Fig. 2

T cell exosomes shuttle mtDNA and genomic DNA. a Representation of the number of sequence reads aligned with the mitochondrial genome in EVs (EVs) and mitochondrial extracts (mtDNA) isolated from human primary T cells. b Upper panel: Diagram of the mitochondrial genome, indicating the mtDNA regions analyzed by PCR (marked in red: HVRII, tRNALeu, COXII, and 16 S rRNA). Lower panel: Agarose gel showing the amplification products of the different mtDNA regions and the genomic DNA (β2-MICROGLOBULIN) analyzed in EVs purified from primary human T cells. c Agarose gel electrophoresis showing the detection of DNA in purified EVs. EVs obtained from the culture supernatant of J77 T were treated with DNase or DNase and Tx-100. Levels of mtDNA and genomic DNA were assessed by PCR amplification (HVRII and β2-MICROGLOBULIN, respectively). Isolated DNA from J77 T cells treated or not with DNase (DNA + DNase and DNA, respectively) is shown as control for DNase activity. d Distribution of mtDNA and TSG101 and CD63 in sucrose fractions. EVs from human Jurkat T cells were laid on a discontinuous sucrose gradient and floated by overnight centrifugation. DNA from gradient fractions was PCR-amplified for the HVRII mtDNA region. Proteins from gradient fractions were analyzed by immunoblot for TSG101 and CD63. A representative gel is shown (n = 3). e Exosomes from primary mouse T lymphoblasts were purified, coupled to aldehyde sulfate beads, and analyzed by flow cytometry by staining with the anti-DNA Ab under permeabilizing and non-permeabilizing conditions. Histograms show DNA staining in exosomes. Numbers are mean fluorescence intensities of the positive population from a representative experiment (n = 3). f Left: Histogram shows oxidized DNA staining in exosomes coupled to aldehyde sulfate beads with anti-8-hydroxydeoxyguanosine (8-OHdG)Ab under non-permeabilizing conditions. Right: qPCR analysis showing the enrichment of the indicated mitochondrial genes in the DNA obtained from exosomes and mitochondrial fractions from the same exosome-producing cells. DNA from both fractions was immunoprecipitated with 8-OHdG and total DNA Abs. Graph shows the ratio between oxidized DNA and total DNA in exosomes relative to their content in the mitochondrial fractions from the producing cells in duplicates from two independent experiments. t-test: *P-value < 0.05, ***P-value < 0.0001