Identification and Characterization of Human Aspergillus Fumigatus-Specific Tr1-(Like) Cells

The ubiquitous mold Aspergillus fumigatus (A. fumigatus) induces two forms of pathogenesis: invasive aspergillosis in neutropenic patients and allergic aspergillosis in patients with chronic obstructive lung disease as well as in immunosuppressed patients. Mouse models of aspergillosis suggest that not only effector T cells (T_eff) but also regulatory T cells (T_reg) play a crucial role for the regulation of a protective T cell-mediated immunity to A. fumigatus. However, it is little-known about the involvement of T_reg during A. fumigatus infection in humans. In order to develop new therapeutical strategies for the treatment of aspergillosis this project aims to understand the influence of regulatory T cells on A. fumigatus infection in humans.

Material/Methods: A. fumigatus-specific CD4⁺ T cell clones were established from PBMC of healthy donors. Based on this clone pool T_reg clones were identified due to their inability to proliferate in the absence of costimulation assessed by ³[H]-TdR incorporation as well as their Ag-specific cytokine production and phenotype determined by flow cytometry. T_reg function was analyzed by their ability to suppress proliferation of autologous CD4⁺ T cells using CFSE dilution.

We identified A. fumigatus-specific T cell clones that exhibited marginal detectable proliferation after restimulation with immobilized anti-CD3 mAb in the absence of costimulation. However, these T cell clones vigorously proliferated in response to restimulation with their cognate antigen. A more detailed characterization showed that these suppressor T cell clones produced high amounts of IL-10 and moderate levels of IFN-gamma upon Ag-specific restimulation and expressed low amounts of Foxp3 but not Helios, a transcription factor that had recently been linked to natural occurring T_reg. Most importantly, these T cell clones suppressed Ag-specific expansion of CD4⁺ T_eff. This effect was contact-independent since suppression of Ag-specific CD4⁺ T cell expansion detected in transwell experiments was comparable to cocultures that enabled cellular-contact. Furthermore, anti-CD3/CD28-induced proliferation of naïve CD4⁺ T cells was not reduced in the presence of culture supernatants obtained from suppressor T cell clones after their antigen-specific restimulation in the absence of DCs.

We identified for the first time A. fumigatus-specific CD4⁺ T cell clones with a Tr1(-like) IL-10⁺IFN-gamma⁺Foxp3^lowHelios⁻ phenotype. These cells suppressed expansion of A. fumigatus-specific T_eff in an Ag-specific manner mediated by soluble factors released from Tr1(-like) cell clones. Since these factors did not affect CD4⁺ T cell proliferation in the absence of DCs our data suggest, that Tr1(-like) cell clones rather negatively regulate the stimulatory capacity of DCs leading to a reduced expansion of Ag-specific CD4⁺ T cells. Therefore these Tr1(-like) cells might play a protective role during A. fumigatus infection in humans. Thus, adoptive transfer of A. fumigatus-specific T_reg could be useful to enhance protective immunity in patients with chronic A. fumigatus infection.

Topp:Micromet: Consultancy, Honoraria.