Cancer Immunology, Immunotherapy

, Volume 68, Issue 11, pp 1791–1804 | Cite as

Lenalidomide improves the therapeutic effect of an interferon-α-dendritic cell-based lymphoma vaccine

  • Caterina LapentaEmail author
  • Simona Donati
  • Francesca Spadaro
  • Laura Lattanzi
  • Francesca Urbani
  • Iole Macchia
  • Paola Sestili
  • Massimo Spada
  • Maria Christina Cox
  • Filippo Belardelli
  • Stefano M. SantiniEmail author
Original Article


The perspective of combining cancer vaccines with immunomodulatory drugs is currently regarded as a highly promising approach for boosting tumor-specific T cell immunity and eradicating residual malignant cells. The efficacy of dendritic cell (DC) vaccination in combination with lenalidomide, an anticancer drug effective in several hematologic malignancies, was investigated in a follicular lymphoma (FL) model. First, we evaluated the in vitro activity of lenalidomide in modulating the immune responses of lymphocytes co-cultured with a new DC subset differentiated with IFN-α (IFN-DC) and loaded with apoptotic lymphoma cells. We next evaluated the efficacy of lenalidomide and IFN-DC-based vaccination, either alone or in combination, in hu-PBL-NOD/SCID mice bearing established human lymphoma. We found that lenalidomide reduced Treg frequency and IL-10 production in vitro, improved the formation of immune synapses of CD8 + lymphocytes with lymphoma cells and enhanced anti-lymphoma cytotoxicity. Treatment of lymphoma-bearing mice with either IFN-DC vaccination or lenalidomide led to a significant decrease in tumor growth and lymphoma cell spread. Lenalidomide treatment was shown to substantially inhibit tumor-induced neo-angiogenesis rather than to exert a direct cytotoxic effect on lymphoma cells. Notably, the combined treatment with the vaccine plus lenalidomide was more effective than either single treatment, resulting in the significant regression of established tumors and delayed tumor regrowth upon treatment discontinuation. In conclusion, our data demonstrate that IFN-DC-based vaccination plus lenalidomide exert an additive therapeutic effect in xenochimeric mice bearing established lymphoma. These results may pave the way to evaluate this combination in the clinical ground.


Cancer vaccines Immunotherapy Lymphomas Dendritic cells Combination therapy Lenalidomide 



Antibody-dependent cell cytotoxicity


Confocal laser scanning microscopy


Dendritic cells


Follicular lymphoma


Forkhead box P3


Granulocyte macrophage colony-stimulating factor


IFN-α-conditioned dendritic cells


Interferon alpha


Interferon gamma




Natural killer


Non-Hodgkin lymphoma


Nonobese diabetic/severe combined immunodeficiency


Peripheral blood lymphocytes


Tumor necrosis factor α



The authors thank Mr. Daniele Macchia for extensive help with animal care and technical assistance in studies with xenochimeric mice.

Author contributions

Conception and design: SMS, CL; Development of methodology: SMS, CL, FU, IM; Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): SMS, CL, SD, FS, PS, FU, IM, LL, MS; Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): SMS, CL, SD, FS, PS, FU; Writing, review, and/or revision of the manuscript: SMS, CL, FB; Administrative, technical, or material support (i.e., reporting or organizing data, constructing databases): SMS, CL; Study supervision: SMS, CL, FB, MCC.


The research was supported by Grants from Celgene Corporation (Stefano M. Santini) (Grant no. ITA-017) and the Italian Association for Research against Cancer (AIRC IG16891) (Filippo Belardelli).

Compliance with ethical standards

Conflict of interest

Stefano M. Santini received research funding from Celgene. All other authors declare that they have no conflict of interest. Celgene had no role in study design, data collection, data interpretation, writing of the manuscript.

