Figure 5.

Targeting fungal Idos by CTLA-4 Ig reduces Aspergillus pathogenicity

(A and B) Clinical isolates were grown overnight in liquid GMM at 37°C rotating at 200 rpm. Clinical isolates were obtained from CF patients, patients with acute invasive aspergillosis, and patients with chronic pulmonary aspergillosis. RT-PCR of Aspergillus genes involved in the catabolic pathway (Figure 2C) and (B) relative abundance (2^−ΔCT) of gene expression by qPCR analysis.

(C) idoA, idoB, and idoC Aspergillus mRNA expression in Aspergillus akuB^KU80 strain exposed to CTLA4-Ig, isotype control IgG3, or mutated CTLA-4 Ig (CTLA-4 104Y) overnight at 37°C.

(D) Histopathological analyses (PAS) in infected C57BL/6 and Ido1^–/– mice (n = 9) upon infection with the indicated Aspergillus strains in lung tissue at 7 dpi. Mice were treated intranasally with 50 μg/mouse of CTLA-4-Ig or IgG3 for 2 days, commencing on the second day of infection. Scale bars, 10 μm.

(E) FACS analysis of Aspergillus akuB^KU80 resting conidia (RC) exposed to CTLA-4-Ig or IgG3 and reacted with secondary anti-IgG3-fluorescein isothiocyanate (FITC) antibody (continuous black line). The gray histogram denotes unstained cells.

(F) Representative transmission electron microscopy (TEM) images of Aspergillus akuB^KU80 RC or swollen conidia (SC) treated with CTLA-4-Ig or IgG3 followed by secondary antibodies conjugated with gold particles.

(G) Fluorescence-activated cell sorting (FACS) analysis of Aspergillus akuB^KU80 RC treated with CTLA-4-Ig in the presence of anti-B71 antibody. The gray histogram denotes unstained cells.

(B and C) Data are represented as mean ± SD. Plots are representative of data collected from three independent replicate experiments. (C) Statistical significance (^∗p < 0.01) was determined with one-way ANOVA and Bonferroni post hoc test. (D–G) Micrographs and FACS overlay analysis are representative of three replicate experiments.

See also Figure S8.