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Mesenchymal Stromal Cell Secretion of Programmed Death-1 Ligands Regulates T Cell Mediated Immunosuppression - PubMed

. 2017 Mar;35(3):766-776.

doi: 10.1002/stem.2509. Epub 2016 Oct 26.

Affiliations

Mesenchymal Stromal Cell Secretion of Programmed Death-1 Ligands Regulates T Cell Mediated Immunosuppression

Lindsay C Davies et al. Stem Cells. 2017 Mar.

Abstract

Mesenchymal stromal cells (MSCs) exert broad immunosuppressive potential, modulating the activity of cells of innate and adaptive immune systems. As MSCs become accepted as a therapeutic option for the treatment of immunological disorders such as Graft versus Host Disease, our need to understand the intricate details by which they exert their effects is crucial. Programmed death-1 (PD-1) is an important regulator in T cell activation and homeostatic control. It has been reported that this pathway may be important in contact-dependent mediated immunomodulation by MSCs. The aim of this study was to establish whether MSCs, in addition to their cell-surface expression, are able to secrete PD-1 ligands (PD-L1 and PD-L2) and their potential importance in modulating contact-independent mechanisms of MSC immunosuppression. Here we report that MSCs express and secrete PD-L1 and PD-L2 and that this is regulated by exposure to interferon γ and tumor necrosis factor α. MSCs, via their secretion of PD-1 ligands, suppress the activation of CD4+ T cells, downregulate interleukin-2 secretion and induce irreversible hyporesponsiveness and cell death. Suppressed T cells demonstrated a reduction in AKT phosphorylation at T308 and a subsequent increase in FOXO3 expression that could be reversed with blockade of PD-L1. In conclusion, we demonstrate for the first time, that MSCs are able to secrete PD-1 ligands, with this being the first known report of a biological role for PD-L2 in MSCs. These soluble factors play an important role in modulating immunosuppressive effects of MSCs directly on T cell behavior and induction of peripheral tolerance. Stem Cells 2017;35:766-776.

Keywords: Adult human bone marrow; Adult stem cells; Cytokines; Immunomodulation; Marrow stromal stem cells; Programmed-death 1; T cells.

© 2016 The Authors STEM CELLS published by Wiley Periodicals, Inc. on behalf of AlphaMed Press.

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Figures

Figure 1
Figure 1

Mesenchymal stromal cell (MSC) cell surface expression and secretion of PD‐L1 and PD‐L2 are potentiated by pro‐inflammatory cytokines, IFNγ and TNFα. MSCs (n = 4) were exposed to 100 U/ml IFNγ and 10 ng/ml TNFα for 3 days in culture. Cell surface expression (MFI) of (A) PD‐L1 and (B) PD‐L2 was assessed by flow cytometry. Secretion of (C) soluble (s)PD‐L1 and (D) sPD‐L2 within the conditioned media of stimulated cells was assessed by ELISA. Bar charts indicate mean ± SEM. Transcriptional regulation of (E) PD‐L1 and (F) PD‐L2 were assessed by qRT‐PCR. mRNA data are expressed as fold change compared to unstimulated, resting MSCs ± SEM. *, p < .05; **, p < .01. Abbreviations: IFNγ, Interferon γ; MFI, mean fluorescence intensity; PD‐L1 and PD‐L2, programmed death 1 ligands 1 and 2; TNFα, tumor necrosis factor α.

Figure 2
Figure 2

MSCs suppress CD4+ T cell activation and downregulate T cell surface PD‐1 expression via the secretion of PD‐1 ligands. (A): MSCs (n = 5) were co‐cultured with purified CD3+ T cells in direct contact and under transwell conditions. Cell surface expression of PD‐1 was downregulated in CD4 + CD25+ activated T cells by MSCs to the same extent under both contact and transwell conditions. Further experiments were therefore conducted in transwell only to assess the role of MSC secreted soluble PD‐1 ligands on CD4+ T cell function. (Ai): Representative histograms are shown for one donor. (Aii): MFI was used to quantify changes in expression between treatment groups. Data are normalized to activated T cell controls. (B): Blocking of the PD‐1 pathway inhibited MSC‐mediated suppression of T cell activation (CD25+) via the secretion of PD‐L1 and PD‐L2, whereas (C) specific blockade of soluble PD‐L2 inhibited suppression of PD‐1 cell surface expression by MSCs. Bar charts indicate mean ± SEM. *, p < .05. Abbreviations: IFNγ, interferon γ; MSC, mesenchymal stromal cell; MFI, median fluorescence Intensity; PD‐L1 and PD‐L2, programmed death 1 ligands 1 and 2; TNFα, tumor necrosis factor α.

