Cancer Immunol Immunother (2015) 64:213–224 DOI 10.1007/s00262-014-1623-y
Immune impairments in multiple myeloma bone marrow mesenchymal stromal cells Thibaud André · Mehdi Najar · Basile Stamatopoulos · Karlien Pieters · Olivier Pradier · Dominique Bron · Nathalie Meuleman · Laurence Lagneaux
Received: 13 November 2013 / Accepted: 4 October 2014 / Published online: 24 October 2014 © Springer-Verlag Berlin Heidelberg 2014
Abstract In multiple myeloma (MM), bone marrow mesenchymal stromal cells (BM-MSCs) play an important role in pathogenesis and disease progression by supporting myeloma cell growth and immune escape. Previous studies have suggested that direct and indirect interactions between malignant cells and BM-MSCs result in constitutive abnormal immunomodulatory capacities in MM BM-MSCs. The aim of this study was to investigate the mechanisms that underlie these MM BM-MSCs abnormalities. We demonstrated that MM BM-MSCs exhibit abnormal expression of CD40/40L, VCAM1, ICAM-1, LFA-3, HO-1, HLA-DR and HLA-ABC. Furthermore, an overproduction of IL-6 (1,806 ± 152.5 vs 719.6 ± 18.22 ng/mL; p = 0.035) and a reduced secretion of IL-10 (136 ± 15.02 vs 346.4 ± 35.32 ng/mL; p = 0.015) were quantified in culture medium when MM BM-MSCs were co-cultured with T lymphocytes compared to co-cultures with healthy donor (HD) BM-MSCs. An increased Th17/Treg ratio was Thibaud André and Mehdi Najar have contributed equally to this work. Electronic supplementary material The online version of this article (doi:10.1007/s00262-014-1623-y) contains supplementary material, which is available to authorized users. T. André (*) · M. Najar · B. Stamatopoulos · K. Pieters · L. Lagneaux Laboratory of Clinical Cell Therapy, Institut Jules Bordet Université Libre de Bruxelles (ULB), 808, Route de Lennik, 1070 Brussels, Belgium e-mail: [email protected]
O. Pradier Laboratory of Hematology, Erasmus Hospital, Brussels, Belgium D. Bron · N. Meuleman Hematology Department, Institut Jules Bordet, Brussels, Belgium
observed when T cells were co-cultured with MM BMMSCs compared to co-cultures with HD BM-MSCs (0.955 vs 0.055). Together, these observations demonstrated that altered immunomodulation capacities of MM BMMSCs were linked to variations in their immunogenicity and secretion profile. These alterations lead not only to a reduced inhibition of T cell proliferation but also to a shift in the Th17/Treg balance. We identified factors that are potentially responsible for these alterations, such as IL-6, VCAM-1 and CD40, which could also be associated with MM pathogenesis and progression. Keywords MSCs · Myeloma · Immunomodulation · Th17/Treg Abbreviations BM Bone marrow BrdU 5-Bromo-2-deoxy-uridine CCL5 Chemokine (C–C motif) ligand 5 CFSE Carboxyfluorescein succinimidyl ester CM Conditioned medium ELISA Enzyme-linked immunosorbent assay FOXP3 Forkhead box P3 HD Healthy donors HGF Hepatocyte growth factor HLA Human leukocyte antigen HO-1 Hemeoxygenase-1 ICAM-1 Intercellular adhesion molecule-1 IFN Interferon IL Interleukin IL-23R Interleukin-23 receptor LFA-3 Lymphocyte function-associated antigen-3 MCP-1 Monocyte chemotactic protein-1 MIP-1α Macrophage inflammatory protein-1 alpha MLR Mixed lymphocyte reactions
MM Multiple myeloma MMP Matrix metalloproteinase MSC Mesenchymal stromal cell PBMC Peripheral blood mononuclear cells PCs Plasma cells PGE2 Prostaglandin E2 PHA Phytohemagglutinin RORγt RAR-related orphan receptor gamma t SEM Standard error of the mean TGF Tumor growth factor Th T helper cells TNF Tumor necrosis factor Treg T regulatory cells VCAM-1 Vascular cell adhesion molecule-1
