Determination of Optimal Target Antigen and Immune Modulatory Approaches to Improve and Standardize Epstein-Barr Virus-Specific Adoptive T Cell Therapy
Authors: Mitchell Monica L , Patton John T. , O'Donnell Lynn , Baiocchi Robert A. 
Published: October 2019

Abstract
Epstein-Barr Virus (EBV) is a ubiquitous gamma herpesvirus that infects lymphocytes and epithelial cells, establishing lifelong latency. Immunosuppression is associated with a transition from a quiescent, latent EBV infection to either a lytic or active latent life cycle, which involves transformation of memory B cells to rapidly growing lymphoblastoid cell lines (LCLs). Due to the immunogenicity of EBV viral proteins, cells expressing most latent or lytic peptides are eradicated through immune surveillance mechanisms. EBV-associated lymphoproliferative disorders (LPDs) arise in immunocompromised individuals and are facilitated by the expression of the same viral proteins that are expressed in the immune tolerant environment. Furthermore, EBV-LPDs acquire various mechanisms to suppress immune function, such as expression of the programmed death ligand (PD-L1), CTLA-4, amino acid depletion, and propagation of suppressive immune cell subsets, including tumor-associated macrophages (TAMs) that attenuate T cell receptor signaling and cytokine production. These immunosuppressive mechanisms increase the risk of acquiring EBV viremia and can facilitate the development of LPDs. In other work reported by our group (Patton et al, ASH abstract 2014), a TAM-like population has recently been discovered to spontaneously expand in peripheral blood mononuclear cell (PBMC)/LCL co-cultures. These TAM-like macrophages are capable of potent cytotoxicity against EBV-specific T cells, thus novel strategies to counteract this negative regulatory network are needed.
One effective strategy developed to reestablish EBV-specific immunity is adoptive T cell therapy. However, the optimal combination of target antigens required to protect against EBV-LPD is not well characterized. It remains plausible that expansion of cytotoxic and/or helper T cell populations specific for multiple EBV antigens will improve the efficiency of the T cell response. The suppression of negative immune regulators may further enhance the effectiveness of the adoptive therapies to provide optimal control of viremia and effectively treat EBV-driven malignancies.
Here, we generated EBV-specific T cell preparations by culturing PBMCs supplemented with interleukins 4 and 7 (IL4, IL7) in the presence of latent (EBNA1, EBNA3, LMP1, LMP2) or lytic (BZLF1, BRLF, BMLF1, BMRF1) PepMixes (complete protein-spanning pools of overlapping peptides). Cells were separated into two groups, a test and expansion group, to determine optimal ex vivo expansion. Activity of T cells was measured by direct flow-based cytotoxicity against LCL targets and interferon gamma (IFNγ) production via Enzyme-Linked Immuno Spot (ELISpot).
Preliminary ELISpot assays from two donors in the test group showed that BZLF1-specific T cells produced as much as 34-fold more IFNγ in comparison to other EBV antigens (standardized to a negative control). T cells produced in culture with a peptivator PepMix, a collection of 13 latent and lytic peptide pools, on average exhibited the second highest levels of IFNγ release, expressing as much as 1.4-fold more IFNγ than the next highest antigen-specific response, depending on the donor. Additionally, LMP1 produced as much as 24-fold higher levels of IFNγ. Therefore, given the heterogeneous EBV viral gene expression profiles and variation of immune dominance across human leukocyte antigen (HLA) types, use of polyclonal immunogen-specific T cells may be more effective in improving survival and long-term protection from EBV-driven complications via improved memory T cell surveillance. Additional ex vivo work is currently underway examining combinations of lytic and latent antigens based on immunodominance analysis across HLA types. Finally, use of immune modulatory mechanisms to address newly discovered TAM-like populations contributing to reduced immune effector function against EBV-LPD are currently being evaluated. We are testing strategies to promote PD-L1 and CTLA-4 blockade, inhibition of amino acid depletion, and down regulation of regulatory T cells, to allow for enhanced expansion of antigen-specific T cell populations. Confirmation of these hypotheses will be directed toward developing streamlined methods for efficient, rapid, and personalized T cell preparation that can be standardized for translation into clinical trials.

Disclosures
No relevant conflicts of interest to declare.