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Biomedical subjects

A Nisonoff

Publications and source records attributed to A Nisonoff.

At least 109 records · Page 6Linked to original sources
PubMed1977

Segregation at a locus determining an immunoglobulin genetic marker for the light chain variable region affects inheritance of expression of an idiotype.

Previous investigations have demonstrated close genetic linkage between loci governing expression of strain-specific idiotypes and immunoglobulin heavy (H) chain allotype (i.e., the C(H) locus). This linkage is presumed to reflect polymorphism of V(H) genes (or of their expression) linked to the polymorphic C(H) locus. That there was no apparent involvement of light (L) chain loci (thought to be unlinked to H chain) in inheritance of the idiotype-positive (Id(+)) phenotype was surprising, because the L chain is required for formation of each of the idiotypes studied at the chemical level. However, previous studies involving backcrosses of F(1)(Id(+) x Id(-)) mice to the Id(-) parental strain have never employed as the Id(-) parent one of several inbred strains shown by G. M. Edelman and P. D. Gottlieb [(1970) Proc. Natl. Acad. Sci. USA 67, 1191-1199] to express a V(L)-region polymorphism. Among backcrosses performed in the present study, one involved the A/J strain as the Id(+) parent and the PL/J strain, one of the several strains with and L chain polymorphism, as the Id(-) parent. Whereas in three other backcrosses performed, idiotype expression segregated with H chain allotype, in the backcross to PL/J, all mice producing the characteristic A/J Id(+) phenotype were A/J allotype(+), but not all A/J allotype(+) mice were Id(+). Typing of backcross progeny for the Ly-3 thymocyte alloantigens, controlled by a locus closely linked to that governing the L chain polymorphism (called the VK-1 locus), indicated that only the Ly-3 heterozygotes expressed the characteristic A/J Id(+) phenotype. Thus, all Id(+) backcross mice inherited both the H chain allotype and the Ly-3 locus (and closely linked L chain-related locus) from the Id(+) A/J strain. This suggests that, when strains with L chain polymorphisms are included in genetic studies of idiotype expression, segregation of loci governing L chain expression may be found to contribute to inheritance of the Id(+) character. In addition, these studies may be taken as further evidence that the V(L)-region repertoire of the PL/J strain may differ considerably from that of most other inbred strains of mice.

Animals↗
PubMed1977

Binding to idiotypic determinants of large proportions of thymus-derived lymphocytes in idiotypically suppressed mice.

All A/J mice immunized with a conjugate of p-azophenylarsonate groups to keyhole limpet hemocyanin produce antibodies against azophenylarsonate, some of which share a crossreactive idiotype. The appearance of the idiotype can be suppressed, without reducing the response against azophenylarsonate, by injecting rabbit anti-idiotypic antibodies prior to immunization. We have now observed that mice suppressed in this way, or by adoptive transfer of leukocytes from other suppressed mice, and then immunized with the hemocyanin-azophenylarsonate conjugate, possess high proportions (up to 14%) of lymphocytes that form rosettes with A/J erythrocytes coated with Fab fragments possessing the idiotype. Idiotypic specificity was demonstrated by various experiments. Most of all of the rosette-forming lymphocytes appear to be thymus-derived lymphocytes (T cells). Treatment of T cells with trypsin eliminated the capacity to form rosettes, which was restored on standing overnight in medium. Thus, the receptors are synthesized by the cells and are not passively adsorbed. Treatment of mice with anti-idiotypic antiserum without antigenic stimulation did not elicit substantial numbers of rosette-forming cells. The requirement for antigen suggests that antigen-idiotype complexes may be a stimulatory agent. A prolonged rest period after immunization of suppressed mice was required for the induction of high percentages of rosette-forming cells. Rosette formation provides a convenient method for studying factors that induce the formation of idiotype-specific T cells.

Animals↗
PubMed1977

Interaction with homologous erythrocytes of rat T cells which act as aggressors in the mixed lymphocyte reaction.

