The T-cell receptor is necessary and sufficient for recognition of peptides presented by major histocompatibility complex molecules. Other adhesion molecules, like CD4 or CD8, play an auxiliary role in antigen recognition by T cells. Here we analyse T-cell receptor (TCR) binding using a soluble rather than a cell-bound receptor molecule. A TCR-immunoglobulin chimaera is constructed with the variable and the first constant regions of both the TCR alpha- and beta-chains linked to the immunoglobulin light-chain constant regions. This soluble TCR is expressed, assembled and secreted as an alpha beta heterodimer by a myeloma cell line transfected with the recombinant genes. Furthermore, the soluble TCR is biologically active: it specifically inhibits antigen-dependent activation of the relevant T-cell clones and thus discriminates between proper and irrelevant peptides presented by major histocompatibility complex molecules.

Interaction of CD4 with MHC class II molecules plays a crucial role during thymlc development and activation of single-positive CD4 T lymphocytes. The quantltatlon of this Interaction is, therefore, Important for understanding the role of CD4 during these events. To this end, we have developed a rosette assay, which enabled us to study this molecular Interaction. By coupling soluble mouse CD4 onto beads, we could show specific binding of CD4 to MHC class II molecules on A20 B lymphoma cells. These binding studies revealed an extremely low affinity (/C, ^ 10 4 M" 1 ) between CD4 and MHC class II molecules in mouse.The CD4 co-receptor is expressed on T cells which are MHC class II restricted and mostly of T helper (Th) cell type (1,2). CD4 is known to bind to a non-polymorphic determinant on MHC class II (2-6). Interaction of CD4 with MHC class II is crucial during thymic ontogeny (for review see 7) and plays a dual role in T cell activation. First, by strengthening the overall interaction between T h cells and antigen presenting cells (APCs) (8-10), and, second, by transducing signals to T cells (for review see 11).The quantitation of this interaction would provide further insights into the complex interactions occuring in a TCR -CD3 -CD4 -MHC plus peptide complex during antigen recognition by T cells and is of specific interest since the affinity of mouse TCR to MHC class II plus peptide was recently resolved to be about 10 5 M" 1 (12,13).The direct binding of CD4 and MHC class II has been studied in the human system (3,6), but no such studies are available for the corresponding murine molecules. Here, we report the development of an in vitro assay, with which direct and specific binding of mouse soluble CD4 (sCD4) to mouse MHC class II on the cell surface is demonstrated. This assay enabled us to estimate the affinity of mouse CD4 for MHC class II to be lower than 10 4 M"'. This further supports the hypothesis that the role of CD4 as a co-receptor probably is more important for signal transduction than adhesion.To generate soluble CD4 molecules, i.e. CDA-bC/i and CD4-Cx, Ig expression vectors containing the cDNA for the extracellular part of the mouse CD4 gene and the C 2 -C 4 exons of human /i heavy chain gene or the mouse Cx exon of light chain genes were transfected into the myeloma cell line J558/L by protoplast fusion (18,19). Stable transfectants were used for mass culture. CD4-hC/t was purified from concentrated supernatants by gel filtration, as described in Fig. 1, and CD4-Cx by using affinity chromatography (19).SDS -PAGE analysis of the materials used in this study showed that chimeric sCD4s of predicted molecular nature were produced. Non-reducing SDS-PAGE analysis revealed that CD4-hC/i assembled into a typical pentameric structure of IgM (Fig. 1), corresponding to a valency of 10 for the CD4 part, while CD4-Cx behaved as a monomer.In addition, all tested mAbs (GK1.5 (20); H129 (21); and RM4-4 and RM4-5, both Pharmingen, San Diego, CA) against different CD4 epitopes recognized the CD4-Qi as...

Interactions of CD4 with the major histocompatibility complex (MHC) class II molecules are crucial during thymic development and subsequently for the function of single-positive CD4+CD8- T lymphocytes. Here, we have investigated the potential effects of soluble CD4 (sCD4) on the immune system. We generated two different transgenic mouse lines, which constitutively expressed either approximately 100 micrograms/ml of monovalent or approximately 20 micrograms/ml of decavalent mouse sCD4 molecules in their sera. Analysis of these mice revealed no differences compared to control littermates, e.g. the single-positive CD4+ cells developed normally and these cells responded to allogeneic and anti-CD3 antibody stimuli like the cells from control mice. Furthermore, the T helper cell function for antibody responses in vivo were not affected. Our data provide evidence that, in mouse, the CD4-MHC class II-interaction has very low affinity. Since sCD4 is considered to be a therapeutical agent for human immunodeficiency virus infection, these findings are not only of basic, but also of clinical interest.

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