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LETTERS TO THE EDITOR Impact of lenalidomide on immune functions in the setting of maintenance therapy for multiple myeloma

Leukemia (2015) 29, 2098–2100; doi:10.1038/leu.2015.64 polarization and the cytokine secretion profile in patients receiving lenalidomide in the maintenance setting (MM-Len group) compared with patients treated for MM without any maintenance (MM group) Lenalidomide is an effective and widely used therapeutic agent for and to healthy donors (HD group). Ten patients belonged to the MM-Len group. Their median age multiple myeloma (MM). The combination of its anti-neoplastic – effect on myeloma cells1,2 and immunomodulatory properties3,4 was 60.3 years (49.1 64.4) and, at the time of analysis, they had makes lenalidomide a major therapeutic drug in the treatment received between 4 and 12 cycles of lenalidomide alone (one algorithms for MM patients. This is supported by its ability to cycle = 28 days) at a dose ranging between 5 and 15 mg/day. Five confer a positive impact on outcome in the setting of of them had previously received 3 cycles of RVD (lenalidomide, maintenance therapy in young MM patients receiving autologous bortezomib and dexamethasone), autologous stem cell transplant stem cell transplantation.5–7 An important area of lenalidomide (ASCT), conditioning by melphalan, then consolidation by 3 cycles investigation is the study of both its reversible effects on of RVD, followed by maintenance therapy with continuous oral myeloma-mediated immune suppression and its positive effects lenalidomide alone. The other five patients received induction on antitumor immunity. Previous studies have demonstrated that with the RVD combination, then maintenance with lenalidomide. lenalidomide promotes T-cell proliferation and suppresses inhibi- Seven patients were in very good partial response (VGPR) and tory factors such as program death receptor 1, in addition to three in complete response (CR) at the time of analysis. The MM enhancing T-8 and NK-cell function.9 However, its immune effects group included eight patients after ASCT without any main- in MM patients treated with lenalidomide alone remain to be tenance at the time of analysis (Supplementary Table S1). The two elucidated. This pilot prospective study aimed to assess T-cell groups were comparable in terms of age, sex, interval between

Figure 1. Intracellular cytokine secretion by CD4+ T cells from MM patients under lenalidomide maintenance (MM-Len; n = 10), from MM patients without maintenance treatment (MM; n = 8) and from healthy donors (HD; n = 6). (a) Representative plots showing intracellular expression of IFN-γ, TNF-α and IL-21. (b) Proportion of CD4+ T cells secreting IFN-γ, TNF-α, IL-4, IL-10, IL-17 and IL-21 after PMA-ionomycin stimulation. *Po0.05.

Accepted article preview online 9 March 2015; advance online publication, 14 July 2015 Letters to the Editor 2099 diagnosis and time of analysis, and disease status (data not shown). All patients treated for MM were in CR or VGPR at the inclusion in the study. The HD group comprised six healthy donors. Peripheral blood mononuclear cells (PBMCs) were isolated by Ficoll-Hypaque and an aliquot was cryopreserved for storage. After thawing, PBMCs were cultured in RPMI-1640 medium containing 2 mML-glutamine, 100 UI/ml penicillin, 0.1 mg/ml streptomycin and either 8% human serum for the detection of intracellular cytokines or 10% fetal calf serum (Sigma, St Quentin Fallavier, France) for flow cytometry analysis. PBMCs were stimulated with 25 ng/ml phorpbol-12-myristate-13-acetate (PMA; Sigma), 1 μg/ml ionomycin (Sigma) and 10 μg/ml brefeldin A (Sigma) for 5 h. Cells were first stained with anti-CD3 APC-H7, anti-CD4 Pe-Cy7 and anti-CD8 APC, permeabilized using Cytofix/ cytoperm, and were then incubated with anti-TNFα FITC, anti-IL-10 PE, anti-IFNγ FITC, anti-IL-4 PE, anti-IL-21AF647 and anti-IL-17 V450. Viability markers (TO-PRO3 or Vivid (Invitrogen, Illkirch, France)) were used to gate on viable cells. Data were acquired on a CANTO-II flow cytometer and analyzed using Flow-Jo software (Tree Star, Inc., Ashland, OR, USA). In parallel, a total of 34 cytokines were analyzed in the serum collected simultaneously from the MM-Len and HD groups. Concentrations of serum cytokines, chemokines and growth factors were determined in the MM-Len and the HD groups by the bead-based multiplex protein array technology (Luminex assay from Millipore, St Charles, MO, USA) according to the manufacturer instructions (Supplementary Table S2). For validation, IL-8, IFN-γ-inducible protein 10 (IP-10) and MIP-1β serum levels were tested by ELISA in these two groups and also in the MM group. Statistical analyses were performed using GraphPad Prism 5.0 (Graphpad