Aspergillus proteins CcpA and Shm2 were recombinantly expressed as previously described (9). Briefly, the PCR-amplified open reading frame of shm2 cDNA or a synthetic gene encoding for CcpA of A. fumigatus were amplified and cloned into the expression vector pET43.1H6 and pET28aH6TEV, respectively, for recombinant expression as His-tagged proteins. Proteins were purified by metal chelate affinity chromatography (Äkta explorer purification system, GE Healthcare). Protein concentrations were determined by measuring absorbance at 280 nm (NanoDrop 1000, Thermo Scientific) or by a Bradford protein assay (Biorad). Endotoxin contamination was determined (LAL Chromogenic Kit, Thermo Scientific) and reduced to <0.1 EU/ml using resin columns (Thermo Scientific). Research grade A. fumigatus lysate (AfuLy) was purchased from Miltenyi Biotec. To generate A. fumigatus germ tubes, 2×10⁷ conidia of strain ATCC 46645 were incubated in 20 mL RPMI, supplemented with 120 µg/mL gentamicin, for 9 h at 25°C in a shaking incubator (Infors HT, 200 rpm), pelleted at 5000 g for 10 min, and resuspended in RPMI + 5% FCS.

Human peripheral blood mononuclear cells (PBMC) were obtained from leukocyte reduction chambers using Ficoll-Hypaque (Biochrome AG) density gradient centrifugation. Thereafter, mDCs were isolated from PBMCs by CD19 depletion followed by CD1c positive selection (Myeloid Dendritic Cell Isolation Kit, Miltenyi Biotec). Monocyte isolation from PBMCs was performed using MACS CD14 positive selection (Miltenyi Biotec). The moDCs were generated by incubation of monocytes with 20 ng/mL IL-4 and 100 ng/mL GM-CSF for 6 days as previously described (12). A minimum purity of ≥ 95% CD1c⁺ mDCs or CD14^- CD1a⁺ moDCs was confirmed by flow cytometry (FACS Calibur, Becton Dickinson, antibodies from Miltenyi Biotec). Furthermore, we confirmed at least 4-fold induction of the maturation markers CD80 and CD86 upon overnight stimulation with ultrapure LPS to validate the functionality of our DC preparations.

After obtaining informed consent, 15 mL venous EDTA blood was collected from healthy adult donors. PMNs were isolated using PolymorphPrep (Axis-Shield) gradient centrifugation according to the manufacturer’s instructions, except that Buffer EL (Qiagen) was used instead of Solution C. PMNs were suspended in RPMI 1640 + 10% FCS at a concentration of 3×10⁶/mL and the purity of the PMN suspension was verified by flow cytometry (CD3-PerCP^-/CD66b-FITC⁺/CD14-PE^low cells > 90%, antibodies from Miltenyi Biotec).

6×10⁵ mDCs or moDCs were cultured for 18 h at 37°C in 200 µL CellGro GMP DC medium (CellGenix, Cat. No.: 20801-0500) supplemented with 50 µg/mL AfuLy, 1-5 µg/mL Aspergillus proteins CcpA or Shm2, 1 µg/mL LPS (Sigma, positive control), or plain medium (negative control). Thereafter, DCs were stained with anti-CD1a-APC (moDCs) or anti-CD1c-APC (mDCs), anti-CD14-FITC, anti-CD80-APC, anti-CD83-PE, anti-CD86-FITC, anti-CD40-FITC, anti-HLA-ABC-PE, anti-HLA-DR-PE (BD), and anti-CCR7-APC (Miltenyi Biotec) in three different panels. Samples were analyzed using a FACS Calibur cytometer (BD), Cell Quest Pro software (BD), and Flow Jo software (version 10.6.2.). Dead cells were excluded from analysis by light scatter (FSC/SSC) properties.

6×10⁵ moDCs or mDCs were incubated for 18 h in plain CellGro medium (unstimulated control) or in 200 µL medium containing 50 µg/ml AfuLy, 5 µg/mL of A. fumigatus proteins CcpA or Shm2, 1 µg/mL ultrapure LPS (Invivogen), 100 µg/mL beta-glucan (Merck Millipore), or 6×10⁵ ethanol-inactivated A. fumigatus germ tubes. The supernatants were then collected and analyzed using a ProcartaPlex Human 8-plex plate assay (Thermo Scientific).