Ethical approval

All experiments utilizing PBMC from healthy donors were conducted in accordance with the ethical standards of the Ethics Committee of Istituto Superiore di Sanità and the Declaration of Helsinki. Institutional Review Board approval was not required for this kind of study. Buffy coat supply for our studies was approved by Azienda Ospedaliera Policlinico Umberto I on 24/02/2014 (aut.6802). All experiments utilizing blood samples from FL patients were conducted in accordance with the declaration of Helsinki. Study and procedures were approved by the Ethics Committee of Azienda Ospedaliera Sant’Andrea (aut.169/2011). All experiments on mice were executed in compliance with the Istituto Superiore di Sanità Service for Animal Welfare guidelines and after approval from the Italian Ministry of Health (aut. 296/2015-PR). Mice were housed according to Legislative Decree 26/2014 guideline.

Informed consent

PBMC were freshly isolated from peripheral blood samples of anonymous volunteer healthy donors at the Transfusion Center of of Policlinico Umberto I—University “La Sapienza”, Rome. Written informed consent was obtained from all blood donors to the use of their blood for research and scientific purposes. Blood samples from FL patients were anonymously provided by the Hematology Unit at the Azienda Ospedaliera Sant’Andrea Rome, Italy. Written informed consent was obtained from FL patients for the use of blood and lymph node specimens in IFN-DC-based vaccine researches.

Animal source

NOD/SCID (NOD.CB17-Prkdcscid/NCrHsd) female mice were purchased from Envigo (Italy), used at 3–4 weeks of age.

Cell line authentication

Karpas-422 FL cell line was purchased from the cell bank Interlab Cell Line Collection (ICLC). Human K562 erythroleukemic cell line was purchased from the European Collection of Authenticated Cell Culture (ECACC). Both cell lines were authenticated by the suppliers by short tandem repeat (STR) profiling. The cell lines were initially grown and cryopreserved into multiple aliquots. All the experiments were performed with cells at low passage numbers (≤ 10).


