Cancer Immunol Immunother DOI 10.1007/s00262-015-1708-2

ORIGINAL ARTICLE

Impact of the immunomodulating peptide thymosin alpha 1 on multiple myeloma and immune recovery after hematopoietic stem cell transplantation Marilène Binsfeld1 · Muriel Hannon1 · Eléonore Otjacques1 · Stéphanie Humblet‑Baron2 · Etienne Baudoux3 · Yves Beguin1,3 · Frédéric Baron1 · Jo Caers1 

Received: 12 February 2015 / Accepted: 4 May 2015 © Springer-Verlag Berlin Heidelberg 2015

Abstract  Multiple myeloma (MM) is characterized by the accumulation of monoclonal plasma cells in the bone marrow and causes several immune alterations in patients. Thymosin α1 (Tα1) is a thymic peptide that has been associated with immuno-stimulating properties. In addition, this peptide exerts anti-tumor effects in several cancer types. Beneficial effects of Tα1 administration have also been shown on immune reconstitution after hematopoietic stem cell transplantation (HSCT), a current treatment modality Previous publication of this work as poster and abstract Binsfeld M, Otjacques E, Hannon M, Dubois S, Beguin Y, Baron F, Caers J. The immunomodulating peptide thymosin alpha 1 has no effects on multiple myeloma evolution and on immune reconstitution. 28th General Annual Meeting of the Belgian Hematological Society (BHS). Gent (Belgium), 24th–26th January 2013. Abstract Book p. 41, P73. Binsfeld M, Hannon M, Humblet-Baron S, Beguin Y, Baron F, Caers J. Effects of the immunomodulating peptide thymosin alpha 1 in multiple myeloma and immune reconstitution after hematopoietic stem cell transplantation in murine models. 30th General Annual Meeting of the Belgian Hematological Society (BHS) La Hulpe (Belgium), 31st January–1st February 2015. Abstract Book p. 29, P3.14. Electronic supplementary material  The online version of this article (doi:10.1007/s00262-015-1708-2) contains supplementary material, which is available to authorized users. * Marilène Binsfeld [email protected] 1

Laboratory of Hematology, GIGA‑Research, University of Liège, Bat. B34, CHU of Liège, Avenue de l’Hôpital, 1, 4000 Liège, Belgium

2

Laboratory of Genetics of Autoimmunity, Katholieke Universiteit Leuven, 3000 Louvain, Belgium

3

Laboratory of Cell and Gene Therapy, University of Liège and CHU of Liège, 4000 Liège, Belgium





in hematological malignancies including MM. In this study, we observed a slight reduction in the proliferation of murine and human MM cell lines in the presence of Tα1 in vitro. However, using two immunocompetent murine MM models (5TGM1 and MOPC315.BM), we did not observe any impact of Tα1 administration on MM development in vivo. Furthermore, no beneficial effects of Tα1 treatment were observed on lymphocyte immune reconstitution after transfusion of human hematopoietic stem cells into immunodeficient mice. In conclusion, despite direct effects of Tα1 on human MM cell line proliferation in vitro, Tα1 did not exert anti-myeloma effects in vivo in the two murine models tested. Moreover, Tα1 failed to improve immune recovery in a xenogeneic HSCT model. Keywords  Multiple myeloma · Thymosin alpha 1 · Hematopoietic stem cell transplantation · Immune recovery List of abbreviations Allo-HSCT  Allogeneic hematopoietic stem cell transplantation Allo-MLR Allogeneic mixed lymphocyte reaction Auto-HSCT  Autologous hematopoietic stem cell transplantation BM Bone marrow CTL Cytotoxic T lymphocytes FBS Fetal bovine serum GvHD Graft-versus-host disease HSCT Hematopoietic stem cell transplantation IL Interleukin i.v. Intravenous MHC Major histocompatibility complex MM Multiple myeloma NSCLC Non-small cell lung carcinoma NSG NOD/SCID/IL2rγnull