Introduction Multiple myeloma (MM) is a hematopoietic neoplasm characterized by a monoclonal expansion of secreting plasma cells (PCs) in the bone marrow (BM), resulting in skeletal destruction, renal failure, anemia, hypercalcemia and recurrent infections. MM represents approximately 1 % of all malignant tumors, 10 % of hematopoietic neoplasms and 2 % of cancer deaths . One of the characteristic specific to MM is that BM constitutes a required microenvironment for disease development and progression. Within this BM microenvironment, the malignant clone is able to elude immune surveillance by inducing abnormalities in immune cells (natural killer, dendritic and T cells) and by enhancing the release of immunoregulatory cytokines by microenvironmental cells [2–4]. Patients with MM exhibit a variety of numerical and functional abnormalities of T cells: abnormal T cell subsets (i.e., CD4:CD8 and Th1:Th2 ratios), reduced T cell diversity and abnormal functional responses. Tumor cells also affect the frequency and the inhibitory capacities of T regulatory (Treg) cells in MM patients [5, 6]. Finally, the proportion of T helper (Th) 17 cells and the plasma concentrations of Th17-associated cytokines are increased in MM patients . Among these bone marrow microenvironmental cells, the mesenchymal stromal cells (MSCs) play a crucial role in pathogenesis through their contacts with MM-PCs. The constitutive abnormalities that are observed in MM BM-MSCs, such as a reduced inhibition of T lymphocyte proliferation, are one result of these contacts [8, 9]. MSCs possess immunomodulatory capacities and can modulate most immune effectors, particularly T cells. MSCs can inhibit the activation and the proliferation of activated T cells and induce a state of anergy. These capacities are mediated by the secretion of many soluble factors (e.g., HGF, TGF-β, IL-6, IL-10, PGE2), which requires a dynamic crosstalk between MSCs and T cells, as well as by direct cellular contacts .
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MSCs can also impact two CD4+ T cell subpopulations: Th17 cells and Treg cells. Th17 cells are pro-inflammatory and are defined by a preferential secretion of interleukin (IL)-17. Tumor growth factor-β (TGF-β) and IL-6, with or without IL-21, IL-23 and IL-1, are necessary for the induction and expansion of Th17 cells from naïve CD4+ precursors. It has been demonstrated that MSCs induce regulatory characteristics in Th17 cells in inflammatory environments by downregulating RAR-related orphan receptor γt (RORγt) . However, some evidence also exists for a Th17 cell-promoting effect of MSCs . Treg cells have suppressor functions that are essentials for the prevention of autoimmunity and the resolution of inflammatory processes. These cells are characterized by surface expression of CD25 and by intracellular expression of forkhead box P3 (FOXP3). The development and proliferation of Treg cells are induced by IL-10, IL-2 and TGF-β. Many studies demonstrated an enhancement of Treg numbers and activity by MSCs . Previous studies from our group and other groups [8, 9, 14] reported that the immunomodulatory functions of BMMSCs derived from MM patients are impaired. A reduced inhibition of T lymphocyte proliferation, a lower ability to silence mitogen-stimulated T cells in G0/G1 phase, a reduced inhibition of T cell activation and a reduced rate of T cell apoptosis have been observed during co-culture of T cells with MM BM-MSCs compared with co-cultures with healthy donor (HD) BM-MSCs. The aim of this study was to investigate the mechanisms that underlie the MM BM-MSCs immunomodulatory impairment observed in MM BM-MSCs and the impacts of these impairments on MM. We analyzed MM BM-MSCs expression of a variety of adhesion molecules and immune effectors in constitutive and inflammatory conditions. We also measured the secretion of immunoregulatory cytokines by MM BM-MSCs that were co-cultured with activated T cells. Finally, we evaluated the fate of activated T cells that were co-cultured with MM BM-MSCs.
