Human cardiac stem cells inhibit lymphocyte proliferation through paracrine mechanisms that correlate with indoleamine 2,3-dioxygenase induction and activity

Fig. 1. Schematic representation of immunomodulatory assay experiments. Carboxyfluorescein succinimidyl ester (CFSE)-labeled and phytohemagglutinin (PHA)-activated human peripheral blood mononuclear cells (hPBMCs) were cultured with mitomycin-treated human cardiac/stem progenitor cells (hCSCs)/human adipose-derived mesenchymal stem cell (hASCs) either in direct contact (DC), in a transwell support (TW), or in contact with mitomycin-C-treated hCSC/hASC conditioned medium (Cond.M.). Viable T lymphocyte proliferation was accessed by CFSE labeling of the CD3+ 7AAD– hPBMC population

Fig. 2. Human cardiac/stem progenitor cells (hCSCs) display a favorable immune-suppressive phenotype. Representative expression of the immune relevant molecules indoleamine 2,3-dioxygenase (IDO) and programmed death ligand 1 (PDL-1) after 24 and 48 h in untreated (black line histograms) and interferon (IFN)-γ-activated (red line histograms) cells against isotype controls (gray-filled histograms). The percentages (%) of positive cells are indicated. Human adipose-derived mesenchymal stem cells (hASCs) were used as a positive control for IDO expression. hCSC results are shown for donor hCPC8. Other donors presented similar results

Fig. 3. Human cardiac/stem progenitor cells (hCSCs) inhibit T lymphocyte proliferation in a time- and hCSC concentration-dependent manner. CFSE-labeled hPBMCs were stimulated with PHA and cultured alone or in direct contact with hCSCs (ratios 1:10, 1:17, and 1:25 hCSCs:hPBMCs). After 72 h (white bars) and 96 h (black bars), proliferation of the viable population of CD3-viable T lymphocytes (CD3+/7AAD–) was assayed by loss of CFSE staining. Percentage of inhibition of proliferation was determined using FSC Express software against proliferation of activated hPBMCs alone. Human adipose-derived mesenchymal stem cells (hASCs) were used as a positive control for T cell proliferation inhibition (ratio 1:25 hASCs:hPBMCs). Adjusted p values are shown

Fig. 4. Human cardiac/stem progenitor cells (hCSCs) inhibit T lymphocyte proliferation via a paracrine mechanism. a CFSE-labeled hPBMCs were stimulated with PHA and cultured alone, in direct contact (DC), or in a transwell setting (TW) with hCSCs (ratio 1:10 hCSCs:hPBMCs). b Concentrations of tryptophan (Trp) and kynurenine (Kyn) were determined by HPLC in the supernatants. c CFSE-labeled hPBMCs were stimulated with PHA and cultured alone or in conditioned medium (Cond.M.) from hCSCs cultures activated or not with interferon (IFN)-γ. Conditioned media were generated for 24 h (white bars), 36 h (grey bars), and 48h (black bars). d Concentrations of Trp and Kyn were determined by HPLC in the conditioned media. Proliferation of the viable population of CD3 T lymphocytes (CD3+/7AAD–) was assayed by loss of CFSE staining after 72 h (white bars) and 96 h (black bars) for TW and DC experiments (a) and after 96 h for Cond.M. experiments (c). Percentage of cells per generation and percentage of inhibition of proliferation was determined using FSC Express software against proliferation of activated hPBMCs alone. Human adipose-derived mesenchymal stem cells (hASCs) were used as a positive control…