Human mesenchymal stromal cells from adult and neonatal sources: a comparative in vitro analysis of their immunosuppressive properties against T cells

Abstract

Bone marrow-mesenchymal stromal cells (BM-MSCs) have immunosuppressive properties and have been used in cell therapies as immune regulators for the treatment of graft-versus-host disease. We have previously characterized several biological properties of MSCs from placenta (PL) and umbilical cord blood (UCB), and compared them to those of BM-the gold standard. In the present study, we have compared MSCs from BM, UCB, and PL in terms of their immunosuppressive properties against lymphoid cell populations enriched for CD3(+) T cells. Our results confirm the immunosuppressive potential of BM-MSCs, and demonstrate that MSCs from UCB and, to a lesser extent PL, also have immunosuppressive potential. In contrast to PL-MSCs, BM-MSCs and UCB-MSCs significantly inhibited the proliferation of both CD4(+) and CD8(+) activated T cells in a cell-cell contact-dependent manner. Such a reduced proliferation in cell cocultures correlated with upregulation of programmed death ligand 1 on MSCs and cytotoxic T lymphocyte-associated Ag-4 (CTLA-4) on T cells, and increased production of interferon-γ, interleukin-10, and prostaglandin E2. Importantly, and in contrast to PL-MSCs, both BM-MSCs and UCB-MSCs favored the generation of T-cell subsets displaying a regulatory phenotype CD4(+)CD25(+)CTLA-4(+). Our results indicate that, besides BM-MSCs, UCB-MSCs might be a potent and reliable candidate for future therapeutic applications.

Figures

FIG. 1.

BM-MSCs and UCB-MSCs decrease both CD4+ and CD8+ T-cell proliferation in a cell contact-dependent manner. Anti-CD3/CD28-activated CD3+ T cells were cocultured in the presence of BM-MSCs, UCB-MSCs, or PL-MSCs at a 1:1 ratio of MSCs:T cells. Cocultures were prepared in the presence (black bars) or absence (white bars) of cell–cell contact; the latter were performed with transwell chambers. Proliferation of activated CD3+ T cells cultured in absence of MSCs was the positive control (100% proliferation, n=10, gray bars). The percentage of BrdU incorporation was determined in the CD3+, CD4+, and CD8+ populations after 3–4 days of culture. (A) Representative dot plots from an experiment. (B) Data are shown as the mean±SEM for the percentages of proliferation (BM-MSCs: n=10 with cell–cell contact, n=5 transwell; UCB-MSCs: n=10 with cell–cell contact, n=5 transwell; and PL-MSCs: n=10 with cell–cell contact, n=5 transwell. Individual experiments). *Indicates a statistically significant difference with P<0.05. BM-MSCs, bone marrow mesenchymal stromal cells; PL, placenta; UCB, umbilical cord blood; BrdU, 5-bromo-2-deoxyuridine; SEM, standard error of mean.

FIG. 2.

MSCs effects on CD25, CD69, and CTLA-4 expression. Anti-CD3/CD28-activated CD3+ T cells were cocultured in the absence or presence of BM-MSCs, UCB-MSCs, or PL-MSCs at a 1:1 ratio of MSCs:T cells (black bars). Cocultures were performed with cell–cell contact. Molecule expression on activated T cells cultured in absence of MSCs was the positive control (Control: CD25 n=7, CD69 n=5, and CTLA-4 n=10, gray bars). The percentages of marker expression were determined in the CD3+ population after 48 h (CD25) or 24 h (CD69 and CTLA-4). (A) Representative histograms for CD25, CD69, and CTLA-4. (B) Data are shown as the mean±SEM of the percentages of expression (CD25: BM-, UCB-, or PL-MSCs n=7 individual experiments for each cell source; CD69: BM-, UCB-, or PL-MSCs n=7 individual experiments for each cell source and CTLA-4 BM-, UCB-, or PL-MSCs n=10 individual experiments for each cell source). *Indicates a statistically significant difference with P<0.05.

FIG. 3.

BM-MSCs and UCB-MSCs increase the frequency and intensity of CTLA-4 expression on CD4+CTLA-4+ T cells. Anti-CD3/CD28-activated CD3+ T cells were cocultured in the absence or presence of BM-MSCs, UCB-MSCs, or PL-MSCs at a 1:1 ratio of MSCs:T cells. Cocultures were performed with and without (Transwell, white bars) cell–cell contact (black bars). CTLA-4 expression on activated T cells cultured in absence of MSCs was the expression control (Control, n=7, gray bars). The percentages of CTLA-4+ cells and the fold changes the CTLA-4 MFI were determined in the CD4+ population after 3 days of culture. (A) Representative dot plots from an experiment. (B) Data are shown as the mean±SEM of the percentage of CD4+CTLA-4+ and (C) the fold increases in the CTLA-4 MFI in the CD4+CTLA-4+ population. BM-MSCs: n=7 with cell–cell contact, n=7 transwell; UCB-MSCs: n=7 with cell–cell contact, n=7 transwell; and PL-MSCs: n=7 with cell–cell contact, n=7 transwell (Individual experiments). *Indicates a statistically significant difference with P<0.05. MFI, mean fluorescence intensity.