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PBS Phosphate-buffered saline RTE Recent thymic emigrants Tα1 Thymosin α1 TCM Central memory T cells TEM Effector memory T cells TEMRA CD45RA re-expressing effector memory T cells Treg Regulatory T cells

Introduction Multiple myeloma (MM) is a hematological malignancy characterized by the accumulation of monoclonal plasma cells in the bone marrow (BM) and usually associated with the presence of a monoclonal immunoglobulin (paraprotein) in blood and/or urine. MM is the second most frequently detected hematological malignancy and has a poor prognosis [1]. In patients, the occurrence of several immune alterations caused by MM cells leads to decreased anti-myeloma and general immune responses [2]. Standard treatment of MM currently includes the use of the proteasome inhibitor bortezomib and immunomodulatory drugs combined with chemotherapy and autologous hematopoietic stem cell transplantation (auto-HSCT), but the disease eventually recurs in virtually all patients. The use of allogeneic hematopoietic stem cell transplantation (allo-HSCT) is controversial in MM because of still high myeloma relapse rates and a high treatment-related morbidity and mortality [2]. Thus, in the context of MM treatment, there is a need for new immunomodulating agents that could improve antimyeloma and general immune response before and after HSCT. Thymosin α1 (Tα1) is a naturally occurring thymic peptide which consists of 28 amino acids [3] and is derived through cleavage from its precursor proTα [4]. Tα1 has been associated with several immunomodulating properties, such as restoration of NK cell activity [5] and protection from viral infections [6] or fungal infections after HSCT through dendritic cell activation [7]. Regarding T cells, Tα1 has been shown to increase the production of interleukin (IL)-2 and the expression of IL-2 receptors by T lymphocytes [8], to reduce the sensitivity of thymocytes to apoptosis [9], to stimulate the maturation of CD4+ T cells [10] and to directly increase in vitro T cell proliferation [11, 12]. In patients who received allo-HSCT followed by Tα1 administration for 16 weeks, an earlier appearance and increased cell counts of pathogen-specific T cells have been observed [13]. In addition to its immunomodulating properties, Tα1 could exert direct anti-tumor effects in several cancer types. Tα1 has been shown to increase class I major histocompatibility complex (MHC I) expression on murine and human cancer cells (melanoma and hepatocellular

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Cancer Immunol Immunother

carcinoma, respectively) [14]. As MHC I downregulation is most likely an immune escape mechanism of tumor cells, reducing their recognition by cytotoxic T lymphocytes (CTL) [15], Tα1 could increase the recognition and elimination of tumor cells by CTL in vivo. The administration of Tα1 reduced the number of lung adenomas in mice [16], prevented mammary carcinoma incidence in rats [17], inhibited the growth of non-small cell lung carcinoma (NSCLC) in vitro and in vivo (in mice) [18] and improved the relapse-free and overall survival in NSCLC patients following radiotherapy in a randomized trial [19]. In addition, Tα1 could act as an adjuvant by improving the antitumor effects of chemotherapy in rat glioblastoma [20] and those of chemo-immunotherapy in murine melanoma [21], in patients with metastatic melanoma [22] and in rats with liver metastases from colorectal cancer [23, 24]. In view of all the biological properties that have been described, Tα1 appears as a promising candidate for antimyeloma treatment by potentially promoting anti-myeloma immune response while directly inhibiting myeloma growth. Moreover, this peptide could exert beneficial effects on immune reconstitution after auto- or allo-HSCT in MM patients and thus reduce the treatment-related mortality and increase the anti-myeloma immune response. In this work, we first studied the direct effects of Tα1 on murine and human myeloma cell proliferation in vitro and on MM development in vivo using two immunocompetent murine models. Further, we were also interested in the potential immuno-stimulating role of Tα1 in the context of HSCT.

