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Ibrutinib impairs anti-Aspergillus responses
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Figure 5. Engulfment and killing of Aspergillus fumigatus by neutrophils is impaired in patients receiving ibrutinib. Neutrophil-Aspergillus fumigatus interactions were tracked in real-time by video-microscopy during co-culture for 16 h. Three thousand Aspergillus fumigatus conidia were first allowed to germinate in a 96-well plate before 48,000 purified neutrophils were added. For opsonized conditions, autologous serum was added for 15 min and then removed before neutrophils were added. (A) The time sequence shows elongation of one germinating conidium (black arrows; time: 0 to 36 min after the start of experiment) before engulfment by a neutrophil from a patient with lymphoid malignancy prior to ibrutinib therapy (white arrow; time: 45 min). The neutrophil kills the Aspergillus conidium (as indicated by the fungus stained with Sytox green) after approximately 80 min of close non-discontinuous interactions. Approximately 4 h after killing the fungus, the neutrophil dies (time: 363 min). (B) Percentage of Aspergillus fumigatus engulfed by neutrophils in patient samples before ibrutinib therapy and 1 or 3 months after the start of ibrutinib therapy, with or without previous opsonization with autologous serum. The Kruskal-Wallis test with the Dunn multiple comparison post-test were applied; *P<0.05. (C) Comparison of two different time sequences of a sample from a single patient with chronic lymphoid leukemia before ibrutinib therapy (upper) and after 34 days of ibrutinib therapy (lower). In the sample taken after ibrutinib treatment, the inability of the neutrophil to kill the germinating conidia led to uncontrolled fun- gal growth. (D) Rate of killing of Aspergillus fumigatus germinating conidia by neutrophils from patients before ibrutinib therapy and 1 or 3 months after the start of ibrutinib therapy, with or without previous opsonization with autologous serum. The Kruskal-Wallis test with the Dunn multiple comparison post-test were applied; **P<0.005.
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