Poster #006, Stanford University
Liberating EBV-reactive T cells from Immunosuppression to Prevent PTLD
Mentors: Masato Ogishi, PhD and Mark M. Davis, PhD
The immunosuppressant tacrolimus prevents T-cell activation by binding to the intracellular protein FKBP12. Used in pediatric transplant recipients to prevent rejection, it broadly suppresses CD8⁺ cytotoxic T lymphocytes (CTLs), permitting Epstein-Barr virus (EBV) reactivation. In nearly 20% of patients, post-transplant lymphoproliferative disorder (PTLD) also occurs, which is the second most common post-transplant malignancy. No targeted prophylactic strategy for PTLD currently exists.
We hypothesized that selectively liberating EBV-reactive CTLs from tacrolimus suppression through FKBP12 knockdown could prevent EBV-driven malignancies while maintaining systemic immunosuppression to protect the transplanted organ.
We measured tacrolimus-mediated CTL suppression using a human spleen organoid system, where primary spleen-derived immune cells were co-cultured with autologous EBV-transformed B (EBV-B) cells. We then tested whether we could knockdown FKBP12 through lentiviral shRNA transduction. Finally, we tested whether three major EBV-derived peptide constructs could be presented on the cell surface as a step toward antigen-specific delivery of genetic material to EBV-reactive CTLs.
Here we show that 1) tacrolimus partially suppresses CTL effector function in a dose-dependent manner in spleen-EBV-B co-culture assays; 2) lentiviral shRNA transduction achieved a partial but specific reduction of FKBP1A mRNA (encoding FKBP12) relative to scramble-shRNA controls, without affecting other FKBP family genes; and 3) surface expression was dependent on the different EBV-derived peptides, with one construct stably presented on the cell surface. Together, these results represent initial steps toward a therapeutic modality that modulates tacrolimus sensitivity in antigen-specific CTLs.
Going forward, we plan to optimize FKBP12 suppression, potentially through full knockout, and expand our EBV epitope panel to find additional candidates for specific delivery to EBV-reactive CTLs. Ultimately, we aim to combine these components into a single targeted delivery system: a starting point for a drug candidate that restores CTL function in the presence of tacrolimus, without disrupting the systemic immunosuppression needed to prevent organ rejection.