Investigating the Role of Dendritic Cells in Extracorporeal Photopheresis Using an In Vitro Model.

Abstract

Graft versus Host disease (GvHD) is the major limitation to successful allogeneic hematopoietic stem cell transplantation and contributes significantly to transplant related mortality and morbidity. Steroid refractory or steroid depending GvHD in particular is linked to poor survival or life quality. Conventional immunosuppression has very limited success in these conditions and increases susceptability for infection and relapse.Extracorporeal Photopheresis (ECP) is a promising therapy for acute and chronic GvHD not responding to conventional immunosuppressive therapy. ECP treatment seems not to result in a pan-immunosuppression but has quite selective effects on the pathogenic process in GvHD.The mechanisms of action of ECP in GvHD known so far include lymphocyte apoptosis, cytokine modulation and increased regulatory T cell numbers. It has been suggested that monocytes and dendritic cells (DC) were more resistant to ECP induced apoptosis, so that they might either be directly modulated by ECP or modulated by encounter with apoptotic cells and this way aquiring immunomodulating properties. Here we tested the first hypothesis analysing direct effects of ECP on monocyte derived dendritic cells in vitro.Direct in vitro PUVA treatment leads to activation of monocyte derived immature DCs (upregulation of CD83, CD86, HLA-DR, reduced endocytosis capacity, increased migratory capacity), whereas mature DCs are not affected. However, immature and mature DCs undergo apoptosis later on within 24h-72h. Prestimualtion with either antigen (KLH), IL-12 or co-culture with CD40L expressing cells could not prevent apoptosis. Further DC stimulation through CD40L is abrogated 24h after treatment. DC cytokine analysis (IL-12, TNFα) is pending. After in vitro PUVA primary dermal DCs and Langerhans Cells migrated from skin biopsies undergo apoptosis similarly to monocyte derived DCs.Going along with the early activation, the stimulatory capacity of in vitro PUVA treated immature DCs is enhanced in an MLR on naive T cells. IL-10 levels are decreased as compared to untreated or UVA treated cells. Secondary stimulation through either CD3/CD28 beads or mature DCs leads to reduced proliferation of naive T cells that were primarily stimulated with in vitro PUVA treated as compared to untreated immature DCs. Cytokine analysis after secondary stimulation is pending.We conclude that in our model in vitro PUVA modulates the character especially of immature dendritic cells. However, immunostimulating and immunosuppressive characteristics could be described depending on the assay. Cytokine results after second stimulation (Th1/Th2) are pending. Apoptosis is a major finding and occurs both in monocytes and DCs after 24–72h. Our findings indicate that there might be direct ECP effects on monocytes and DCs going beyond the process of merely depleting antigen presenting cells. Further relevance is currently being investigated.