title: Identification of a murine CD28 dileucine motif that suppresses single-chain chimeric T-cell receptor expression and function.
abstract: Recent preclinical and clinical trials have demonstrated the therapeutic potential of T lymphocytes redirected with genetically engineered T-cell receptor (TCR) surrogates against infected, cancerous, or autoreactive cells. These surrogate TCRs link a ligand-recognition domain to signaling regions from the TCR. We previously compared the function of surrogate TCRs that include TCR or TCR and CD28 signaling regions. We found that primary murine T cells modified to specifically target Kb-restricted CD8+ T cells using either Kb-zeta or Kb-CD28-zeta receptors had similar functional activities, although the CD28-zeta receptor showed a 2-fold to 4-fold decreased expression. We have now identified a previously unrecognized dileucine motif in the murine CD28 signaling domain that accounts for this reduced expression. Inactivation of this motif increased chimeric receptor surface expression 2- to 5-fold. T cells expressing the dileucine-mutated CD28-zeta chimeric receptor demonstrated enhanced proliferation, cytokine production, and cytolytic activities. Further, cells expressing this dileucine-mutated receptor were highly effective in eliminating antigen-specific CD8+ T lymphocytes in vivo. These results therefore identify a critical motif limiting the function of receptor-modified T lymphocytes, demonstrate that inactivation of this motif enhances chimeric receptor function, and illustrate a potential novel application of receptor-modified T lymphocytes in the induction of immune tolerance.
authors.affiliation: Department of Pathology, St Jude Children's Research Hospital, 332 N Lauderdale St, DT-4047E, Memphis, TN 38105, USA.
authors.forename: Phuong
authors.initials: P
authors.lastname: Nguyen
authors.forename: Ioana
authors.initials: I
authors.lastname: Moisini
authors.forename: Terrence L
authors.initials: TL
authors.lastname: Geiger
mesh_terms: D020816:Amino Acid Motifs; D000818:Animals; D018106:CD28 Antigens / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics; D003602:Cytotoxicity, Immunologic; D005786:Gene Expression Regulation; D006183:H-2 Antigens / Q000737:chemistry / Q000235:genetics; D007108:Immune Tolerance; D007371:Interferon-gamma / Q000378:metabolism; D007930:Leucine / Q000737:chemistry; D008565:Membrane Proteins / Q000096:biosynthesis / Q000235:genetics*; D051379:Mice; D008822:Mice, Transgenic; D011948:Receptors, Antigen, T-Cell / Q000096:biosynthesis / Q000235:genetics*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000276:immunology; D013602:T-Lymphocytes, Cytotoxic / Q000276:immunology*
chemical_list: D018106:CD28 Antigens; D006183:H-2 Antigens; C032876:H-2Kb protein, mouse; D008565:Membrane Proteins; D011948:Receptors, Antigen, T-Cell; D011993:Recombinant Fusion Proteins; C054979:antigen T cell receptor, zeta chain; D007371:Interferon-gamma; D007930:Leucine