Fig. 1

Allogeneic bone marrow-derived MSCs suppress HNT CD4⁺ T cell responses and transfer mitochondria. A Naïve purified HNT CD4⁺ T cells were activated with anti-CD3 and anti-CD28 mAbs and co-cultured with C57BL/6 bone marrow-derived MSCs (MSC) or TNFα/IFNγ-activated MSCs (MSC-A). Control activated HNT CD4⁺ T cells were left untreated without any MSCs (Non-Tr). After 4 days, CD4⁺ T cells were harvested and enumerated. Absolute numbers of cultured HNT CD4⁺ T cells are shown. Values are represented as mean ± SEM. Data from 5 independent experiments is presented. B CFSE-labeled HNT CD4⁺ T cells were activated and co-cultured with MSC-A or left untreated. At day 4, 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 5 independent experiments is presented. Values are represented as mean ± SEM. C Day 4 activated HNT CD4⁺ T cells cultured in the presence or absence of MSC-A were harvested and expression of CD25 and CD40L activation markers was assessed by FACS. Mean fluorescence intensity (MFI) minus that of the isotype controls is indicated. Data from 4 independent experiments is presented. Values are represented as mean ± SEM. D Day 4 activated HNT CD4⁺ T cells were restimulated with PMA and ionomycin in the presence of brefeldin A and production of intracellular IFNγ and IL-2 was assessed by FACS. Percentage of cytokine-producing T cells is indicated. Data from 4 independent experiments is presented. Values are represented as mean ± SEM. E HNT CD4⁺ T cells were activated and cultured with MSC-A or Mitotracker Deep Red-labeled MSC-A. As control HNT CD4⁺ T cells were activated and cultured with the supernatant of Mitotracker Deep Red-labeled MSC-A that underwent the same process of labeling, washing and culturing time. After 12 h, harvested T cells were analyzed by FACS. Data from one representative experiment out of three is presented. Values represent MFI ± SEM. F HNT CD4⁺ T cells were stained with CFSE and cultured with MitoTracker Red CMXRos-labeled MSC-A for 12 h. Three-dimensional reconstruction of confocal microscopy images of T cells (left, whole cells; right, cell sections). Scale bar 10 μm