Abstract
CD4^+^ T cells specific for human cytomegalovirus (HCMV) IE1 protein are potential effectors of the control of HCMV infection through cytokine production. Better knowledge of major histocompatibility complex (MHC)‐peptide‐T cell receptor (TcR) interactions in the CD4^+^ T cell response should result in a better design of immunizing peptides and is a prerequisite for the development of vaccines or anti‐cytomegalovirus therapy. In this study, the recombinant protein comprising residues 86–491 encoded by exon 4 of IE1 (GST‐e4) was cleaved by enzymatic digestion and analyzed by high pressure liquid chromatographymass spectroscopy (HPLC‐MS). We identified the 14‐residue epitope 162‐DKREMWMACIKELH‐175 recognized by an HLA‐DR8‐restricted clone, BeA3. Synthetic elongated, truncated and di‐Ala‐substituted peptides of the 18‐mer IE1 158‐IVPEDKREMWMACIKELH‐175 sequene were used to analyze the amino acid motifs involved in binding to HLA‐DR8 and recognition by the BeA3 clone. Substitutions which abolished (MW → AA), or decreased (RE → AA and MA → AA) T cell clone proliferation, cytokine production and cytotoxicity were identified. Loss of T cell function induced by the MW → AA substitution was associated with poor HLA‐DR8 binding. Decreased T cell function (RE → AA and MA → AA) was associated with good HLA‐DR8 binding, which suggested that these motifs were involved in TcR binding. Other substitutions induced potentiation of the T cell clone response: the IV → AA substitution induced stronger proliferation, but equivalent cytokine production, when compared with the reference peptide IE1 (158–175). CI → AA substitution induced strong potentiation of HLA‐DR8 binding, proliferation and interferon‐γ and interleukin‐4 production, possibly due to the removal of negative effects of Cys, Ile, or both side chains. Cytotoxicity was not improved by any substitution. Our results show modulation of the CD4^+^ T cell response according to the peptide residues involved in the HLA‐DR8‐peptide‐TcR interaction.