Multipotent stromal cells (MSC) have been shown to possess immunomodulatory capacities and are therefore explored as a novel cellular therapy. One of the mechanisms through which MSC modulate immune responses is by the promotion of regulatory T cell (Treg) formation. In this study, we focused on the cellular interactions and secreted factors that are essential in this process. Using an in vitro culture system, we showed that culture-expanded bone marrow-derived MSC promote the generation of CD41 T cells in human PBMC populations and that these populations are functionally suppressive. Similar results were obtained with MSC-conditioned medium, indicating that this process is dependent on soluble factors secreted by the MSC. Antibody neutralization studies showed that TGF-b1 mediates induction of Tregs. TGF-b1 is constitutively secreted by MSC, suggesting that the MSC-induced generation of Tregs by TGF-b1 was independent of the interaction between MSC and PBMC. Monocyte-depletion studies showed that monocytes are indispensable for MSC-induced Treg formation. MSC promote the survival of monocytes and induce differentiation toward macrophage type 2 cells that express CD206 and CD163 and secrete high levels of IL-10 and CCL-18, which is mediated by as yet unidentified MSC-derived soluble factors. CCL18 proved to be responsible for the observed Treg induction. These data indicate that MSC promote the generation of Tregs. Both the direct pathway through the constitutive production of TGF-b1 and the indirect novel pathway involving the differentiation of monocytes toward CCL18 producing type 2 macrophages are essential for the generation of Tregs induced by MSC.

Adipose tissue-derived multipotent stromal cells (AT-MSCs) are studied as an alternative to bone marrow-derived multipotent stromal cells (BM-MSCs) for immunomodulatory treatment. In this study, we systematically compared the immunomodulatory capacities of BM-MSCs and AT-MSCs derived from age-matched donors. We found that BM-MSCs and AT-MSCs share a similar immunophenotype and capacity for in vitro multilineage differentiation. BM-MSCs and AT-MSCs showed comparable immunomodulatory effects as they were both able to suppress proliferation of stimulated peripheral blood mononuclear cells and to inhibit differentiation of monocyte-derived immature dendritic cells. However, at equal cell numbers, the AT-MSCs showed more potent immunomodulatory effects in both assays as compared with BM-MSCs. Moreover, AT-MSCs showed a higher level of secretion of cytokines that have been implicated in the immunomodulatory modes of action of multipotent stromal cells, such as interleukin-6 and transforming growth factor-␤1. This is correlated with higher metabolic activity of AT-MSCs compared with BM-MSCs. We conclude that the immunomodulatory capacities of BM-MSCs and AT-MSCs are similar, but that differences in cytokine secretion cause AT-MSCs to have more potent immunomodulatory effects than BM-MSCs. Therefore, lower numbers of AT-MSCs evoke the same level of immunomodulation. These data indicate that AT-MSCs can be considered as a good alternative to BM-MSCs for immunomodulatory therapy. STEM CELLS TRANSLATIONAL MEDICINE 2013;2:455-463

Multipotent stromal cells have immunomodulatory capacities and have been used in transplantation and autoimmune diseases. One of the effects of multipotent stromal cells involves the inhibition of dendritic cell differentiation. Since interleukin-6 and interleukin-10 are known to play a role in inhibiting immature dendritic cell differentiation, we hypothesized that these cytokines may also mediate the inhibitory effect of human multipotent stromal cells in immature dendritic cell differentiation. In order to test this hypothesis monocytes were cultured with interleukin-4 and granulocyte-monocyte colony-stimulating factor in the presence or absence of culture-expanded bone marrow-derived multipotent stromal cells. Neutralization and cytokine-depletion strategies were applied to reveal the cellular source and effect of interleukin-6 and interleukin-10. Addition of multipotent stromal cells to monocyte cultures significantly reduced the generation of immature dendritic cells (CD14 -CD1a + ) and resulted in the generation of CD14 + CD1a -cells that displayed a significantly reduced immunostimulatory effect. We found that culture supernatants of co-cultures of multipotent stromal cells and monocytes contained higher concentrations of interleukin-6 and interleukin-10. Multipotent stromal cells produced interleukin-6 and neutralizing this interleukin-6 reversed the inhibitory effect of the multipotent cells. Interleukin-10 was not produced by multipotent stromal cells, but exclusively by monocytes after exposure to multipotent stromal cell-produced interleukin-6. In conclusion, through constitutive production of interleukin-6, multipotent stromal cells prevent the differentiation of monocytes towards antigen-presenting immunogenic cells and skew differentiation towards an antiinflammatory interleukin-10-producing cell type.Multipotent stromal cells skew monocytes towards an anti-inflammatory interleukin-10-producing phenotype by production of interleukin-6 Sara M. Melief, Sacha B. Geutskens, Willem E. Fibbe, and Helene Roelofs Department of Immunohematology and Blood Transfusion, Leiden University Medical Center, Leiden, The Netherlands ABSTRACT Introduction

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Adoptive T cell transfer (ACT) with ex vivo-expanded tumor-reactive T cells proved to be successful for the treatment of metastatic melanoma patients. Mixed lymphocyte tumor cell cultures (MLTC) can be used to generate tumor-specific T cells for ACT; however, in a number of cases tumor-reactive T cell, expansion is far from optimal. We hypothesized that this is due to tumor intrinsic and extrinsic factors and aimed to identify and manipulate these factors so to optimize our clinical, GMP-compliant MLTC protocol. We found that the tumor cell produced IDO and/or galectin-3, and the accumulation of CD4+CD25hiFoxP3+ T cells suppressed the expansion of tumor-specific T cells in the MLTC. Strategies to eliminate CD4+CD25hiFoxP3+ T cells during culture required the depletion of the whole CD4+ T cell population and were found to be undesirable. Blocking of IDO and galectin-3 was feasible and resulted in improved efficiency of the MLTC. Implementation of these findings in clinical protocols for ex vivo expansion of tumor-reactive T cells holds promise for an increased therapeutic potential of adoptive cell transfer treatments with tumor-specific T cells.Electronic supplementary materialThe online version of this article (doi:10.1007/s00262-017-1995-x) contains supplementary material, which is available to authorized users.

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The presence of tumor-infiltrating immune cells is associated with longer survival and a better response to immunotherapy in early-stage melanoma, but a comprehensive study of the in situ immune microenvironment in stage IV melanoma has not been performed. We investigated the combined influence of a series of immune factors on survival and response to adoptive cell transfer (ACT) in stage IV melanoma patients. Metastases of 73 stage IV melanoma patients, 17 of which were treated with ACT, were studied with respect to the number and functional phenotype of lymphocytes and myeloid cells as well as for expression of galectins-1, -3, and -9. Single factors associated with better survival were identified using Kaplan-Meier curves and multivariate Cox regression analyses, and those factors were used for interaction analyses. The results were validated using The Cancer Genome Atlas database. We identified four parameters that were associated with a better survival: CD8þ T cells, galectin-macrophage ratio, and the expression of galectin-3 by tumor cells. The presence of at least three of these parameters formed an independent positive prognostic factor for long-term survival. Patients displaying this four-parameter signature were found exclusively among patients responding to ACT and were the ones with sustained clinical benefit.

<p>Legends for supplementary Figure 1 through 5</p>