Date Published: March 1, 2019
Publisher: Public Library of Science
Author(s): Marie Laulhé, Sylvie Lefebvre, Delphine Le Broc-Ryckewaert, Maxime Pierre, Aurélie Ferry, Bruno Delorme, Senthilnathan Palaniyandi.
Extracorporeal photopheresis (ECP) is an autologous immunomodulatory cell therapy that consists of the ex vivo collection of mononuclear cells (MNCs), which are irradiated with UVA in the presence of the photosensitizing agent 8-methoxypsoralen (8-MOP) to induce cell apoptosis. This photoactivated cell preparation is then reinfused into the patient. While the clinical benefits of ECP are well-demonstrated, no study has yet characterized the influence of variations in the composition of the cell preparation on the efficacy of ECP in vitro. Here, we describe a standardized methodology for the in vitro assessment of ECP that uses the human lymphoma T-cell line and mimics the clinical procedure. By quantifying cell apoptosis, inhibition of cell proliferation, and 8-MOP consumption, we used this approach to characterize the specific influence of key variables on the cellular response to ECP. We found that (i) increases in hematocrit and plasma concentrations attenuated the cellular response to ECP; (ii) plasma concentration was the only variable tested that influenced 8-MOP consumption; and (iii) the loss of efficacy due to variations in the concentration of certain blood components could be counteracted by modulating the UVA dose. This methodology may enable evaluation of other leukapheresis preparation protocols and better determination of the optimal working parameters for ECP.
Extracorporeal photopheresis (ECP) is an immunomodulatory cell therapy in which patients receive autologous infusions of apoptotic mononuclear cells (MNCs) for the treatment of a variety of diseases involving immune dysfunction [1–3]. Although effective and used for several decades, the precise in vivo mode of action of ECP is poorly understood [2, 4, 5]. ECP is carried out in 3 stages: 1) MNCs are withdrawn from the patient by apheresis; 2) the cells are then incubated with a photosensitizing agent (8-methoxypsoralen or 8-MOP) and exposed to UVA; and 3) irradiated MNCs are reinfused into the patient [6–8]. 8-MOP passively enters the cells where it intercalates into the DNA within 2 minutes [9, 10]. Upon irradiation with UVA photons, 8-MOP induces DNA cross-linking, which interferes with the cell replication machinery [11–14]. ECP treatment inhibits cell proliferation and induces T-cell apoptosis and the release of immunomodulatory cytokines [6–8]. Irradiation-activated dendritic cells (DCs) transform into antigen-presenting cells (APCs), which recognize and phagocytose ECP-treated apoptotic T-cells and ultimately induce regulatory T-cell (Treg) activation. In graft-versus-host disease (GvHD), Tregs attenuate immune reactions by secreting tolerogenic cytokines [15, 16], while in cutaneous T-cell lymphoma (CTCL), ECP-induced Tregs secrete inflammatory cytokines, which exert immunostimulatory effects on neoplastic cells [6, 17, 18]. Two ECP techniques are currently used to treat immunological disorders: “on-line” and “off-line” ECP, depending on whether MNCs are collected and irradiated using 1 or 2 devices, respectively.
In this study, we present a standardized methodical approach for the in vitro assessment of ECP, and use this approach to quantify for the first time the influence of individual components of the cell preparation and of UVA dose on in vitro ECP efficacy. Experiments were performed using the immortalized JURKAT cell line, thereby avoiding the response to treatment variability and mitogen-induced proliferation efficacy associated with PBMCs obtained from different donors, and allowing standardization of the treatment to ensure a more precise analysis. Apoptosis of JURKAT cells increased in a time-dependent manner over a 2-day period after UVA irradiation, irrespective of variations in the composition of the cell preparation in the irradiation container. This apoptotic process may account for the progressive immunomodulatory effects observed in patients after ECP treatment [7, 40].