Substantial percentages of T-enriched spleen lymphocytes or thymocytes of inbred rats were found to form rosettes with the RBC of homologous strains. When an excess of RBC was used, essentially all of the rosette-forming subpopulation of lymphocytes was removed when the rosettes were separated by centrifugation. After depletion of the lymphocytes, reactive with RBC of one strain, most of the lymphocytes reactive with RBC of other strains could be recovered in the supernatant. A very large percentage of lymphocytes of the BN strain formed rosettes when a mixture of the RBC of five other strains was tested; the percentage was, however, somewhat lower than that predicted on the basis of complete additivity. Rosettes dissociated when warmed to 37 degrees C. The lymphocytes recovered were unable to form rosettes again. In nearly all instances, the subpopulation that formed rosettes with RBC of a given strain included essentially all of the lymphocytes that acted as aggressors against peripheral leukocytes or mytomycin C-treated thymocytes of that strain; the lymphocytes in the supernatant always retained activity as aggressors against the leukocytes of one or more other strains and retained their responsiveness to PHA. Lymphocytes recovered from rosettes, by warming to 37 degrees C, were highly reactive as aggressors in the MLR against the strain providing the RBC. Varying degrees of reactivity were noted against leukocytes of other strains.

Animals↗
PubMed1977

Relation of a cross-reactive idiotype to genetic control of the immune response.

Antibodies elicited in strain A mice by immunization with keyhole limpet hemocyanin-p-azophenylarsonate (KLH-Ar) produce anti-Ar antibodies, some of which share a cross-reactive idiotype (CRI); in general, 20 to 70% of the antihapten antibody population carries the idiotype. Large amounts of antibody can be produced by the induction of ascitic fluids, using a 9:1 ratio of complete Freund's adjuvant (CFA) to antigen. Antibodies with the CRI can be isolated by isoelectric focusing from selected mice that have produced a high concentration of the CRI. The H chains exhibit a single homogeneous sequence through the first hypervariable region and, when isolated from a large number of individual mice, appear to be invariant in the first framework region. These findings indicate that somatic mutation is not a significant factor in the determination of framework sequences. Appearance of the CRI can be suppressed in adult A/J mice by administration of rabbit anti-idiotypic antiserum prior to immunization. Such suppressed mice produce normal concentrations of anti-Ar antibodies lacking the CRI. Anti-idiotypic antibodies produced against such antibodies failed to show cross-reactivity with anti-Ar antibodies arising in idiotypically suppressed or nonsuppressed A/J mice. The great sensitivity of the assay indicates that the number of such "private" idiotypes, all present on anti-Ar antibodies of a single strain, must be extremely large; this supports a somatic mechanism for the generation of diversity. The "private" idiotypes arising in suppressed, hyperimmunized mice can be adoptively transferred into multiple, irradiated (200 R) recipients by injections of spleen cells or of cells from ascitic fluids. The use of ascitic fluids permits the rapid production of a colony of mice bearing the idiotype. This should facilitate structural studies of a variety of idiotypically different molecules sharing the same (anti-Ar) specificity, as well as studies of the mechanism of suppression.

Amino Acid Sequence↗
PubMed1977

Structural studies on induced antibodies with defined idiotypic specificities. V. The complete amino acid sequence of the light chain variable regions of anti-p-azophenylarsonate antibodies from A/J mice bearing a cross-reactive idiotype.

The complete amino acid sequence of the variable regions of light chains derived from anti-p-azophenylarsonate antibodies from A/J mice bearing a cross-reactive idiotype is reported. At least two and probably more than three distinct light chains are associated with this idiotypically characterized antibody. The antibodies have several differences in their "framework" structures but evidence is presented indicating that all three light chain hypervariable regions have a homogeneous sequence. The data are discussed in relation to the various theories of antibody diversity. In addition, the findings support the view that hypervariable regions, idiotypic determinants, and the antibody-combining site involve, to a large extent, the same molecular structures.

