of several anti-arsonate antibodies of the Ars-A family in the A/J, C.AL-20, and BALB/c strains. These structures have been compared to certain BALB/c anti-phenyloxazolone and anti-levan antibodies previously sequenced and to the A/J V kappa 10-Ars-A genomic sequence (where V kappa = kappa chain variable). These primary structural studies were complemented by Southern filter hybridization analyses utilizing V kappa and kappa chain joining (J kappa) molecular probes. We found a surprising uniformity of structure among these antibody light chains derived from different murine strains. Thus, in contrast to the heavy chain variable (Vh) regions of the Ars-A antibody family where the BALB/c strain lacks the VH gene segment utilized in the A/J Ars-A response, the light chain variable region gene segments at the V kappa 10-Ars-A locus appear to be identical between the two strains.

Alleles↗

PubMed1987

Enhancement and suppression of an intrastrain cross-reactive idiotype.

The effects of a copolymer of monoclonal anti-idiotype (7B7.10) with keyhole limpet haemocyanin (KLH), designated 7-K, on an ongoing immune response were investigated. It was found that the response could be diverted to the production of higher titres of anti-p-azobenzenearsonate (Ar) antibodies, of which nearly 100% carry an intrastrain cross-reactive idiotype (CRIA). The effect was observed only in mice that had received a pre-inoculation of KLH-Ar, or KLH plus bovine gamma globulin-Ar (BGG-Ar). The effect was also observed, however, when cross-linked 7B7.10 was mixed, rather than conjugated with KLH, suggesting that the role of KLH was to induce the production of a B-cell growth factor. Cross-linked 7B7.10 was not effective in the absence of KLH. A primary inoculation of 7-K together with KLH-Ar did not result in significant suppression or enhancement of CRIA. Also, pre-inoculation of 7-K alone did not suppress a subsequent idiotypic response to KLH-Ar, whereas monomeric anti-Id was suppressive. This supports a possible role for the unmodified Fc segment in the suppressive mechanism. In mice primed with KLH-Ar, before administration of 7-K, CRI+A molecules lacking anti-Ar activity were present in very low concentrations in the immune sera. Larger quantities of such molecules were present in the sera of mice that received 7-K alone. The methods described permit the reproducible production of large amounts of CRI+A anti-Ar antibodies.

Animals↗

PubMed1986

Inhibition of an IgE response by secondary B cells of a different isotype.

We found that the synthesis of IgE anti-Ars antibodies is strongly inhibited by the presence of secondary non-IgE-producing cells that are specific for the Ars hapten. Such B cells can be induced by inoculation of a protein-Ars conjugate in CFA. The effect is seen after inoculation of OVA-Ars in CFA followed by KLH-Ars in alum, or, more convincingly, after adoptive transfer of B cells induced by antigen in CFA. Dose-response data indicated that inhibition can be effected by B cells containing noninhibitory numbers of contaminating T cells. Possible synergistic effects of carrier-specific regulatory T cells were ruled out by using a different protein carrier for immunization of donor and recipient mice. The effect was shown to be specific for the hapten used for immunization of donor mice.

Animals↗

PubMed1986

Primary structure of IgE monoclonal antibodies expressing an intrastrain crossreactive idiotype.

We have obtained amino acid sequences (by mRNA and amino acid sequencing) for two IgE kappa mAb that have specificity for the Ars hapten group and are related to the major idiotypic family, CRIA (crossreactive idiotype A), in the A strain of mouse. One mAb, SE20.2, fully expresses CRIA; the other, SE1.3, possesses some but not all of the characteristic idiotopes. Both IgE proteins contain VH and V kappa segments that are closely related to those associated with CRIA. The D segment of SE20.2 is also typical of CRIA+ mAb, but that of SE1.3 is one amino acid residue longer. Chain recombination experiments indicated that the L chain of SE1.3 is fully capable of supporting CRIA expression. Its deficiency with respect to idiotopes of CRIA was attributed to the extra amino acid in the D region and/or substitutions in the VH segment. A major objective was to ascertain the frequency of somatic mutations in IgE. For the VH segment (amino acids 1-98) of SE20.2, there are only three nucleotide differences and one uncertainty with respect to the nucleotide sequence of the germline gene associated with CRIA. A somewhat higher frequency of substitutions is present in the VH segment of SE1.3. The VK amino acid sequences of the IgE proteins are nearly identical to those of a prototype of the CRIA family, mAb R16.7. The results are discussed with reference to the mechanism of the IgM to IgE switch.

