Background: Aspergillus-specific T-cells were described as a novel biomarker for early diagnosis of
invasive aspergillosis (IA) in patients undergoing hematological stem cells transplant (HSCT). These
patients receive T-cell suppressive GvHD prophylaxis regimes often containing ciclosporin A and/or
mycophenolate-mofetil (MMF). Corticosteroids are commonly used for the initial therapy of acute
GvHD. This study sought to determine, whether ciclosporin A, MMF, and prednisone influence the
sensitivity of the quantification of A. fumigatus specific T-cells.
Material/methods: 54 ml of heparinized blood were collected from 11 healthy adult donors. PBMCs
were isolated and plated into a 6 Well-plate at a concentration of 5 x 106 cells/ml. Mycophenolatemofetil
(2 μg/ml), ciclosporin A (200 ng/ml), and/or prednisolone (200 ng/ml) were added. Cells were
incubated at 37 °C, 5% CO2 for 3 h. Subsequently, the cells were harvested and resuspended in RPMI
1640 supplemented with 5% autologous serum at a concentration of 1 x 107 cells/ml. 100μl of the cell
suspension were plated into a 96 well-plate. Cells were stimulated with a commercially available A.
fumigatus mycelial lysate, treated with Brefeldin A, and stained as previously described. The
percentage of CD154+ cells among CD4+ cells was quantified by flow cytometry.
Results: In absence of immunosuppressive drugs a mean frequency of 0.10 % ± 0.05 % A. fumigatus
specific T-cells was observed. If PBMCs were pre-treated with therapeutic concentrations of
mycophenolate-mofetil and ciclosporin A for 3 h, the mean frequency declined to 0.04 % ± 0.02 (p <
0.01, Figure A). Sole exposure of PBMCs to mycophenolate-mofetil (0.05 %, p = 0.02) or ciclosporin A
(0.03 %, p = 0.02) was also associated with a significantly decreased mean frequency of specific Tcells
(Figure B). Combined pre-treatment of the cells with prednisolone plus MMF resulted in a further
reduction of assay sensitivity (0.01%, p = 0.03).
Conclusions: These findings demonstrate that the quantification of A. fumigatus specific T-cells is
significantly impaired by cell exposure to therapeutic concentrations of T-cell suppressive agents.
Hence, test results should be interpreted carefully when using the assay in HSCT patients receiving
GvHD prophylaxis or therapy. In particular, false-negative test results need to be considered in these
Full conference title:
- ECCMID 26th (2016)