Amino Acid Sequence↗
PubMed1976

Production of large amounts of antibodies in individual mice.

A method is described for the elicitation of substantial amounts of anti-hapten and anti-protein antibodies in ascitic fluids of individual mice of various strains. The method utilizes repeated i.p. inoculations of u.5-mg quantities of antigen in complete Freund's adjuvant. A high volume ratio of adjuvant to antigen solution, and the volume used per inoculation, were shown to be critical factors. Ascitic fluids developed after three to five inoculations and were subsequently tapped repeatedly over a period of several weeks. All strains tested produced substantial amounts of anti-KLH antibodies, but marked differences were noted with respect to response to a hapten. Titers were enhanced in low responders by priming before the induction of ascites. The method is also useful for the production of "nonspecific" IgG and other serum components.

Animals↗
PubMed1975

Isolation from individual A/J mice of anti-rho-azophenylarsonate antibodies bearing a cross-reactive idiotype.

Immuization of A/J mice with a KLH-p-azophenylarsonate conjugate induces the formation of antihapten antibodies, some of which share idiotypic specificity common to all recipients. The subpopulation carrying the idiotype generally comprises 20-70% of the total antibody content. Large quantities of antihapten antibody (occasionally over 100 mg) were obtained from individual mice through the induction of an ascites fluid. This facilitated isolation of antibodies with the cross-reactive idiotype by isoelectric focusing. Most of this subpopulation has pI values between 6.65 and 6.95 and essentially all is of the IgG1 subclass. Two peaks, near pI 6.7 and 6.9, were frequently observed. Upon refocusing, the protein artifact of the procedure, but indicates microheterogeneity. The antibodies in the two peaks were found to be idiotypically identical by measurements of cross-inhibition. Preliminary studies have indicated that it is feasible to initiate investigations of primary structure with antibodies from individual inbred mice.

Animals↗
PubMed1975

Induction of an immune response through interaction of helper cells with an immune complex bound to the surface of B cells.

Adoptive anti-trinitrophenyl (Tnp) responses were elicited from Tnp-hemocyanin (Tnp-KLH)-primed cells by challenge with an immune complex of KLH and a Tnp conjugate of the Fab fragment of rabbit anti-KLH. Removal of T cells by treatment with anti-Thy 1.2 (phi C3H) + complement abolished this effect. Tnp-KLH primed cells that had been incubated with a Tnp conjugate of pneumococcal polysaccharide type III (Tnp-SIII), and which were then unable to respond to Tnp-KLH, made an anti-Tnp response upon incubation with rabbit anti-Dnp when transferred together with cells primed with rabbit gamma globulin. Since Tnp-KLH was not added in these experiments, it would appear that the cells were triggered by immune complexes on their surfaces consisting of Tnp-SIII and rabbit anti-Dnp with the help of T cells primed with rabbit gamma globulin; the presence of free antigen was evidently not required. Therefore, antigen-antibody complexes, when bound to the surface of B cells, may mediate T-B cell cooperation in the humoral immune response.

Animals↗
PubMed1975

Constancy of amino-terminal amino-acid sequences of antibodies of defined specificity and shared idiotype from individual inbred mice.

Amino-terminal amino-acid sequences were determined for the heavy (H) chains of anti-p-azophenylarsonate antibodies with shared idiotype, isolated from nine individual A/J mice. Mice chosen for investigation were those producing high titers of antibody with the cross-reactive idiotype. The antibodies were further enriched for the idiotype by isoelectric focusing. The H chains are largely or entirely unblocked at the N-terminus and appear by sequence analysis to be greatly restricted in heterogeneity. All N-terminal sequences were found to be identical. The results suggest that the framework (nonhypervariable) sequence is determined by a germ line gene and is not influenced by somatic processes.

Amino Acid Sequence↗
PubMed1975

Structural studies on induced antibodies with defined idiotypic specificities. II. The light chains of anti-p-azophenylarsonate antibodies from A/J mice bearing a cross-reactive idiotype.

