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Updated: Aug 11, 2026
Culture of myeloid dendritic cells from bone marrow precursors
Published on: July 26, 2008
Calcium ionophore-treated peripheral blood monocytes and dendritic cells rapidly display characteristics of activated
B J Czerniecki1, C Carter, L Rivoltini
1Department of Surgery, University of Pennsylvania Medical Center, Philadelphia 19104, USA.
Insights
Calcium ionophore (CI) rapidly converts human monocytes and immature dendritic cells (iDC) into activated dendritic cells (DC). This method efficiently generates autologous antigen-presenting cells (APC) for T cell sensitization strategies.
Area of Science:
- Immunology
- Cell Biology
Background:
- Peripheral blood contains immature dendritic cells (iDC) and monocytes, differing in CD14 expression.
- Both cell types are crucial for immune responses but require activation for optimal function.
Purpose of the Study:
- To investigate the potential of calcium ionophore (CI) to induce monocyte and iDC activation.
- To evaluate the efficacy of CI-treated cells as antigen-presenting cells (APC) for T cell sensitization.
Main Methods:
- Isolation of myeloid origin mononuclear cells (MOMC), including monocytes and iDC, using leukapheresis and elutriation.
- Treatment of cells with calcium ionophore (CI), recombinant human granulocyte-monocyte colony-stimulating factor (rhGM-CSF), or endotoxin.
- Immunophenotypic analysis (CD14, CD83, MHC, costimulatory molecules) and morphologic assessment.
- Assessment of T cell sensitization capacity using keyhole limpet hemocyanin (KLH) and MART-1 peptide.
Main Results:
- CI treatment rapidly induced dendritic cell (DC) morphology, down-regulated CD14, and upregulated MHC and costimulatory molecules in monocytes and iDC.
- CI treatment led to de novo CD83 expression, an activation marker, within 20 hours.
- CI-treated monocytes and iDC demonstrated enhanced T cell-sensitizing efficiency, enabling CD4+ and CD8+ T cell sensitization in a single stimulation.
Conclusions:
- Circulating monocytes and iDC can be effectively induced into activated DC by CI treatment.
- CI provides a rapid and consistent method for generating large numbers of potent, autologous APC for T cell-based immunotherapies.
Abstract:
Human peripheral blood contains a small subpopulation of immature dendritic cells (iDC) distinguished from circulating monocytes by their low expression of CD14. We utilized leukapheresis and countercurrent centrifugal elutriation to obtain myeloid origin mononuclear cell (MOMC) fractions of monocytes and iDC for study. These subpopulations were ultrastructurally and immunophenotypically similar before culture. After a 20- to 96-h culture either alone, with recombinant human granulocyte-monocyte CSF, or with endotoxin, greater up-regulation of costimulatory molecule expression was observed among iDC than among monocytes, and only iDC expressed the activation molecule CD83. Treatment with rhIL-4 caused many MOMC to develop morphologic properties of dendritic cells within 96 h, but costimulatory molecule up-regulation and CD14 down-regulation were heterogeneous, and CD83 expression was infrequent. In contrast, calcium ionophore (CI) treatment induced rapid and consistent effects in MOMC from both healthy volunteers and cancer patients, including down-regulated CD14 expression, acquisition of dendritic cell morphologic properties, up-regulated MHC and costimulatory molecule expression, and de novo CD83 expression. Many such effects occurred within 20 h of treatment. CI treatment activated purified CD14+ monocytes and also enhanced the spontaneous activation of purified CD14-/dim iDC in culture. Unfractionated MOMC, purified monocytes, and purified iDC displayed equivalently enhanced T cell-sensitizing efficiency following CI treatment. CD4+ T cell sensitization to keyhole limpet hemocyanin and CD8+ T cell sensitization to MART-1 melanoma-associated peptide were achieved in a single culture stimulation. Therefore, circulating monocytes and iDC can be induced by CI to manifest properties of activated DC, providing large numbers of efficient, nontransformed autologous APC for T cell sensitization strategies.
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