Cryopreserved ECP-treated lymphocytes maintain apoptotic response and anti-proliferative effect.

Abstract

The ability to cryopreserve a portion of the cells treated during extracorporeal photopheresis (ECP) would improve therapy logistics, particularly for pediatric patients, by allowing multiple therapeutic doses to be collected from a single apheresis session. However, the effect of cryopreservation on ECP-treated cells is unknown (e.g., ECP-induced lymphocyte apoptosis and inhibition of proliferation). Mononuclear cell (MNC) apheresis products collected from healthy subjects were ECP-treated using offline methods. Fresh samples of ECP-treated and control cells were placed immediately in culture. The remainder of the cells were frozen in cryovials (n = 8) or cryobags (n = 8) at -80°C. After 1 week of -80°C storage, ECP-treated and control cells were thawed rapidly and samples were placed in culture. Lymphocyte apoptosis was assessed by phosphatidylserine exposure using Annexin V/7-AAD labeling. Lymphocyte proliferation after 3 days culture was measured using the carboxyfluorescein succinimidyl ester labeling technique. On Day 0, apoptosis levels were <5% in fresh ECP-treated and control cells and approximately 20% on thawing of cryopreserved ECP-treated and control cells. Apoptosis levels were comparable between the two cryopreserved groups immediately on thawing, indicating that ECP-treated cells were no more sensitive to the cryopreservation process than control cells. During 72-h culture, apoptosis levels increased to >80% in fresh and cryopreserved ECP-treated cells but remained near constant in both control groups. Inhibition of lymphocyte proliferation was >95% in all ECP-treated cells with no significant difference between fresh and cryopreserved cells (P = 0.12). Cryopreservation did not impair the apoptotic response or anti-proliferative effect of ECP-treated lymphocytes, thereby demonstrating early feasibility of this approach.