Figure 1. A. fumigatus–reactive conventional CD4⁺ T cell responses in pwCF.

(A) Dot plot examples for the ex vivo detection of A. fumigatus–reactive CD4⁺ T cells by ARTE. PBMCs (1 × 10⁷) were stimulated with A. fumigatus or left unstimulated. Cell counts before and after magnetic CD154⁺ enrichment are indicated in the plots. (B) Frequencies of A. fumigatus–reactive CD154⁺CD4⁺ T cells in healthy donors (n = 220) and pwCF (n = 200). (C) CD45RA and Ki-67 staining of A. fumigatus–reactive CD154⁺ T cells. The percentage of marker-positive cells within the CD154⁺ population is indicated. (D) Frequencies of A. fumigatus–reactive CD154⁺CD45RA^–memory CD4⁺ T cells (Tmem) in healthy donors (n = 220) and pwCF (n = 200). (E) Ki-67 expression of A. fumigatus–reactive CD154⁺ T cells (healthy individuals, n = 65; pwCF, n = 200). (F and G) TCR-β sequence analysis of the top 50 expanded A. fumigatus–specific T cell clones from healthy individuals and pwCF (n = 5 for both). (F) Gini index depicting the distribution of TCR sequences (0 is total equality, i.e., all clones have the same proportion; 1 is total inequality, i.e., a population dominated by a single clone). (G) Rényi diversity profiles. Values of α = 0, 1, 2, and infinite, correspond to the richness, Shannon diversity, Simpson diversity, and Berger-Parker index, respectively. The sample with the highest value at α = 0 has the highest richness, but the lower value at α = infinite indicates a higher proportion of the most abundant sequence, i.e., lower population diversity. A sample with a profile that is overall higher than the profiles of other samples is therefore more diverse. (H and I) A. fumigatus–reactive CD154⁺ Tmem cells from healthy donors (n = 11) and pwCF (n = 8) were FACS purified, expanded, and restimulated in the presence of autologous FastDCs derived from blood monocytes. (H) Dose-response curves of expanded T cell lines, restimulated with decreasing antigen concentrations. (I) EC₅₀ values were calculated from dose-response curves. Each symbol in B, D–F, and I represents 1 donor; horizontal lines indicate the mean in B, D, and E and the geometric mean in I. Box-and-whisker plots display quartiles and range in F. Data indicate the mean ± SEM in G and H. Statistical differences were determined by 2-tailed Mann-Whitney U test in B, D, E, and I; 2-tailed, unpaired t test in F; and Kruskal-Wallis test in G.