Genetically Modified EBV-Specific Cytotoxic T Lymphocytes Induce Regression Of EBV Lymphoproliferation Despite Immunosuppression In Xenografted Mice: A Novel Strategy For EBV-Associated Post-Transplant Lymphoproliferative Disease
Authors: Ricciardelli Ida , Brewin Jenny , Blundell Mike , Pule Martin , Amrolia Persis 
Published: October 2019

Abstract

Background
EBV associated lymphoproliferative disease (PTLD) remains a major cause of morbidity and mortality after stem cell (SCT) or solid organ (SOT) transplant. Adoptive transfer of ex vivo-derived EBV-specific cytotoxic T lymphocytes (EBV-CTL) to reconstitute immunity to the oncogenic virus EBV has been highly effective for both the prophylaxis and treatment of PTLD after SCT, where immunosuppression could be withdrawn before transfer of EBV-CTLs. In contrast, the application of this approach for the treatment of PTLD in SOT patients, although feasible, has been challenging with restricted expansion, persistence and efficacy of the adoptive transferred T cells. This difference is likely to reflect the need for the on-going immunosuppression to prevent graft-rejection post SOT which inhibits the virus-specific T cell responses. Our group has previously genetically engineered EBV-CTL to enable them to function in vitro in the presence of the calcineurin inhibitor Tacrolimus (FK506) through retroviral transfer of a calcineurin mutant (CNA12).

Aim
To examine the ability of genetically engineered EBV-CTLs resistant to FK506 to control EBV+ B cell lymphoma progression in vivoin a xenogeneic mouse model in the presence of FK506.

Methods
NOD/SCID/IL2rgnull (NSG) mice were inoculated subcutaneously with 5×106 EBV-transformed lymphoblastoid B cell lines (LCL) labelled with F-Luc to develop human EBV+ lymphoma. To evaluate in vivo antitumor activity, 5×106 CTLs retrovirally transduced with CNA12 or eGFP control CTLs were injected intravenously after 7 days in the presence or absence of FK506 (10mg/kg/day). Tumour growth was analysed using the Xenogen-IVIS system.

Results
Adoptive transfer of autologous CNA12 transduced CTLs induced EBV+ lymphoma regression in the presence of FK506, as assessed both by IVIS and tumour size, whereas FK506 treated mice receiving eGFP control CTLs had tumour progression (P<0.05). This resulted in significantly improved survival of mice treated with CNA12-CTL in the presence of FK506 than eGFP-CTLs treated animals (P<0.0001). CNA12 transduced EBV CTLs persisted longer and expanded more (P<0.0001) than eGFP-CTLs in peripheral blood of mice treated with FK506 demonstrating a selective growth advantage and enrichment of CTLs resistant to immunosuppression. Immunohistochemical staining showed that adoptively transferred CTLs home to the tumour and an increase of tumour infiltrating T cells in CNA12-CTL compared with eGFP-CTLs treated mice in the presence of FK506 was observed.

Conclusions
Our results demonstrate that CNA12 modified EBV-CTL can induce regression of EBV-associated tumours in vivo in the face of on-going immunosuppression with FK506. Clinical application of this novel approach may enhance the efficacy of adoptive transfer of EBV-CTL in SOT patients developing PTLD without the need for reduction in immunosuppressive therapy.

Disclosures:
No relevant conflicts of interest to declare.