Presentation of Epstein Barr Virus (EBV) Epitopes by Activated Human γδ T Cells Induces Peptide-Specific Cytolytic CD8+ T Cell Expansion.
Authors: Landmeier Silke , Altvater Bianca , Pscherer Sibylle , Varnholt Lena , Bollard Catherine M. , Juergens Heribert , Rossig Claudia 
Published: October 2019

Abstract
Efficient antigen presentation is an important prerequisite for the induction of T cell mediated immunity against viral and tumor antigens. The functional heterogeneity, limited availability and poor expansion of dendritic cells (DC) has motivated a search for alternative sources of antigen-presenting cells (APC). Bisphosphonate-activated human γδ T cells were shown to function as APC by inducing primary T cell responses to allogeneic and microbial antigens. Here, we extended these observations by investigating the capacity of activated γδ T cells to present Epstein Barr virus lytic cycle and latency antigens in a peptide-specific manner and induce expansion of fully functional, virus-specific cytotoxic αβ T cells. Peripheral blood-derived γδ T cells were expanded from three individual healthy donors by stimulation with the aminobisphosphonate zoledronic acid (1 μg/ml) in the presence of rhIL-2 (100 U/ml) and rhIL-15 (10 ng/ml). Under these conditions, γδ T cells (Vγ9+Vδ2+) acquired a phenotype characteristic for APC, including upregulation of HLA-DR as well as the costimulatory ligands CD80, CD83, CD86, CD40, and 4-1BBL (CDw137L). Whereas expression of most markers decreased after peak levels were reached on day 5, CD86 and HLA-DR remained upregulated on >80% of the cells for prolonged culture periods of >14 days. We next assessed the APC function of activated γδ T cells by testing their ability to stimulate expansion of antigen-specific cytotoxic T cells in vitro. Autologous peripheral blood lymphocytes (PBMC) were incubated with activated γδ T cells pulsed with the HLA-B8-restricted epitope of the EBV lytic cycle antigen BZLF1 (RAKFKQLL). We demonstrated selective expansion of RAK-pentamer-specific CD3+ CD8+ T cells to a mean of 15% (range 6–24%) on day 10. BZLF-1 represents a highly immunogenic virus antigen, whereas the EBV latency-associated antigen LMP-2a is less T cell-stimulatory and, due to its expression on Hodgkin′s lymphoma cells, is an important target for tumor-specific immune therapy. Stimulation of PBMC with γδ T cells pulsed with the HLA-A2-restricted LMP2a epitope FLYALALLL also resulted in a substantial increase of pentamer-reactive T cells to a mean of 3.5% (1.4–5.5%), which was comparable to the increase obtained when peptide-loaded autologous DC were used (mean of 3.5%; range 1.2–5.7%). γδ T cell stimulation in the absence of peptide failed to induce specific T cell expansion (mean of 0.1%; 0.03–0.15%). CD8+ T cells expanded against EBV-peptide-pulsed activated γδ T cells functionally interacted with peptide-loaded autologous DC or lymphoblastoid cell lines (LCL), as demonstrated by efficient and specific MHC class I-restricted cytolysis of 41–65% at an effector-target ratio of 40:1. Peptide-specific cytotoxic T cell functionality was comparable to that obtained with T cells expanded in the presence of pulsed autologous DC from the same donors. In summary, bisphosphonate-activated γδ T cells are potent antigen-presenting cells for reproducible expansion of disease antigen-specific CTL. Thus, they represent a novel source of highly efficient professional APC for antigen-specific immunotherapy of viral or malignant disease.