Generation of Combined CD8+ and CD4+ T Cell Lines with High Specificity for Adenovirus Hexon Epitopes for Adoptive Immunotherapy after Allogeneic Stem Cell Transplantation.
Authors: Zandvliet Maarten L. , Falkenburg J.H. Frederik , Veltrop-Duits Louise A. , Schilham Marco W. , Willemze Roelof , Guchelaar Henk-Jan , Meij Pauline 
Published: April 2020

Abstract
Human Adenovirus (HAdV) can cause serious morbidity in immunocompromised patients, in particular in pediatric recipients of allogeneic stem cell transplantation (alloSCT). Progression to disseminated adenoviral disease is associated with a high mortality, despite treatment with antiviral agents such as ribavirin and cidofovir. It has been demonstrated that reconstitution of HAdV-specific T cells is essential to control adenoviral infection after alloSCT. Adoptive transfer of donor-derived HAdV-specific T cells may therefore be a strategy to provide long-term protection from HAdV. In healthy individuals, T cells directed against HAdV are only detected at low frequencies and are predominantly directed to the HAdV hexon protein. Only recently, a number of immunodominant CD8+ and CD4+ epitopes of HAdV hexon have been defined. Since these epitopes are largely conserved between the different HAdV subgroups, T cells specific for these immunodominant epitopes may provide protection from a wide range of adenoviral serotypes. The aim of this study was to develop a method for the generation of combined CD8+ and CD4+ T cell lines with high and well defined specificity for the HAdV hexon protein. We first analyzed the frequencies of HAdV hexon-specific CD8+ and CD4+ T cells in healthy individuals using sensitive measurement by peptide-MHC tetramers, and intracellular cytokine staining combined with CD154 or peptide-MHC tetramer staining, after stimulation with defined MHC class I peptides, 30-mer peptides containing class II epitopes, or a HAdV hexon protein-spanning pool of overlapping 15-mer peptides (Miltenyi Biotec, Germany). We demonstrated that the frequencies of HAdV hexon-specific T cells were very low in most healthy individuals tested. HAdV hexon-specific CD8+ T cells were detectable in only 3/15 individuals (range 0.16–0.43% of CD8+ T cells), and hexon-specific CD4+ T cells were detected in all individuals with a median of 0.07% (range 0.004–0.38% of CD4+ T cells). The highest frequencies were found after stimulation with the hexon protein-spanning 15-mer peptide pool, indicating activation of both known and unknown epitopes. Kinetic analysis showed highest levels of IFNg production after 4–8 hours of stimulation for HAdV-specific CD8+ T cells, and after 4–48 hours of stimulation for HAdV-specific CD4+ T cells. The phenotype of these HAdV hexon-specific T cells corresponded to an early memory phenotype, CD27+, CD28+, CD62L+, CD45RO+. Despite these low or undetectable frequencies of HAdV-specific T cells, IFNg-based enrichment 4 hours after activation with the HAdV hexon protein-spanning peptide pool resulted in efficient isolation of CD8+ and CD4+ T cells recognizing both known and unknown HAdV hexon epitopes. Following a short culture period of 7 days, the T cell lines consisted of 49–80% CD8+ T cells and 13–15% CD4+ T cells. Restimulation by autologous EBV-LCL loaded with HAdV hexon peptide pool followed by intracellular IFNg staining showed that the frequency of HAdV-specific T cells was increased to 65–95% of CD8+ T cells, and 38–72% of CD4+ T cells. The frequency of HAdV-tetramer-positive cells was increased to 32–76% of CD8+ T cells, indicating that part of HAdV-specific CD8+ T cells recognized known epitopes. After 14 days, the frequency of HAdV-specific T cells had further increased to 89–94% of CD8+ T cells and 61–91% of CD4+ T cells. Starting with only 25x106 donor peripheral blood mononuclear cells, this strategy yielded T cell lines containing 1.3–2.7x106 HAdV-specific combined CD8+ and CD4+ T cells in 14 days. We conclude that we developed a GMP-grade method for the fast generation of highly HAdV-specific CD8+ and CD4+ T cell lines from all healthy donors tested, irrespective of HLA-restriction, for the treatment HAdV infection after alloSCT, with very limited risk of graft-versus-host disease.