Invasive aspergillosis is a major cause of morbidity and mortality in patients undergoing allogeneic hematopoietic SCT. There is a growing body of evidence that T cells are important in the host defense against Aspergillus, and adoptively transferred anti-Aspergillus T-helper 1 (TH) 1 cells might reduce infectious mortality in hematopoietic transplant recipients. Here we present for the first time a simple and rapid method for the clinical-scale generation of functionally active anti-Aspergillus T cells according to good manufacturing practice conditions. A total of 1.1 × 109 WBCs derived from a leukapheresis product were incubated with Aspergillus antigens. Stimulated cells were selected by means of the IFN-γ secretion assay and expanded. In three independent experiments, a median number of 2 × 107 CD3+CD4+cells (range, 0.9–3.2 × 107) were obtained within 13 days. The cultured CD3+CD4+ cells exhibited almost exclusively a memory activated T-helper cell phenotype. Upon restimulation, the generated T cells produced IFN-γ, but no IL-4 or IL-10, indicating a TH1-cell population. Additionally, the cells proliferated upon restimulation and showed reduced alloreactivity compared to unselected CD4+ cells. This method of generating is suitable for future prospective trials designed to evaluate the effect of adoptive immunotherapy in hematopoietic transplant recipients with invasive aspergillosis.
Invasive aspergillosis remains a major cause of morbidity and mortality in patients undergoing allogeneic hematopoietic SCT.1 Whereas neutropenia and defects in the phagocyte cell function are well-established risk factors for invasive aspergillosis,2 there is an increasing body of evidence that the adaptive immune response plays a critical role in the secondary host defense against Aspergillus fumigatus, the most frequent cause of invasive aspergillosis. For example, in up to two-thirds of patients with invasive aspergillosis after allogeneic SCT, the infection occurred after neutrophil recovery, but at a time when the patients still suffered from a low number of functional T cells.3 Conversely, patients surviving aspergillosis show significant antigen-specific proliferation of IFN-γ producing T cells, supporting the crucial role of a T-helper (TH) 1 reactivity in the control of infection.4 Over the past decade, significant progress has been made in reconstituting adoptive cell immunity in hematopoietic transplant patients with ex vivo generated antigen-specific T cells to restore essential responses for protection against a specific pathogen. For example, eight transplant recipients who lacked CMV-specific T-cell immunity and did not respond to antiviral chemotherapy for CMV disease, received 1 × 107 CMV-specific T cells per m2.5 Despite cessation of antiviral medication, the CMV load decreased significantly in seven evaluable patients. In contrast to various protocols for the clinical-scale generation of virus-specific cytotoxic T cells,6, 7, 8 no single method for the generation of anti-Aspergillus T cells based on clinical-scale conditions has been reported to date. The availability of such a method, however, is mandatory for clinical trials evaluating the effect of adoptively transferred T cells in patients with aspergillosis. Therefore, the present study was designed to develop a simple and rapid method for the generation of functionally active anti-Aspergillus T cells according to good manufacturing practice (GMP) conditions.
Materials and methods
A total of 1.1 × 109 WBCs of unstimulated leukapheresis products obtained from three healthy donors were used for the generation of anti-Aspergillus T cells. All donors had been tested negative for CMV, HIV, hepatitis B virus, hepatitis C virus, Treponema pallidum and Toxoplasma gondii. The products were processed on the day of leukapheresis. All donors gave their informed consent. The study was approved by the local Ethical committee.
Aspergillus fumigatus antigens
A water soluble cellular extract of A. fumigatus (strain: CBS 144-89), grown on Sabouraud agar plates, was prepared at the Institute Pasteur (Paris, France) as described before.9, 10 Antigens of one preparation were kept in aliquots at −80 °C until use. The extract was tested for sterility and endotoxin concentration in an external certified laboratory (L+S AG, Bad Bocklet-Grossenbrach, Germany).