Software, San Diego, CA, USA). All comparisons were made by either the parametric t-test or the nonparametric Mann–Whitney U-test for unpaired data. A P-value of o0.05 was considered statistically significant. We observed that CD4+ T cells derived from the MM-Len group exhibited a significantly higher secretion of interferon-gamma (IFN-γ; P = 0.01) and interleukin-21 (IL-21; P = 0.04) and a slightly higher secretion of tumor necrosis factor-alpha (TNF-α; P = 0.056), compared with stimulated CD4+ T cells obtained from the MM and HD groups (Figure 1a and b). Focusing on CD8+ T cells, there was a trend towards an increased number of IFN-γ+ CD8+ T cells in LEN patients compared with HD donors (P = 0.057). There was also a trend for an increased number of TNF-α+ CD8+ T cells in LEN-MM patients compared with MM patients without lenalido- mide (P = 0.09; Supplementary Figure S3). Secretion levels of IL-10, IL-4 and IL-17 by CD4+ T cells were comparable between the three groups. In the multiplex bead assay, we observed that IP-10 Figure 2. Levels of IP-10 (a), IL-8 (b) and MIP-1β (c) in the serum of (P = 0.005), IL-8 (P = 0.02), and macrophage inflammatory protein- MM patients under lenalidomide maintenance (MM-Len; n = 10), of 1β (MIP-1β; P = 0.03) serum levels were also significantly increased MM patients without maintenance treatment (MM; n = 8) and in in the MM-Len versus the HD group (Supplementary Table S2). healthy donors (HD; n = 6). *Po0.05; **Po0.01. These results were confirmed by ELISA. IP-10 was significantly increased in serum of patients from the MM-Len group as compared with healthy donors (P = 0.03), but not in relation to MM mouse model, that myeloma immunosurveillance is mediated by patients (P = 0.12). We observed a significant increase of the IL-8 Th1 cells and particularly by IFN-γ-producing tumor-specific serum levels in the MM-Len group as compared with MM and HD Th1 cells. Th1-derived IFN-γ was shown to render macrophages groups (P = 0.04 and 0.004, respectively). The same held true for directly cytotoxic to MM cells and to secrete the angiostatic β MIP-1 (P = 0.001 and 0.004, respectively; Figure 2). chemokines IP-10. We also found that CD4+ T cells from MM Overall, these results suggest that the use of lenalidomide as patients maintained with lenalidomide secreted higher amounts maintenance therapy in myeloma can trigger some potent of IL-21 compared with MM patients without treatment and to immunostimulatory activities through induction of a chronic inflammatory reaction. Interestingly, IP-10 is a major Th1-related healthy donors. IL-21 is a cytokine with broad pleiotropic effects chemokine involved in antitumor responses, and CD4+T cells from that has been described to regulate both innate and adaptive MM patients under lenalidomide maintenance expressed signifi- immune responses, to exert a key role in antitumour responses cantly higher amounts of IFN-γ and also a trend to higher and to also promote the development of inflammatory responses. secretion of TNF-α. These findings are consistent with in vitro Only limited data are available on the role of IL-21 in B-cell studies of lenalidomide effects on PBMC of MM patients.8,10 Our malignancy. Ahearne et al.12 have shown that IL-21, in combina- results demonstrate that lenalidomide polarizes T-cell responses tion with Il-4, can promote the proliferation of chronic lympho- towards a Th1 phenotype. Haabeth et al.11 have described, in a cytic leukemia and, interestingly, that IL-21 expression led to an

© 2015 Macmillan Publishers Limited Leukemia (2015) 2098 – 2116 Letters to the Editor 2100 increase in resistance to chemotherapy. IL-21 was reported to be a 2INSERM UMRs 938, Paris, France; growth factor for a subset of MM cells that are CD45-negative.13 3Hématologie Clinique, CHU Hôtel-Dieu, Nantes, France; The role of Il-21 on tumor cells is still under investigation and the 4CRCNA, UMR 892 INSERM- 6299 CNRS, Nantes, France; effect of IL-21 on CD4+ T cells under lenalidomide needs to be 5INSERM UMR 1098, Besançon, France; explored. However, even if the antitumoral role of CD4+ T cells has 6Université de Franche-Comté, Besançon, France; been demonstrated in some cancers, there is no clear evidence of 7EFS Bourgogne Franche-Comté, Besançon, France and CD4+ T-cell function in MM patients, which warrants further 8Service d’Hématologie Clinique et de Thérapie Cellulaire, Hôpital functional investigations. In our study, we found that patients Saint Antoine, APHP, Paris, France maintained with lenalidomide had higher serum levels of IL-8 and E-mail: [email protected] MIP-1β. It is noteworthy that these two chemokines have been described to positively influence NK cell functions (maturation, migration and increase of their cytolytic