Specific Gamma Delta T Cells for Cellular Immunotherapy of EBV-Associated Diseases after Allo-HSCT By T-Cell Receptor Gene Modification
Authors: Wu Xiuli , Wang Xinyu , Jin Zhenyi , Xu Ling , Chen Shaohua , Yang Lijian , Grabarczyk Piotr , Przybylski Grzegorz K. , Schmidt Christian A , Li Yangqiu 
Published: October 2019

Abstract
Epstein-Barr virus (EBV) infection may result in a spectrum of diseases in recipients of allogeneic hematopoietic stem cell transplantation (allo-HSCT), especially post-transplant lymphoproliferative disease (PTLD). Furthermore, the clinical course of EBV-associated diseases often superimposes with graft-versus-host disease (GVHD) and that increases the difficulty of treatment. Adoptive immunotherapy with EBV-specific cytotoxic T lymphocytes (EBV-CTL) is recommended as one of the first-line therapy for PTLD. Engineering of T cell receptor (TCR)-transferred T lymphocytes could be an attractive strategy to obtain sufficient T cells with an antigen specificity of choice. But TCR-modified alpha beta T cells are restricted by major histocompatibility (MHC) class I expression and low affinity. Because gamma delta T cells (GD T cells) recognize target cells independent of human leukocyte antigen (HLA), the alpha beta TCR engineering of GD T cells forms a feasible strategy to generate antigen-specific effector T cells without HLA restriction. And regulatory GD T cells (GD Tregs) (one of the novel subset of GD T cells), which express Foxp3 and primarily belong to CD27+CD25high phenotype, might have application prospect in the treatment of GVHD. In order to investigate a novel strategy with potential therapeutic benefits in EBV-associated diseases after allo-HSCT, we obtained the monoclonal EBV-CTL clone HLA-A*0201/GLCTLVAML from the PROIMMUNE company (U.K.) and detected peptide-specific CTL activity in chromium release assays, then identified the EBV-CTL associated - TCR Valpha 15 and TCR Vbeta 1 chain genes, and subsequently cloned into the eukaryotic expression vector pIRES to construct the recombinant plasmid; We transferred the EBV specific recombinant plasmid to GD T cells and GD Tregs (generated in vitro by stimulating with anti-TCR gamma delta in the presence of TGF-beta and IL-2). The TCR gene-transduced groups had special TCR Vbeta 1 expression in fluorescence detection, quantitative PCR and western Blot analysis. TCR gene-transduced GD T cells had significant cytotoxicity against the EBV+ target cell lines (including toledo cells (HLA-A2 positive) (38.17±0.86%) and daudi cells (HLA-A2 negative) (61.74±1.84%)) compared with empty vector transfected GD T cells (toledo cells (30.15±0.89%); daudi cells (53.91±4.33%)) (P<0.001, P=0.006). TCR gene-transduced GD Tregs also had significant cytotoxicity against toledo cells (56.21±5.34%) and daudi cells (68.41±2.30%) compared with empty vector transfected GD Tregs (toledo cells (48.47±0.35%); daudi cells (56.23±2.04%)) (P=0.012, P<0.001). Both TCR gene-transduced GD T cells and GD Tregs had high levels of IFN-gamma in culture supernatants. Moreover, TCR gene-transduced GD Tregs had high levels of IL-10 in culture supernatants. Interestingly, TCR-modified GD Tregs showed significant suppressive effect on autologous CD4+ responder cells in CCK8 detection and CFSE assays. In conclusion, GD T cells or GD Tregs co-transduced with TCR alpha beta genes (from EBV CTL clone) could acquire antiviral capacity. Immunomodulatory function of GD Tregs could remain after TCR gene transfection. TCR modified GD Tregs might be an alternative and attractive strategy with potential therapeutic benefits in EBV-associated diseases after allo-HSCT, especially with GVHD.

Disclosures
Wu: National Natural Science Foundation of China (No. 81200388), the Science and Technology Planning Project of Guangdong Province of China (No. 2012B031800403), the project of the Zhujiang Science & Technology Star of Guangzhou(No. 2013027): Research Funding. Li:National Natural Science Foundation of China (No. 81200388), the Science and Technology Planning Project of Guangdong Province of China (No. 2012B031800403), the project of the Zhujiang Science & Technology Star of Guangzhou(No. 2013027): Research Funding.