Materials and methods Patients Samples were obtained after receiving written informed consent from patients and HD volunteers and after approval from the Ethical Committee of the Jules Bordet Institute and Erasmus Hospital (Brussels, Belgium). Thirty-four patients with MM and twelve HDs were included in this study, and the characteristics of these participants are listed in Supplementary Table 1. All MM patients who underwent treatment were in remission at the time of BM harvesting and did not receive grafts.
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Isolation, culture and characterization of MSCs BM was harvested from the sternum or iliac crest of the patients. BM-MSCs were isolated by using the classical adhesion method and cultured as previously described . Adherent cells in the culture were identified as MSCs when they fulfilled the International Society for Cellular Therapy (ISCT) criteria . BM-MSCs were cultured for 24 h in the presence or absence of inflammatory medium (i.e., Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 2 mM l-glutamine, 50 U/mL of penicillin, 50 µg/mL of streptomycin, 1,000 U/mL of interferon-γ (IFN-γ), 3,000 U/mL of IFN-α, 50 ng/mL of tumor necrosis factor-α (TNF-α) and 25 ng/mL of IL-1β) (R&D Systems, Abingdon, UK). The harvested cells were analyzed using flow cytometry with the following markers to establish the immunological profile of the MSCs: members of the human leukocyte antigen (HLA) family, co-stimulatory molecules, cell adhesion molecules and immunoregulatory factors. Briefly, the cells were washed with phosphate-buffered saline (GmbH, Bergisch, Germany) and incubated for 30 min with the following monoclonal antibodies: CD105FITC (Ancell Corporation, Bayport, MN, USA), CD229PE (Imtec Diagnostics, Antwerp, Belgium), CD58-FITC (BD Biosciences Pharmingen, Erembodegen, Belgium), CD106-PC5 (BD Biosciences Pharmingen), CD54-PE (BD Biosciences Pharmingen), CD95-FITC (Miltenyi Biotec, Leiden, the Netherlands), CD274-PE (BD Biosciences Pharmingen), HLA-ABC-PC5 and HLA-DRPC5 (BD Biosciences Pharmingen), HLA-G-PE (EXBIO Praha, Vestec, Czech Republic), CD4-PE (Miltenyi Biotec), CD8-FITC (Miltenyi Biotec), CD40-PE (Miltenyi Biotec), CD154-PC5 (Imtec Diagnostics, Antwerp, Belgium), CD200R-PE (Imtec Diagnostics), CD200-PC5 (Imtec Diagnostics), CD70-PE (Imtec Diagnostics), CD27-PerCP (Imtec Diagnostics), CD80-PE (Imtec Diagnostics), CD86PC5 (Imtec Diagnostics), CD134-FITC (Imtec Diagnostics), CD166 (BD Biosciences Pharmingen), SDF1-APC (R&D Systems, Abingdon, UK), MMP9-FITC (R&D Systems), CD44-FITC (Miltenyi Biotec), CD56-PE (ANALIS, Suarlée, Belgium), CD252-PE (Imtec Diagnostics), CD49c-PE (BD Biosciences Pharmingen), CD49d-PE (BD Biosciences Pharmingen), CD49b-PE (BD Biosciences Pharmingen), CD183-PE (BD Biosciences Pharmingen), CD184-PE (BD Biosciences Pharmingen), HO1-PE (ENZO Life Sciences, Farmingdale, NY, USA), CD138-PE (BD Biosciences Pharmingen), RORγt-APC (R&D Systems), IL-6-PE (ImmunoTools, Friesoythe, Germany), IFNγ-FITC (Imtec Diagnostics), IL-4-PerCP-Cy5.5 (Imtec Diagnostics) and IL-23R-PE (R&D Systems). After washing with MACSQuant Running Buffer (Miltenyi Biotec), the cells were fixed with 4 % formaldehyde solution. Data were acquired using a MACSQuant Analyzer (Miltenyi
