Investigation of the role of monocytes/macrophages in allogeneic immune response

Abstract

Hematopoietic stem cell transplantation (HSCT) is a therapy for many diseases, however, development of graft versus host disease (GVHD) severely limits the successful use of HSCT. Pre-transplant conditioning including total body irradiation (TBI) causes tissue damage and cytokine production that activates antigen presenting cells (APCs). This change triggers monocyte differentiation to macrophages that present alloantigen to T cells during early GVHD. A better understanding of monocytes/macrophages activation during the early phase of alloresponse is needed. This study evaluated monocyte/macrophage function during the early phase alloresponse by measuring surface marker expression in whole blood mixed leukocytes reaction (MLR). To simulate GVHD inflammatory conditions, IFN-γ or IL-4 were added. In response to the allogeneic cells in MLR culture (day 1 and 2), monocytes/macrophages showed significant up-regulation of CD86, HLA-DR, CD64, C3aR and CD204 demonstrating strong monocyte/macrophages activation, accompanied by secretion of TNF-α, IFN-γ, IL-2 and IL-6 (day 2), and T cell activation (day 3). Unexpectedly, IFN-γ addition to MLR culture did not affect co-stimulatory potential or antigen presentation, it instead induced significant up-regulation of CD64 expression, suggesting stimulation of phagocytosis and pathogen defence. Whereas, IL-4 addition significantly up-regulated co-simulation and Ag presentation (day 2), reducing T cell alloreactivity on day 3. Spleen tyrosine kinase (SYK) phosphorylation regulates monocytes/macrophages activation, thus the SYK pathway is a potential therapeutic target for MLR and GVHD. Data in this study showed that the highly selective SYK inhibitor, PRT0603, modulated monocyte/macrophage activation in MLR culture by significantly reducing CD64 (FcγRI) expression, antigen presentation and T cell alloreactivity. In MLR IFN-γ and MLR IL-4 the anti-SYK effects on monocyte/macrophages function were modest. However, a subsequent decrease in T cells alloreactivity was observed in MLR IFN-γ culture due to PRT0603 treatment. More importantly, a significant reduction in cytokine production was observed in all treated cell cultures. This reduction could stop the inflammatory cycle and prevent the development of allogeneic response in GVHD.