Isolation of anti-Aspergillus T cells
Each step during the selection of anti-Aspergillus T cells and the following period of cell culture was performed under GMP conditions in the Institute for Transfusion Medicine and Immunohematology, Frankfurt, Germany. In a first step, a total of 1.1 × 109 WBCs of a leukapheresis product were washed twice using X-vivo 10 medium without phenolred and antibiotics (BioWhittacker, Verviers, Belgium), resuspended in 100 ml X-vivo 10 medium containing 10% heat-inactivated GMP-grade human serum (HS; Center for Clinical Transfusion Medicine, Tübingen, Germany) and transferred into a T-cell expansion bag (Miltenyi Biotech, Bergisch Gladbach, Germany). Then, cells were stimulated with 10 μg/ml A. fumigatus antigens for 12–16 h in a humidified incubator at 37 °C. On the following day, anti-Aspergillus T cells were enriched using the clinical-scale CliniMACS Cytokine Capture System (IFN-γ; CCS IFN-γ; Miltenyi Biotech) on the CliniMACS device (Miltenyi Biotech) according to the manufacturer's instructions. In brief, the cells were washed after the stimulation period in X-vivo 10 medium containing 2% HS and adjusted at a final volume of 10 ml. Then, 7.5 ml Catchmatrix Reagent (Miltenyi Biotech) was added to the suspension, followed by a first incubation time on ice for 5 min. The cells were resuspended in 1000 ml X-vivo 10 medium containing 10% HS, evenly spread on four cell bags and incubated for 45 min in a humidified incubator at 37 °C. Then, the cells were washed, recombined in one bag at a volume of 10 ml and incubated with the IFN-γ enrichment reagent (Miltenyi Biotech) for 15 min on ice. After another washing step, cells were finally resuspended in 100 ml CliniMACS buffer containing 2% HS and immunomagnetically separated using the enrichment program 3.1 on the CliniMACS device. The whole isolation procedure was performed in a closed system using 500 ml cell bags (Fresenius, Bad Homburg, Germany), spike connectors (Baxter, Lessines, Belgium), three-way stopcocks and extension tubings for infusion lines (Braun, Melsungen, Germany) and a sterile tubing welder (Terumo, Tokio, Japan).
Generation of A. fumigatus-loaded APCs
Autologous monocytes were purified by adherence method using the negative fraction from the immunomagnetic cell separation procedure. Briefly, the cells were washed, resuspended in 50 ml X-vivo 10 medium containing 2% HS, evenly distributed on two 80 cm3 cell-culture flasks (Nunc, Roskilde, Denmark) and incubated overnight in a humidified incubator at 37 °C On the next day, nonadherent cells were removed, and A. fumigatus antigens (10 μg/ml) were added. As preliminary experiments in a small volume setting indicated a higher yield of anti-Aspergillus T cells when monocyte-derived dendritic cells (moDCs) were used for restimulation, monocytes were replaced by moDCs after the first experiment. MoDCs were generated within 3 days using a protocol previously described with some modifications.11 In brief, monocytes were incubated with A. fumigatus antigens (10 μg/ml), 1000 IU/ml GM-CSF and 800 IU/ml IL-4. On the next day, GM-CSF (1000 IU/ml), IL-4 (800 IU ml), tumor necrosis factor (TNF)-α (1000 IU/ml), IL-1β (500 IU/ml) and IL-6 (500 IU/ml) were added. All recombinant human GMP-grade cytokines were purchased from CellGenix (Freiburg, Germany). For the stimulation of anti-Aspergillus T cells on days 4 and 7, APCs (monocytes or moDCs as indicated) were harvested using a cell scraper (Sarstedt, Newton, NC, USA).
Expansion of isolated anti-Aspergillus T cells
The isolated anti-Aspergillus T cells were resuspended in 20 ml X-vivo 10 medium containing 10% HS and evenly distributed on two 25 cm3 cell-culture flasks (Corning Inc., Corning, NY, USA) in the presence of 1 × 107 irradiated autologous feeder cells taken from the negative fraction after immunomagnetic cell separation. The cells were cultured between 11 and 13 days, and supplemented with fresh medium on days 4, 7 and 10. GMP-grade recombinant human IL-2 (50 IU/ml; Proleukin; Chiron, Ratingen, Germany) was added on days 0, 2, 5, 8 and 10. Irradiated autologous A. fumigatus-loaded APCs were added on days 4 and 7. Whereas in the first donor, restimulation was performed with monocytes (1.7 × 106 and 2.4 × 106, respectively), moDCs highly expressing CD83, CCR-7 and HLA-DR were used in the two other donors (median number (range), 3.8 × 105 (3.5 × 105–5 × 105)). Expanded anti-Aspergillus T cells were aliquoted and cryopreserved in X-vivo 10 medium containing 10% dimethylsulfoxide (Sigma, Steinheim, Germany).
