Development of a Simple and Robust Whole Blood Assay with Dual Co-Stimulation to Quantify the Release of T-Cellular Signature Cytokines in Response to Aspergillus fumigatus Antigens

Abstract

Deeper understanding of mold-induced cytokine signatures could promote advances in the diagnosis and treatment of invasive mycoses and mold-associated hypersensitivity syndromes. Currently, most T-cellular immunoassays in medical mycology require the isolation of mononuclear cells and have limited robustness and practicability, hampering their broader applicability in clinical practice. Therefore, we developed a simple, cost-efficient whole blood (WB) assay with dual α-CD28 and α-CD49d co-stimulation to quantify cytokine secretion in response to Aspergillus fumigatus antigens. Dual co-stimulation strongly enhanced A. fumigatus-induced release of T-cellular signature cytokines detectable by enzyme-linked immunosorbent assay (ELISA) or a multiplex cytokine assay. Furthermore, T-cell-dependent activation and cytokine response of innate immune cells was captured by the assay. The protocol consistently showed little technical variation and high robustness to pre-analytic delays of up to 8 h. Stimulation with an A. fumigatus lysate elicited at least 7-fold greater median concentrations of key T-helper cell signature cytokines, including IL-17 and the type 2 T-helper cell cytokines IL-4 and IL-5 in WB samples from patients with Aspergillus-associated lung pathologies versus patients with non-mold-related lung diseases, suggesting high discriminatory power of the assay. These results position WB-ELISA with dual co-stimulation as a simple, accurate, and robust immunoassay for translational applications, encouraging further evaluation as a platform to monitor host immunity to opportunistic pathogens.

Chronic Occupational Mold Exposure Drives Expansion of Aspergillus-Reactive Type 1 and Type 2 T-Helper Cell Responses

“…Accordingly, ELISPOT and WB-based Luminex assays revealed marked induction of A. fumigatus antigen-reactive Th1-and Th2-cell-derived cytokines in organic farmers, whereas differences in IL-17 levels were much smaller between the two cohorts. In contrast to these findings in our healthy mold-exposed cohort of agricultural workers, we and others previously reported that patients with Aspergillus-associated hypersensitivity diseases, such as ABPA, display strongly elevated Th17 cytokine responses to A. fumigatus antigens [12,28]. Although important in the initial stage of fungal clearance [29][30][31][32], enhanced Th17-cell activation has been associated with Aspergillus airway colonization and fungal persistence and is considered a driving factor in the establishment of hypersensitivity pneumonitis, a potential complication of chronic occupational mold exposure [28,[33][34][35][36][37].…”

“…Compared to PBMC-based assays, WB provides a more physiological stimulation environment, allowing us to capture feedback loops between T cells and innate immune cells that can enhance cytokine responses [12]. This might explain why Crf1 and Aspf4, which we selected for our WB-based Luminex assay due to their known allergenic properties [19][20][21][22][23][24], elicited stronger differences in cytokine responses between organic farmers and controls in the Luminex panel than in the ELISPOT assay.…”

“…An A. fumigatus mycelial lysate (AfuLy) and proteinaceous antigens were generated as described before [12]. Primer sequences for the amplification of fungal cDNA are provided in Table S1.…”

“…Anticoagulant-free stimulation tubes were prepared as detailed in Table S2, following a previously published protocol [12], and cryopreserved at −20 • C for up to four weeks. Prior to whole-blood (WB) stimulation, the ready-to-use stimulation tubes were brought to room temperature.…”

“…Therefore, we employed flow cytometry, enzyme-linked immunospot assays (ELISPOT), and a recently developed whole-blood-based cytokine release assay [12] with dual T-cellular co-stimulation in order to compare T-helper (Th) cell responses to mold antigens in a defined occupationally mold-exposed cohort and non-occupationally exposed controls. We selected agricultural workers as our occupationally exposed cohort due to their known risk for extensive Aspergillus exposure, mold sensitization, and development of asthma and hypersensitivity pneumonitis ("farmer's lung") [5,6,13].…”