Fig. 2

Transfer of MSC mitochondria to HNT CD4⁺ T cells inhibits their expansion and gain of effector function. A Isolated mitochondria from MSC-A labeled with Mitotracker Deep Red were transferred by mitoception to naïve HNT CD4⁺ T cells. Control HNT CD4⁺ T cells were mock mitocepted without mitochondria. 12 h later CD4⁺ T cells were analyzed by FACS. Data from one representative experiment out of three is presented. Values represent MFI ± SEM. B Isolated mitochondria from MSC-A labeled with Mitotracker Deep Red were transferred by mitoception to CFSE-labeled HNT CD4⁺ T cells. After 12 h cells were prepared for microscopy. Three-dimensional reconstruction of confocal microscopy images of T cells (left, whole cells; right, cell sections). Scale bar 10 μm. C Isolated mitochondria from MSC-A were transferred by mitoception to naïve HNT CD4⁺ T cells. 12 h later, mitocepted or mock mitocepted HNT CD4⁺ T cells were activated with anti-CD3 and anti-CD28 mAbs and cultured during 4 days. Absolute numbers of cultured HNT CD4⁺ T cells are shown. Values are represented as mean ± SEM. D Mitocepted or mock mitocepted CFSE labeled HNT CD4⁺ T cells were activated and cultured. After 4 days, CD4⁺ T cells were harvested and CFSE fluorescence analyzed by FACS. Viability was assessed by 7-AAD uptake. Proliferation index was calculated using FlowJo software. Data from 3 independent experiments is presented. Values are represented as mean ± SEM. E Activated HNT CD4⁺ T cells were restimulated with PMA and ionomycin in the presence of brefeldin A and production of intracellular IFNγ was assessed by FACS. Percentage of cytokine-producing T cells is indicated. F Expression of CD25 and CD40L in activated HNT CD4⁺ T cells. Mean fluoresce intensity (MFI) minus that of the isotype controls is indicated. Values are represented as mean ± SEM. Data from 5 independent experiments is presented in panels B, C, E and F