Generation of Epstein Barr Virus Specific Cytotoxic T Lymphocytes (EBVCTLs) Resistant to the Immunosuppressive Drug Tacrolimus (FK506)
Authors: De Angelis Biagio , Dotti Gianpietro , Quintarelli Concetta , Huye Leslie E , Zhang Lan , Zhang Ming , Heslop Helen E. , Brenner Malcolm K , Rooney Cliona M , Savoldo Barbara 
Published: October 2019

Abstract
Adoptive transfer of autologous EBV-CTLs to hematopoietic stem cell transplant (HSCT) and solid organ transplant (SOT) recipients is a safe and often effective means for prevention and treatment of EBV-associated post transplant lymphoproliferative disorders (PTLD). Although immunosuppressive drugs can be tapered in patients developing PTLD, they often cannot be completely withdrawn because of the risk of graft rejection, a particular concern in lung, heart or liver transplant recipients. These immunosuppressive drugs may limit the expansion, persistence and efficacy of transferred EBV-CTLs. One of the most widely used immunosuppressive agents is FK506 whose effects are highly dependent on binding FKBP12 proteins, since T cells generated from FKBP12 knockout mice are completely resistant to the inhibitory effects of FK506. We have generated EBV-CTLs resistant to FK506 by knocking down FKBP12 using a small interfering RNA (siRNA) stably expressed from a retroviral vector. After extensively screening potential target sequences, we identified one, designed as siRNA4, that knocks down >90% of FKBP12 protein expression in T cell lines and also in EBV-CTLs, as assessed by Western blotting. We then generated two retroviral vector encoding for siRNA4/eGFP and irrelevant siRNA/eGFP, respectively. These vectors were used to transduce established EBV-CTL lines generated from 7 EBV-seropositive donors. Transduction efficiency was 46.3±22.5% and 55.4±27.5% for siRNA4 and irrelevant-siRNA, respectively. We measured the proliferation of transduced CTLs in the presence of FK506, in short term and long term cultures. Using a thymidine uptake assay, we found that the inhibiton of proliferation by increasing concentrations of FK506 was significantly diminished in siRNA4+ CTLs compared to control CTLs (41±4% inhibition for siRNA4+ CTLs vs 74± 2% for control CTLs). To evaluate the effects of knocking down FKBP12 in long-term cultures, control and siRNA4+ CTLs were stimulated weekly with the antigen (autologous EBV-LCL) with or without the addition of FK506 (5ng/ml) and low dose IL-2 (20U/mL). We found that the proportion of siRNA4+ CTLs increased over time not only as a percentage of GFP+ cells (from 46±22% to 89.4±5.3% after 5 stimulations) but also numerically (median fold expansion: 34.3, range 5–60). In contrast, control EBV-CTLs did not show any selection in culture, since the percentage of GFP+ cells remained unchanged (from 56±27% to 57±23.1%) and CTLs ceased to proliferate (median fold expansion: 2, range 0–5). Finally, we found that siRNA4+ CTLs retained their antigen specificity, having MHC-restricted cytotoxic activity against EBV-targets (66±22% vs 16±12% for autologous and allogenic LCL, respectively at an E:T ratio of 20:1 for siRNA4+ CTLs; 61±12% vs 15±12% for autologous and allogenic LCL, respectively, for control CTLs). Modified CTLs also maintained their production of IFNγ in response to specific EBV-peptides, as assessed by ELIspot assays. We are currently evaluating the in vivo expansion of genetically modified EBV-CTLs in the presence of FK506. In conclusion, we have developed a strategy that produces EBV-CTLs resistant to FK506. This strategy may be beneficial to improve EBV immune reconstitution in patients at high risk of developing post transplant lymphoma.