Elsevier

Cytotherapy

Volume 18, Issue 5, May 2016, Pages 621-629
Cytotherapy

Mesenchymal Stromal Cells
Indoleamine 2,3-dioxygenase mediates inhibition of virus-specific CD8+ T cell proliferation by human mesenchymal stromal cells

https://doi.org/10.1016/j.jcyt.2016.01.009Get rights and content

Abstract

Background aims

Mesenchymal stromal cells (MSCs) exert broad immunomodulatory functions. We recently demonstrated a strong suppressive effect of MSCs on virus-specific CD8+ T-cell proliferation. Here, we further explored the underlying mechanism.

Methods

The role of indoleamine 2,3-dioxygenase (IDO) in inhibition of virus-specific CD8+ T-cell proliferation by human MSCs was investigated using a mixed lymphocyte peptide culture assay, IDO intracellular staining and IDO inhibition through 1-methyl-DL-tryptophan (1-MT). Moreover, the influence of the number of passages and the seeding density of MSCs on their IDO expression and immunosuppressive ability were investigated.

Results

MSCs with low IDO expression exhibited a reduced suppressive effect on both allo-antigen- and cytomegalovirus (CMV)-specific CD8+ T-cell proliferation. 1-MT could partially abrogate the suppressive effect. MSCs inhibited CMV-specific CD8+ T-cell proliferation regardless of the number of passages and the seeding density. IDO expression of MSCs was not significantly affected by the number of passages or the seeding density. In addition, the interferon (IFN)-γ level in the culture system was crucial for MSCs to inhibit the proliferation of CMV-specific CD8+ T cells.

Summary

MSCs inhibit virus-specific CD8+ T-cell proliferation through IFN-γ-induced IDO activity, resolving current conflicting reports on this issue and indicating the potential need for prophylaxis and surveillance of virus infection in patients treated with MSCs. In addition, our study makes a contribution to the development of MSC potency assay for clinical use.

Introduction

Mesenchymal stromal cells (MSCs) are fibroblast-like plastic-adherent cells that have the potential to differentiate into osteoblasts, adipocytes and chondroblasts [1]. They were first identified in the bone marrow [2] and have been found in a variety of other tissues, such as umbilical cord blood [3], adipose tissue [4] and dental pulp [5]. In 2006, the International Society for Cellular Therapy proposed minimal criteria for defining multipotent MSCs, in an attempt to eliminate the ambiguities and inconsistencies in the research field of MSCs that might be caused by lack of a proper characterization [6]. MSCs have a strong immunomodulatory impact on various cells of the adaptive and innate immune systems [7]. Therefore, MSCs have been extensively investigated in clinical trials for steroid-refractory graft-versus-host disease (GvHD), as well as multiple sclerosis and other autoimmune diseases [8]. Therapeutic potential of MSCs has been demonstrated in animal models and human clinical trials [9], [10]. However, a large randomized phase III clinical trial using an industrial MSC product (Prochymal, Osiris Therapeutics) failed to show significant benefit for patients with steroid-refractory acute GvHD. Galipeau made a comprehensive analysis and defined several possible influencing factors, for example, MSC donor heterogeneity and differential culture conditions [11]. Therefore, further intensive investigation is needed for a better understanding of the immunoregulatory properties of MSCs.

In addition to contradictory data on the clinical outcome of patients receiving MSCs, another important concern about MSC therapy is the potential of MSCs to disturb immune responses against pathogens, such as viruses. After allogeneic hematopoietic stem cell transplantation, viral infections are one of the major causes of morbidity and mortality. Third-party MSCs were reported to inhibit allogeneic and mitogen-induced T-cell responses [12], whereas their ability to suppress virus-specific T cells is still up for debate [13], [14], [15]. Karlsson et al. reported that MSCs could not effectively suppress cytomegalovirus (CMV)-specific and more general virus-specific T-cell responses and concluded that MSCs exert differential effects on allo-antigen- and virus-specific T-cell responses [14]. This would be an ideal scenario for the application of MSC therapy. However, our previous data showed a high anti-proliferative effect of MSCs on virus-specific T cells [13], but the mechanisms and possible factors influencing such MSC-mediated inhibition of virus-specific T-cell responses remain unclear.

