Generation of highly purified and functionally active human TH1 cells against Aspergillus fumigatus

Abstract

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...

Clinical-scale generation of human anti-Aspergillus T cells for adoptive immunotherapy

“…22 Although we did not assess the capacity to damage hyphae of A. fumigatus, the CD4 þ T cells generated according to GMP conditions produced IFN-g upon restimulation with Aspergillus antigens, and thus most likely enhance the antifungal activity of phagocytes. 10 Our in vitro data also indicate a reduced alloreactivity of the generated antiAspergillus T cells and support the in vivo findings by Perruccio et al 16 who transferred ex vivo cultured and pathogen-specific T cells without triggering GVHD. In summary, we present a simple and rapid method for the clinical-scale generation of functionally active antiAspergillus T H 1 cells according to GMP conditions.…”

“…We recently described a simple and rapid generation of a high number of functionally active T H 1 cells against A. fumigatus by means of the IFN-g secretion assay and demonstrated that these cells significantly increased the damage of Aspergillus hyphae by phagocytes. 10 However, in none of the studies published to date, the generation of anti-Aspergillus T cells had been performed according to current GMP conditions, which include criteria such as the use of GMP-grade materials, the restriction to an autologous setting and the inclusion of extensive controls, and are necessary for prospective clinical trials designed to evaluate benefit and side effects of adoptively transferred anti-Aspergillus T cells.…”

“…These data corroborated our previous results of immunomagnetically selected and cultured antiAspergillus T H 1 cells. 10 Influence of cryopreservation on anti-Aspergillus T cells As anti-Aspergillus T cells may be an option for prophylactic strategies in high-risk patients undergoing allogeneic SCT, we assessed the influence of cryopreservation in liquid nitrogen for up to 5 months on the generated T cells. Thawing resulted in a loss of less than half of the original anti-Aspergillus T cells (median (range) 45.5% (28.2-78.9)).…”

“…10,12 Specific cytokine secretion upon restimulation was assessed by means of intracellular cytokine (ICC) flow cytometry. Briefly, the generated anti-Aspergillus T cells were stimulated for 6 h with autologous A. fumigatus-loaded APCs (effector-stimulator cell ratio, 5:1) and costimulated with the moAbs CD28 (BD Biosciences) and CD49d (Coulter Immunotech; 2 mg/ml each).…”

“…9,10 Antigens of one preparation were kept in aliquots at À80 1C until use. The extract was tested for sterility and endotoxin concentration in an external certified laboratory (L þ S AG, Bad Bocklet-Grossenbrach, Germany).…”