title: TCR-engineered T cells: a model of inducible TCR expression to dissect the interrelationship between two TCRs.
abstract: TCR gene modified T cells for adoptive therapy simultaneously express the Tg TCR and the endogenous TCR, which might lead to mispaired TCRs with harmful unknown specificity and to a reduced function of TCR-Tg T cells. We generated dual TCR T cells in two settings in which either TCR was constitutively expressed by a retroviral promoter while the second TCR expression was regulable by a Tet-on system. Constitutively expressed TCR molecules were reduced on the cell surface depending on the induced TCR expression leading to strongly hampered function. Besides that, using fluorescence resonance energy transfer we detected mispaired TCR dimers and different pairing behaviors of individual TCR chains with a mutual influence on TCR chain expression. The loss of function and mispairing could not be avoided by changing the TCR expression level or by introduction of an additional cysteine bridge. However, in polyclonal T cells, optimized TCR formats (cysteineization, codon optimization) enhanced correct pairing and function. We conclude from our data that (i) the level of mispairing depends on the individual TCRs and is not reduced by increasing the level of one TCR, and (ii) modifications (cysteineization, codon optimization) improve correct pairing but do not completely exclude mispairing (cysteineization).
authors.affiliation: Max-Delbrück-Center for Molecular Medicine, Berlin, Germany.
authors.forename: Simone
authors.initials: S
authors.lastname: Reuß
authors.forename: Zsolt
authors.initials: Z
authors.lastname: Sebestyén
authors.forename: Niels
authors.initials: N
authors.lastname: Heinz
authors.forename: Rainer
authors.initials: R
authors.lastname: Loew
authors.forename: Christopher
authors.initials: C
authors.lastname: Baum
authors.forename: Reno
authors.initials: R
authors.lastname: Debets
authors.forename: Wolfgang
authors.initials: W
authors.lastname: Uckert
mesh_terms: D060846:Cell Engineering; D019281:Dimerization; D031541:Fluorescence Resonance Energy Transfer; D005786:Gene Expression Regulation; D005822:Genetic Vectors; D006801:Humans; D016219:Immunotherapy, Adoptive / Q000379:methods*; D019169:Jurkat Cells; D015202:Protein Engineering; D011948:Receptors, Antigen, T-Cell / Q000235:genetics / Q000378:metabolism*; D012097:Repressor Proteins / Q000235:genetics / Q000378:metabolism; D012190:Retroviridae / Q000235:genetics; D013329:Structure-Activity Relationship; D013601:T-Lymphocytes / Q000276:immunology* / Q000637:transplantation; D019076:Transgenes / Q000235:genetics
chemical_list: D011948:Receptors, Antigen, T-Cell; D012097:Repressor Proteins; C034811:tetracycline resistance-encoding transposon repressor protein
keywords: Fluorescence resonance energy transfer (FRET); Inducible gene expression; TCR gene optimization; TCR gene therapy; TCR mispairing