Pulsing of moDCs and mDCs with antigens was performed for 18 h in 200 µL CellGro medium as described above. After pulsing, DCs were washed twice with CellGro to remove residual antigens. An ELISpot culture plate (Millipore) was washed 3 times with 200 µL PBS and coated with an IFN-γ capture antibody (1-D1K, BD), then incubated overnight at 4°C. The plate was then washed 5 times with PBS and blocked with CellGro medium for 3 h at 37°C. After removal of the medium, 5×10⁵ freshly isolated autologous T-cells (pan T-cell Isolation Kit, Miltenyi Biotec) were seeded in each well. Cells were stimulated with SEB + α-CD3 (1 µg/ml respectively, as a positive control) or 5×10⁴ autologous, A. fumigatus antigen-pulsed mDCs (T-cell-DC ratio 10:1). The ELISpot plate was incubated for 18 h at 37°C and subsequently washed 6 times with PBS. IFN-γ producing cells were stained for 2 h with an IFN-γ detection antibody (7-B6-1 biotin, BD) at RT, followed by another 5 PBS washing steps. After 1 h of incubation with a streptavidin-linked phosphatase (Southern Biotec) at RT, plates were washed 8 times with PBS and developed for 25 min with an NBT/BCIP substrate (Sigma). The staining process was stopped by rinsing the plate with water and spot counts were determined with an Immunospot S2A Core Analyzer ELISpot reader (CTL).

The capacity of antigen-pulsed mDCs and moDCs to activate T-cell proliferation was evaluated by a carboxyfluorescein succinimidyl ester (CFSE)-based flow cytometry assay. DCs were pulsed with Aspergillus antigens for 18 h and washed twice with CellGro medium as described above. 5×10⁴ mDCs were co-cultured with 5×10⁵ autologous pan T-cells (1:10), stained with CFSE (5 µM, Life Technologies), in 550 µL CellGro medium for 6 days at 37°C. During this incubation, the remaining antigen-pulsed DCs were cryopreserved in 5CS-CryoStor medium (Stem Cell Technologies) at -80°C. Following restimulation with thawed antigen-pulsed DCs, T-cells were cultured for 6 additional days at 37°C and subsequently stained with fluorescently-labeled α-CD4 and α-CD8 antibodies (Miltenyi Biotec). The fraction of T-cells displaying reduced CFSE content compared to an unstimulated control was determined by flow cytometry.

6×10⁵ mDCs or moDCs were incubated in 200 µL CellGenix medium containing 50 µg/mL AfuLy, 5 µg/mL CcpA or 5 µg/mL Shm2, or in antigen-free medium for 18 h. Culture supernatants were sterile-filtered (0.2 µm) and cryopreserved at -80°C until further use.

In a 96-well plate, 1×10⁵ PMNs were combined with 5×10⁴ A. fumigatus ATTC 46645 germ tubes (in a total of 50 µL RPMI + 5% FCS) or were seeded in 50 µl RPMI + 5% FCS without addition of fungal cells. Thereafter, 33.3 µL of plain CellGenix medium or thawed DC supernatants diluted 1:4 with fresh CellGenix medium were added, containing the metabolites released by 2.5×10⁴ DCs (PMN-DC-ratio of 4:1). To quantify the release of reactive oxygen species, 16.7 µL dichlorofluorescein (DCF) solution was added (final concentration, 15 µg/mL). Thereafter, the plate was incubated for 2 hours in a temperature-controlled (37°C) SpectraMax iD3 Microplate Reader (Molecular Devices) and fluorescence was read every 5 min at an excitation and emission wavelength of 485 nm and 535 nm, respectively. Area-under-the-curve of time/relative fluorescence curves was calculated as a surrogate for the anti-Aspergillus oxidative burst. DC supernatants from three donors were combined with PMNs from three allogeneic donors for nine DC/PMN combinations. PMNs stimulated with phorbol 12-myristate 13-acetate (PMA, 10 ng/mL) were used as a positive control. PMN seeding density, DCF concentration, and multiplicity of infection were optimized in preceding experiments (unpublished data).

Significance testing was performed using the two-tailed, paired Student’s t-test or one-way repeated measures analysis of variance (RM ANOVA) with Dunnett’s post-hoc test, as detailed in the figure legends. Data were analyzed and visualized using Microsoft Excel and GraphPad Prism 8.

Maturation of DCs is necessary for efficient activation of adaptive immune cells (13). Therefore, we first assessed the ability of CcpA, Shm2, and AfuLy to induce MHC upregulation and maturation of mDCs and moDCs. After 18 h of stimulation, limited induction of CD40, CD80 and CD86 was found in moDCs stimulated with the proteinaceous antigens, whereas stimulation with the lysate did not alter surface expression of the maturation markers and co-stimulatory molecules that were tested ( Figure 1A ). While the lysate showed minimal stimulatory capacity in mDCs, Shm2 and CcpA induced greater upregulation (1.8-25.2-fold MFI) of all maturation markers tested compared with the unstimulated control ( Figure 1A ). CcpA led to a stronger induction of the co-stimulatory molecules CD80 and CD83 than Shm2 and elicited an 11-fold upregulation in the cell surface expression of homing receptor CCR7 on mDCs. For both CcpA and Shm2 stimulation, upregulation of maturation marker expression on the surface of mDCs was concentration-dependent ( Figure 1B ). For Shm2, we also evaluated the temporal kinetics of maturation marker expression by comparing mDCs exposed to 1 and 5 µg/mL Shm2 for 6 versus 18 hours. Except for the early activation marker CD83 (14), all differentially expressed markers showed more pronounced upregulation after overnight Shm2 stimulation ( Figure 1C ).