At various time points of the immunomagnetic separation procedure and after cell culture, phenotypic characteristics and absolute cell numbers were determined by means of five-color flow cytometry (FC 500; Beckman Coulter, Krefeld, Germany) in a single platform approach using moAbs against CD3, CD4, CD8, CD14, CD19, CD45, CD56 CD45RA, CD45RO, CD69, HLA-DR, 7-AAD (Coulter Immunotech, Marseille, France), IFN-γ (Miltenyi Biotech) and Flow-Count beads (Beckman Coulter). Additionally, moAbs against CD83 (Invitrogen, Karlsruhe, Germany) and CCR-7 (BD Biosciences, San Jose, FL, USA) were used for characterization of moDCs. All settings included isotype controls.
Functional characterization of generated anti-Aspergillus T cells
The generated anti-Aspergillus T cells were functionally characterized regarding cytokine secretion, proliferation upon restimulation and alloreactivity as previously described in detail.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 μg/ml each). Positive controls were performed using phorbol-12-myristate-13-acetate (Sigma) and ionomycin (Sigma), negative controls using unloaded APCs. Brefeldin A (Sigma) was added for the past 5 h of incubation. Cells were then permeabilized with BD FACS Permeabilizing Solution 2 (BD Biosciences) for ICC staining.
Carboxy-fluorescein-diacetate-succinimidyl ester (CFSE; Molecular Probes, Eugene, OR, USA) labeling was performed for the assessment of proliferation upon restimulation and of alloreactivity. For testing proliferation upon restimulation, CFSE-labeled anti-Aspergillus T cells were cocultured with irradiated autologous A. fumigatus-loaded APCs. Unloaded APCs served as negative controls. For testing alloreactivity, donor-derived CFSE-labeled WBCs from the original leukapheresis product as well as highly purified anti-Aspergillus T cells were coincubated with irradiated third-party APCs. The proliferation index was estimated by flow cytometric analyses gated for CD4+ T cells on day 7. All analysis concerning ICC and CFSE were performed using the FACSCalibur flow cytometer (Becton Dickinson, Heidelberg, Germany) and moAbs against CD3, CD8, IFN-γ, TNF-α, IL-4, IL-10 (BD Biosciences) and CD4 (Dako Cytomation, Glostrup, Denmark).
Sterility of the leukapheresis products, the supernatants after immunomagnetic separation and at the end of the culture period were assessed using aerobic and anaerobic blood culture bottles according to the manufacturer's instruction (BD Bactec, Becton Dickinson, Sparks, MD, USA). The final cell-culture products were additionally tested for fungal and bacterial pathogens including Mycoplasma spp by universal PCR (Institute of Medical Microbiology and Infection Control, University of Frankfurt) and for endotoxin concentration by a turbidimetric kinetic method (L+S AG, Bad Bocklet-Grossenbrach, Germany).
Preliminary small-scale experiments
Before performing the evaluation of the clinical-scale generation of human anti-Aspergillus T cells, the method was established in preliminary small-scale experiments. These experiments are identical with the clinical-scale generation regarding GMP-grade media and antigens used, stimulation and culturing period, supplementation of GMP-grade IL-2 and restimulation with A. fumigatus-loaded moDCs, but differ regarding the number of cells used and the device for enrichment. In brief, a total of 1.1 × 108 PBMCs, obtained by density-gradient centrifugation from 150 ml blood of healthy donors, were stimulated with A. fumigatus antigens. Activated cells were isolated using the IFN-γ CSA MiniMACS device (Miltenyi Biotech) and placed in 2 ml medium in a 24-well plate (Nunc, Wiesbaden, Germany). After culturing the anti-Aspergillus T cells, phenotyping and functional tests were performed as described above. In addition, moDCs for restimulation were generated with the same method as described above in 2 ml medium using six-well plates (Nunc, Wiesbaden, Germany).
Preliminary small-scale experiments
With the established protocol, two small-scale experiments were performed. Using 1.1 × 108 stimulated PBMCs, enrichment resulted in both trials in a positive fraction containing 1 × 105 cells. During the culturing period, the cells were restimulated twice with a median number of 9.5 × 105 (range, 6–15 × 105) irradiated autologous A. fumigatus-loaded moDCs, which yielded after 13 days of culturing in a total number of 7.1 × 106 and 4.1 × 106 CD3 and CD4 double-positive cells, respectively (Table 1). The cells uniformly coexpressed HLA-DR and CD45RO (Table 1). The percentage of CD3+CD8+ cells, CD14+ cells and CD19+ cells were negligible. On restimulation, cultivated CD3+CD4+cells produced IFN-γ (8.5 and 7.9%, respectively) and TNF-α (9.3 and 22.7%; Table 1). As there was no detectable production of IL-4 and IL-10, the vast majority of generated T cells were activated TH1 cells.
