Abstract
Background and aims. Aspergillus fumigatus infections are the leading cause of invasive fungal infection-related deaths in stem cell transplant patients, and may be amenable to correction with adoptive immunotherapy providing T lymphocytes specific for A. fumigatus. However, a clinically usable source of antigen and a reliable procedure for the generation of large numbers of Aspergillus-specific T lymphocytes to clinical-grade standards is not available. Methods. An environmental strain of A. fumigatus (WMAfES) was isolated and cultured using materials and reagents suitable for clinical manufacture. Water-soluble lysate from germinated conidia of WMAfES was used as the antigen source. Peripheral blood mononuclear cells were stimulated with antigen-pulsed autologous dendritic cells on days 0 and 7. Cells were expanded with a cocktail of interleukin (IL)-2, IL-7 and IL-15 from days 7 to 21. Results. We obtained a mean 32.8-fold increase in cell numbers over 21 days of culture (n = 8). Resultant cultures were predominantly effector and central memory CD4+ T cells, which produced T-helper (h)1 and Th17 cytokines when restimulated with A. fumigatus antigen derived from environmental or clinically isolated A. fumigatus. Cultured cells exhibited a high level of specific expansion and chemokine production when restimulated. Moreover, cultured cells cross-reacted with antigens from other fungi, including Penicillium, Candida albicans and other non-fumigatus Aspergillus species. Conclusions. We describe a simple, robust, reproducible and clinically applicable procedure using a clinically appropriate antigen preparation for the expansion of polyfunctional A. fumigatus-specific T cells from normal donors of varying HLA types.
Acknowledgments
We wish to thank Dr Karen Byth, Westmead Hospital, for help with statistical analysis, Dr Catriona Halliday, Centre for Infectious Diseases and Microbiology, Westmead Hospital, for kindly gifting pure strains of fungi for cross-reactivity experiments, and Professor Tania Sorrell and A/Professor Sharon Chen, Centre for Infectious Diseases and Microbiology, Westmead Hospital, for helpful discussions.
Authorship contributions: SG isolated WMAfES from the environment, designed and performed all experiments, performed data analysis and wrote the manuscript. LC had intellectual input. EB obtained consent from donors to participate in the study. WM performed molecular identification of WMAfES and edited the manuscript. DG initiated the study, provided intellectual input, assisted with experimental design and edited the manuscript.
Conflict of interest disclosures: The authors disclose no conflict of interest.