Compositional and functional analyses of ECP-elicited soluble factors

Extracorporeal photopheresis (ECP) is a therapy for different T cell-mediated diseases that can exert both immune-stimulatory and immune-regulatory effects. However, its mechanism of action is poorly understood. Although previous studies have identified cellular mediators elicited by ECP, little is known about the implication of cell-free soluble components. Here, we studied the composition of ECP-elicited soluble factors and their functional effect on different immune cells in vitro.

Human peripheral blood mononuclear cells (PBMCs) of healthy blood donors were treated with 8-methoxypsoralen (8-MOP) for 30 min and UVA in vitro. 12 different combinations of 8-MOP (0, 200, 300, or 400 ng/mL) and UVA (0, 2, or 3 J/cm²) were tested. Cell-free supernatants (ECP-SNs) were collected after 48h to measure cytokine and chemokine concentrations using a cytometric bead-based immunoassay. The impact of ECP-SNs on survival of immune cell subsets was tested by exposing PBMCs to ECP-SNs for 48h followed by Annexin V and Sytox staining by flow cytometry. The effect of ECP-SN on T cell proliferation and activation and was analyzed by stimulating magnetically enriched T cells with anti-CD3 in the presence or absence of ECP-SNs.

ECP-SNs collected after 48h contained significantly higher levels of IL-6 and reduced levels of MCP-1 in an 8-MOP-dependent manner compared to untreated PBMCs. IL-1β and IFN-? were also increased and decreased respectively, though not significantly. IL-8, IL-10, IL-2, and TNF-α remained unchanged. Regarding the functional characterization of ECP-SNs, ECP-SNs did not affect survival of major immune cell subsets like B cells, T cells, monocytes, and NK cells upon 48h. Moreover, ECP-SNs did not alter T cell proliferation nor CD25, CD69, nor CD71 activation marker expression within 5 days.

We showed that different ECP conditions can alter the composition of soluble factors that could be relevant to the mechanism of action of ECP. However, deeper compositional analyses such as metabolomics are needed. Moreover, this data suggests that soluble factors might not affect allogeneic T cell proliferation nor activation. Nevertheless, here we used ECP-SNs generated by unstimulated PBMCs that do not mimic inflammation present in patients. Further experiments testing ECP-SNs on additional immune cell subsets (e.g. monocytes and B cells) could unveil unknown mechanisms. This could help discover new biomarkers of potency of ECP and broaden its clinical application.

There are no financial conflicts of interest to disclose