Protection from experimental autoimmune thyroiditis conferred by a monoclonal antibody to T cell receptor from a cytotoxic hybridoma specific for thyroglobulin.

A clonotypic mAb, AG7, has been prepared from splenocytes of CBA/J mice immunized with a cytotoxic T cell hybridoma, HTC2, specific for a pathogenic epitope of the thyroglobulin molecule in association with class I MHC Ag. AG7 binds to HTC2 cells but not to the other T cell hybridomas tested. Moreover, when used in functional studies, AG7 blocks the HTC2 capacity of specific target lysis. It also reacts with a determinant that comodulates with the CD3 Ag present on the surface of HTC2 cells. Immunoprecipitation of 125I-labeled solubilized HTC2 membranes demonstrated two bands located at 90 and 72 kDa under nonreducing conditions, which became a 46-kDa band under reducing conditions. Finally, when AG7 is injected into CBA/J mice, on day -1 before immunization with the pathogenic tryptic fragments of the thyroglobulin molecule, experimental autoimmune thyroiditis is abrogated. Thus, one of the multiple potential mechanisms of the protective immunity against EAT induced by HTC2 cells that we previously proposed, i.e., the generation of anti-clonotypic antibodies to HTC2 TCR, seems apparent.

Bordetella pertussis adenylate cyclase. Purification, characterization, and radioimmunoassay.

The extracellular adenylate cyclase of Bordetella pertussis was purified either as a free enzyme or as a complex with calmodulin. The purified enzyme has a specific activity of 1600 mumol of cAMP min-1 X mg-1 and exists under two molecular forms of 45 and 43 kDa which are apparently structurally related. Calmodulin increased considerably the resistance of adenylate cyclase to inactivation by trypsin. Although trypsin cleaved the adenylate cyclase-calmodulin complex, the digested fragments remained associated by noncovalent interactions in an active conformation. Specific mouse anti-adenylate cyclase antibodies inhibit adenylate cyclase activity and were used to develop a specific radioimmunoassay that allows detection of as little as 5 ng of adenylate cyclase in culture supernatants.

Differentiation antigens of human hemopoietic cells: patterns of reactivity of two monoclonal antibodies.

Two mouse anti-human monoclonal antibodies (S3.13 and S5.7) raised against cells of acute myelogenous leukemia were found to react with antigens expressed on the surface of subsets of monocytes and lymphocytes. S3.13 precipitates a peptide of Mr 29,000, and S5.7 precipitates a peptide of Mr 20,000 present on the surface of all the cell types tested. These two surface antigens were distributed on discrete subpopulations of normal hemopoietic cells. The antibodies reacted with all (S5.7) or a subpopulation (S3.13) of peripheral blood T-lymphocytes, and with a subset of monocytes. Both antibodies reacted with bone marrow blast cell progenitors of the myelomonocytes and erythroid lineage. S5.7 also reacted with non-T-lymphocytes and with cells of the eosinophilic lineage. Both antigens disappeared from the cell surface during normal myeloid and erythroid differentiation. Thus, these monoclonal antibodies define the molecular characteristics and the cellular distribution of two differentiation antigens present on cells of the hemopoietic lineage.

Structural correlates to the rabbit immunoglobulin heavy chain a100 allotype.

Three a100/a100 homozygous rabbits immunized with Micrococcus lysodeikticus produced large amounts of anti-polysaccharide antibodies of restricted heterogeneity. These antibodies were purified by either immunoabsorption or ion exchange chromatography. The almost complete sequence of one heavy chain spanning residues 1-123, with the exception of 10 residues (66-67 and 79-86), was determined. Partial sequence data were also obtained for the two other heavy chains. The identity of these three sequences in the first framework region unraveled a prototype sequence of the a100 allotype that differs from homologous sequence of VH regions that determine other allotypic specificities. The gradient of sequence conservation was found to be a100 greater than a3 greater than a1 greater than a- greater than a2. Homologies in sequence paralleled previously described serological cross-reactions observed between a100, a3 and a1 specificities. This remarkable conservation of framework residues suggests that the VH regions of the rabbit immunoglobulins represent a paucigene system, in which each basic allotypic specificity might be encoded a discrete subgroup of genes.

Control of isotype expression by helper T-cell clones and suppressor cells.

