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We have previously reported that different putative CD4 ligands (anti-CD4 antibody, gp160 from HIV, synthetic peptides analogous to the residues 35-46 of HLA class II β1 chain and residues 134-148 of HLA class II β2 chain) down-regulate LFA-1-dependent adhesion between CD4⁺ T cells and HLA class II⁺ B cells, and also activate p56lck and the phosphatidylinositol-3 kinase (P13-kinase) associated with the CD4-p56^Ick complex. It was demonstrated that the latter activation was dependent on the CD4-p56^Ick association. Since these results suggest a relationship between p56^Ick and PI3-kinase, we investigated whether P13-kinase was tyrosine phosphorylated after CD4 binding and whether this phosphorylation was also dependent on the CD4-p56^Ick association. We show herein that CD4 binding increased tyrosine phosphorylation of the catalytic subunit p110 of P13-kinase but not of the p85 subunit. Association between p56^Ick and P13-kinase was constitutive, and was not modified after CD4 binding. In contrast, p110 tyrosine phosphorylation was inducible, transient and dependent on the CD4-p56^Ick association. The role of the tyrosine phosphorylation of p110-P13-kinase following ligand binding to CD4 is unknown. We speculate that this event could link the activation of p56^Ick and of P13-kinase after CD4 binding.

We studied the ability of a peptide mimicking the major binding site of HLA-DR beta 2 for CD4 (i.e. amino acids 134-148) to inhibit the adhesion of CD4+ T cells to B cells and ICAM-1-DR-expressing fibroblasts, as well as the proliferation of TCR-CD3-triggered CD4+ T cells. Peptide DR134-148 blocked CD4+ T cell (but not CD8+ T cell) binding to B cells and to DR+ ICAM-1+ fibroblasts in a concentration-dependent manner. A peptide composed of randomly associated identical amino acid residues had no effect. This inhibitory activity was not additive with the effect of an anti-CD4 antibody, peptide DR35-46 (mimicking another potential binding site of HLA-DR beta 1 to CD4) or an anti-LFA-1 antibody. Adhesion of a T cell line (HUT78) expressing a mutated form of CD4 unable to bind p56lck cytosine kinase was not inhibited by peptide DR134-148. In addition, herbimycin A, a tyrosine kinase inhibitor, abrogated the inhibitory activity of DR134-148. Since CD4-MHC class II interactions have been shown to play no detectable role in mediating antigen-independent adhesion in this assay, peptide interactions with CD4 may trigger an off signal down-regulating LFA-1-mediated adhesion. Indeed, adhesion of CD4+ T cells to ICAM-1- fibroblasts was not inhibited by peptide DR134-148, while the same peptide inhibited antigen (protein-pure derivative)- and anti-CD3 antibody-induced CD4 T cell proliferation. These findings suggest that the major sequence involved in the MHC class II interaction with CD4 is sufficient to induce a downstream negative regulatory signal that is mediated by p56lck, independently of antigen-specific TCR triggering.

We have previously reported that different putative CD4 ligands (anti-CD4 antibody, gp160 from HIV, synthetic peptides analogous to the residues 35-46 of HLA class II beta1 chain and residues 134-148 of HLA class II beta2 chain) down-regulate LFA-1-dependent adhesion between CD4+ T cells and HLA class II+ B cells, and also activate p56lck and the phosphatidylinositol-3 kinase (PI3-kinase) associated with the CD4-p56lck complex. It was demonstrated that the latter activation was dependent on the CD4-p56lck association. Since these results suggest a relationship between p56lck and PI3-kinase, we investigated whether PI3-kinase was tyrosine phosphorylated after CD4 binding and whether this phosphorylation was also dependent on the CD4-p56lck association. We show herein that CD4 binding increased tyrosine phosphorylation of the catalytic subunit p110 of PI3-kinase but not of the p85 subunit. Association between p56lck and PI3-kinase was constitutive, and was not modified after CD4 binding. In contrast, p110 tyrosine phosphorylation was inducible, transient and dependent on the CD4-p56lck association. The role of the tyrosine phosphorylation of p110-PI3-kinase following ligand binding to CD4 is unknown. We speculate that this event could link the activation of p56lck and of PI3-kinase after CD4 binding.