Abstract

Invasive pulmonary aspergillosis is a primary cause of morbidity and mortality in immunocompromised patients, including hematopoietic progenitor cell transplant patients. Studies in patients with allergic bronchopulmonary aspergillosis as well as murine models have demonstrated the importance of a CD4+ Th1 T cell response in conferring protection from infection or preventing disease progression. A. fumigatus allergens have been identified based on reactivity to IgG and IgE antibodies in serum from patients with allergic bronchopulmonary aspergillosis. One allergen, Asp f16 was shown to induce a protective Th1 T cell response to infection with aspergillus conidia in murine studies. We prepared a series of 104 overlapping pentadecapeptides spanning the entire 427 aa coding region of Asp f16. Each 15 aa peptide overlaps the preceding peptide by 11 aa. Autologous monocytes from healthy donors were treated with GM-CSF and IL-4 for 2–3 days to generate immature dendritic cells (fast DC), pulsed with a pool containing 1 μg of each pentadecapeptide, then matured with inflammatory cytokines (IL-1β, IL-6, PGE2 and TNF-α) for 2 days. Mature, pulsed fast DC were used to prime proliferative and CTL responses (weekly primings). T cells from 5 of 5 donors proliferated to the peptide pool. CTL lines were obtained from each of the first two donors that were primed. As early as 3 weeks the T cell line from donor #1 had a high frequency of IFNγ-producing CD4+ T cells (24.5±5.4%) in response to the peptide pool, was cytotoxic to autologous peptide pool-pulsed and aspergillus culture extract pulsed DC, and peptide pool pulsed autologous BLCL. Supernatant from this culture killed fresh aspergillus conidia. Screening of 21 smaller pools containing 4–11 peptides showed reactivity to three smaller pools and could be narrowed by screening individual peptides shared by the pools to a single candidate sequence of TWSIDGAVVRT that elicited IFNγ production and lytic activity equal to the entire pool. Peptide pulsed BLCL matched with the donor for only 1 or 2 HLA alleles were used to demonstrate that CTL activity and IFNγ production was restricted by HLA-DRB1- 0301. 23% of the CD4+ cells in the culture expressed CD107a in response to peptide pool indicating degranulation. PBMC from DRB1-0301+ individuals proliferated strongly in response to peptide TWSIDGAVVRT. Our data show that DC pulsed with a pentadecapeptide pool from Asp f16 are capable of inducing a CD4+, HLA-DR-restricted aspergillus-specific Th1 type T cell response directed to a single peptide contained within the pool. Further characterization of this system is in progress to identify other immunogenic peptides from Asp f16 that might be useful in clinical immunotherapy protocols to prime protective immune responses to prevent or treat aspergillus infection.

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