We report here experiments demonstrating the profound influence of T lymphocytes on isotype expression by B lymphocytes. It was shown that in a secondary in vitro response, T helper cells from primed spleen predominantly induced an IgG1 plaque-forming cell (PFC) response, while T helper cells from primed lymph node induced IgG1, IgG2a and IgG2b PFC responses. Under the same experimental conditions, T helper cell clones were able to induce IgG1, IgG2a and IgG2b responses; therefore, T helper cells are not involved in controlling preferential isotype expression. Stimulated spleen cells were shown to contain T suppressor cells which were able to limit the expression of IgG2a and IgG2b responses. Under certain circumstances, IgG1-specific suppressor cells were also demonstrated. A T-cell hybridoma, T2D4, spontaneously produced the IgG-binding factor, thereby suppressing the expression of the three subclasses studied. Exposure of these cells to IgG1 myeloma protein led to selective enhancement for the production of molecules binding to IgG1, and suppression of the IgG1 PFC response. Similar observations were made with IgG2a and IgG2b myeloma proteins, and the specificity of these isotype suppressive factors was demonstrated. The general significance of these observations is discussed.

Isotype regulation of antibody production: T-cell hybrids can be selectively induced to produce IgG1 and IgG2 subclass-specific suppressive immunoglobulin-binding factors.

T2D4, a T-cell hybrid, spontaneously secretes suppressive immunoglobulin factor(s); when incubated with purified monoclonal mouse immunoglobulins, this hybrid produces high levels of immunoglobulin-binding factors specific for the subclass of the inducing immunoglobulin. Thus, we were able to induce the production of IgG1- or IgG2-specific inhibitory factors by the same T2D4 T-cell hybrid. These subclass-specific suppressive factors bind selectively to the IgG1 or IgG2 subclasses and inhibit specifically the secretion of antibodies of the corresponding subclass. Our results favor a model of negative regulation of isotype expression in which a given isotype triggers suppressor mechanism(s) specifically inhibiting its production.

Organization of the diversity--joining region in rabbit immunoglobulin heavy chains as revealed by cleavage of a specific methionine residue in a100 allotype.

Three anti-micrococcus antibodies of restricted heterogeneity have been isolated from the antisera of homozygous a100/a100 rabbits. Heavy chains contained an unusual methionine residue at position 87 that may correlate with the a100 specificity. From this position, the sequence of a stretch of 35-50 amino acid residues was determined, permitting the definition of variable (V), diversity(D), and joining (J) segments in rabbit Ig heavy chains, with their most probable boundaries. Rabbit D regions so defined appear to be highly variable, both in sequence and in length, which varies, in the heavy chains analyzed, between 6 and 11 residues. The J regions are highly homologous to the mouse J2 segment.

Allotypy of rabbit immunoglobulins: a fifth allele at the a locus.

Studies in wild rabbits (Oryctolagus cuniculus) allowed the detection of a fifth allele (a101) at the a locus of rabbit immunoglobulins. The structures responsible for the A101 allotypic specificity have been found on both IgG and IgM. Their location is on the Fab fragment, on the H chain. None of the rabbits possessing A101 allotypic specificity possessed two known allotypic specificities of the a series. Genetic evidence by breeding studies agree with assignment of A101 to the a series and allow to designate this allotypic specificity as a101. a101 has been found in wild rabbits in different areas in France. It has no detectable cross-reactivity with the known allotypes. The genetic polymorphism of immunoglobulins is much larger in the Oryctolagus cuniculus species than the one described in the domestic form of the species.

A and b allotypy in Oryctolagus and Lepus species.

It had been previously shown by the description in wild rabbits Oryctolagus cuniculus of unknown allotypic specificities belonging to the a series and the b series, that the genetic polymorphism found in domestic rabbits was only a part of the genetic polymorphism of the species O. cuniculus. To the known a1, a2 and a3 allotypic specificities have been added in the last years a100 and a101 which are under the control of allelic genes at the a locus. Studies of cross-reactivities in the a system is a way to detect variants of a given allotypic specificity. The phylogenetic studies in the hare Lepus capensis demonstrate that a2 and a3 determinants are allotypic, apparently without polymorphism in this species; indeed, all the tested hares have either a2 or a3 determinants or both. Four presumably "new" allotypic specificities of the b series, A93, A94, A95 and A96 have been found in a population of wild rabbits O. cuniculus from an Island in Tunisia. A95 allotypic specificities against which specific antisera have been prepared, shows cross-reactivities with b4, b5 and b6 allotypes of domestic rabbit. Genetic data demonstrate that A95 is indeed under the control of an allele at the b locus. If schematic mechanisms involving duplication and mutations of genes are suitable to understand the presence of variants and their cross-reactivities, one has to understand how so many genes are involved in allotypy and how it remains stable during evolution.