Clinical-scale generation of anti-Aspergillus T cells
Anti-Aspergillus T cells were isolated from unstimulated leukapheresis products of three healthy donors. A total of 1.1 × 109 WBCs containing a median of 53.9% CD3+ cells (range, 25.1–68.6%) were stimulated overnight with Aspergillus antigens, which resulted in 0.4% (0.3–0.9%) CD3+ cells staining positive for IFN-γ on the cell surface (‘Pre-isolation’, Table 2). After selection, the positive fraction consisted of a median number of 2.7 × 106 WBCs (range, 2.0–7.8 × 106; Table 2). Among these cells, 19.4% (11.8–39.3%), 11.3% (3–16%) and 1.5% (1.5–7.5%) stained positive for CD14, CD19 and CD56, respectively, (data not shown) whereas most of these WBCs expressed CD3 (median (range), 52.2% (29–61.2%); Table 2). Further analysis revealed that 28% (25.2–42.7%) of the CD3+ cells stained positive for cell surface IFN-γ. The following culturing period of up to 13 days yielded in a median number of 2.2 × 107 cells (range, 1.3–3.7 × 107), which almost exclusively expressed both CD3 and CD4 (86.8% (73–88.4%)). The median percentage of CD3+CD8+ cells was 3.2% (range, 0.4–5.8%), whereas the number of CD14+ cells, CD19+ cells and CD56+ cells were negligible. In addition, the cultured anti-Aspergillus CD4+ T cells stained homogenously positive for CD45RO and HLA-DR, indicating a memory, activated T-helper cell population (Table 1). Upon restimulation with Aspergillus antigens, a median of 5.1 and 10.3% of these cells secreted IFN-γ and TNF-α, respectively (range, 4–12.5 and 7.3–27.7%; Figure 1 and Table 1). In contrast, no IL-4 or IL-10 production was detectable, indicating that the vast majority of generated T cells were activated TH1 cells (data not shown).
Functional characteristics of anti-Aspergillus T cells
To estimate whether the generated functionally active anti-Aspergillus T cells can appropriately divide after being stimulated with an endogenously processed antigen, generated T cells of one donor were labeled with CFSE and cocultured with either autologous Aspergillus-APCs or unloaded APCs. Whereas restimulation with Aspergillus-antigens resulted in the proliferation of the majority of generated anti-Aspergillus T cells, considerable fewer cells expanded when coincubated with unloaded APCs (Figure 2). Adoptive transfer of donor-derived T cells into allogeneic transplant recipients may induce GVHD. We therefore assessed in two available donors whether the generated functionally active T cells have reduced alloreactivity compared with the original cell fraction. In both donors, unspecific and unselected CD4+ T cells elicited a strong proliferation response against third-party APCs, as measured by loss of intensity in the CFSE signal (Figure 3, bottom a and b). In contrast, functionally active anti-Aspergillus T cells coincubated with allogeneic APCs revealed a considerably weaker expansion in the same time period (Figure 3, top a and b), which was comparable with the expansion observed when the purified anti-Aspergillus T cells were cocultured with unloaded autologous APCs (Figure 2, top). These data corroborated our previous results of immunomagnetically selected and cultured anti-Aspergillus TH1 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)). Assessment of viability of CD3+CD4+ cells did not reveal a significant difference between cells before and after cryoconservation (Table 1). In addition, cryopreservation did not significantly affect the phenotype or the function of the generated anti-Aspergillus T cells, in particular, IFN-γ production and proliferation upon restimulation (data not shown).
None of the cell-culture products were contaminated by fungal or bacterial pathogens. In addition, endotoxin concentration of all samples tested was less than 0.1 IU/ml, indicating that all culture products were suitable for clinical application from a microbiological point of view.
Invasive fungal infection remains a life-threatening complication in patients undergoing allogeneic SCT, showing an increasing incidence over the past decade.1 Although neutrophil-mediated immunity is well described as important host defense against filamentous fungi, such as A. fumigatus, it recently became clear that CD4+ T cells provide a critical secondary defense against these organisms. Animal models suggest that the reconstitution of Aspergillus-specific immune responses after allogeneic SCT is protective against the development of invasive aspergillosis.13, 14 In humans, the approach of adoptively transferring antigen-specific T cells after allogeneic SCT is well established for patients suffering from severe viral disease, such as CMV disease or EBV-associated post transplantation lymphoproliferative disease,5, 15 but there are only few data on patients with invasive fungal infection. In one clinical trial, 10 hematopoietic transplant recipients with evidence of invasive aspergillosis received anti-Aspergillus adoptive therapy after allogeneic SCT.16 In all of them, galactomannan antigenemia fell within 6 weeks of infusion to the normal range whereas it persisted in all 13 patients who did not receive adoptive immunotherapy; 9 out of the 10 patients who received anti-Aspergillus T cells survived. However, the donor-derived pathogen-specific T cells that were characterized as TH1 cells were generated by limiting dilution, which needed a minimum of 25 days. We recently described a simple and rapid generation of a high number of functionally active TH1 cells against A. fumigatus by means of the IFN-γ 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.
