© 2012. Published by The Company of Biologists Ltd.
Human mesenchymal stem cells shift CD8+ T cells towards a suppressive phenotype by inducing tolerogenic monocytes
Journal of Cell Science
Accepted manuscript
Running title: hMSCs downregulate CD8 via monocytes
Irit Hof-Nahor,1 Lucy Leshansky,1 Shoham Shivtiel,1 Liron Eldor,2 Daniel Aberdam,1 Joseph Itskovitz-Eldor,1,3 and Sonia Berrih-Aknin4 1
INSERTECH, Bruce Rappaport Department of Medicine, Technion, Haifa, Israel; 2Department
of Plastic Surgery, Rambam Health Care Campus, Haifa, Israel; 3Department of Obstetrics and Gynecology, Rambam Health Care Campus, Haifa, Israel; 4UMRS 974 - UPMC Univ. Paris 6 / U974 - Inserm / UMR7215 - CNRS
Corresponding author: Sonia Berrih-Aknin UMRS 974 - UPMC Univ. Paris 6 / U974 - Inserm / UMR7215 - CNRS Hôpital La Pitié Salpêtrière 105 Bd de l’hôpital, 75013 Paris
[email protected] Tel: +33 1 40 77 81 28; Fax: +33 1 40 77 81 29
Key words: Human Mesenchymal Stem Cells, immunoregulation, CD8+ cells, monocytes, humanized mice.
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JCS online publication date 5 July 2012
SUMMARY The mechanisms underlying the immunomodulatory effects of mesenchymal stem cells (MSCs) have been essentially studied in conditions of strong T cell activation that represents extreme situation and induces rapid death of activated lymphocytes. The objective of this study was to investigate these mechanisms in absence of additional polyclonal activation. In cocultures of peripheral mononuclear blood cells with hMSC, we observed a striking decreased expression of CD8 level on CD8+ cells, together with decreased CD28 and CD44 expression and impaired
Journal of Cell Science
Accepted manuscript
IFN-gamma and Granzyme B production. This effect was specific to hMSCs, since it was not observed with several other cell lines. Down-regulation of CD8 expression required CD14+ monocytes in direct contact with the CD8+ cells, while the effects of hMSCs on the CD14+ cells were essentially mediated by soluble factors. The CD14+ monocytes exhibited a tolerogenic pattern when co-cultured with hMSCs, with a clear decrease in CD80 and CD86 co-stimulatory molecules, and an increase in the inhibitory receptors ILT-3 and ILT-4. MSC-preconditioned CD8+ cells had similar effects on monocytes and were able to inhibit lymphocyte proliferation. Injection of human MSCs in humanized NSG mice showed similar trends, in particular decreased CD44 and CD28 on human immune cells. Altogether, our study demonstrates a new immunomodulation mechanism of action of hMSCs through the modulation of CD8+ cells towards a non-cytotoxic/suppressive phenotype. This mechanism of action has to be taken into account in clinical trials, where it should be beneficial in grafts and autoimmune diseases, but potentially detrimental in malignant diseases.
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Introduction Mesenchymal stem cells (MSCs) are non-hematopoietic stem cells first identified in the bone marrow (BM) cavity by Fridenshtein (Fridenshtein, 1982); their ability to differentiate into various kinds of mesoderm tissues were then demonstrated (Pittenger et al., 1999; Reyes et al., 2001; Wakitani et al., 1995). MSCs may be isolated from BM, skeletal muscle, adipose tissue, and synovial membrane, as well as cord blood (da Silva Meirelles et al., 2006) and are defined by using a combination of phenotypic markers.
Accepted manuscript
subsets of immune cells, namely T and B cells, dendritic cells (DCs), and natural killer cells
Journal of Cell Science
It is well established that human MSCs (hMSCs) possess suppressive capabilities on several
lymphocytes and alloreactive T cells, reported that hMSCs prevent the expression of CD25,
(NK), in response to polyclonal stimulation (Uccelli et al., 2007). MSCs have a unique immunophenotype, with a low expression of major histocompatibility complex (MHC) class I and an absence of co-stimulatory molecules. Although several groups have demonstrated that hMSCs have the capacity to inhibit T-cell proliferation (Benvenuto et al., 2007; Glennie et al., 2005 ), controversy remains regarding their effect on T-cell activation. Le Blank et al. and Groh et al., who conducted their research on cultures of phytohaemagglutinin (PHA)-stimulated
CD38, and CD69 activation markers on T lymphocytes (Groh et al., 2005; Le Blanc et al., 2004). However, in a similar model, Aggarwal et al. argued that hMSCs actually induce a slight increase in the proportion of CD25+ T cells (Aggarwal and Pittenger, 2005). In addition, some studies have claimed that the inhibitory effect of MSCs on T cells is confined to cellular proliferation rather than its effector function (Ramasamy et al., 2008). CD8 is commonly used as a cytotoxic T-cell marker, whereas its down-regulation has been suggested as one of the mechanisms for peripheral tolerance (Rocha and von Boehmer, 1991; Schonrich et al., 1991; Zhang et al., 1995). Xiao et al. have argued that the down-regulation of CD8 expression and the loss of specific peptide/MHC tetramer binding during the immune response following bacterial infection are subjected to detuning during normal immune responses (Xiao et al., 2007). Different CD8+ T-cell subsets have been identified based on the expression of cell surface markers, such as the CD28 co-stimulatory molecule, which is necessary for the initiation of most T-cell responses. CD8+CD28- cells are defined as suppressor T cells (Ts) that have been shown to down-modulate the antigen-presenting cell (APC) function by inducing 3
immunoglobulin-like transcript 3 and 4 (ILT-3 and ILT-4) inhibitory receptors, leading to inhibition of CD4+ T-cell proliferation by antigen-presenting cells (Chang et al., 2002). The difficulty in achieving long-term allograft survival has been attributed to the resistance of effector CD8+ cells (Trambley et al., 1999) and/or memory T cells (Lakkis and Sayegh, 2003; Valujskikh et al., 2002) to co-stimulatory blockade. Pre-clinical studies reveal that hMSCs are capable of preventing graft rejection in a rat model for cardiac allograft (Zhou et al., 2006) and in rat kidney transplantation, as well as reducing the CD8+ cell number in the infiltrates (De
Accepted manuscript
Martino et al.). hMSCs injection prolonged the survival of skin transplant in a baboon model (Bartholomew et al., 2002) and extended heart allograft survival when administered in a mouse model, with 33% of recipients showing long-term tolerance (Casiraghi et al., 2008). The mechanisms underlying the immunomodulatory effects of MSCs are still poorly understood, and most studies have used strong T cell activators that represent extreme situations and induce rapid death of activated lymphocytes. The objective of this study was to investigate the effect of human MSCs on peripheral mononuclear blood cells without additional exogenous stimulation.
Journal of Cell Science
Our findings indicate that hMSCs shift the CD8+ cytotoxic cells towards a suppressive phenotype, an effect depending on CD14+ monocytes whose phenotype is strikingly regulated by hMSC.
Results hMSCs decrease expression of CD8 on CD8+ T cells We evaluated whether hMSC lines originating from adipose tissue had the potency to modify the balance between the main T-cell subsets in direct co-cultures without additional stimulating molecules. Kinetics analysis in living cells showed that CD8, but not CD4 expression was significantly decreased on day 6 of incubation (p
