Healthy human T-Cell Responses to Aspergillus fumigatus antigens

Abstract

Background: Aspergillus fumigatus is associated with both invasive and allergic pulmonary diseases, in different hosts. The organism is inhaled as a spore, which, if not cleared from the airway, germinates into hyphal morphotypes that are responsible for tissue invasion and resultant inflammation. Hyphae secrete multiple products that function as antigens, evoking both a protective (T(H)1-T(H)17) and destructive allergic (T(H)2) immunity. How Aspergillus allergens (Asp f proteins) participate in the development of allergic sensitization is unknown.

Methodology/principal findings: To determine whether Asp f proteins are strictly associated with T(H)2 responses, or represent soluble hyphal products recognized by healthy hosts, human T cell responses to crude and recombinant products were characterized by ELISPOT. While responses (number of spots producing IFN-gamma, IL-4 or IL-17) to crude hyphal antigen preparations were weak, responses to recombinant Asp f proteins were higher. Recombinant allergens stimulated cells to produce IFN-gamma more so than IL-4 or IL-17. Volunteers exhibited a diverse CD4+ and CD8+ T cell antigen recognition profile, with prominent CD4 T(H)1-responses to Asp f3 (a putative peroxismal membrane protein), Asp f9/16 (cell wall glucanase), Asp f11 (cyclophilin type peptidyl-prolyl isomerase) and Asp f22 (enolase). Strong IFN-gamma responses were reproduced in most subjects tested over 6 month intervals.

Conclusions: Products secreted after conidial germination into hyphae are differentially recognized by protective T cells in healthy, non-atopic individuals. Defining the specificity of the human T cell repertoire, and identifying factors that govern early responses may allow for development of novel diagnostics and therapeutics for both invasive and allergic Aspergillus diseases.

Figure 1. Recombinant Asp f proteins expressed in E. coli.

SDS-PAGE of Asp f3, Asp f6, Asp f9/16, Asp f11, Asp f12, Asp f17, and Asp f22 produced in E. coli with C-terminal c-myc and His₆ tags. Each lane contains one microgram of protein. Triangles identify Asp f proteins. Asp f22 appears as a doublet due to loss of the C-terminal tags in the faster migrating protein species. Molecular size standards (SeeBlue Plus2 Pre-Stained Standards, Invitrogen) are shown at left in kD.

Figure 2. ELISPOT assays using A. fumigatus crude hyphal extract (CHE) and purified recombinant antigens.

(a) IFN-γ (b) IL-4 and (c) IL-17 ELISPOT assay. Blood was collected from 20 healthy donors and triplicate samples of 1×10⁵ (IFN-γ) or 2×10⁵ PBMCs (IL-4 or IL-17) were stimulated with medium, 5 µg/ml phytohemoagglutinin (PHA) or 1 µg/ml CHE or recombinant A. fumigatus antigens for 24–48 hrs at 37°C/5%CO₂. The mean antigen-specific spot forming cells, SFC (after background subtraction of control wells with no antigen) is plotted. The median for each set of healthy individuals is indicated by a bar. The figures show one representative data from three independent experiments. ^*** P<0.001, ^** P<0.01 versus CHE stimulated PBMC.

Figure 3. A. fumigatus antigens recognition by 20 healthy donors.

(a) IFN-γ (b) IL-4 and (c) IL-17 responses. Blood was collected from 20 healthy donors and triplicate samples of 1×10⁵ PBMCs were stimulated with medium, 5 µg/ml PHA or 1 µg/ml CHE or recombinant A. fumigatus antigens for 24–48 hr at 37°C/5%CO₂. The mean antigen-specific spot forming cells, SFC (after background subtraction of control wells with no antigen) is plotted.

Figure 4. IFN-γ stability assay with rAsp f3 and rAsp f9 after period of 6 months.

To determine whether the IFN-γ responses are stable over time, blood was collected from high responders after six month interval of previous assay and triplicate samples of 1×10⁵ IFN-γ PBMC were stimulated with medium and 1 µg/ml rAsp f3 (a) or rAsp f9 (b) for 18–24 hrs at 37°C/5%CO₂. The mean antigen-specific spot forming cells, SFC (after background subtraction of control wells with no antigen) ± SD is shown. The figures show one representative data from three independent experiments.

Figure 5. IFN-γ ELISPOT assay with purified CD4⁺ and CD8⁺ T-cells.

CD4⁺ or CD8⁺ T-cells (1×10⁵) were isolated from strong responders' PBMC, incubated with 1 µg/ml of (a) rAsp f3 and (b) rAsp f9 with autologous irradiated PBMC (5×10⁴) in triplicates for 24 hr at 37°C/5% CO₂. PHA (5 µg/ml) was used as a positive control. The mean antigen-specific spot forming cells, SFC (after background subtraction of control wells with no antigen) ± SD is shown.