activity). Furthermore, REFERENCES many studies have reported that lenalidomide immunomodula- 1 Verhelle D, Corral LG, Wong K, Mueller JH, Moutouh-de Parseval L, tory properties enhance both NK- and antibody-dependent cell Jensen-Pergakes K et al. Lenalidomide and CC-4047 inhibit the proliferation of cytotoxicity.14 However, in our study, there was no available data malignant B cells while expanding normal CD34+ progenitor cells. Cancer Res 67 – on NK cells, and further studies on NK cells functions in the setting 2007; :746 755. of lenalidomide in maintenance would be of great interest. In spite 2 Gandhi AK, Kang J, Capone L, Parton A, Wu L, Zhang LH et al. Dexamethasone synergizes with lenalidomide to inhibit multiple myeloma tumor growth, but of the limited number of patients in our study, the strict selection reduces lenalidomide-induced immunomodulation of T and NK cell function. criteria for their inclusion has allowed to limit biases introduced by Curr Cancer Drug Targets 2010; 10: 155–167. association of diverse chemotherapy regimens. Howbeit, the small 3 Neuber B, Herth I, Tolliver C, Schoenland S, Hegenbart U, Hose D et al. number of patients included in this study was an important Lenalidomide enhances antigen-specific activity and decreases CD45RA expres- limitation and further investigation on independent larger cohorts sion of T cells from patients with multiple myeloma. J Immunol 2011; 187: should be performed, especially that lenalidomide is nowadays 1047–1056. widely used in MM therapy. Altogether, our data shed new light 4 Noonan K, Rudraraju L, Ferguson A, Emerling A, Pasetti MF, Huff CA et al. on the immunostimulatory properties of lenalidomide in the Lenalidomide-induced immunomodulation in multiple myeloma: impact on 18 – setting of long-term maintenance therapy for myeloma, high- vaccines and antitumor responses. Clin Cancer Res 2012; :1426 1434. 5 Attal M, Lauwers-Cances V, Marit G, Caillot D, Moreau P, Facon T et al. lighting the need for detailed immunomonitoring in large Lenalidomide maintenance after stem-cell transplantation for multiple myeloma. multicenter randomized trials. N Engl J Med 2012; 366: 1782–1791. 6 McCarthy PL, Owzar K, Hofmeister CC, Hurd DD, Hassoun H, Richardson PG et al. Lenalidomide after stem-cell transplantation for multiple myeloma. N Engl J Med CONFLICT OF INTEREST 2012; 366: 1770–1781. MM, PM and SLG have received research support and lecture fees for work outside of 7 Roussel M, Lauwers-Cances V, Robillard N, Hulin C, Leleu X, Benboubker L et al. the current study from Celgene and Janssen, whose product is discussed in this Front-line transplantation program with lenalidomide, bortezomib, and article. The remaining authors declare no conflict of interest. dexamethasone combination as induction and consolidation followed by lenalidomide maintenance in patients with multiple myeloma: a phase ii study by the Intergroupe Francophone du Myelome. JClinOncol2014; 32: ACKNOWLEDGEMENTS 2712–2717. 8 Luptakova K, Rosenblatt J, Glotzbecker B, Mills H, Stroopinsky D, Kufe T et al. MM would like to thank Pr J.V. de Melo (Adelaide, Australia) for critical reading of the Lenalidomide enhances anti-myeloma cellular immunity. Cancer Immunol manuscript. We acknowledge the technical and logistical support of V Dehame. EB was Immunother 2013; 62:39–49. supported by educational grants from the ‘Association for Training, Education and 9 Davies FE, Raje N, Hideshima T, Lentzsch S, Young G, Tai YT et al. Thalidomide and Research in Hematology, Immunology and Transplantation’ (ATERHIT) and by Fulbright immunomodulatory derivatives augment natural killer cell cytotoxicity in multiple Foundation. We also thank the ‘Région Pays de Loire’,the‘Association pour la Recherche myeloma. Blood 2001; 98: 210–216. sur le Cancer (ARC; grant #3175 to MM and BG)’,the‘Fondation de France’,the 10 De Keersmaecker B, Fostier K, Corthals J, Wilgenhof S, Heirman C, Aerts JL et al. ‘Fondation contre la Leucémie’,the‘Agence de Biomédecine’,the‘Association Immunomodulatory drugs improve the immune environment for dendritic CentpourSang la Vie’,the‘Association Laurette Fuguain’, the IRGHET and the ‘Ligue cell-based immunotherapy in multiple myeloma patients after auto- Contre le Cancer’ (Comités Grand-Ouest) for their generous and continuous support of logous stem cell transplantation. Cancer Immunol Immunother 2014; 63: our clinical and basic research work. Our transplant programs are supported by several 1023–1036. grants from the French National Cancer Institute (PHRC, INCa to MM). 11 Haabeth OA, Lorvik KB, Hammarstrom C, Donaldson IM, Haraldsen G, Bogen B et al. Inflammation driven by tumour-specific Th1 cells protects against AUTHOR CONTRIBUTIONS B-cell cancer. 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