Amino Acid Sequence↗

PubMed1986

Influence of helper T cells on the expression of a murine intrastrain crossreactive idiotype.

The requirement for idiotype-specific helper T (Th) cells in the generation of a major intrastrain crossreactive idiotype was investigated. This idiotype, designated CRIA, is associated with a large proportion of anti-p-azobenzenearsonate (anti-Ar) antibodies in A/J mice. Secondary in vitro responses were studied. Using carrier-primed heterogeneous Th-cell populations, it was found that CRIA expression is determined by the mouse strain that provides the responding B cells and is independent of the strain of the Th cells functioning in vitro. Thus, A/J or A.BY (Ighe) B-plus-accessory-cell populations, primed in vivo to keyhole limpet hemocyanin-Ar (KLH-Ar), generated CRIA-dominant responses in vitro in the presence of KLH-Ar regardless of whether the KLH-primed Th cells were derived from CRIA+ strains (A/J or A.BY, Ighe) or CRIA- strains (B10.A or C57BL/10, Ighb). Further, when major histocompatibility complex-restricted, KLH-specific Th-cell clones were used, the CRIA dominance of the Ar-specific responses was again determined by the strain providing B plus accessory cells. Similar levels of expression of CRIA in Ar-specific antibodies were generated in the presence of heterogeneous or cloned Th cells. The results suggest that there is no absolute requirement for idiotype-specific Th cells in generating an Ar-specific secondary antibody response in vitro.

Animals↗

PubMed1986

Relationship of VH and VL genes encoding three idiotypic families of anti-p-azobenzenearsonate antibodies.

We describe here an intrastrain, cross-reactive idiotype (CRI), CRID, associated with anti-p-azobenzenearsonate antibodies of the A/J strain of mouse and distinguishable, by some but not by all of its idiotopes, from the major anti-p-azobenzenearsonate idiotype (CRIA). Molecules carrying the CRID idiotype have heavy chain variable-segment sequences that are identical or nearly identical to that of the germ-line-encoded heavy chain variable sequence of CRIA. Their light chain variable sequences are very similar to those present in a third idiotypic family, CRIC, that is a minor CRI in the A/J strain but a major CRI in BALB/c. This appears to represent a form of combinational diversity, in which the heavy and light chain variable region genes of two unrelated idiotypic families interact to form a third family, all involving antibodies of the same antigen-binding (anti-p-azobenzenearsonate) specificity. The D region of CRID, in the six monoclonal representatives studied (three IgMs, three IgGs), is unusual in that it comprises a single amino acid--arginine or serine; there are eight amino acids in the D region of CRI+A antibodies. Three different heavy chain joining regions, JH1, JH2, and JH3, are utilized. A serological reagent was developed that identifies CRID; it was used to show that the idiotype is present in relatively high concentration, comparable to that of CRIA, in anti-p-azobenzenearsonate antibodies taken soon after primary immunization. In serum taken later CRIA greatly predominates, but CRID persists at low levels.

Animals↗

PubMed1985

Quantitation of IgE antibodies by radioimmunoassay in the presence of high concentrations of non-IgE antibodies of the same specificity.

