Generation of dendritic cells from positively selected CD14+ monocytes for anti-tumor immunotherapy

Antonio Curti, Alessandro Isidori, Elisa Ferri, Carolina Terragna, Paolo Neyroz, Claudia Cellini, Marina Ratta, Michele Baccarani, Roberto M. Lemoli

Research output: Contribution to journalArticlepeer-review


Peripheral blood CD14+ monocytes from multiple myeloma (MM) patients can be induced to differentiate into fully functional, mature, CD83+ dendritic cells (DCs) which are highly efficient in priming autologous T lymphocytes in response to the patient-specific tumor idiotype (Id). We have recently scaled up our manufacturing protocol for application in a phase I-II clinical trial of anti-Id vaccination with DCs in MM patients. Elegible patients received a series of by-monthly immunizations consisting of three subcutaneous and two intravenous injections of Id-keyhole limpet hemocyanin (KLH)-pulsed DCs (5 × -, 10 × -, 50 × 106 cells and 10 × -, 50 × 106 cells, respectively). To generate DCs, monocytes were labeled with clinical grade anti-CD14 conjugates and positively selected by immunomagnetic separation. Cells were then cultured, according to Good Manufacturing Practice guidelines, in FCS-free medium in cell culture bags, and differentiated to DCs with GM-CSF plus IL-4 followed by TNF-α or, more recently, by a cocktail of IL-1β, IL-6, TNF-α and prostaglandin-E2. Before maturation, Mo-DCs were pulsed with the autologous Id as whole protein or Id (VDJ)-derived HLA class I restricted peptides. Ten MM patients, who had been treated with two courses of high-dose chemotherapy with peripheral blood stem cell support, entered into the clinical study. CD14+ monocytes were enriched from 16.1 ± 5.7% to 95.5 ± 3.2% (recovery 67.9 ± 15%, viability > 97%). After cell culture, phenotypic analysis showed that 89.6 ± 6.6% of the cells were mature DCs. We obtained 2.89 ± 1 × 108 DCs/leukapheresis which represented 24.5 ± 9% of the initial number of CD14+ cells. Notably, the cytokine cocktail induced a significantly higher percentage and yield (31 ± 10.9 of initial CD14+ cells of DCs than TNF-α alone, secretion of larger amounts of IL-12, potent stimulatory activity on allogeneic and autologous T cells. Storage in liquid nitrogen did not modify the phenotype or functional characteristics of pre-loaded DCs. The recovery of thawed, viable DCs, was 78 ± 10%. Thus, positive selection of CD14+ monocytes allows the generation of a uniform population of mature pre-loaded DCs which can be cryopreserved with no effects on phenotype and function and are suitable for clinical trials. Based on these results, a DCs-based phase II trial of anti-Id vaccination with VDJ-derived HLA class I-restricted peptides and KLH is underway for lymphoma patients.

Original languageEnglish
Pages (from-to)1419-1428
Number of pages10
JournalLeukemia and Lymphoma
Issue number7
Publication statusPublished - Jul 2004


  • CD14 cells
  • Cellular
  • Dendritic cells
  • Multiple myeloma
  • Tumor-associated antigens

ASJC Scopus subject areas

  • Cancer Research
  • Hematology
  • Oncology


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