Method to generate clinical grade fungus-specific T cells targeting different fungal species. (A) Diagram of the method used for selection and enrichment of fungus-specific T cells from stem cell apheresis product. Representative flow cytometric analysis of the CD137-selected product after activation with fungal lysate (showing the content of CD3⁺CD137⁺ and CD4⁺ or CD8⁺ from this population). (B) Total cell number of fungus-specific T cells selected using different fungal lysates at the start of culture (CD137⁺-selected cells, day 0) and after expansion (end of culture, day 11). (C) Total cell number of T cells (FUN T cells) at the start of culture, after expansion, and after restimulation for 7 or 8 days with irradiated PBMCs pulsed with A terreus and C krusei lysates. Results are means of 3 to 5 experiments (±SEM) and shown total cell count. (D) TNF-α expression in fungus-specific T cells selected and expanded using different fungal lysates (shown on the top x-axis) after 6-hour coculture with DCs pulsed with a range of fungal species (shown on the y-axis) analyzed by flow cytometry. Results are expressed in terms of heat map in which each square represents the mean value percentage of CD4⁺ TNF-α⁺ cells from 3 to 6 experiments. Results are expressed in terms of heat map in which each square represents the mean value percentage of the indicated marker from 3 to 6 experiments. (E) Representative flow cytometric analysis showing TNF-α expression determined by flow cytometry in panfungal T cells after 6-hour coculture with DCs pulsed with individual fungal lysates, a mix of all fungal lysates, or pp65 cytomegalovirus (CMV) pepmix as irrelevant control antigen. Results are means of 6 experiments and shown as percentage of CD4⁺ T cells (*P ≤ .001 DC control vs fungal lysate–pulsed DC; paired, 2-tailed Student t test). AT, A terreus; CK, C krusei; CTR, control; RO, R oryzae.