Work

Generation Of Virus-Specific Cytotoxic T Lymphocytes (CTLS) Resistant to the Immunosuppressive Drug Tacrolimus (FK506)

Year: 2009

Type: article

Abstract: Viral infections (CMV, EBV and Adenovirus) can be fatal complications post allogeneic hematopoietic stem cell and solid organ transplantation. Administration of virus-specific CTLs, targeting individual- or multiple-viral antigens is effective in these patients. However, in those whose immunosuppressive treatment needs to be maintained to prevent GvHD or organ rejection, expansion and persistence of adoptively transferred virus-specific CTLs are impaired. FK506 is frequently used as immunosuppressive drug. Its effects depend on binding to FKBP12 proteins. Since T cells generated from FKBP12 knockout mice are resistant to the inhibitory effects of FK506, we knocked down FKBP12 using a small interfering RNA (siRNA) to generate virus- specific CTLs resistant to FK506. As a model we used EBV+ tumor and EBV-CTLs. We identified one siRNA sequence (siRNA4), that knocked down >90% of FKBP12 expression in T cell lines and virus-CTLs. We then generated 2 retroviral vectors encoding for siRNA4/eGFP and irrelevant siRNA/eGFP and used them to transduce CTLs generated from 7 EBV-seropositive donors. Transduction efficiency was 46 ± 22% for siRNA4 and 55 ± 27% for irrel.-siRNA. We measured the proliferation of transduced CTLs in the presence of FK506, in short and long term cultures. We found that in the presence of FK506, proliferation of control CTLs was significantly inhibited (by 74± 2%) as compared to siRNA4+ CTLs (41 ± 4%). In long term cultures, modified CTLs were stimulated weekly with EBV+ cells, with or without FK506 (5ng/ml) and low dose IL-2 (20U/mL). The proportion of siRNA4+ CTLs increased over time not only as a percentage of GFP+ cells (from 46 ± 22% to 89 ± 5% after 5 stimulations) but also numerically (34 median fold expansion, range 5–60). In contrast, control CTLs did not show any selection in culture, as the percentage of GFP+ cells remained unchanged (from 56 ± 27% to 57 ± 23%) and CTLs ceased to proliferate (2 median fold expansion, range 0–5). Finally, we found that siRNA4+ CTLs retained their MHC-restricted cytotoxicity against EBV+ cells (66 ± 22% lysis at an E:T ratio of 20:1). Modified CTLs also maintained their production of IFN-γ in response to EBV-peptides, as assessed by ELIspot assays. In conclusion, we have developed a strategy that produces virus-specific CTLs resistant to FK506. This strategy may be beneficial to improve virus immune reconstitution in patients post transplant, despite ongoing immunosuppresion.

Source: Transplantation and Cellular Therapy

DOI: 10.1016/j.bbmt.2008.12.413