Improving TCR affinity on 293T cells

Rieko Ohta, Ayako Demachi-Okamura, Yoshiki Akatsuka, Hiroshi Fujiwara, Kiyotaka Kuzushima

Research output: Contribution to journalArticle

2 Citations (Scopus)

Abstract

This study presents an efficient method to improve TCR affinity, comprising 1) CDR-directed saturation mutation of TCR cDNA, 2) transient TCR display on CD3-expressing HEK293T (CD3-293T) cells by simple plasmid transfection, 3) staining with HLA-tetramers, and 4) multi-round sorting of cells with CD8-independent tetramer binding on a flow cytometer. Using these procedures, we successfully identified mutant TCRs with enhanced binding from an HLA-A*24:02-restricted, human telomerase reverse transcriptase (hTERT)-specific TCR. Two such clones, 2A7A and 2D162, harboring mutations in CDR1 and CDR2 of TCRβ, respectively, were isolated with both showing sequential four amino acid substitutions. When expressed on CD3-293T cells along with wild-type TCRα, the TCR molecules of these mutants as well as their combinatory mutation, bound to HLA-A24/hTERT-tetramers more strongly than the wild-type TCRs, without binding to control tetramers. Besides, in order to facilitate a functional study of TCR, we established an artificial T cell line, designated as CD8I-J2, which expresses a human CD8 and IFN-γ producing cassette by modifying Jurkat-derived J.RT3-T3.5 cells. CD8I-J2 cells expressing wild-type or affinity-enhanced hTERT-specific TCRs were analyzed for their recognition of serially diluted cognate peptide on HLA-A*24:02-transduced T2 cells. CD8I-J2 cells expressing each mutant TCR recognized the hTERT peptide at lower concentrations than wild-type TCR. The hierarchy of peptide recognition is concordant with tetramer binding on CD3-293T cells and none of these mutant TCRs were cross-reactive with irrelevant peptides reported to be present on HLA-A*24:02 molecules as far as tested. These methods might thus be useful for obtaining high affinity mutants from other TCRs of interest.

Original languageEnglish
Pages (from-to)1-8
Number of pages8
JournalJournal of Immunological Methods
Volume466
DOIs
Publication statusPublished - 01-03-2019

Fingerprint

HEK293 Cells
Peptides
Mutation
HLA-A24 Antigen
Artificial Cells
Amino Acid Substitution
Transfection
Plasmids
Complementary DNA
Clone Cells
Staining and Labeling
T-Lymphocytes
Cell Line
human TERT protein

All Science Journal Classification (ASJC) codes

  • Immunology and Allergy
  • Immunology

Cite this

Ohta, R., Demachi-Okamura, A., Akatsuka, Y., Fujiwara, H., & Kuzushima, K. (2019). Improving TCR affinity on 293T cells. Journal of Immunological Methods, 466, 1-8. https://doi.org/10.1016/j.jim.2018.11.010
Ohta, Rieko ; Demachi-Okamura, Ayako ; Akatsuka, Yoshiki ; Fujiwara, Hiroshi ; Kuzushima, Kiyotaka. / Improving TCR affinity on 293T cells. In: Journal of Immunological Methods. 2019 ; Vol. 466. pp. 1-8.
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Ohta, R, Demachi-Okamura, A, Akatsuka, Y, Fujiwara, H & Kuzushima, K 2019, 'Improving TCR affinity on 293T cells', Journal of Immunological Methods, vol. 466, pp. 1-8. https://doi.org/10.1016/j.jim.2018.11.010

Improving TCR affinity on 293T cells. / Ohta, Rieko; Demachi-Okamura, Ayako; Akatsuka, Yoshiki; Fujiwara, Hiroshi; Kuzushima, Kiyotaka.

In: Journal of Immunological Methods, Vol. 466, 01.03.2019, p. 1-8.

