Functional Characterization of Aspergillus Fumigatus Specific T-Cells Clones.

Thomas Lehrnbecher, Ulrike Koehl, Emmanuel Roilides, Maria Simitsopoulou, Mitra Hanisch, Lars Tramsen, Jean-Paul Latge, Max S. Topp, Hermann Einsele, Thomas Klingebiel, Olaf Beck

Author address: 

Pediatric Hematology and Oncology, J.W.Goethe University, Frankfurt/Main, Germany; 3rd Department of Pediatrics, University of Thessaloniki, Thessaloniki, Greece; Institut Pasteur, Paris, France; Medizinische Poliklinik, University of Wuerzburg, Wuer


Invasive aspergillosis (IA) remains a major cause of morbidity and mortality in patients with hematological malignancies, in particular in patients who have undergone allogeneic hematopoietic stem cell transplantation (SCT). There is a growing body of evidence that T-cells play an important role in the immunological response to Aspergillus fumigatus. Using the Aspergillus fumigatus antigen extract EC SAB and the IFN-γ secretion assay (Miltenyi Biotec, Germany), we generated Aspergillus fumigatus specific T-cell clones by limiting dilution (n=4). Flow cytometry revealed a cell population of CD3+/CD4+ cells (mean±SEM, 98.2±1.2%). Functional assessment by ICC revealed that an average of 8.7% of these cells (range, 6.6%-18.5%) specifically secreted IFN-γ on stimulation with EC SAB, which supports the TH1 response of the generated cells to Aspergillus fumigatus antigens. The antigenic components of EC SAB are one or more proteins, since the addition of proteinase completely suppressed the stimulating effect of this preparation. The percentage of IFN-γ producing CD3+/CD4+ cells was less than 1% upon activation with antigen extracts from Aspergillus flavus, Aspergillus niger, Alternaria alternata, Mucor racemosus, Penicillium notatum and Candida albicans, indicating that the generated T-cell clones are specific for Aspergillus fumigatus. A strong proliferation of the generated Aspergillus fumigatus specific T-cells was seen after re-stimulation with EC SAB, whereas alloreactivity was reduced compared to CD4+ T-cells of the original fraction. Hyphal damage of Aspergillus fumigatus was assessed by means of an XTT assay. Polymorphonuclear leukocytes (PMNs) showed a similar hyphal damage when tested alone (mean±SEM, 14.2±2.1%), in combination with antigen presenting cells (APCs) (15.1±1.4%), or in combination with Aspergillus fumigatus specific T-cells (15.0±2.0%). A comparable hyphal damage was seen when Aspergillus fumigatus specific T-cells were co-incubated with APCs (14.2±1.7%). In contrast, the combination of APCs and Aspergillus fumigatus specific T-cells with PMNs resulted in a significantly higher hyphal damage compared to all other settings (23.3±2.8%; P<.0001 interestingly="" apcs="" alone="" or="" aspergillus="" fumigatus="" specific="" t-cells="" showed="" a="" weak="" but="" significant="" capacity="" to="" induce="" hyphal="" damage="" and="" respectively="" before="" considering="" clinical="" application="" however="" further="" studies="" need="" focus="" on="" defining="" the="" optimal="" antigen="" which="" reproducibly="" th1="" response="" elicit="" high="" antifungal="" activity="" as="" well="" characterize="" subpopulation="" of="" patients="" undergoing="" allogeneic="" sct="" who="" ultimately="" benefit="" from="" either="" prophylactic="" therapeutic="" adoptive="" transfer="" t-cells.="">

abstract No: 


Full conference title: 

47th American Society for Haematology
    • ASH 47th (2005)