Apoptosis kinetics of purified and PBMC-associated lymphocytes after photopheresis-like 8-methoxypsolaren/UVA treatment.

Abstract

Studies of apoptosis kinetics in extracorporeal photopheresis (ECP) contribute to identifying modifications that can positively impact the configuration of ECP offline in a clinical setting. This work compared for the first time the apoptosis kinetics of human peripheral blood mononuclear cell (PBMC)-associated T lymphocytes versus the isolation of high-purity T cells using the MacoGenic G2 system, and times with predictive value were identified through ROC-AUC analysis, which facilitates quality control of the ECP. PBMC and T lymphocytes from healthy donors were exposed to 300 ng/mL of 8-methoxypsoralen combined with 2 J/cm² of ultraviolet A light (320-400 nm) using the MacoGenic G2 system. The cells were then cultured in vitro, and immunophenotyping and apoptosis induction were assessed by flow cytometry analysis. Apoptosis levels in monocyte controls reached 47% at 24 h, while treated monocytes maintained high apoptosis at 90.4%. Both PBMC-associated lymphocytes and isolated T cells exhibited sigmoidal apoptosis kinetics; however, PBMCs exhibited a lower apoptosis percentage (65.2%) compared with isolated T cells (73.8%); this difference was not statistically significant (p = 0.8229). Early apoptosis was faster in NKT, NK, and Tc cells, while B and Th cells exhibited slower kinetics, peaking at 24 h. Isolated T cells showed a faster early apoptosis peak after irradiation (baseline measure), with NKT cells most affected. AUC analysis at 24 h demonstrated high predictive accuracy for apoptosis in PBMCs (AUC = 0.97, p = 0.0004) and isolated T cells (AUC = 1, p = 0.0003). Nearly all treated PBMC-associated lymphocytes and isolated T lymphocytes underwent apoptosis within 72 h. Apoptosis kinetics were comparable, though slightly accelerated in T lymphocytes. Predictions of ECP effects showed high sensitivity and specificity at 24 h for PBMCs and at 4 h after treatment (baseline) for isolated T lymphocytes, with distinct apoptosis susceptibility patterns.