Various soluble and membrane-bound molecules were proposed to contribute to MSC immunoregulatory function, for example, B7-H4, programmed death-ligand 1 (PD-L1), transforming growth factor-β (TGF-β), cyclooxygenase-2 (Cox-2), inducible nitric oxide synthase (iNOS) and indoleamine 2,3-dioxygenase (IDO) [16], [17]. Among them, IDO is considered to play a major role in inhibition of allogeneic and mitogen-induced T-cell proliferation by human MSCs [18], [19], [20]. IDO is an intracellular enzyme that catalyzes the degradation of the amino acid tryptophan to kynurenine. Both tryptophan depletion and the presence of its metabolite kynurenine are able to suppress T-cell proliferation [21], [22]. Moreover, IDO is also considered to be involved in the generation of regulatory T cells and tolerogenic dendritic cells induced by MSCs [23], [24], [25]. MSCs do not constitutively express IDO, but interferon-γ (IFN-γ) induces IDO expression in a dose-dependent manner [18]. So far, it has not been reported whether IDO is also involved in the anti-proliferative effect of human MSCs on virus-specific T cells. In addition, although heterogeneity of IDO expression has been observed for MSCs [19], [20], it remains unclear whether IDO induction in MSCs can be influenced by in vitro expansion.

In the present study, we investigated the involvement of IDO in the anti-proliferative effect of human MSCs on virus-specific CD8+ T cells. Furthermore, in an attempt to resolve conflicting reports, we explored the impact of factors on the outcome of the MSC/T cell coculture system, such as the number of passages and the seeding density of MSCs as well as the IFN-γ level.

Section snippets

Isolation and culture of human bone marrow MSCs

Human MSCs were isolated and cultured as previously described [26]. Briefly, bone marrow aspirates of healthy voluntary donors (HDs) were collected after obtaining informed written consent and according to guidelines of Heidelberg University on the Use of Human Subjects approved by its Ethics Committee. Mononuclear cells were isolated by density gradient centrifugation at 2000 rpm at 20°C for 30 min without brake using Ficoll-Hypaque (Biochrom), subsequently seeded at a density of 2 × 105

MSCs with low IDO expression exhibit lower suppressive ability on virus-specific T cell proliferation

MSCs from six HDs were cultured at a high (5000 cells/cm2) seeding density until P5 or P6, and five of these six donors were also cultured at a low (1000 cells/cm2) seeding density from P2 to P5. All six MSC populations at early passage (P2) and eleven (both cultured at high and low seeding densities) at late passage (P5 or P6) were added to MLR and pp65-MLPC assays to assess the ability to suppress allo-antigen- and virus-specific CD8+ T-cell proliferation. All MLRs and pp65-MLPCs were

Discussion

In our previous work, we demonstrated a pronounced anti-proliferative effect of MSCs on virus-specific T cells in a dose-dependent manner [13]. In this study, we sought to investigate the mechanisms lying behind these observations. Among the mechanisms reported to be involved in the immunosuppressive function of MSC, IDO was considered to play a pivotal role in MSC-mediated inhibition of allogeneic T-cell responses [19]. Therefore, we focused on IDO in MSCs and established a flow

Author contributions

JH designed and performed all experiments, analyzed the data and wrote the original manuscript; AH, LW and AS discussed experimental design, the data and the manuscript; JT performed statistical data analysis; PW established the MSC culture protocol and discussed the manuscript; KB provided human platelet lysate and discussed the manuscript; CK provided PBMCs of healthy donors and discussed the manuscript; ADH discussed the manuscript; and MS initiated the study and discussed the research and

Acknowledgments

The authors thank the National Institutes of Health Tetramer Core Facility for provision of CMVpp65(495–503), EBV-BMLF1(280–288) and influenza virus matrix protein IMP(58–66) monomers, as well as Ulrike Gern, Anke Diehlmann and Stefanie Mechler for excellent technical support.

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