N-terminal amino acid sequence analyses have been performed on three preparations of light chains of A/J mice. Light chains derived from the IgG of unimmunized animals were compared to light chains of anti-p-azo-phenylarsonate (anti-Ar) antibodies possessing a cross-reacting idiotype (CRI); the latter were derived from the ascites fluid of a single A/J mouse, or from the pooled ascites fluids of 18 A/J mice. The heavy chains of these same two antibody preparations had previously been shown to comprise a single, homogeneous sequence to position 40. With few exceptions, the first 26 positions of light chains derived from unimmunized animals were extremely heterogeneous; the heterogeneity is comparable to that observed in a composite of sequence data on light chains of BALB/c myeloma proteins. Although the light chains obtained from anti-Ar antibodies possessing the CRI (whether from the pool of 18 A/J mice or from a single mouse) were more restricted in their sequence, at several positions as many as four alternative amino acids were detected. These studies indicate that an antibody population with defined idiotypic specificity, and very possibly identical heavy chain sequences, may contain at least four distinct light chains. The feasibility of structural studies on antibodies induced in individual mice is further demonstrated.

Amino Acid Sequence↗
PubMed1975

Competition for antigen by cell populations having receptors with the same specificity but of different idiotype.

All normal A/J mice immunized with keyhole limpet hemocyanin-p-azophenylarsonate (KLH-Ar) produce anti-Ar antiboides, some of which share a cross-reactive idiotype. The present results indicate that the biosynthesis of antibody molecules bearing this idiotype does not occur in mice having an excess of lymphoid cells with receptors for Ar that lack the idiotype. This was shown, first, by introducing into normal, nonirradiated mice lymphoid cells from mice which had been suppressed with respect to production of the idiotype and then immunized with KLH-Ar. The recipients failed to express the idiotype upon immunization. Alternatively, cells from suppressed, mice were transferred into lethally irradiated syngeneic recipients, followed 17 days later by normal cells. All recipients expressed the idiotype upon immunization. If, however, the recipients were challenged with antigen between the two cell transfers, antibodies bearing the idiotype were not produced during subsequent immunization. Arguing against active, cell-mediated suppression was the production of the idiotype by normal cells in the presence of cells from a suppressed animal. However, the possibility of active suppression by hyperimmune suppressed cells was not ruled out. On the basis of the present data, the simplest interpretation is that cells with anti-Ar receptors from immune, suppressed animals, being present in larger numbers, compete successfully for antigen with nonimmune cells and thus prevent the expression of the idiotype. The system may provide a basis for quantitative studies of competition among cells for a limited supply of antigen, particularly if B cell populations are utilized.

Animals↗
PubMed1975

Structural studies on induced antibodies with defined idiotypic specificities. I. The heavy chains of anti-p-azophenylarsonate antibodies from A/J mice bearing a cross-reactive idiotype.

Amino acid sequence analysis has been performed on three groups of heavy (H) chains of A/J mice. H chains derived from unimmunized animals were compared to anti-p-azophenylarsonate (anti-Ar) antibodies which were further subdivided into those possessing and those depleted of a cross-reacting idiotype (CRI). It was found that anti-Ar antibodies bearing the CRI are homogeneous through the first hypervariable region of the H chain. The same sequence was obtained for pooled antibody isolated from the ascites fluid of 18 A/J mice or from a single mouse. The H chains appear to belong to a minor V-H subgroup. In the first 30 positions Anti-Ar antibodies depleted of the CRI had the same sequence as those containing the CRI (with small amounts of heterogeneity at some positions), but contained a mixture of sequences in the first hypervariable region of the H chain. These studies indicate that antibodies with similar specificity and with identical framework sequences, but which differ in their hypervariable regions, contain different idiotypic determinants, and support the concept that the idiotypic determinants reside primarily within hypervariable regions.

Amino Acid Sequence↗