Promiscuous Behavior of HPV16E6 Specific T Cell Receptor Beta Chains Hampers Functional Expression in TCR Transgenic T Cells, Which Can Be Restored in Part by Genetic Modification

Kirsten B J Scholten,Chris J L M Meijer,Stephen Man,Erik Hooijberg,Janneke J Ruizendaal,Marcus Graf,Duco Kramer,Thomas Schoedl

doi:10.1155/2010/616497

Kirsten B J Scholten, Chris J L M Meijer + Show 6 more

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https://doi.org/10.1155/2010/616497

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Abstract

Background: T cell receptor gene transfer is a promising strategy to treat patients suffering from HPV induced malignancies. Therefore we isolated the TCRαβ open reading frames of an HPV16E6 specific CTL clone and generated TCR transgenic T cells. In general low level expression of the transgenic TCR in recipient human T cells is observed as well as the formation of mixed TCRs dimers. Here we addressed both issues employing three different expression platforms.Methods: We isolated the HVP16E6 specific TCRα and TCRβ open reading frames and retrovirally transduced human T cells with either wild-type (wt), or codon-modified (cm) chains to achieve enhanced TCR expression levels, or used codon-modification in combination with cysteinization (cmCys) of TCRs to facilitate preferential pairing of the introduced TCRα and TCRβ chains.Results: Careful analysis of recipient T cells carrying the HPV16E6 TCRβ and endogenous TCR chains revealed the transgenic TCRβ chain to behave very promiscuously. Further analysis showed that the percentage of tetramer positive T cells in codon-modified/cysteinized TCR transgenic T cells was four-fold higher compared to wild-type and two-fold higher compared to codon-modification only. Functional activity, as determined by IFN-γ production, was high in cmCysTCR transgenic T cells, where it was low in cm and wt TCR transgenic T cells. Recognition of endogenously processed HPV16E6 antigen by cmCysTCR transgenic T cells was confirmed in a cytotoxicity assay.Conclusions: Promiscuous behavior of the HPV16E6 specific TCRβ chain can in part be forced back into specific action in TCR transgenic T cells by codon modification in combination with the inclusion of an extra cysteine in the TCR chains.

Highlights

Human Papilloma viruses (HPV) are DNA containing viruses belonging to the family of papovaviridae.K.B.J
In the current study we describe the isolation of the αβTCR ORFs encoding the TCR from CTL clone 7E7 [13], which is directed against HPV16E629–38, and detail the functional expression in recipient CD8 positive T cells
JurMA cells, which are devoid of endogenous TCRβ chains, were cultured in IMDM supplemented with 8% foetal calf serum (FCS) and antibiotics

Summary

Introduction

Human Papilloma viruses (HPV) are DNA containing viruses belonging to the family of papovaviridae.K.B.J. Infection with “low” risk HPV types, including HPV6 and 11, may result in benign epithelial lesions like warts. “high” risk or oncogenic HPV types, of which HPV16 and HPV18 are the most predominant, are associated with precancerous lesions [32,52]. An association between infection with HPV and cancer has been shown for a number of other anogenital carcinomas, including vulvar and penile carcinomas [11, 37], and for some head and neck squamous cell carcinomas [5,44]. In general low level expression of the transgenic TCR in recipient human T cells is observed as well as the formation of mixed TCRs dimers. We addressed both issues employing three different expression platforms

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