title: Polymorphisms of the T cell receptor CD3delta and CD3epsilon chains affect anti-CD3 antibody binding and T cell activation.
abstract: T cell receptor (TCR) structure and function have been thoroughly studied for decades. Production and analyses of knock-out and knock-in mice with mutations in the CD3 chains have contributed significantly to these studies. The generation of such gene-modified mice relies on the availability of suitable embryonic stem (ES) cell lines. Traditionally, ES cell lines from the 129 mouse strains have been used followed by backcrossing to the C57BL/6 strain. In the present study, we demonstrate the existence of polymorphisms in the CD3 genes from mice of the 129 and C57BL/6 strains. These polymorphisms result in amino acid substitutions in the ectodomains of both the CD3delta and CD3epsilon chains in 129 mice compared to C57BL/6 mice. The amino acid substitutions do not change the stoichiometry or surface expression level of the TCR complex in 129 T cells but cause reduced anti-CD3 antibody binding to 129 T cells. Further, when stimulated with mitogenic anti-CD3 antibodies, T cells from the 129 strains show reduced expression of the activation marker CD69, Ca(2+) flux, IL-2 production and proliferative responses compared to C57BL/6 T cells. These findings demonstrate that polymorphisms of the CD3delta and epsilon ectodomains exist in mice, and that some of these polymorphisms lead to amino acid substitutions which cause structural changes and affect anti-CD3 antibody binding. Thus, functional T cell studies should be interpreted with caution when anti-CD3 antibodies are used for stimulation of T cells derived from gene-modified mice originating from 129 ES cell lines.
authors.affiliation: Department of International Health, Immunology and Microbiology, Faculty of Health Sciences, University of Copenhagen, Blegdamsvej 3C, DK-2200 Copenhagen, Denmark.
authors.forename: Lasse
authors.initials: L
authors.lastname: Boding
authors.forename: Martin Weiss
authors.initials: MW
authors.lastname: Nielsen
authors.forename: Charlotte Menné
authors.initials: CM
authors.lastname: Bonefeld
authors.forename: Marina Rode
authors.initials: MR
authors.lastname: von Essen
authors.forename: Bodil Lisbeth
authors.initials: BL
authors.lastname: Nielsen
authors.forename: Jens Peter H
authors.initials: JP
authors.lastname: Lauritsen
authors.forename: Ann Kathrine
authors.initials: AK
authors.lastname: Hansen
authors.forename: Morten Milek
authors.initials: MM
authors.lastname: Nielsen
authors.forename: Martin
authors.initials: M
authors.lastname: Kongsbak
authors.forename: Maria
authors.initials: M
authors.lastname: Rubin
authors.forename: Marie Torp
authors.initials: MT
authors.lastname: Vennegaard
authors.forename: Niels
authors.initials: N
authors.lastname: Odum
authors.forename: Carsten
authors.initials: C
authors.lastname: Geisler
mesh_terms: D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D017252:CD3 Complex / Q000235:genetics* / Q000378:metabolism; D002460:Cell Line; D053595:Embryonic Stem Cells; D055879:Gene Knock-In Techniques; D008213:Lymphocyte Activation; D051379:Mice; D008810:Mice, Inbred C57BL; D008815:Mice, Inbred Strains; D018345:Mice, Knockout; D008969:Molecular Sequence Data; D016853:Muromonab-CD3 / Q000378:metabolism; D020641:Polymorphism, Single Nucleotide; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D016415:Sequence Alignment; D013329:Structure-Activity Relationship; D013601:T-Lymphocytes / Q000378:metabolism*
chemical_list: D017252:CD3 Complex; C472562:CD3delta antigen; C485468:Cd3e protein, mouse; D016853:Muromonab-CD3