Abstract
Delayed natural killer (NK) cell reconstitution after allogeneic stem cell transplantation (alloSCT) is associated with a higher risk of developing invasive aspergillosis. The interaction of NK cells with the human pathogen Aspergillus (A.) fumigatus is mediated by the fungal recognition receptor CD56, which is relocated to the fungal interface after contact. Blocking of CD56 signaling inhibits the fungal mediated chemokine secretion of MIP-1α, MIP-1β, and RANTES and reduces cell activation, indicating a functional role of CD56 in fungal recognition. We collected peripheral blood from recipients of an allograft at defined time points after alloSCT (day 60, 90, 120, 180). NK cells were isolated, directly challenged with live A. fumigatus germ tubes, and cell function was analyzed and compared to healthy age and gender-matched individuals. After alloSCT, NK cells displayed a higher percentage of CD56brightCD16dim cells throughout the time of blood collection. However, CD56 binding and relocalization to the fungal contact side were decreased. We were able to correlate this deficiency to the administration of corticosteroid therapy that further negatively influenced the secretion of MIP-1α, MIP-1β, and RANTES. As a consequence, the treatment of healthy NK cells ex vivo with corticosteroids abrogated chemokine secretion measured by multiplex immunoassay. Furthermore, we analyzed NK cells regarding their actin cytoskeleton by Structured Illumination Microscopy (SIM) and flow cytometry and demonstrate an actin dysfunction of NK cells shown by reduced F-actin content after fungal co-cultivation early after alloSCT. This dysfunction remains until 180 days post-alloSCT, concluding that further actin-dependent cellular processes may be negatively influenced after alloSCT. To investigate the molecular pathomechansism, we compared CD56 receptor mobility on the plasma membrane of healthy and alloSCT primary NK cells by single-molecule tracking. The results were very robust and reproducible between tested conditions which point to a different molecular mechanism and emphasize the importance of proper CD56 mobility.
Highlights
Following allogeneic stem cell transplantation, patients are characterized by a period of profound T and B cell deficiency until the completion of immune system reconstitution, which can take up to 2 years [1]
Supernatants of NK cells from allogeneic stem cell transplantation (alloSCT) patients and healthy age and gender-matched controls treated with A. fumigatus germ tubes or non-treated NK cells were analyzed by multiplex immunoassay regarding the presence of the chemokines macrophage inflammatory protein (MIP)-1α, MIP-1β, and RANTES using the BioPlex 200 System (Bio-Rad)
NK cells derived after alloSCT significantly displayed lower amounts of CD56dimCD16++ cells, whereas the proportion of CD56brightCD16+ and CD56brightCD16− cells was higher (Figure 2B), which is in accordance with previous findings [12]
Summary
Following allogeneic stem cell transplantation (alloSCT), patients are characterized by a period of profound T and B cell deficiency until the completion of immune system reconstitution, which can take up to 2 years [1]. Confirming the importance of NK cells in humans, Stuehler et al monitored recipients of an allograft over 12 months and found a clear correlation between reduced NK cell counts and delayed NK cell reconstitution with a higher risk of developing IA in patients receiving HSCT [19]. These studies highlighted the role of NK cells during fungal infections in immunocompromised hosts and were influential for our study. CD56 receptor mobility within the plasma membrane of alloSCT NK cells was not influenced as probed by single-molecule tracking experiments which indicates defects in CD56 signaling rather than its mobility
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