[Phylogeny of the e allotypic system of rabbit immunoglobulins: study of determinants recognized on ochotona IgG by anti-e15 antisera]. / Sur la phylogénie du système allotypique e des immunoglobulines du lapin: étude des déterminants reconnus sur les IgG d'ochotone par les immunsérums anti-e15

The phylogenetic studies of Cgamma allotypes already made in the Leporidae was extended to another family of lagomorphs, the Ochotonidae. The d allotypic system is only found in rabbits; thus, it appeared late in the lagomorphs evolution. The e system has two alleles (e14 and e15, present in rabbits and in another Leporidae genus (Sylvilagus). Previous studies on hare IgG showed that e15 pattern is composed of at least two " determinants " (or group of determinants) i and j. The hare has three allotypic patterns i+j+, i+j- and i-j-. All the studied ochotonae (from Afghanistan and Iran) possess the i determinant. From comparisons between results obtained with various genera of lagomorphs, and hypothetical scheme is proposed for the evolution of the Cgamma gene on the basis of the series allotypes.

Cross-reaction of a minor variant of the a1 allotypic specificity with anti-a2 antibodies.

Certain samples of hare IgG can combine with cross-linked anti-a2 antisera prepared in the a3/a3 rabbit. This cross-reaction permitted the isolation, on hare IgG immunoadsorbent, of anti-a2 cross-reacting antibodies (anti-a2(Lv) antibodies). The binding of labeled rabbit a2 IgG to insolubilized anti-a2(Lv) antibodies is inhibited by a1 IgG, demonstrating a cross-reactivity with a2. The percentage of a1 IgG cross-reacting with anti-a2 antiserum (a1(2) IgG) is about 0.5% of total a1 IgG. The a1(2) molecules represent another variant (or set of variants) of the a1 specificity. Demonstration of this variant of a1 IgG brings to seven the minimum number of described a1 variants, and its low concentration among a1 IgG (0.5%) is in favor of a larger number of variants of allotypic specificities. Arguments in favor of common ancestor genes for allotypy in lagomorphs are given.

Allotypes of the a series and their variants in rabbit immunoglobulins.

Six allotypic specificities of the a series are found on rabbit immunoglobulins: a1, a2 and a3 are found both in domestic and wild rabbits Oryctolagus cuniculus; a100, a101 and a102 seem to be present only in wild rabbits. Each of these specificities is a family of variants always present together in a given serum. These variants can be studied through the cross-reactivities detected between the patterns of the a series. The results of studies of cross-reactivities between a1, a3 and the two specificities a100, and a102 and also the cross-reactivity between a2 and a minor variant of the a1 specificity suggested a hypothetical scheme. This hypothesis attempts to take into account the evolution of the specificities of the a series and their variants. This hypothesis also postulates the existence of a set of closely linked genes which control the synthesis of the variants of a given specificity. One could suppose that primordial allelic genes might have appeared from an ancestor gene. By duplication each allele would have led to the appearance of a set of genes coding for a given specificity. These genes might have evolved through mutations and recombinations. In wild rabbits, the observation of an allotype which seems to result from a recombination between the group of genes coding for the a2 variants and the group of genes coding for a3 variants argues in favors of the genetic recombination mechanism.

Study of A100 allotypic specificity of rabbit immunoglobulins: the cross-reactivity between A100, a1 and a3.

Evidence for the genetic determination of A100 allotypic specificity suggests that it is the product of an allele at the alpha locus. The A100 allotypic specificity was detected in a wild rabbit lacking all the known specificities of the a series. None of the 8 rabbits possessing the A100 allotypic specificity possessed two allotypic specificities of the a series. The concentration of A100+ molecules is larger in the serum of a presumably homozygous rabbit than in that of a presumably heterozygous rabbit. In the sera of heterozygous A100+ rabbits, the A100 allotypic specificity and the known specificity of the a series are carried by different molecules. The determinants responsible for the A100 allotypic specificity are present both on IgG and IgM. They are located on the heavy chain and the Fab fragment of IgG. A family of variants of A100 specificity has been detected and cross-reactivity between A100, a1 and a3 specificities has been found. This was detected and studied with anti-a3 antisera. It seems that A100 allotypic specificity is closer to a3 specificity than a1 is to a3 specificity.