Materials and methods Mice NOD/SCID/IL2rγnull (NSG) mice were purchased from Jackson Laboratory (Bar Harbor, ME, USA), and C57BL/ KaLwRijHsd and Balb/c OlaHsd mice from Harlan laboratories B.V. (Horst, Netherlands). All the strains were kept and bred at the animal facility of our institute. Mice were used for experiments when they were between 10 and 14 weeks old. All experimental procedures and protocols used in this investigation were approved by the Institutional Animal Care and Use Ethics Committee of the University of Liège (Belgium). Animal welfare was assessed at least once per day, and all efforts were made to strictly control animal suffering during the experiments. Thymosin α1 Synthetic human Tα1 was kindly provided by SciClone Pharmaceuticals (Foster City, CA, USA). Lyophilized

Cancer Immunol Immunother

Tα1 was reconstituted in sterile phosphate-buffered saline (PBS) for all the experiments. Mice treated with Tα1 received 0.4 mg/kg/day by subcutaneous injection. Myeloma cell lines and models Two murine myeloma cell lines that can be maintained in vitro were used. The selection of the Balb/c-derived MOPC315.BM cell line has been described previously [25, 26]. Firefly luciferase-transfected MOPC315.BM cells [26], kindly provided by B. Bogen (University of Oslo, Oslo, Norway), were used for all experiments. The establishment of the 5TGM1 cell line, originating from C57BL/KaLwRij mice, and eGFP-expressing 5TGM1 cells has been described previously [27, 28]. eGFP-transfected 5TGM1 cells, kindly provided by G. R. Mundy and C. M. Edwards (Vanderbilt University, Nashville, TN, USA), were used for all experiments. In vivo experiments and monitoring of MOPC315.BM tumor development by bioluminescence were performed as previously described [29, 30]. The human MM cell lines used for in vitro experiments were U266, OPM-2 and LP-1 cells. The last two cell lines were a kind gift from H. Jernberg-Wiklund (Uppsala University, Sweden). All cell lines were maintained in culture at 37 °C in 5 % CO2 using RPMI 1640 medium or Dulbecco’s modified Eagle’s medium for 5TGM1 cells, supplemented with 10 % heat-inactivated fetal bovine serum (FBS) and Penicillin/Streptomycin (100 U/ml) (=complete medium). All cell culture products were purchased from Lonza (Verviers, Belgium). Humanized murine HSCT model This model has been adapted from a humanized murine model described by Ishikawa et al. [31]. Human hematopoietic stem cells were collected from peripheral blood after granulocyte colony-stimulating factor mobilization and signed consent by the donor. The collected cells were further enriched in CD34+ cells using the CliniMACS device (Miltenyi Biotec, Bergisch Gladbach, Germany) according to the manufacturer’s instructions. The purity of CD34+ hematopoietic stem cells was 98.1 %, and cells were cryopreserved until use. NSG mice received total body irradiation (2.5 Gy) using a 137Cs source (GammaCell 40, Nordion, Ontario, Canada) 24 h before transplantation. For the transplantation, human CD34+ cells were thawed, washed, suspended in PBS (5 × 105 cells/200 µl/mouse) and immediately transplanted to NSG mice by intravenous (i.v.) injection. Starting from the day after HSCT, animals received subcutaneous