  1. 1.
    Kahl BS, Yang DT (2016) Follicular lymphoma: evolving therapeutic strategies. Blood 127:2055–2063. CrossRefGoogle Scholar
  2. 2.
    Freedman A (2018) Follicular lymphoma: 2018 update on diagnosis and management. Am J Hematol 93:296–305. CrossRefGoogle Scholar
  3. 3.
    Federico M, Bellei M, Marcheselli L et al (2009) Follicular Lymphoma International Prognostic Index 2: a new prognostic index for follicular lymphoma developed by the international follicular lymphoma prognostic factor project. J Clin Oncol 27:4555–4562. CrossRefGoogle Scholar
  4. 4.
    Sarkozy C, Trneny M, Xerri L et al (2016) Risk factors and outcomes for patients with follicular lymphoma who had histologic transformation after response to first-line immunochemotherapy in the PRIMA trial. J Clin Oncol 34:2575–2582. CrossRefGoogle Scholar
  5. 5.
    Garg AD, Coulie PG, Van den Eynde BJ, Agostinis P (2017) Integrating next-generation dendritic cell vaccines into the current cancer immunotherapy landscape. Trends Immunol 38:577–593. CrossRefGoogle Scholar
  6. 6.
    Garg AD, More S, Rufo N et al (2017) Trial watch: immunogenic cell death induction by anticancer chemotherapeutics. Oncoimmunology 6:e1386829. CrossRefPubMedPubMedCentralGoogle Scholar
  7. 7.
    Weinstock M, Rosenblatt J, Avigan D (2017) Dendritic cell therapies for hematologic malignancies. Mol Ther Methods Clin Dev 5:66–75. CrossRefPubMedPubMedCentralGoogle Scholar
  8. 8.
    Di Nicola M, Zappasodi R, Carlo-Stella C et al (2009) Vaccination with autologous tumor-loaded dendritic cells induces clinical and immunologic responses in indolent B-cell lymphoma patients with relapsed and measurable disease: a pilot study. Blood 113:18–27. CrossRefGoogle Scholar
  9. 9.
    Kolstad A, Kumari S, Walczak M et al (2015) Sequential intranodal immunotherapy induces antitumor immunity and correlated regression of disseminated follicular lymphoma. Blood 125:82–89. CrossRefGoogle Scholar
  10. 10.
    Santini SM, Lapenta C, Belardelli F (2005) Type I interferons as regulators of the differentiation/activation of human dendritic cells: methods for the evaluation of IFN-induced effects. Methods Mol Med 116:167–181. CrossRefGoogle Scholar
  11. 11.
    Lapenta C, Santini SM, Spada M et al (2006) IFN-alpha-conditioned dendritic cells are highly efficient in inducing cross-priming CD8+ T cells against exogenous viral antigens. Eur J Immunol 36:2046–2060. CrossRefGoogle Scholar
  12. 12.
    Parlato S, Santini SM, Lapenta C et al (2001) Expression of CCR-7, MIP-3beta, and Th-1 chemokines in type I IFN-induced monocyte-derived dendritic cells: importance for the rapid acquisition of potent migratory and functional activities. Blood 98:3022–3029CrossRefGoogle Scholar
  13. 13.
    Lapenta C, Santini SM, Logozzi M et al (2003) Potent immune response against HIV-1 and protection from virus challenge in hu-PBL-SCID mice immunized with inactivated virus-pulsed dendritic cells generated in the presence of IFN-alpha. J Exp Med 198:361–367. CrossRefPubMedPubMedCentralGoogle Scholar
  14. 14.
    Santini SM, Lapenta C, Donati S et al (2011) Interferon-α-conditioned human monocytes combine a Th1-orienting attitude with the induction of autologous Th17 responses: role of IL-23 and IL-12. PLoS One 6:e17364. CrossRefPubMedPubMedCentralGoogle Scholar
  15. 15.
    Spadaro F, Lapenta C, Donati S et al (2012) IFN-α enhances cross-presentation in human dendritic cells by modulating antigen survival, endocytic routing, and processing. Blood 119:1407–1417. CrossRefGoogle Scholar
  16. 16.
    Lattanzi L, Rozera C, Marescotti D et al (2011) IFN-α boosts epitope cross-presentation by dendritic cells via modulation of proteasome activity. Immunobiology 216:537–547. CrossRefGoogle Scholar
  17. 17.
    Lapenta C, Donati S, Spadaro F et al (2016) NK cell activation in the antitumor response induced by IFN-α dendritic cells loaded with apoptotic cells from follicular lymphoma patients. J Immunol 197:795–806. CrossRefGoogle Scholar
