Ionomycin, which induces strong Ca2+ flux, was used as a positive control. with TEX. All exosomes induced Ca2+ influx in T cells, with TEX and EXO isolated from malignancy patients’ plasma delivering the strongest, sustained signaling to Treg. Such sustained signaling resulted in the significant upregulation of the conversion of extracellular ATP to inosine (adenosine metabolite) by Treg, suggesting that TEX signaling could have functional effects in these recipient cells. Thus, modulation of Treg suppressor functions by TEX is usually mediated by mechanisms dependent on cell surface signaling and does not require TEX internalization by recipient cells. values denote significant differences. Differences in the exosome uptake at 24?h between T cells and the other MNC subsets were highly significant (Fig.?3A). Clearly, the uptake of exosomes by immune cells depended on the type of recipient immune cells: T cells did not internalize exosomes, while the other MNCs did. To determine whether pre-activation of the recipient cells influences exosome uptake, we co-incubated resting or activated T cells with PKH26-labeled TEX or DEX. As shown in Fig.?S2A, the activation of the recipient T cells had no effect on the uptake of either TEX or DEX, which was equally low and not significantly different for these two exosome types. In contrast to T cells, resting or activated B cells, effectively internalized TEX or DEX, and the uptake by activated B cells was greater (= 0.03) than that by resting B cells (Fig.?S2B). Also, activated NK cells and monocytes internalized TEX or DEX with significantly greater efficiency (< 0.0001) than activated recipient T cells (Fig.?S2C). In aggregate, the Amnis-generated results showed that TEX and DEX are equally well internalized by MNC, except for T cells that did not internalize either. Pre-activation of recipient cells appears to improve the uptake of TEX and Roflumilast DEX by monocytes and NK cells as well as B cells. Exosome interactions with Treg We have previously reported that this co-incubation of CD4+CD25hiCD39+ Treg with TEX or DEX induced changes in the transcriptome of the recipient cells.17 Therefore, it was of interest to determine whether Treg internalized any TEX or DEX relative to CD8+ or CD4+Tconv cells. As shown in Fig.?3B, pre-activated or resting CD8+ T cells did not take Roflumilast up labeled exosomes during 24?h co-incubation, with CD4+ Tconv cells demonstrating only poor positivity by Amnis, and Treg showing significantly better but still very low uptake at 24?h (compare with the uptake by other MNC in Fig.?3A). The activation of Treg via the T cell receptor (TcR) did not improve exosome uptake, and the uptake of EXO obtained from malignancy patients’ or normal control’s (NC’s) plasma was equivalent to that of TEX or DEX (data not shown). Fig.?4 presents representative Amnis images of the TEX uptake by monocytes and various T cell subsets following 48?h and 72?h co-incubation with labeled exosomes. The images clearly show that in comparison to unfavorable CD8+T cells and CD4+Tconv, poor but detectable remnants of PKH26+ exosomes can be encountered in some but not all Treg. Thus, interactions of TEX, DEX, or EXO with T lymphocytes did not involve their internalization, except in the case of Treg, where the binding of exosomes to the cell surface was followed by Roflumilast poor and reluctant internalization. Open in a separate window Physique 4. Amnis-generated representative images of recipient MNC co-incubated with PKH26-labeled TEX for 48 or 72?h. Immune cell subsets were isolated from healthy donors’ plasma and analyzed by Amnis Image Stream as explained in Methods. The presented images are representative results of four experiments performed with MNC of different donors and show results obtained by a triple overlay (PKH26-stain in yellow, surface stain in reddish, and a brightfield image) as explained in Methods. Exosomes induce Ca2+ influx in T cells The data we previously reported showed that TEX MDA1 induced significant changes in the phenotype and functions of T lymphocytes, including Treg.15 The Amnis uptake data for TEX described above suggest that.

Ionomycin, which induces strong Ca2+ flux, was used as a positive control