Development of an in vitro assay to assess circulating monocyte phagocytosis of Aspergillus fumigatus conidia from patients with chronic graft-versus-host disease

Claire Kenny*, Oliver Morton, Eibhlin Conneally, Anne Atzberger, Anthony Davies, Thomas Rogers

Author address: 

Dublin, IE


Objectives: Invasive aspergillosis (IA) is a frequently life-threatening complication of allogeneic haematopoietic stem cell transplantation. Recent epidemiologic studies suggest an incidence of 5-10%. It is now recognised that in addition to cases occurring during the neutropenic post-transplant phase there is a late peak of cases who have chronic graft-versus-host disease (GVHD) and are receiving steroid or other immunosuppressive therapy. There has been an increasing focus on what host immune factors may account for individuals’ susceptibility to IA. We investigated circulating monocytes from this cohort of patients compared with circulating monocytes from healthy controls in a pilot study to determine their ability to phagocytose A. fumigatus (Af) conidia. 
Methods: After providing informed consent 7 adult patients with grade II-III chronic GVHD, requiring steroid or other immunosuppression, and 7 healthy volunteers respectively donated 30ml blood each from which monocytes were harvested using the method described by Serbina NV et al, J Immunology 2009, vol 183,pp 2678-87. Briefly, monocytes were purified and selected using MACS reagents and CD14 magnetic beads and next stained with anti-CD14 label. Af were differentially labelled with AlexaFluor 647 succinimidyl ester dye. Conidia and monocytes were co-incubated for 3 hours in a ratio of 2:1 following which the numbers of conidia ingested was determined using FACS analysis and confocal microscopy. Viability of the ingested conidia was determined by standard plating on agar medium and counting the number of colony forming units (CFU). Supernatants from each interaction were collected at times 0h and 3h, frozen, and later analysed for pro-inflammatory cytokines using a 7-plex assay (Meso Scale Discovery). 
Results: There was no difference between donors and patients in the number of conidia ingested by monocytes. However an analysis of the ability of monocytes to kill Af conidia showed there was a reduced killing (expressed as median% conidia killed for each cohort) by patients’ monocytes compared with donors’ monocytes. The cytokine experiment revealed that patients’ pro-inflammatory responses were impaired; in particular interferon gamma and IL-12 responses were reduced at 3h compared to controls. 
Conclusion: The study of circulating monocytes’ responses to Af deserves further investigation as a potential biomarker of susceptibility to IA in patients with chronic GVHD. 


Full conference title: 

Annual Meeting of European Society for Blood and Marrow Transplantation
    • EBMT 39th (2013)