title: T-cell synapse formation depends on antigen recognition but not CD3 interaction: studies with TCR:ζ, a candidate transgene for TCR gene therapy.
abstract: T-cell receptors (TCRs) can be genetically modified to improve gene-engineered T-cell responses, a strategy considered critical for the success of clinical TCR gene therapy to treat cancers. TCR:ζ, which is a heterodimer of TCRα and β chains each coupled to complete human CD3ζ, overcomes issues of mis-pairing with endogenous TCR chains, shows high surface expression and mediates antigen-specific T-cell functions in vitro. In the current study, we further characterized TCR:ζ in gene-engineered T cells and assessed whether this receptor is able to interact with surface molecules and drive correct synapse formation in Jurkat T cells. The results showed that TCR:ζ mediates the formation of synaptic areas with antigen-positive target cells, interacts closely with CD8α and MHC class I (MHCI), and co-localizes with CD28, CD45 and lipid rafts, similar to WT TCR. TCR:ζ did not closely associate with endogenous CD3ε, despite its co-presence in immune synapses, and TCR:ζ showed enhanced synaptic accumulation in T cells negative for surface-expressed TCR molecules. Notably, synaptic TCR:ζ demonstrated lowered densities when compared with TCR in dual TCR T cells, a phenomenon that was related to both extracellular and intracellular CD3ζ domains present in the TCR:ζ molecule and responsible for enlarged synapse areas.
authors.affiliation: Department of Biophysics and Cell Biology, Medical and Health Science Center, University of Debrecen, Debrecen, Hungary.
authors.forename: János
authors.initials: J
authors.lastname: Roszik
authors.forename: Zsolt
authors.initials: Z
authors.lastname: Sebestyén
authors.forename: Coen
authors.initials: C
authors.lastname: Govers
authors.forename: Yakir
authors.initials: Y
authors.lastname: Guri
authors.forename: Arpád
authors.initials: A
authors.lastname: Szöor
authors.forename: Zsuzsanna
authors.initials: Z
authors.lastname: Pályi-Krekk
authors.forename: György
authors.initials: G
authors.lastname: Vereb
authors.forename: Peter
authors.initials: P
authors.lastname: Nagy
authors.forename: János
authors.initials: J
authors.lastname: Szöllosi
authors.forename: Reno
authors.initials: R
authors.lastname: Debets
mesh_terms: D019264:Adoptive Transfer; D018106:CD28 Antigens / Q000276:immunology / Q000378:metabolism; D017252:CD3 Complex / Q000235:genetics / Q000276:immunology; D016827:CD8 Antigens / Q000276:immunology / Q000378:metabolism; D005434:Flow Cytometry; D015316:Genetic Therapy; D015395:Histocompatibility Antigens Class I; D006801:Humans; D007111:Immunity, Cellular; D054992:Immunological Synapses / Q000502:physiology*; D019169:Jurkat Cells; D017493:Leukocyte Common Antigens / Q000276:immunology / Q000378:metabolism; D021962:Membrane Microdomains / Q000378:metabolism; D017260:Receptor-CD3 Complex, Antigen, T-Cell / Q000235:genetics / Q000276:immunology*; D016693:Receptors, Antigen, T-Cell, alpha-beta / Q000235:genetics / Q000276:immunology*; D013601:T-Lymphocytes / Q000276:immunology* / Q000378:metabolism; D019076:Transgenes
chemical_list: D018106:CD28 Antigens; D017252:CD3 Complex; C121704:CD3 antigen, zeta chain; D016827:CD8 Antigens; D015395:Histocompatibility Antigens Class I; D017260:Receptor-CD3 Complex, Antigen, T-Cell; D016693:Receptors, Antigen, T-Cell, alpha-beta; D017493:Leukocyte Common Antigens