Amniotic membrane mesenchymal cells-derived factors skew T cell polarization toward Treg and downregulate Th1 and Th17 cells subsets

Abstract

We previously demonstrated that cells derived from the mesenchymal layer of the human amniotic membrane (hAMSC) and their conditioned medium (CM-hAMSC) modulate lymphocyte proliferation in a dose-dependent manner. In order to understand the mechanisms involved in immune regulation exerted by hAMSC, we analyzed the effects of CM-hAMSC on T-cell polarization towards Th1, Th2, Th17, and T-regulatory (Treg) subsets. We show that CM-hAMSC equally suppresses the proliferation of both CD4(+) T-helper (Th) and CD8(+) cytotoxic T-lymphocytes. Moreover, we prove that the CM-hAMSC inhibitory ability affects both central (CD45RO(+)CD62L(+)) and effector memory (CD45RO(+)CD62L(-)) subsets. We evaluated the phenotype of CD4(+) cells in the MLR setting and showed that CM-hAMSC significantly reduced the expression of markers associated to the Th1 (T-bet(+)CD119(+)) and Th17 (RORγt(+)CD161(+)) populations, while having no effect on the Th2 population (GATA3(+)CD193(+)/GATA3(+)CD294(+)cells). T-cell subset modulation was substantiated through the analysis of cytokine release for 6 days during co-culture with alloreactive T-cells, whereby we observed a decrease in specific subset-related cytokines, such as a decrease in pro-inflammatory, Th1-related (TNFα, IFNγ, IL-1β), Th2 (IL-5, IL-6), Th9 (IL-9), and Th17 (IL-17A, IL-22). Furthermore, CM-hAMSC significantly induced the Treg compartment, as shown by an induction of proliferating CD4(+)FoxP3(+) cells, and an increase of CD25(+)FoxP3(+) and CD39(+)FoxP3(+) Treg in the CD4(+) population. Induction of Treg cells was corroborated by the increased secretion of TGF-β. Taken together, these data strengthen the findings regarding the immunomodulatory properties of CM-hAMSC derived from human amniotic membrane MSC, and in particular provide insights into their effect on regulation of T cell polarization.

Fig. 1

Inhibitory effects of CM-hAMSC on CD4⁺ and CD8⁺ proliferation. T cell proliferation was stimulated either with allogeneic PBMC in mixed lymphocyte reactions (MLR, circles in top panel) or by T cell receptor stimulation using anti-CD3/CD28 (squares in bottom panel). Proliferation of CD4⁺ (left panel) and CD8⁺ (right panel) was measured in the absence (empty circles/squares) or presence (black-filled circles/squares) of conditioned medium (CM-hAMSC). Data represent the mean and SD of at least four experiments. Asterisks indicate statistically significant differences between MLR-CM-hAMSC and MLR; *p < 0.05; **p < 0.01

Fig. 2

Effects of CM-hAMSC on the proliferation of CD4+ and CD8+ subsets. Panel (A) shows the gating strategy used for analysis. CD4 and CD8 T cell proliferation in mixed lymphocyte reactions (MLR) or co-cultured with CM-hAMSC is expressed as a percentage of CFSE diluting cells (Proliferative Fraction, PF) or by the Proliferation Index (PI). The following phenotypes were analyzed after 6 days of co-culture: (B) CD4 Effector Memory (EM, CD4⁺CD45RO⁺CD62L⁻); (C) CD4 Central Memory (CM, CD4⁺CD45RO⁺CD62L⁺); (D) CD4 Naïve (CD4⁺CD45RO⁻CD62L⁺); (E) CD8 Effector Memory (EM, CD8⁺CD45RO⁺CD62L⁻); (F) CD8 Central Memory (CM, CD8⁺CD45RO⁺CD62L⁺); (G) CD8 Effector (EMRA, CD8⁺CD45RO⁻CD62L⁻); (H) CD8 Naïve (CD8⁺CD45RO⁻CD62L⁺ cells). The frequency of the different subsets (reported as %) gated on the CD4 (I) and CD8 cells (J). The figure is representative of three independent experiments which showed statistically significant differences between MLR-CM-hAMSC and MLR for PF in CD4 CM, CD4 EM, CD8 CM, CD8 EM, CD8 Naïve, CD8 EMRA; and for PI in CD4 CM, CD4 EM, CD8 CM, CD8 EM

