Fig. 4.

Exosomal transfer of mitochondria by MDRCs to T cells. Transferred exosomal mitochondria merge with T cell mitochondrial network. (a) Schematic for experimental approach to validate exosomal transfer of mitochondria in co-cultures of exosomes and peripheral CD4⁺ T cells. (b) ImageStream flow cytometry images showing MitoT-Green⁺ BAL-derived exosomes within peripheral autologous CD4⁺ T cells (c) Quantitation of % CD4⁺ T cells in which MitoT- Green⁺ airway exosomes were transferred following co-culture (no statistical significance observed; Student's T-test) (b & c: n = 4 healthy, n = 4asthmatics, 3 technical replicates each) (d) ImageStream flow cytometry images of autologous peripheral CD4⁺ T cells following transfer of MDRC-derived MitoT-Green⁺ exosomes (n = 4 healthy, n = 3 asthmatics; 3 technical replicates each) (e) Quantitation of % CD4⁺ T cells in co-cultures with transferred MDRC-derived Mito-GFP⁺ exosomes (no statistical significance observed; Student's T-test) (n = 6 healthy, n = 6 asthmatics; 3 technical replicates each) (f) ImageStream flow cytometry images showing overlap of host T cell MitoT-Red signal with transferred Mito-GFP from MDRC-derived exosomes, validating merging of these transferred exosomes with the mitochondrial network of the host cells (f & g: n = 3 healthy, n = 3 asthmatics; 3 technical replicates each). (g) ImageStream Flow cytometry images of MitoSox labeling indicating ROS in peripheral autologous CD4⁺ T cells that partially overlaps with transferred MDRC-derived Mito-GFP⁺ exosomes.