  18. 18.
    Witzig TE, Nowakowski GS, Habermann TM et al (2015) A comprehensive review of lenalidomide therapy for B-cell non-Hodgkin lymphoma. Ann Oncol 26:1667–1677. CrossRefGoogle Scholar
  19. 19.
    Ahmadi T, Chong EA, Gordon A et al (2014) Combined lenalidomide, low-dose dexamethasone, and rituximab achieves durable responses in rituximab-resistant indolent and mantle cell lymphomas. Cancer 120:222–228. CrossRefPubMedPubMedCentralGoogle Scholar
  20. 20.
    Chong EA, Ahmadi T, Aqui NA et al (2015) Combination of lenalidomide and rituximab overcomes rituximab resistance in patients with indolent B-cell and mantle cell lymphomas. Clin Cancer Res 21:1835–1842. CrossRefGoogle Scholar
  21. 21.
    Nguyen-Pham T-N, Jung S-H, Vo M-C et al (2015) Lenalidomide synergistically enhances the effect of dendritic cell vaccination in a model of murine multiple myeloma. J Immunother 38:330–339. CrossRefGoogle Scholar
  22. 22.
    Vo M-C, Nguyen-Pham T-N, Lee H-J et al (2017) Combination therapy with dendritic cells and lenalidomide is an effective approach to enhance antitumor immunity in a mouse colon cancer model. Oncotarget. CrossRefPubMedPubMedCentralGoogle Scholar
  23. 23.
    Vo M-C, Anh-NguyenThi T, Lee H-J et al (2017) Lenalidomide enhances the function of dendritic cells generated from patients with multiple myeloma. Exp Hematol 46:48–55. CrossRefGoogle Scholar
  24. 24.
    Epron G, Ame-Thomas P, Le Priol J et al (2012) Monocytes and T cells cooperate to favor normal and follicular lymphoma B-cell growth: role of IL-15 and CD40L signaling. Leukemia 26:139–148. CrossRefGoogle Scholar
  25. 25.
    Mourcin F, Pangault C, Amin-Ali R et al (2012) Stromal cell contribution to human follicular lymphoma pathogenesis. Front Immunol 3:280. CrossRefPubMedPubMedCentralGoogle Scholar
  26. 26.
    Amé-Thomas P, Tarte K (2014) The yin and the yang of follicular lymphoma cell niches: role of microenvironment heterogeneity and plasticity. Semin Cancer Biol 24:23–32. CrossRefGoogle Scholar
  27. 27.
    Montico B, Lapenta C, Ravo M et al (2017) Exploiting a new strategy to induce immunogenic cell death to improve dendritic cell-based vaccines for lymphoma immunotherapy. Oncoimmunology 6:e1356964. CrossRefPubMedPubMedCentralGoogle Scholar
  28. 28.
    Macchia I, Urbani F, Proietti E (2013) Immune monitoring in cancer vaccine clinical trials: critical issues of functional flow cytometry-based assays. Biomed Res Int. CrossRefPubMedPubMedCentralGoogle Scholar
  29. 29.
    Santini SM, Lapenta C, Santodonato L et al (2009) IFN-alpha in the generation of dendritic cells for cancer immunotherapy. Handb Exp Pharmacol. CrossRefGoogle Scholar
  30. 30.
    Galustian C, Meyer B, Labarthe M-C et al (2009) The anti-cancer agents lenalidomide and pomalidomide inhibit the proliferation and function of T regulatory cells. Cancer Immunol Immunother 58:1033–1045. CrossRefGoogle Scholar
  31. 31.
    Morschhauser F, Fowler NH, Feugier P et al (2018) Rituximab plus lenalidomide in advanced untreated follicular lymphoma. N Engl J Med 379:934–947. CrossRefGoogle Scholar
  32. 32.
    Palma M, Hansson L, Mulder TA et al (2018) Lenalidomide as immune adjuvant to a dendritic cell vaccine in chronic lymphocytic leukemia patients. Eur J Haematol 101:68–77. CrossRefGoogle Scholar
  33. 33.
    Lee B-N, Gao H, Cohen EN et al (2011) Treatment with lenalidomide modulates T-cell immunophenotype and cytokine production in patients with chronic lymphocytic leukemia. Cancer 117:3999–4008. CrossRefPubMedPubMedCentralGoogle Scholar
  34. 34.
    Minnema MC, van der Veer MS, Aarts T et al (2009) Lenalidomide alone or in combination with dexamethasone is highly effective in patients with relapsed multiple myeloma following allogeneic stem cell transplantation and increases the frequency of CD4 + Foxp3 + T cells. Leukemia 23:605–607. CrossRefGoogle Scholar
  35. 35.
