Rapid Generation of Full Clinical-Grade Human Antiadenovirus Cytotoxic T Cells for Adoptive Immunotherapy

Abstract

Adenovirus (ADV) infections are one of the major causes of morbidity and mortality after hematopoietic stem cell transplantation, despite new antiviral treatment strategies. We describe here a complete clinical-grade generation of human anti-ADV cytotoxic T cells to propose an adoptive immunotherapy. Peripheral blood mononuclear cells (PBMC) from 7 healthy donors, known for their good cellular immunity against ADV, were stimulated for 6 hours with a synthetic peptide pool covering the ADV5 Hexon protein interferon-gamma (IFN-gamma) secreting cells were isolated on a clinical device. After immunoselection, a mean number of 1.01 +/- 0.84 x 10(6) total nucleated cells was obtained. The isolated ADV-specific T cells were mainly CD4+ (mean=56% +/- 20.8%, yield=51% +/- 32.4%) but also CD8+ (mean=42% +/- 27%, yield = 56% +/- 39.3%). Isolated T lymphocytes (CTL) were expanded to carry out functional tests. Ability of the expanded CTL to secrete IFN-gamma and to proliferate after restimulation with the ADV peptide pool was confirmed. A high cytotoxicity against autologous target cells loaded with ADV antigens was observed but not against nonloaded target cells. We observed a decrease of 1.27 log of the allogeneic reaction against non HLA identical healthy donor PBMC with CTL compared with the PBMC before selection. Clinical-grade generation of ADV-specific T cells was achieved with a synthetic antigen. This technology has the advantage of being fast, and is sufficiently reactive to be proposed for immunotherapy if antiviral treatment fails.

Adenovirus-specific T-cell Subsets in Human Peripheral Blood and After IFN-γ Immunomagnetic Selection

“…Our group previously reported a frequency of 0.04%-0.58% for ADV-specific CD4 + T cells and 0.02%-0.72% for ADV-specific CD8 + T cells in 10 healthy donors. 23 Among CD3 IFN-g + T cells, the T EM memory compartment was the most abundant, as expected. .…”

“…After isolation, virus-specific CD4 + T cells were the immunodominant compartment after PepTivator-ADV5 Hexon stimulation as previously reported [23][24][25] (Fig. 3C).…”

“…These cells were subsequently processed using the IFN-g-based immunomagnetic technology as previously described. 22,23 Finally the VSTs were obtained. Then after immediate phenotypic analysis, the remaining VSTs were cultured for 2-4 weeks in RPMI-1640 supplemented with 10% human AB serum and 100 IU/mL of IL-2 (Pepro-Tech, TEBU, Paris, France) in a culture flask.…”

“…Effector cells were added at an effector-to-target cell ratio of 10:1 in triplicate, as previously defined in our preclinical report. 23 After a 2-hour incubation at 371C, 5% CO 2 , plates were centrifuged (500g, 5 min) and 20 mL of the supernatant was transferred to a DELFIA yellow 96-well plate (Perkin Elmer). Two hundred microliters of DELFIA Europium Solution was added to each well and incubated for 15 minutes at room temperature.…”

How I treat adenovirus in hematopoietic stem cell transplant recipients

“…AdV-activated CD4 ϩ T cells have been shown to produce interferon-␥ (IFN-␥) and can be cytotoxic, being able to lyse AdV-infected cells in vitro. 3,4 Approximately one-third of the AdV genome is devoted to counteract innate and adaptive immune defenses compromising the development of a protective immune response. 5 The frequent reinfections and persistence of the virus in children on the one hand and the presence of AdV-specific CD4 ϩ T cells in asymptomatic adults on the other hand are a sign that the development of an adequate specific cross-reactive immune protection against AdV takes many years to develop.…”