Extended Data Fig. 7: Cross-reactivity of yeast-responsive T cells.

(a) α/β-TCR constructs of the most expanded cross-reactive TCRs identified from the single cell data set were inserted into primary human CD4+ T cells by orthotopic T cell receptor replacement using CRISPR/Cas. Dot plot examples showing CD4+ T cells with pulse only, knock-out of the endogenous TCR, combined knock-out of the endogenous TCR and knock-in of the transgenic TCR containing a murine β-chain constant region used as tracking marker. Transduced cells were further FACS purified and expanded to high purity. (b) TCR-α/β sequences selected from the scRNAseq data set for transgenic T cell generation. (c) S. cerevisiae-reactive Tmem from an ASCA+ CD patient were expanded and re-stimulated with various yeast species. Percentages of reactive CD154+ TNFα+ cells are indicated. (d) Cross-reactivity of expanded yeast-reactive T cells in ASCA- CD patients (C. albicans n = 4, S. cerevisiae, C. tropicalis n = 3) and ASCA+ CD patients (C. albicans, S. cerevisiae n = 4, C. tropicalis n = 3). Cross-reactivity is plotted as percentage in relation to total reactivity after stimulation with the initially used yeast species. (e) CMV serum antibody status of ASCA- CD patients (n = 39) and ASCA+ CD patients (n = 49) was determined by ELISA and percentage of CMV negative and CMV positive patients is indicated. (f) Yeast-reactive T cell frequencies of ASCA+ patients according to their CMV status (C. albicans CMVneg n = 32, CMVpos n = 17; C. tropicalis CMVneg n = 24, CMVpos n = 14; S. cerevisiae CMVneg n = 17, CMVpos n = 11). (g) TCR transgenic T cells (n = 7) were restimulated with common viral antigens. Cross-reactivity in relation to stimulation with S. cerevisiae is shown. Each symbol in (d, f) represents one individual donor and in (g) one TCR transgenic T cell line. Truncated violin plots with quartiles and range are shown in (d). Horizontal lines indicate geometric mean values in (f). Statistical differences: two-tailed Mann-Whitney test in (f).