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.
Pityrosporum orbiculare
‐Reactive T‐Cell Lines in Atopic Dermatitis Patients and Healthy Individuals
