In Vitro Study of New Photochemotherapeutic Compounds for Extracorporeal Photopheresis.
Authors: Garcia-Bernal David , Blanquer Miguel , del Rio Jose Antonio , Correal Enrique , Lopez Javier , Majado Maria Juliana , Moraleda Jose M 
Published: October 2019

Abstract
Abstract 2144

Introduction:
Extracorporeal photopheresis (ECP) is a cell-based immunomodulatory therapy involving the separation of autologous mononuclear cell fraction followed by ex-vivo administration of 8-methoxypsoralen (8-MOP) and UVA irradiation before reinfusion. ECP is efficient for the treatment of cutaneous T-cell lymphomas, multiple skin disorders, autoimmune diseases, solid organ transplant rejection and graft versus host disease (GVDH). During UVA irradiation phase, 8-MOP binds covalently to leukocytes' DNA leading to cell cycle arrest and apoptosis. These pre-apoptotic leukocytes are reintroduced into the peripheral circulation where they are phagocytosed by immature dendritic cells, which play a crucial role in the induction of peripheral tolerance through the induction of T regulatory cells subsets, production of anti-inflamatory cytokines such as IL-10 and TGF-b and the deletion of effector antigen-specific CD4+ and CD8+ T cells into lymph nodes. Our aim in the present work was to compare the therapeutic effectiveness of 8-MOP with other four new compounds (BB01 to BB04).

Methods:
Mononuclear cells (MNC) were isolated by ficoll density gradient, incubated with increasing concentrations of 8-MOP, BB01, BB02, BB03 and BB04 and irradiated with UVA light (2 J/cm2). MNC apoptosis percentage was measured by flow cytometry (Annexin-V and 7-AAD staining) after 48h of culture at 37°C. Mixed lymphocyte cultures (MLC) with either myeloid immature (iDC) or mature dendritic cells (mDC) and UVA irradiated MNC treated with the different compounds were performed. After 48h of MLC CCL21-stimulated migration of iDC and mDC was studied. In other assays, after 6 days of MLC the proliferation of treated MNC (BrdU incorporation) and the production of pro-inflammatory and anti-inflammatory cytokines were analyzed.

Results:
After incubation of MNC with the different compounds + UVA there was a significative increase of apoptosis percentage using BB02 (from 50 ng/ml, p<0.001) and BB01 (from 200 ng/ml, p<0.001) over that achieved using 8-MOP, while BB03 and BB04 induced significatively less apoptosis. Furthermore, in comparation with 8-MOP, there was a significant upregulation of the CCL21-promoted migration of iDC (p<0.05) and lower migration of mDC (p<0.001) respectively, when these DC were co-cultured with MNC treated with BB02 + UVA. On the other hand, MNC proliferation achieved after MLC with allogeneic iDC or mDC was partially inhibited after treatment with all tested compounds + UVA. These inhibition was higher using BB02 and BB01, although the difference was no statistically significant when compared to 8-MOP. In addition, in MLC using treated MNC and stimulator iDC there was a reduction in the release of pro-inflammatory cytokines (IFN-g, IL-6 and IL-17A) and an increase of anti-inflammatory cytokines such as IL-10 and TGF-b that was higher using BB02 + UVA.

Conclusions:
Compounds BB01 and specially BB02 were more efficient than 8-MOP in the induction of apoptosis and the upregulation of chemokine-promoted migration of iDC. They were also better in the inhibition of the proliferation of MNC stimulated by iDC and mDC, and in the induction of anti-inflammatory cytokine production. This higher in vitro activity might be relevant to increase the therapeutic potential of ECP.

Acknowledgments:
Work financed by the Spanish Ministry of Science and Innovation (Ref: BFU2010–19599). Spanish Net of Cell Therapy (TerCel) Carlos III Institute.

Disclosures:
No relevant conflicts of interest to declare.