Objectives: One of the major life threatening fungal infections associated with allogeneic stem cell or bone marrow transplantation is invasive aspergillosis (IA). Dendritic cells represent an important element of the innate immune system in the defence against Aspergilli; they acquire antigens in the periphery, migrate into secondary lymphoid tissues, activate T-cells and maturate and secrete cytokines and chemokines. Up until now, the infl uence of distinct Aspergillus fumigatus molecules on the human innate immune system is largely unexplored. Methods: We have characterized the interaction between the A. fumigatus antigens Aspf1 and Crf1 and human monocytederived immature dendritic cells (iDC), respectively. Antigens were recombinantly expressed in the Pichia pastoris system and subsequently used for stimulation experiments, which allowed quantifying gene expression (by qRT-PCR) and secretion of cytokines and chemokines (by ELISA assays) in iDC, to localize microscopically Aspf1 and to determine its capacity to induce apoptosis in iDCs. Furthermore, by mixed lymphocyte reactions (MLR), we have analyzed whether iDCs loaded with Aspf1 are able to induce T-cell proliferation. Results: Compared to Crf1, Aspf1 showed a high ability to specifi cally stimulate iDCs as demonstrated by an increased expression of genes encoding for pro-infl ammatory cytokines (e.g. IL-12p35, TNFa) and chemokines (CXCL10, CCL20, IL- 8). By using germlings of a deletion mutant for Aspf1 (dAspf1) in confrontation assays with iDCs, increased protein secretion compared to stimulation with wild type morphologies was detectable. Apoptosis of iDCs was markedly induced after cocultivation with Aspf1. MLR revealed that iDCs were not able to present Aspf1 to autologous T-cells. Conclusion: This study demonstrates that recombinant A. fumigatus antigens are able to effi ciently stimulate iDCs in vitro; these observations might potentially lead to the development of a new therapeutic and/or prophylactic option to prevent and treat this devastating infection in high risk patients after allogeneic stem cell transplantation.
Full conference title:
Annual Meeting of the EBMT, 36th
- EBMT 36th (2010)