Comparable to the approach of other groups, we used a cellular extract from A. fumigatus for the stimulation of donor-derived PBMCs.4, 16 Importantly, several extracts prepared under identical conditions resulted in comparable numbers and function of the generated cells, indicating the reproducibility of our system (data not shown). In addition, extensive controls after stimulation, isolation and culture of anti-Aspergillus T cells did not reveal contamination with a pathogen or endotoxin. The use of a cellular extract consisting of several antigens may even be of advantage in the clinical setting. First, corroborating our previous data, the T cells generated with a cellular extract of A. fumigatus not only respond upon restimulation with A. fumigatus, but also upon restimulation with other filamentous fungi, but not upon Candida spp, suggesting a limited cross-reactivity by which immunity against other pathogens than A. fumigatus may be enhanced (data not shown). Furthermore, in the majority of patients with an invasive mycosis it is not possible to isolate the fungal pathogen and to specify the antigen for generating antigen-specific T cells. Last, the antigenic properties of Aspergillus spp are variable in vivo.17 Therefore, the fungus may escape the immunologic response of generated T cells that are specific for a certain Aspergillus antigen only. In this respect, antigen lysates have also been used in the adoptive immunotherapy against various tumors.18, 19
Out of a total of 1.1 × 109 WBCs from the leukapheresis product, a median number of 12 × 107 anti-Aspergillus TH1 cells was generated within 1–13 days (Table 1). In contrast to TH2 reactivity that is associated with susceptibility to invasive mycoses, TH1 cells play a crucial role in the control of Aspergillus infection, but the factors that determine the differential activation of TH1 or TH2 cells are not fully understood.20, 21 In a pilot study, Perruccio et al.16 transferred between 1 × 105 and 1 × 106 anti-Aspergillus TH1 cells per kg body weight to the transplant recipient between days +17 and +37 after SCT. The assessment of post-immunotherapy T-cell responses revealed stable frequencies of anti-Aspergillus T cells for up to 36 weeks after SCT, which were associated with control of Aspergillus antigenemia. Corroborating our results, the generated T cells did not represent terminally differentiated CD4+ T cells, but proliferated upon restimulation with Aspergillus antigens. It is unclear to date that numbers of anti-Aspergillus T cells according to what schedule have to be administered for prophylaxis of high-risk patients or for therapy of patients with invasive aspergillosis. In this respect, our protocol allows the prophylactic preparation of anti-Aspergillus T cells, as the yield after thawing of cryopreserved T cells was similar to those of other primary human cells, and, more importantly, as cryopreservation did not affect the functional activity of the generated cells.22 Although we did not assess the capacity to damage hyphae of A. fumigatus, the CD4+ T cells generated according to GMP conditions produced IFN-γ 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 anti-Aspergillus 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 anti-Aspergillus TH1 cells according to GMP conditions. Upon restimulation with Aspergillus antigens, these T cells produce IFN-γ and proliferate appropriately. In addition, the generated T cells show a reduced alloreactivity compared to unselected lymphocytes. Our protocol is well suited for future clinical trials that are currently designed to evaluate whether adoptive immunotherapy with anti-Aspergillus T cells will reduce infectious mortality in patients undergoing allogeneic SCT.
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We thank Carla Fadler, Nicola Schuely and Frauke Roeger for their technical assistance, and Klaus-Peter Hunfeld for microbiological testing. We also thank Georg Rauser for helpful discussions. This study was supported by the Deutsche Leukämie-Forschungshilfe (DLFH).
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Tramsen, L., Koehl, U., Tonn, T. et al. Clinical-scale generation of human anti-Aspergillus T cells for adoptive immunotherapy. Bone Marrow Transplant 43, 13–19 (2009) doi:10.1038/bmt.2008.271
- invasive aspergillosis
- adoptive immunotherapy
- anti-Aspergillus T cell
- good manufacturing practice
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