Research output: Contribution to journalArticle

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T1 - Improving TCR affinity on 293T cells

AU - Ohta, Rieko

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AU - Akatsuka, Yoshiki

AU - Fujiwara, Hiroshi

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N2 - This study presents an efficient method to improve TCR affinity, comprising 1) CDR-directed saturation mutation of TCR cDNA, 2) transient TCR display on CD3-expressing HEK293T (CD3-293T) cells by simple plasmid transfection, 3) staining with HLA-tetramers, and 4) multi-round sorting of cells with CD8-independent tetramer binding on a flow cytometer. Using these procedures, we successfully identified mutant TCRs with enhanced binding from an HLA-A*24:02-restricted, human telomerase reverse transcriptase (hTERT)-specific TCR. Two such clones, 2A7A and 2D162, harboring mutations in CDR1 and CDR2 of TCRβ, respectively, were isolated with both showing sequential four amino acid substitutions. When expressed on CD3-293T cells along with wild-type TCRα, the TCR molecules of these mutants as well as their combinatory mutation, bound to HLA-A24/hTERT-tetramers more strongly than the wild-type TCRs, without binding to control tetramers. Besides, in order to facilitate a functional study of TCR, we established an artificial T cell line, designated as CD8I-J2, which expresses a human CD8 and IFN-γ producing cassette by modifying Jurkat-derived J.RT3-T3.5 cells. CD8I-J2 cells expressing wild-type or affinity-enhanced hTERT-specific TCRs were analyzed for their recognition of serially diluted cognate peptide on HLA-A*24:02-transduced T2 cells. CD8I-J2 cells expressing each mutant TCR recognized the hTERT peptide at lower concentrations than wild-type TCR. The hierarchy of peptide recognition is concordant with tetramer binding on CD3-293T cells and none of these mutant TCRs were cross-reactive with irrelevant peptides reported to be present on HLA-A*24:02 molecules as far as tested. These methods might thus be useful for obtaining high affinity mutants from other TCRs of interest.

AB - This study presents an efficient method to improve TCR affinity, comprising 1) CDR-directed saturation mutation of TCR cDNA, 2) transient TCR display on CD3-expressing HEK293T (CD3-293T) cells by simple plasmid transfection, 3) staining with HLA-tetramers, and 4) multi-round sorting of cells with CD8-independent tetramer binding on a flow cytometer. Using these procedures, we successfully identified mutant TCRs with enhanced binding from an HLA-A*24:02-restricted, human telomerase reverse transcriptase (hTERT)-specific TCR. Two such clones, 2A7A and 2D162, harboring mutations in CDR1 and CDR2 of TCRβ, respectively, were isolated with both showing sequential four amino acid substitutions. When expressed on CD3-293T cells along with wild-type TCRα, the TCR molecules of these mutants as well as their combinatory mutation, bound to HLA-A24/hTERT-tetramers more strongly than the wild-type TCRs, without binding to control tetramers. Besides, in order to facilitate a functional study of TCR, we established an artificial T cell line, designated as CD8I-J2, which expresses a human CD8 and IFN-γ producing cassette by modifying Jurkat-derived J.RT3-T3.5 cells. CD8I-J2 cells expressing wild-type or affinity-enhanced hTERT-specific TCRs were analyzed for their recognition of serially diluted cognate peptide on HLA-A*24:02-transduced T2 cells. CD8I-J2 cells expressing each mutant TCR recognized the hTERT peptide at lower concentrations than wild-type TCR. The hierarchy of peptide recognition is concordant with tetramer binding on CD3-293T cells and none of these mutant TCRs were cross-reactive with irrelevant peptides reported to be present on HLA-A*24:02 molecules as far as tested. These methods might thus be useful for obtaining high affinity mutants from other TCRs of interest.

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Ohta R, Demachi-Okamura A, Akatsuka Y, Fujiwara H, Kuzushima K. Improving TCR affinity on 293T cells. Journal of Immunological Methods. 2019 Mar 1;466:1-8. https://doi.org/10.1016/j.jim.2018.11.010