Efficient Induction and Isolation of CMV-Specific CD8+ T Cells from CMV Seronegative Donors for the Treatment of CMV Reactivation in CMV Seropositive Patients Transplanted with a CMV Seronegative Donor.
Authors: Jedema Inge , van de Meent Marian , van der Heiden Pim L.J. , Marijt Erik W.A. , Meij Pauline , Willemze Roel , Falkenburg J.H. Frederik 
Published: October 2019

Abstract
Cytomegalovirus (CMV) disease is a significant cause of morbidity and mortality after allogeneic stem cell transplantation (allo-SCT) in CMV seropositive (CMV+) patients. In a recent cohort of CMV+ patients, we investigated the impact of donor CMV serostatus on the severity of CMV reactivation after T cell depleted allo-SCT. A high incidence of CMV related mortality was seen in patients transplanted with a CMV- donor (5/20) whereas no CMV-related deaths occurred after transplantation with a CMV+ donor (0/20). In most CMV+ patients transplanted with a CMV+ donor reconstitution with CMV specific (memory) T cells was found. We recently performed a phase I/II clinical study using isolated CMV-specific CD8+ memory donor T cells for the treatment of patients with persistent CMV reactivation despite seropositivity of the donor. In this study we demonstrated the feasibility of isolating and selecting CMV-specific CD8+ memory T cells from CMV+ donors using the interferon-gamma (IFNg) capture assay and CliniMACS isolation after peptide stimulation of the CMV-specific donor T cells. We have illustrated the in-vivo potential of these T cells after adoptive transfer in 5 patients, resulting in clearance of the CMV load. However, no suitable method was available for the induction of primary immune responses against CMV for the treatment of persistent CMV reactivation in patients transplanted with a CMV- donor. In the current study we investigated the possibility to induce and isolate CMV-specific T cells from CMV- healthy donors by in-vitro priming and selection. We used as responder cells CD14- CD45RO- PBMC from HLA-A1, A2, A3, B7, or B8 positive CMV- healthy donors (n=10). By CD45RO depletion we removed the majority of regulatory T cells (Tregs) capable of inhibiting the initiation of the response. Mature monocyte-derived dendritic cells (DCs) were loaded with a cocktail containing 1μg of each relevant CMV pp65, pp50, or IE1 derived 9-mer peptide, depending on the HLA type of the donor. Naïve donor T cells were cocultured in a 10/1 ratio with peptide loaded DCs. From day 4 on 5 ng/mL IL-7 and IL-15 was added to the culture. At day 10, the responses were specifically restimulated with peptide loaded autologous PBMC. Cytokines were refreshed twice weekly. At day 20 CMV-specific CD8+ T cells were detected and isolated by specific tetramer staining and flowcytometric cell sorting, by specific pentamer staining and immunomagnetic bead isolation, or further enriched by another restimulation, followed by isolation of CD137 expressing T cells at day 21. In 10/10 CMV seronegative donors CMV specific T cells could be detected at day 20 of the immune response in frequencies ranging from 0.01–0.4%. These tetramer positive cells could be isolated and expanded both in bulk cultures and clonally. Functional CMV-specific T cells against all 3 major immunogenic CMV proteins pp65, pp50, and IE1 were detected and isolated with different dominant responses detected in different donors. In conclusion, we have developed a method for the in-vitro induction and isolation of functional CMV-specific CD8+ T cells from CMV- donors. This may allow the treatment of serious CMV-related complications in CMV+ patients transplanted with a CMV- donor.