FIG. 4.

PD-L1 expression is increased in MSCs in the presence of activated CD3+ T cells. BM-MSCs, UCB-MSCs, or PL-MSCs were cocultured in the absence or presence of anti-CD3/CD28-activated CD3+ T cells at a 1:1 ratio of MSCs:T cells. Cocultures were performed with (Contact, black bars) and without (Transwell, white bars) cell–cell contact. PD-L1 expression on MSCs cultured in absence of activated T cells was considered as basal expression of such molecule (Control: BM-MSCs n=6, UCB-MSCs n=5, PL-MSCs n=4, gray bars). The percentages of PD-L1+ cells and the fold increase in the PD-L1 MFI were determined in the CD3-CD90+ population after 3 days of culture. (A) Representative dot plots from an experiment. (B) Data are shown as the mean±SEM of the percentages of CD3-CD90+PD-L1+ cells and (C) the fold increases in PD-L1 MFI in the CD3-CD90+PD-L1+ population. BM-MSCs: n=6 with cell–cell contact, n=6 Transwell; UCB-MSCs: n=5 with cell–cell contact, n=5 transwell; and PL-MSCs: n=4 with cell–cell contact, n=4 transwell (Individual experiments). *Indicates a statistically significant difference with P<0.05. PD-L1, programmed death ligand 1.

FIG. 5.

IFNγ and IL-10 expression are strongly induced in cell-contact cocultures with BM-MSCs and UCB-MSCs. Anti-CD3/CD28-activated CD3+ T cells were cocultured in the absence or presence of BM-MSCs, UCB-MSCs or PL-MSCs at a 1:1 ratio of MSCs:T cells. Cocultures were performed with (black bars) and without (Transwell, white bars) cell–cell contact. IFNγ, IL-10, TNFα and IL-4 concentrations in cell-free supernatants were determined with a cytometric bead array after 3 days of culture. Cytokines concentration detected in conditioned medium of activated T cells cultured in absence of MSCs, were considered as basal expression of such cytokines (Control, n=7, gray bars). Data are shown as the mean±SEM of the cytokine concentrations (BM-MSCs: n=7 with cell–cell contact, n=7 transwell; UCB-MSCs: n=7 with cell–cell contact, n=7 transwell and PL-MSCs: n=7 with cell–cell contact, n=7 transwell. Individual experiments). *Indicates a statistically significant difference with P<0.05. IFNγ, interferon-γ; IL, interleukin; TNFα, tumour necrosis factor alfa.

FIG. 6.

PGE2 expression is induced in BM-MSCs and UCB-MSCs cocultures. BM-MSCs, UCB-MSCs, or PL-MSCs were cocultured in the absence or presence of anti-CD3/CD28-activated CD3+ T cells at a 1:1 ratio of MSCs:T cells. Cocultures were performed with cell–cell contact (black bars). PGE2 concentrations in cell-free supernatants were determined by ELISA after 3 days of culture. PGE2 concentrations detected in conditioned medium of MSCs cultured in absence of activated T cells were considered as basal expression of such molecule (Control, gray bars). Data are shown as the mean±SEM (Control n=3, BM-MSCs n=3, UCB-MSCs n=3, and PL-MSCs n=3). Individual experiments (evaluations were performed in duplicate). *Indicates a statistically significant difference with P<0.05. PGE2, prostaglandin E2; ELISA, enzyme-linked immunosorbent assay.

FIG. 7.

BM-MSCs and UCB-MSCs induce the generation of T-cell subsets displaying a regulatory phenotype. Anti-CD3/CD28-activated CD3+ T cells were cocultured in the absence or presence of BM-MSCs, UCB-MSCs, or PL-MSCs at a 1:1 ratio of MSCs:T cells. Cocultures were performed with cell–cell contact (black bars). The percentages of the CTLA-4+ and CTLA-4high populations and the fold changes in the CTLA-4 MFI were determined in the CD4+CD25+ population after 3 days of culture. CD4+CD25+CTLA-4+ T cells detected in activated T cells cultured in absence of MSCs were considered as control (Control, gray bars). (A) Representative dot plots from an experiment. (B) Data are shown as the mean±SEM of the percentages of CD4+CD25+CTLA-4+, (C) fold changes in the CTLA-4 MFI in CD4+CD25+CTLA-4+ cells and (D) CD4+CD25+CTLA-4high cells. Control n=4, BM-MSCs n=4, UCB-MSCs n=4, and PL-MSCs n=4 (Individual experiments). *Indicates a statistically significant difference with P<0.05.