Fig. 3

CM-hAMSC increases the CD28- population within CD8+ subsets. Flow cytometry analysis was performed on allostimulated T cells in presence or absence of CM-hAMSC after 6 days of co-culture. (A) The percentage of cells expressing CD28 was evaluated in the total CD8⁺ cells (upper panels) or in the CD4⁺ cells (bottom panels). (B) The CD45RO and CD62L positivity was evaluated by gating on CD8⁺CD28⁻ cells. Data represent the mean and SD of at least 3 experiments. Asterisks indicate statistically significant differences between MLR-CM-hAMSC and MLR; *p < 0.05

Fig. 4

Effects of CM-hAMSC on the T helper polarization. The expression of the following T helper transcription factors: T-bet⁺ (Th1) (A) GATA-3⁺ (Th2) (B) RORγt⁺ (Th17) (C) was evaluated on unstimulated T-cells in absence (T, gray-dotted bars) or presence (T + CM-hAMSC, lined bars) of CM-hAMSC, and on alloreactive CD4+ cells in the absence (white bars) or presence (black bars) of CM-hAMSC. The phenotypes of the different CD4 T helper subsets were assessed by double positive populations for both Th-specific transcription factors and surface markers: T-bet⁺CD183⁺ and T-bet⁺CD119⁺ (Th1) (D), GATA-3⁺CD193⁺ and GATA-3⁺CD294⁺ (Th2) (E), RORγt⁺CD161⁺ (Th17) (F), as reported in Table 1. Data represent the mean and SD of at least five experiments. Asterisks indicate statistically significant differences between MLR-CM-hAMSC and MLR; *p < 0.05

Fig. 5

CM-hAMSC induces T lymphocytes with regulatory phenotype. (A) The percentage of CD4⁺FoxP3⁺ cells in unstimulated T cells in absence (T, gray-dotted bars) or presence (T + CM-hAMSC, lined bars) of CM-hAMSC, and in stimulated (MLR) T cells in absence (white bars) or presence (black bars) of CM-hAMSC. Data represent mean and SD of 5 individual experiments. (B) The percentage of proliferating and FoxP3-positive cells in the presence or absence of CM-hAMSC. The plot is representative of 3 experiments. (C) CD4 Treg phenotypes were assessed by double positive populations for both FoxP3 and surface markers (CD25, CD39, CD73). Box and whispers plots were generated using the Tukey method. CD25 Treg and CD39 Treg were characterized by additional surface markers CTLA-4 (D), GARP (E), CD357 (F), and intracellular markers such as TGF-β (G) and Helios (H). The release of TGF-β during 6 days co-culture was evaluated as described in Materials and Methods, the graph represents the mean and standard error mean (SEM) of at least four experiments (F). Asterisks indicate statistically significant differences between MLR + CM-hAMSC and MLR; *p < 0.05

Fig. 6

CM-hAMSC modulates the secretion of Th cytokines. The secretion of cytokines specific for the different T-cell subsets was evaluated each day during 6 days of co-culture in absence (white squares) or presence (black squares) of CM-hAMSC. The amount of cytokine produced by the CM-hAMSC itself was measured on day one only and is represented by a white diamond. The quantification of (A) Th1 cytokines (IFN-γ, TNFα, IL-1β, IL-2, IL-12p70), (B) Th2 cytokines (IL-4, IL-5, IL-6, IL-10, IL-13), (C) Th17 cytokines (IL-17A, IL-22), (D) IL-9 and sIL-2R were evaluated as described in Materials and Methods. Data represent the mean and standard error mean (SEM) of at least three experiments. Asterisks indicate statistically significant differences between MLR-CM-hAMSC and MLR; *p < 0.05; **p < 0.01