Interferon-gamma Expressing EBV LMP2A-Specific T Cells for T Cell Therapy Against EBV-Positive Hodgkin’s Lymphoma.

Hodgkin’s lymphoma (HL) accounts for approximately 67% of lymphomas. Patients with HL refractory to standard intervention have poor prognoses. LMP2A is one of the Epstein-Barr virus (EBV) oncoproteins persistently expressed in approximately 50% of HL cases, providing an ideal target for T cell therapy against EBV-positive HL. However, polyclonal EBV-specific T cells have achieved only limited responses in clinical trials, with eventual disease progression or relapse. While multiple possibilities could underlie the unsatisfactory efficacy, the most obvious shortcoming of the current generation of therapeutic T cells is the low frequency of HL-relevant effectors in the T cell preparations. We designed a strategy to enrich LMP2A-specific T cells from polyclonal EBV-specific T cell preparations. With an “Interferon (IFN) Capture” procedure, LMP2A-specific T cells were isolated from T cell cultures generated with stimulation by autologous EBV immortalized B lymphoblastoid cells based on their antigen-specific expression of IFN-gamma. The enriched LMP2A-specific T cells were subsequently expanded with the anti-CD3 antibody OKT3 to a scale applicable for clinical use. Our results showed that a ten fold enrichment can be achieved for T cell cultures containing as low as 0.5% LMP2A T cells, and 5 fold for those containing higher than 10%. Thus, 2 consecutive expansions increased the LMP2A-specific T cells to nearly 50% from 0.5% in the starting culture. Cytokine expression analysis showed that the enriched T cells retained their antigen specific production of IFN-gamma, and a subpopulation upto 10% of the IFN-gamma producing T cells were capable of expressing interleukin (IL)-2. In contrast, no cells expressing immuno-suppressive cytokines IL-4, IL10 and TGF-beta were detected. The enriched T cells were mostly CD45RO+/CD45RA-, indicating that they were proliferation competent. Chromium release assays confirmed that the enriched T cells maintained antigen-specific cytotoxicity. With a panel of four HLA A02 restricted tetramers, we could further demonstrate that the enriched T cells were polyclonal recognizing multiple LMP2A epitopes and retained their spectrum of specificity throughout the enrichment and expansion. This system provided the foundation for T cell therapy against EBV-positive HL with new generation therapeutic T cells.