    Kneppers E, van der Holt B, Kersten M-J et al (2011) Lenalidomide maintenance after nonmyeloablative allogeneic stem cell transplantation in multiple myeloma is not feasible: results of the HOVON 76 Trial. Blood 118:2413–2419. CrossRefGoogle Scholar
  36. 36.
    Lioznov M, El-Cheikh J, Hoffmann F et al (2010) Lenalidomide as salvage therapy after allo-SCT for multiple myeloma is effective and leads to an increase of activated NK (NKp44+) and T (HLA-DR+) cells. Bone Marrow Transplant 45:349–353. CrossRefGoogle Scholar
  37. 37.
    Busch A, Zeh D, Janzen V et al (2014) Treatment with lenalidomide induces immunoactivating and counter-regulatory immunosuppressive changes in myeloma patients. Clin Exp Immunol 177:439–453. CrossRefPubMedPubMedCentralGoogle Scholar
  38. 38.
    Muthu Raja KR, Kovarova L, Hajek R (2012) Induction by lenalidomide and dexamethasone combination increases regulatory cells of patients with previously untreated multiple myeloma. Leuk Lymphoma 53:1406–1408. CrossRefGoogle Scholar
  39. 39.
    Tzankov A, Meier C, Hirschmann P et al (2008) Correlation of high numbers of intratumoral FOXP3 + regulatory T cells with improved survival in germinal center-like diffuse large B-cell lymphoma, follicular lymphoma and classical Hodgkin’s lymphoma. Haematologica 93:193–200. CrossRefGoogle Scholar
  40. 40.
    Aqui N, Leinbach L, Chong EA et al (2010) Changes in regulatory T-cells in responding and non-responding patients with indolent B-cell or mantle cell lymphomas during treatment with lenalidomide, dexamethasone, and rituximab. J Clin Oncol. CrossRefGoogle Scholar
  41. 41.
    Ramsay AG, Clear AJ, Kelly G et al (2009) Follicular lymphoma cells induce T-cell immunologic synapse dysfunction that can be repaired with lenalidomide: implications for the tumor microenvironment and immunotherapy. Blood 114:4713–4720. CrossRefPubMedPubMedCentralGoogle Scholar
  42. 42.
    Lu L, Payvandi F, Wu L et al (2009) The anti-cancer drug lenalidomide inhibits angiogenesis and metastasis via multiple inhibitory effects on endothelial cell function in normoxic and hypoxic conditions. Microvasc Res 77:78–86. CrossRefGoogle Scholar
  43. 43.
    Song K, Herzog BH, Sheng M et al (2013) Lenalidomide inhibits lymphangiogenesis in preclinical models of mantle cell lymphoma. Cancer Res 73:7254–7264. CrossRefPubMedPubMedCentralGoogle Scholar
  44. 44.
    Ribatti D, Nico B, Ranieri G et al (2013) The role of angiogenesis in human non-Hodgkin lymphomas. Neoplasia 15:231–238CrossRefGoogle Scholar
  45. 45.
    Sozzani S, Rusnati M, Riboldi E et al (2007) Dendritic cell-endothelial cell cross-talk in angiogenesis. Trends Immunol 28:385–392. CrossRefGoogle Scholar
  46. 46.
    Rozera C, Cappellini GA, D’Agostino G et al (2015) Intratumoral injection of IFN-alpha dendritic cells after dacarbazine activates anti-tumor immunity: results from a phase I trial in advanced melanoma. J Transl Med 13:139. CrossRefPubMedPubMedCentralGoogle Scholar
  47. 47.
    Cox MC, Castiello L, Mattei M et al (2019) Clinical and antitumor immune responses in relapsed/refractory follicular lymphoma patients after intranodal injections of IFNα-dendritic cells and rituximab. Clin Cancer Res. CrossRefGoogle Scholar

Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2019

Authors and Affiliations

  1. 1.Reparto di Immunologia dei Tumori, Dipartimento di Oncologia e Medicina MolecolareIstituto Superiore di SanitàRomeItaly
  2. 2.Servizio Grandi Strumentazioni e Core FacilitiesIstituto Superiore di SanitàRomeItaly
  3. 3.Centro nazionale sperimentazione e benessere animaleIstituto Superiore di SanitàRomeItaly
  4. 4.Unità di EmatologiaAzienda Ospedaliera Sant’Andrea, Università La SapienzaRomeItaly
  5. 5.Istituto di Farmacologia TraslazionaleConsiglio Nazionale delle Ricerche (CNR)RomeItaly
  6. 6.Scuola di Dottorato in Biotecnologie Mediche e Medicina TraslazionaleTor Vergata UniversityRomeItaly

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