Cell Recognition Sites Research Articles

16] Preparation of antibody—toxin conjugates

This chapter discusses the two ways of constructing antibody-toxin conjugates with cell-type specificity. The first is to link the antibody by a disulfide bond to the isolated A chain moiety or alternatively to one of the single-chain plant peptides, such as gelonin from Gelonium multiflorum , whose damaging action on eukaryotic ribosomes is apparently identical to that of the A chains of abrin and ricin. The second is to link the intact toxin to the antibody and block the cell recognition site on the B chain to prevent the conjugate from binding to and killing cells nonspecifically. This chapter describes the methods for preparing these two types of conjugate and characterizing them physicochemically and biologically. The chapter presents the advantages and limitations of a chain conjugates to those of intact toxin conjugates as selective cytotoxic agents in vitro and as chemotherapeutic agents in animals.

T cell recognition of lysozyme. IV. Localization and genetic control of the continuous T cell recognition sites by synthetic overlapping peptides representing the entire protein chain.

Recently, this laboratory has developed a comprehensive strategy for the systematic localization of all the 'continuous' antigenic (as well as other binding) sites of complex multivalent protein antigens involved in B and T cell recognition. The strategy depends on the synthesis of consecutive overlapping peptides that together account for the entire protein chain. This strategy was applied here for the localization of the 'continuous' T cell recognition sites of hen egg lysozyme. Eight overlapping peptides encompassing the entire protein chain of lysozyme were synthesized and examined for their ability to stimulate in vitro proliferation of T cells from several mouse strains (A/J, H-2a; BALB/c and DBA/2, H-2d; B10.BR, H-2k; DBA/1, H-2q; SJL, H-2s) that had been primed with native lysozyme. This approach enabled the identification of a full profile of in vitro active lysozyme peptides and the localization of four major T cell recognition sites, three of which were subject to individual control.

Variants of the cell recognition site of fibronectin that retain attachment-promoting activity.

A tetrapeptide sequence, Arg-Gly-Asp-Ser, is the minimal structure recognized by cells in the large, adhesive glycoprotein fibronectin. We now have defined the structural requirements for this cell recognition site by testing several synthetic variants of the active tetrapeptide sequence. The conservative substitutions of lysine for arginine, alanine for glycine, or glutamic acid for aspartic acid each resulted in abrogation of the cell attachment-promoting activity characteristic of the natural sequence. However, in the position of the serine residue, some alterations were compatible with activity. Assay of peptides containing the structure Arg-Gly-Asp-X (where X = another amino acid residue) showed that an Arg-Gly-Asp-Val sequence predicted to be present in some, but not all, fibronectin molecules as a result of alternative RNA splicings could potentially create a second cell attachment site in those fibronectin polypeptide chains carrying that sequence. Other proteins with potentially active Arg-Gly-Asp-X sequences include several proteins that are known to interact with the cell surface. Among these are various types of collagens, thrombin, and discoidin, a slime-mold protein that may be involved in cell aggregation. The result presented here show that the arginine, glycine, and aspartic acid residues are absolutely required for the cell recognition, and that the surrounding amino acids may play a role in the expression of cell attachment activity in fibronectin and other proteins having this sequence. We suggest, based on these data, that this recognition mechanism may be common to a number of biological systems.

An analysis of functional T cell recognition sites on I-E molecules.

The recognition of I-E molecules by antigen-specific T cells was studied to determine if one or multiple topographic sites on the I-E molecules can function as restricting elements for T cells. A panel of 14 I-Ek-specific monoclonal antibodies (mAb) was used to inhibit T cell proliferation induced by antigens, the recognition of which was restricted by I-E-encoded determinants. These antibodies gave patterns of inhibition that were similar for three long-term antigen-specific T cell lines. Multiple distinct patterns of inhibition, however, were observed when a series of antigen-specific I-E-restricted T cell clones was studied. Differences were identified even among clones expressing apparently similar antigen specificities and MHC restriction. The observed inhibition by these antibodies appeared to be caused by specific steric or allosteric interference with T cell recognition of antigen and Ia. Based on the differences in patterns of inhibition, it was possible to infer the existence of distinct sites or conformations on the I-E molecule that are functionally involved in antigen-specific T cell recognition.

The Separate Roles of Hapten and of Carrier in the Conformation of T Cell-Reactive Determinants on Aba-Protein Conjugates

Abstract When new antigenic determinants were formed on a protein by modifying it with p-azobenzenearsonate (ABA), those of the new determinants that were recognized by and activated a T cell-immune response contained functionally separate contributions from the hapten and from the carrier protein. Those parts of the determinant that actually reacted with the T cell recognition site were contributed by the carrier molecule, whereas the hapten contribution was toward specifying the structural arrangement of those parts of the carrier molecule. The indirect nature of the haptenic contribution to the conjugate determinant explains why p-azobenzenearsonic acid on the autologous carrier guinea pig albumin (GPA) cross-reacted with p-azobenzoic acid on the same protein, whereas p-azobenzenearsonic acid in its free form on tyrosine did not cross-react with conjugates of p-azobenzoic acid. Although identity of the hapten components of the autologous albumin conjugates were not necessary for inducing cross-reactive immunity, neither was identity of both hapten and protein moiety sufficient to induce cross-reaction, since ABA-GPA did not induce immunity to ABA-Actyr GPA, and ABA-Actyr-GPA induced only a very weak immunity to ABA-GPA. Thus, the manner or site of attachment of ABA to protein was a major factor in determining the conformation of the determinant that it induced on the protein. In spite of the finding of some cross-reaction between different protein conjugates of ABA, there was no evidence that the conjugate determinants had induced T cell immunity to any structure containing the hapten in an immunologically recognizable form. ABA-protein immune cells did not respond to ABA-amino acids nor to ABA on the amino acid copolymers GT or GL. Enrichment of the cross-reactive population of ABA-OA or ABA-GPA immune T cells by in vitro selection on macrophage pulsed with the cross-reacting antigen did not amplify any small population of hapten-reactive cells that might have been undetectable by direct testing of an unenriched cell population with ABA-amino acids.

Action of concanavalin A on the attachment stage of phagocytosis by macrophages

The plant agglutinin concanavalin A is shown by bacterial adherence phenomena, coupled with specific competitive inhibition studies, to attach by means of its carbohydrate-binding sites to the cell surface of murine macrophages. Attachment of bacteria to the macrophages by means of this lectin, in contrast to their attachment by way of a bovine serum opsonin (IgM). does not result in ingestion. A close topographical relationship between the two sites is likely since bound concanavalin A, the free valency sites of which have been blocked with serum glycoprotein, greatly reduces adherence of motile bacteria opsonised with IgM. Complementary fluorescence microscopy studies using fluorescein-labelled lectin are described and the specific binding of concanavalin A at the surface of the macrophage plasma membrane is confirmed. It is suggested that surface glycoprotein should be considered in any study of the nature of the cell's recognition site for the attachment of foreign particles prior to phagocytosis.