Fig 1. Construction of a GXMR-CAR that targets Cryptococcus spp.

(A) DNA sequence (left) and schematic representation (right) of the GXMR-CAR targeting Cryptococcus spp. has a scFv portion derived from the anti-GXM mAb 18B7. The DNA encodes four domains: the signaling domains of CD3-ς and CD28 (yellow and green regions, respectively), the transmembrane (Tm) domain of CD28 (green region), the constant region of IgG4 (black line), a scFv portion from 18B7 (blue region), and an enhanced GFP (EGFP) portion for detection via fluorescence (white region). The DNA sequence was subcloned into a lentiviral (LV) vector backbone. The LV-GXMR-CAR construct was used for transfection and transduction. Ec, extracellular binding domain; cyto, cytoplasmic signaling region. (B) HEK-293FT cells (1 × 10⁶ cells/ml) were used to make GXMR-CAR⁺ viral particles via transfection with all three plasmids combined: LV-GXMR-CAR, pMD2.G, and psPAX2, using Lipofectamine. Mock- transduced HEK-293FT cells (NoDNA) were used as negative controls. HEK-293FT cells were visualized by using fluorescence microscopy in bright-field (BF) and green (GFP) channels at 100× and 200× magnification 24 h after transfection. (C) GXMR-CAR⁺ viral particles were used to transduce HEK-293FT cells. HEK-293FT cells were evaluated via flow cytometry and expressed in a histogram (left). HEK-293FT cells were visualized by using fluorescence microscopy in brightfield and GFP channels at 100× magnification (right) 3 days after transduction. Mock-transduced negative control cells (NoDNA) did not receive the GXMR-CAR construct.