Imaging of T-cell receptor fused to CD3ζ reveals enhanced expression and improved pairing in living cells

Tao,Changli; Shao,Hongwei; Yuan,Yin; Wang,Hui; Zhang,Wenfeng; Zheng,Wenling; Ma,Wenli; Huang,Shulin

doi:10.3892/ijmm.2014.1839

Imaging of T-cell receptor fused to CD3ζ reveals enhanced expression and improved pairing in living cells

Authors:
- Changli Tao
- Hongwei Shao
- Yin Yuan
- Hui Wang
- Wenfeng Zhang
- Wenling Zheng
- Wenli Ma
- Shulin Huang
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Affiliations: Institute of Genetic Engineering, Southern Medical University, Guangzhou, Guangdong 510515, P.R. China, Guangdong Province Key Laboratory for Biotechnology Drug Candidates, School of Life Science and Biopharmaceutics, Guangdong Pharmaceutical University, Guangzhou, Guangdong 510006, P.R. China
Published online on: July 8, 2014 https://doi.org/10.3892/ijmm.2014.1839
Pages: 849-855

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Abstract

T cell receptor (TCR) gene adoptive therapy is a promising clinical approach for the treatment of malignant tumors and viral diseases. However, the effectiveness of this strategy is hampered by the generation of mixed TCR heterodimers comprising both exogenous and endogenous TCR chains (i.e., mispairing of TCR chains). In the present study, we constructed genetically encoded reporters fused to a pair of fluorescent proteins [enhanced cyan fluorescent protein (ECFP)/enhanced yellow fluorescent protein (EYFP)] to monitor the expression of TCRαζβζ and pairing between TCRαζ and TCRβζ. We demonstrate that these reporters provide accurate images of TCRαζβζ expression, which is markedly stronger with evident microclusters accumulated at the plasma membrane compared to wild-type (wt)TCR. Using fluorescence resonance energy transfer (FRET) analysis, we demonstrate that, in addition to the improved pairing, the expression and assembly of TCRαζβζ are independent of endogenous CD3 subunits. These results suggest that the fusion genes, TCRαζ and TCRβζ, coupled to ECFP and EYFP, respectively can effectively monitor the expression and interaction in cells. Our data suggest a novel strategy with which it is possible to effectively express and pair TCRαζβζ, thus making TCR gene adoptive therapy more effective.