Radioimmunoassays for mouse IgE antibodies, based on adherence to an antigen-coated surface, are precise and sensitive, but errors can be introduced by the presence of relatively high concentrations of non-IgE antibodies of the same specificity. Such errors are caused by competition for the limited number of antigenic determinants on the antigen-coated surface. In this paper we explore further the quantitative aspects of the 'competition effect'. An easily applied method is described, based on preferential precipitation of non-IgE antibodies by ammonium sulfate, that permits analysis of IgE antibodies in the presence of large amounts of non-IgE antibodies (that are principally IgG). For IgE anti-Ar, the maximum permissible ratio is extended from approximately 1500:1 to at least 40,000:1. We have also determined the effect of IgG antibodies or whole mouse serum on PCA reactions of mouse IgE antibodies, carried out in rats.

Ammonium Sulfate↗

PubMed1985

Clearance of IgE from serum of normal and hybridoma-bearing mice.

The half-life of IgE in the mouse was investigated by using radiolabeled and unlabeled monoclonal antibodies of the IgE class. Quantitative serologic assays were used for the unlabeled antibodies. IgE was cleared rapidly upon i.v. inoculation; after 48 hr, less than 0.2% of the initial concentration remained in the serum. The IgE was cleared initially with a half-life of 1 to 2 hr, attaining a relatively constant value of 5 to 8 hr. The corresponding values for IgG1, determined as a control, were 11 to 12 hr and 9 to 11 days, respectively. The initial stage probably reflects equilibration with extravascular spaces. This is supported by experiments with mice in which IgE-secreting tumors were implanted and then resected; IgE was cleared from such mice with an average initial half-life of about 5 hr. The rates of clearance of inoculated IgE were approximately the same in mice bearing an IgE-secreting tumor and in normal mice. This suggests that the initial rapid clearance of IgE from normal mice is not due to adherence of IgE to saturable sites; such sites might be expected to be occupied in mice expressing high serum concentrations of IgE. This conclusion was supported by experiments in which 1-mg quantities of IgE were inoculated i.v. into normal mice daily for 6 days. Additional IgE injected on day 7 was cleared normally. The results obtained with tumor-bearing mice indicate that the reported failure to elicit an IgE response to an antigen in mice bearing IgE-secreting hybridomas cannot be attributed to rapid clearance of newly synthesized IgE in such mice, as compared with normal mice.

Animals↗

PubMed1984

Quantitative measurements of an intrastrain cross-reactive idiotype in IgE antibodies.

This paper describes the development of methods for quantitation of an intrastrain cross-reactive idiotype (CRIA) in IgE antibodies directed to the p-azophenylarsonate (Ar) group and their application to normally immunized and idiotypically suppressed mice. The data were validated by the use of monoclonal and serum anti-Ar antibodies known to possess or lack CRIA. The idiotype was absent in the IgE as well as the total anti-Ar population of idiotypically suppressed mice. A good correlation was noted between the CRIA content of total and IgE anti-Ar with a trend toward a somewhat lower content in the IgE population. Irradiated mice that received immune cells prior to further immunization expressed relatively low concentrations of the idiotype (per weight unit of anti-Ar). The method will be useful in studies of the regulation of the switch to IgE synthesis.

Animals↗

PubMed1984

Crystallization of the fab fragments of monoclonal anti-p-azophenylarsonate antibodies and their complexes with haptens.

We report on the preparation, crystallization, and preliminary x-ray crystallographic study of Fab fragments from monoclonal anti-p-azophenylarsonate antibodies. Several crystalline forms were obtained with the Fab fragment from the R19.9 monoclonal antibody as well as with the complex between the hapten p-aminobenzenearsonic acid and Fab R19.9. The crystals of this hapten-Fab complex are similar to but not always isomorphous with the native Fab crystals. All the native and complex crystals were obtained using polyethylene glycol 6000 as crystallizing agent. Some of these crystalline forms diffract to a 2-A resolution or beyond and are suitable for high resolution x-ray diffraction analysis. A possible interpretation of hapten binding to crystalline Fab fragments from R19.9 and from the R9.3 monoclonal anti-p-azophenylarsonate antibody, implying conformational changes, is discussed.

Animals↗