injections (5 days/week) of Tα1 (0.4 mg/kg) or PBS during 8 weeks. Blood samples were collected 9, 11 and 13 weeks after HSCT, and all mice were killed 15 weeks after transplantation to allow spleen, bone marrow and blood harvesting. White blood cell counts were obtained using XS-800i (Sysmex, Kobe, Japan), and flow cytometric phenotyping was performed for human lymphocyte subsets. Flow cytometry Cell suspensions were obtained from spleen, bone marrow or peripheral blood as follows: spleens and bone marrows (femurs and tibias) were harvested at killing and homogenized in complete medium. Red blood cells were lysed using a RBC lysis buffer (eBioscience, San Diego, USA), and cells were washed, resuspended in PBS containing 3 % FBS and filtered through a 70-µM nylon membrane. Extracellular staining was performed in PBS containing 3 % FBS. Intra-nuclear staining was performed using the Foxp3 Staining Buffer Set (eBioscience, San Diego, CA, USA) for murine cells or the Foxp3 Fix/Perm Buffer set (Biolegend, San Diego, CA, USA) for human cells. Antibodies were incubated for 30 min at 4 °C. The streptavidin complexes and the following antibodies were purchased from eBioscience: anti-mouse CD4/eFluor450 (RM4-5), CD8/PECy7 (53-6.7), CD49b/Biotin (DX5), CD69/APC (H1.2F3), Foxp3/PE (FJK-16s), MHC I/FITC (34-1-2S) and anti-human CD25/PE (BC96), CD4/eFluor450 (RPAT4), CD8/FITC (HIT8a), CD19/APC (HIB19) and CD45/ PECy7 (HI30). The following antibodies were purchased from BD Biosciences (San Jose, CA, USA): anti-mouse CD3e/v500 (500A2) and anti-human CD3/v450 (UCHT1), CD4/PerCP (SK3), CD56/PE (B159), CD45/PerCP (2D1), CD45RA/APC (HI100), CD31/PE (L133.1), CCR7/PeCy7 (3D12), IgD/Biotin (IA6-2), CD27/PE (M-T271), CD19/ APCCy7 (SJ25C1). Anti-human Foxp3/Alexa488 (206D) was purchased from Biolegend. For human cells in blood of NSG mice, absolute counts were obtained by multiplying the percentage of human CD45+ cells in the lymphocyte gate with absolute counts for lymphocytes. Flow cytometric data were acquired using a BD FACSCanto II flow cytometer (BD Biosciences) and the BD FACS DIVA software and analyzed with the FlowJo software (Tree Star, Ashland, OR, USA). Cell proliferation assays Cell proliferation was assessed using the cell proliferation kit I (MTT) from Roche Applied Science (Mannheim, Germany) following the manufacturer’s instructions. Absorbance at 570 nm was measured using a Wallac 1420 Victor2 microplate reader (PerkinElmer, Zaventem, Belgium). For

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all the proliferation assays, cells were cultured in 96-well plates (final culture volume 100 µl/well). Experiments were repeated 3–5 times, and triplicates were performed within each experiment. Lymphocyte proliferation assays Spleens were harvested from C57BL/KaLwRij mice, and sterile cell suspensions were obtained as described above. Splenocytes were cultured in complete RMPI 1640 medium containing 5 µM of 2-mercaptoethanol (Gibco by Life Technologies, Gent, Belgium). T cell proliferation was induced by adding mouse T-activator CD3/CD28 dynabeads (Gibco). Proliferation was assessed after 4 days of culture using the MTT assay as described above. For allogeneic mixed lymphocyte reactions (allo-MLR), splenocytes from C57BL/KaLwRij (H-2b) mice were co-cultured with 20-Gy-irradiated Balb/c (H-2d) splenocytes during 4 days. Proliferation was measured using a 3H-labelled thymidine incorporation assay, by adding 0.17 µCi of [methyl-3H] thymidine (PerkinElmer) to each well for the last 18 h of culture. DNA was harvested on Multiscreen Harvest Plates (Millipore, Carrigtwohill, Co. Cork, Ireland) using Filter Mate Harvester (PerkinElmer). Plates were dried for 3–4 h before adding 25 µl/well of Microscint™ O (PerkinElmer) and measuring radioactivity (c.p.m.) with TopCount NXT Microplate Scintillation counter (PerkinElmer).

Fig. 1  Effects of Tα1 on MM proliferation in vitro. Myeloma cell lines were cultured for 24 h in the presence of various Tα1 concentrations (0.1–100 µM). Proliferation was assessed using a MTT assay, and the percentage of proliferation (mean ± SD) was calculated compared to cells cultured without Tα1 (=100 %). Results represent five independent experiments, and within each experiment, triplicates were performed. *p