Because natural killer (NK) cells kill tumor cells and combat infections, there is growing interest in adoptively transferring NK cells to hematopoietic stem cell recipients. Unfortunately, in humans, the activity of NK cells against Aspergillus species, the major cause of invasive fungal infection in stem cell recipients, are poorly characterized. Our results show that unstimulated and interleukin-2 prestimulated human NK cells kill Aspergillus fumigatus hyphae but do not affect resting conidia. Killing is also induced by the supernatant of prestimulated NK cells and human perforin. The high levels of interferon-γ and granulocyte macrophage colony-stimulating factor produced by prestimulated NK cells are significantly reduced by Aspergillus, indicating an immunosuppressive effect of the fungus. Whereas Aspergillus hyphae activate NK cells, resting, and germinating, conidia and conidia of ΔrodA mutants lacking the hydrophobic surface layer do not. Our results suggest that adoptively transferred human NK cells may be a potential antifungal tool in the transplantation context.

Invasive aspergillosis remains a serious complication in patients undergoing allogeneic stem cell transplantation (SCT). Since it became clear that lymphocytes provide a critical secondary defense against fungi, adoptive transfer of functionally active anti-Aspergillus T cells might be an option to restore adaptive immune effector mechanisms. Using the interferon (IFN)-␥ secretion assay, we isolated human activated T cells upon stimulation with a cellular extract of Aspergillus fumigatus. Culturing this cell population for 14 days, we obtained an average of 1.1 ؋ 10 7 cells from a single 100-mL blood draw in 7 of 7 healthy individuals. Within another 14 days, these cells were expanded to an average number of 2.0 ؋ 10 8 T-helper 1 (T H 1) cells secreting IFN-␥ on stimulation with Aspergillus antigens. Testing various fungal antigen extracts, similar proportions of IFN-␥-producing CD3 ؉ /CD4 ؉ cells were obtained upon activation with antigen extracts of A fumigatus, A flavus, A niger, and Penicillium chrysogenum, whereas no significant IFN-␥ production was observed upon activation with antigen extracts of Alternaria alternata and Candida albicans. In addition, generated T cells were able to induce damage to A fumigatus hyphae, and significantly increased hyphal damage induced by human neutrophils. CD4 ؉ T-cell-mediated alloreactivity of generated anti-Aspergillus T cells was clearly reduced compared with that of the original cell population. In conclusion, we present a simple and feasible strategy for rapid generation of a high number of functional active T cells against Aspergillus from a single blood draw. Our data suggest that functionally active T cells against Aspergillus could be a promising treatment option for patients undergoing allogeneic SCT. IntroductionInvasive aspergillosis (IA) remains a major cause of morbidity and mortality in patients with hematologic malignancies, particularly in patients who have undergone allogeneic stem cell transplantation (SCT). 1 Important risk factors for IA are neutropenia and defects in the phagocyte cell function. 2 On the other hand, there is an increasing body of evidence that adaptive immune response also plays a critical role in the host defense against Aspergillus fumigatus, the most frequent cause of IA. For example, IA has been reported with increasing frequency in patients with advanced AIDS, 3 and up to two-thirds of patients with IA diagnosed after allogeneic SCT are not neutropenic. 1 In contrast to neutrophil recovery, which generally occurs within the first 2 to 3 weeks after SCT, the number of functional T cells and T-cell function increase slowly over the first few months after transplantation. 4 Prolonged immune suppression due either to transplant conditioning and graft-versus-host disease (GVHD) prophylaxis or to treatment of GVHD further increase the risk of IA in SCT patients. 5,6 It has recently been shown that a significant antigen-specific proliferation of interferon (IFN)-␥-producing T cells occurred in healthy individuals and in patients survi...

No abstract

No abstract

No abstract

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 alone showed a weak, but significant capacity to induce hyphal damage (7.4±1.1% and 11.3±1.8%, respectively). Before considering a clinical application, however, further studies need to focus on defining the optimal antigen(s) which reproducibly induce a TH1 response and elicit high antifungal activity, as well as to characterize the subpopulation of patients undergoing allogeneic SCT who ultimately benefit from either a prophylactic or a therapeutic adoptive transfer of Aspergillus fumigatus specific T-cells.

No abstract

No abstract