Figure 3
Figure 3

MSCs suppress IL‐2 secretion in activated T cells via the secretion of PD‐1 ligands. Levels of T cell effector cytokines were assessed within conditioned media derived from transwell co‐culture of MSCs and activated T cells (n = 5) ± blockers for PD‐L1 and PD‐L2 by ELISA. MSCs suppressed the secretion of (A) IFNγ, (B) TNFα, and (C) IL‐2 via the secretion of soluble factors. Blockade of soluble PD‐L1 and PD‐L2 significantly restored the production of (C) IL‐2, but not (A) IFNγ or (B) TNFα. Data are shown as mean ± SEM. Data are compared to activated T cell controls unless otherwise indicated. *, p < .05; **, p < .01. Abbreviations: IFNγ, interferon γ; IL‐2, interleukin‐2; MSC, mesenchymal stromal cell; PD‐L1 and PD‐L2, programmed death 1 ligands 1 and 2; TNFα, tumor necrosis factor α.

Figure 4
Figure 4

MSCs induce hyporesponsivess and apoptosis in T cells via their secretome. MSCs (n = 4) were co‐cultured in transwell with activated T cells for 3 days. T cells were subsequently separated from the transwell MSC co‐cultures and placed in fresh culture media and restimulated with anti‐CD3, anti‐CD2 and anti‐CD28 microbeads. (A): T cell proliferation was significantly inhibited in those cells previously exposed to MSCs and could not be restored by IL‐2 supplementation, indicative of irreversible hyporesponsiveness. Presence of a PD‐1 blocker at the point of initial MSC/T cell co‐culture prevented induction of this hyporesponsive state, with T cells proliferating to levels comparable to controls. Data are expressed as counts per minute ± SEM **, p < .01 (B): Flow cytometry (n = 5 MSC donors) confirmed the proliferation data, demonstrating a reduced ability for T cells to upregulate CD25 and PD‐1 in response to fresh stimulus (±IL‐2) after MSC exposure. Data are expressed as percentage CD4 + CD25 + PD‐1+ T cells ± SEM **, p < .01; ***, p < .001 (C): Annexin V and 7‐AAD staining of T cells post‐MSC co‐culture confirmed induction of apoptosis by exposure to the MSC secretome. Cells are defined as pre‐apoptotic (Av + 7‐AAD‐), apoptotic (Av + 7‐AAD+) or total dead cells (7‐AAD+). Data are expressed as a percentage compared to T cell only controls ± SEM. Dotted line indicates 100%. #, p < .05 compared to T cell only control *, p < .05 **, p < .01 compared to T cells + MSC. Abbreviations: IFNγ, interferon γ; IL‐2, interleukin‐2; MSC, mesenchymal stromal cell; PD‐1, programmed death‐1; PD‐L1 and PD‐L2, programmed death 1 ligands 1 and 2; TNFα, tumor necrosis factor α.

Figure 5
Figure 5

MSC‐mediated induction of the PD‐1 pathway results in reduced AKT phosphorylation and increased FOXO3 expression in T cells. (A): T cells co‐cultured with MSCs (n = 4) in transwell conditions were assessed for mRNA expression of the PD‐1 pathway downstream signaling molecule AKT by qRT‐PCR. No change in the transcriptional level of AKT in response to exposure to MSCs was evident. Data are expressed as fold change compared to activated T cell controls. (B): Phospho‐flow confirmed that prior exposure of the T cells to MSCs render the cells unable to respond to T cell receptor (TCR) simulation with plate bound CD3, with a decrease in AKT phosphorylation at position T308. This ultimately increased the expression of transcription factor (C) FOXO3 within the MSC exposed T cells. MFI was used to quantify changes in expression between treatment groups. Bar charts indicate percentage change in MFI compared to T cell only controls ± SEM. *, p < .05. Abbreviations: MSC, mesenchymal stromal cell; MFI, mean fluorescence Intensity; PD‐L1 and PD‐L2, programmed death 1 ligands 1 and 2.

Figure 6
Figure 6

Proposed model by which mesenchymal stromal cell (MSC) derived soluble PD‐1 ligands modulate the activation status and effector function of CD4+ T cells. Within this study we have demonstrated that MSCs can respond to pro‐inflammatory cytokines secreted by activated T cells. MSCs upregulate their secretion of both PD‐L1 and PD‐L2 in response to this stimulus. These ligands bind directly to the PD‐1 receptor on the surface of activated (CD25+) CD4+ T cells resulting in a downregulation of CD25 and the PD‐1 receptor, with repression of the AKT pathway. A decreased phosphorylation of AKT is seen at T308, leading to an upregulation of the transcription factor FOXO3. Decreased CD25 expression and IL‐2 secretion interferes with the positive auto‐ and paracrine feedback loops maintaining the activated state of the T cell. These downstream effects ultimately culminate in a decrease in T cell activation and PD‐1 cell surface expression, IL‐2 production, proliferation and survival. Abbreviations: IL‐2, interleukin‐2; sPD‐L1 and sPD‐L2, soluble programmed death ligands 1 and 2; TCR, T cell receptor.

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