DCs secrete a broad spectrum of cytokines and chemokines orchestrating the response of other immune cell populations (15). Therefore, we stimulated mDCs and moDCs with Shm2, CcpA, and AfuLy for 18 h and compared cytokine concentrations in the culture supernatants with an 8-plex Luminex assay. Without stimulation, both DC subsets released consistently low amounts of TNF-α, IL-1β, and IL-12 p70, whereas baseline secretion > 10 pg/mL was seen for IL-6, CCL2 (MCP1), and CCL3 (MIP-1α) in moDCs and for IL-8 in both DC populations ( Figure 2 ). AfuLy had no significant impact on cytokine release in either subset. Shm2 and CcpA strongly induced all tested cytokines ( Figure 2 ) except IL-17A (data not shown) in both DC populations. While both protein antigens elicited higher levels of TNF-α (5.1-9.7 ng/mL) and IL-12 p70 (320-841 pg/mL) in moDCs than in mDCs, stronger induction of IL-1β (65-81 pg/mL), IL-6 (85-92 ng/mL), and CCL3 (1.5-2.4 ng/mL) was found in mDCs. Shm2 and CcpA induced greater absolute levels of CCL2 in moDCs than in mDCs, whereas relative induction compared to the unstimulated control was greater in mDCs.

Importantly, these minor quantitative differences in cytokine release by both DC subsets after Shm2 and CcpA stimulation were partially decoupled from cytokine responses elicited by PRR agonists (ultrapure LPS or beta-glucan) and inactivated A. fumigatus germ tubes ( Figure S1 ). For example, higher concentrations of IL-6 and CCL3 (MIP-1α) were found in response to the PRR agonists and germ tubes, whereas Shm2 and CcpA induced stronger release of these cytokines by mDCs.

Next, we assessed the capacity of mDCs and moDCs pulsed with Aspergillus antigens to drive IFN-γ secretion from autologous T-cells, a cytokine that is implicated in protective immunity to fungal infections (16–18). Therefore, DCs were stimulated for 18 h with Shm2, CcpA, or AfuLy, washed, and co-cultured with autologous T-cells in an IFN-γ ELISpot plate ( Figure 3A ). Compared to unstimulated mDCs, pulsing with CcpA, Shm2, and AfuLy caused significant induction of IFN-γ secretion by autologous T-cells ( Figures 3B, C ). Interestingly, despite less pronounced upregulation of maturation markers compared with mDCs ( Figure 1A ), Shm2- and CcpA-pulsed moDCs also stimulated T-cells to release IFN-γ ( Figures 3B, C ). For AfuLy, an even stronger response was seen in moDC- than in mDC-co-cultures ( Figure 3C ). However, the strongest IFN-γ response was elicited by CcpA-pulsed mDCs ( Figure 3C ). Importantly, no direct induction of IFN-γ spot formation was seen in T-cell monocultures stimulated with the antigens (data not shown), precluding that spot formation in co-cultures was due to unprocessed antigens remaining in the medium after harvesting and washing of DCs.

To corroborate that antigen-pulsed mDCs induce autologous T-cell activation, we quantified the frequencies of proliferating CD4⁺ and CD8⁺ cells with a flow cytometric CFSE-based assay ( Figure 3A ). T-cells alone or T-cells co-cultured with unstimulated mDCs showed low proliferation rates of 1.0-5.2%, whereas mDCs pulsed with Shm2, CcpA, and AfuLy induced significant proliferation of CD4⁺ T-helper cells (10.3-24.6%) and cytotoxic CD8⁺ T-cells (10.9-15.5%, Figure 3D ). While unstimulated moDCs induced stronger T-cell proliferation than mDCs, antigen loading of mDCs elicited greater relative changes in T-cell proliferation, particularly for CD8⁺ T-cells.

In addition to their impact on T-cell activation and proliferation, we compared the capacity of supernatants from antigen-pulsed mDCs and moDCs to enhance the anti-Aspergillus oxidative burst of PMNs ( Figure 4A ). Regardless of antigen pulsing, baseline ROS secretion of unstimulated PMNs in the presence of DC supernatants remained consistently low (fold change, 0.94-1.16, Figure 4B ). With germ tube stimulation, we observed at least a 3.6-fold increase in ROS release after 2-hours with all supernatants. Although unstimulated moDC supernatants caused a greater neutrophilic ROS response to the germ tubes than unstimulated mDC supernatants, a stronger relative increase in ROS release was observed with supernatants from mDC when pulsed with protein antigens, especially with CcpA (+62%, Figure 4B , right panel). In contrast, pulsing of DCs with the lysate reduced the stimulatory capacity of their supernatants on the neutrophilic ROS response.