Biotec) and analyzed using FCS Express 4 Flow Cytometry software (De Novo Software, Los Angeles, CA, USA). Preparation of MSC conditioned media We prepared conditioned media (CM) from MSC cultured alone for 3 days in DMEM without serum. The supernatants were collected and frozen at −20 °C until further use. Isolation of T cells and T cell/BM-MSCs co-cultures Peripheral blood samples were collected from HDs after informed consent was obtained. T cells were isolated from human blood by magnetic cell separation using human CD3 MicroBeads (Miltenyi Biotec). The mean percentage of CD3-positive cells was observed using flow cytometry was 95 % (data not shown). (a) Mixed lymphocyte reactions (MLRs) were performed as previously described . Briefly, 105 CD3+ T cells were co-cultured with 2 × 104 irradiated allogeneic peripheral blood mononuclear cells (PBMCs) in a final volume of 250 µL in 96-well plates. The MLRs were prepared in triplicates in the presence or absence of 1.2 × 104 irradiated MSCs. After 4 days, 150 µL of culture medium were harvested for enzymelinked immunosorbent assay (ELISA) analyses. Lymphocyte proliferation was assessed using 5-bromo-2-deoxy-uridine (BrdU) incorporation. On day 4, 50 µM BrdU (Roche Applied Science, Mannheim, Germany) was added to the co-cultures at day 4. T cell proliferation was evaluated using a colorimetric assay to measure BrdU incorporation according to the manufacturer’s instructions. Data were expressed as the % of T cell proliferation. (b) 2.5 × 104 BM-MSCs were co-cultured with 105 CD3+ T cells in 1 mL of DMEM supplemented with 2 mM l-glutamine, 50 U/mL of penicillin, 50 µg/mL of streptomycin, 10 % fetal bovine serum, 1 mg/mL of interleukin 2 (IL-2) (R&D Systems) and 1 mg/mL of phytohemagglutinin (PHA) (Remel Europe, Kent, UK). For blockade experiments, we added 2 µg/mL of anti-hIL-6 antibodies (R&D Systems), 2 µg/mL of anti-hCD40 antibodies (R&D Systems) or 5 µg/ mL of anti-hVCAM-1 antibodies (R&D Systems). After 5 days, the culture medium was harvested for ELISA analyses and flow cytometry analysis. The Th17/Treg ratio was evaluated by flow cytometry using the Human Th17/Treg phenotyping Kit (BD Biosciences Pharmingen). The CD4/ CD8 and Th1/Th2 ratios were evaluated by flow cytometry using the antibodies combinations CD8-FITC/CD4-PE and IFN-γ-FITC/CD4-PE/IL-4-PerCPCy5.5 after cell permeabilization using FIX & PERM® kit (Invitrogen). Lymphocyte stimulation was assessed after carboxyfluorescein succinimidyl ester (CFSE) labeling using the CellTrace CFSE cell proliferation kit (Invitrogen Molecular Probes, Eugene, OR, USA). CFSE fluorescence was visualized by flow
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Fig. 1 Reduced inhibition of T cell proliferation by MM BMMSC. a Percentage of T cell proliferation activated by MLR in co-culture with HD BMMSC (n = 5), BM-MSC from untreated patients (n = 6) and from treated patients (n = 12) compared to a 100 % T cell proliferation without BM-MSC (MLR). Columns represent the mean ± SEM. b Representation of the expansion of T cells unstimulated, activated (PHA/ IL-2), activated and co-cultured with HD BM-MSC and MM BM-MSC
cytometry, and the results were expressed as the percentage of positive T cells.
of the mean). All analyses were performed using GraphPad Prism 5 software (GraphPad Software, San Diego, CA, USA).
Cytokine expression Cytokine concentrations in the culture media from the MLRs were determined using Quantikine ELISAs for IL-6, IL-10, hepatocyte growth factor (HGF) and TGF-β according to the manufacturer’s instructions (R&D Systems). Cytokine concentrations in the culture media from the BM-MSC-/mitogenic-activated T cell co-cultures were determined using ProcartaPlex™ Human Essential Th1/ Th2 Cytokine Panel immunoassays for IL-4/IL-5/IL-12, IFNγ and TNF-α according to the manufacturer’s instructions (eBiosciences, Vienna, Austria). Statistical analysis The Mann–Whitney test was used to analyze differences between groups. All tests performed in this study were twosided. The significance level was set at p