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1	Heemskerk MH, Hoogeboom M, Hagedoorn R, et al: Reprogramming of virus-specific T cells into leukemia-reactive T cells using T cell receptor gene transfer. J Exp Med. 199:885–894. 2004. View Article : Google Scholar : PubMed/NCBI
2	Hughes MS, Yu YY, Dudley ME, et al: Transfer of a TCR gene derived from a patient with a marked antitumor response conveys highly active T-cell effector functions. Hum Gene Ther. 16:457–472. 2005. View Article : Google Scholar : PubMed/NCBI
3	Abad JD, Wrzensinski C, Overwijk W, et al: T-cell receptor gene therapy of established tumors in a murine melanoma model. J Immunother. 31:1–6. 2008. View Article : Google Scholar : PubMed/NCBI
4	Coccoris M, Swart E, de Witte MA, et al: Long-term functionality of TCR-transduced T cells in vivo. J Immunol. 180:6536–6543. 2008. View Article : Google Scholar : PubMed/NCBI
5	de Witte MA, Bendle GM, van den Boom MD, et al: TCR gene therapy of spontaneous prostate carcinoma requires in vivo T cell activation. J Immunol. 181:2563–2571. 2008.PubMed/NCBI
6	Dossett ML, Teague RM, Schmitt TM, et al: Adoptive immunotherapy of disseminated leukemia with TCR-transduced, CD8⁺ T cells expressing a known endogenous TCR. Mol Ther. 17:742–749. 2009. View Article : Google Scholar : PubMed/NCBI
7	Morgan RA, Dudley ME, Wunderlich JR, et al: Cancer regression in patients after transfer of genetically engineered lymphocytes. Science. 314:126–129. 2006. View Article : Google Scholar
8	Johnson LA, Morgan RA, Dudley ME, et al: Gene therapy with human and mouse T-cell receptors mediates cancer regression and targets normal tissues expressing cognate antigen. Blood. 114:535–546. 2009. View Article : Google Scholar
9	Bendle GM, Linnemann C, Hooijkaas AI, et al: Lethal graft-versus-host disease in mouse models of T cell receptor gene therapy. Nat Med. 16:565–570. 2010. View Article : Google Scholar
10	van Loenen MM, de Boer R, Amir AL, et al: Mixed T cell receptor dimers harbor potentially harmful neoreactivity. Proc Natl Acad Sci USA. 107:10972–10977. 2010.PubMed/NCBI
11	Cohen CJ, Zhao Y, Zheng Z, et al: Enhanced antitumor activity of murine-human hybrid T-cell receptor (TCR) in human lymphocytes is associated with improved pairing and TCR/CD3 stability. Cancer Res. 66:8878–8886. 2006. View Article : Google Scholar : PubMed/NCBI
12	Sommermeyer D and Uckert W: Minimal amino acid exchange in human TCR constant regions fosters improved function of TCR gene-modified T cells. J Immunol. 184:6223–6231. 2010. View Article : Google Scholar : PubMed/NCBI
13	Kuball J, Dossett ML, Wolfl M, et al: Facilitating matched pairing and expression of TCR chains introduced into human T cells. Blood. 109:2331–2338. 2007. View Article : Google Scholar : PubMed/NCBI
14	Cohen CJ, Li YF, El-Gamil M, et al: Enhanced antitumor activity of T cells engineered to express T-cell receptors with a second disulfide bond. Cancer Res. 67:3898–3903. 2007. View Article : Google Scholar : PubMed/NCBI
15	Voss RH, Willemsen RA, Kuball J, et al: Molecular design of the Cαβ interface favors specific pairing of introduced TCRαβ in human T cells. J Immunol. 180:391–401. 2008.PubMed/NCBI
16	Willemsen R, Weijtens M, Ronteltap C, et al: Grafting primary human T lymphocytes with cancer-specific chimeric single chain and two chain TCR. Gene Ther. 7:1369–1377. 2000. View Article : Google Scholar : PubMed/NCBI
17	Zhang T, He X, Tsang TC, et al: Transgenic TCR expression: comparison of single chain with full-length receptor constructs for T-cell function. Cancer Gene Ther. 11:487–496. 2004. View Article : Google Scholar : PubMed/NCBI
18	Schaft N, Lankiewicz B, Drexhage J, et al: T cell re-targeting to EBV antigens following TCR gene transfer: CD28-containing receptors mediate enhanced antigen-specific IFNγ production. Int Immunol. 18:591–601. 2006.PubMed/NCBI
19	Wu F, Zhang W, Shao H, et al: Human effector T cells derived from central memory cells rather than CD8⁺ T cells modified by tumor-specific TCR gene transfer possess superior traits for adoptive immunotherapy. Cancer Lett. 339:195–207. 2013. View Article : Google Scholar : PubMed/NCBI
20	Shao H, Zhang W, Hu Q, et al: TCR mispairing in genetically modified T cells was detected by fluorescence resonance energy transfer. Mol Biol Rep. 37:3951–3956. 2010. View Article : Google Scholar
21	Schroers R, Hildebrandt Y, Hasenkamp J, et al: Gene transfer into human T lymphocytes and natural killer cells by Ad5/F35 chimeric adenoviral vectors. Exp Hematol. 32:536–546. 2004. View Article : Google Scholar : PubMed/NCBI
22	Elangovan M, Wallrabe H, Chen Y, et al: Characterization of one-and two-photon excitation fluorescence resonance energy transfer microscopy. Methods. 29:58–73. 2003. View Article : Google Scholar : PubMed/NCBI
23	Wallrabe H and Periasamy A: Imaging protein molecules using FRET and FLIM microscopy. Curr Opin Biotech. 16:19–27. 2005. View Article : Google Scholar : PubMed/NCBI
24	Call ME, Wucherpfennig KW and Chou JJ: The structural basis for intramembrane assembly of an activating immunoreceptor complex. Nat Immunol. 11:1023–1029. 2010. View Article : Google Scholar : PubMed/NCBI
25	Kuhns MS, Girvin AT, Klein LO, et al: Evidence for a functional sidedness to the αβTCR. Proc Natl Acad Sci USA. 107:5094–5099. 2010.
26	Call ME, Schnell JR, Xu C, et al: The structure of the ζζ transmembrane dimer reveals features essential for its assembly with the T cell receptor. Cell. 127:355–368. 2006.