Generation of clinical grade dendritic cells with capacity to produce biologically active IL-12p70

Abstract

Background: For optimal T cell activation it is desirable that dendritic cells (DCs) display peptides within MHC molecules as signal 1, costimulatory molecules as signal 2 and, in addition, produce IL-12p70 as signal 3. IL-12p70 polarizes T cell responses towards CD4+ T helper 1 cells, which then support the development of CD8+ cytotoxic T lymphocytes. We therefore developed new maturation cocktails allowing DCs to produce biologically active IL-12p70 for large-scale cancer vaccine development.

Methods: After elutriation of leukapheresis products in a closed bag system, enriched monocytes were cultured with GM-CSF and IL-4 for six days to generate immature DCs that were then matured with cocktails, containing cytokines, interferon-gamma, prostaglandin E2, and a ligand for Toll-like receptor 8, with or without poly (I:C).

Results: Mature DCs expressed appropriate maturation markers and the lymph node homing chemokine receptor, CCR7. They retained full maturity after culture for two days without maturation cocktails and following cryopreservation. TLR ligand stimulation induced DCs capable of secreting IL-12p70 in primary cultures and after one day of coculture with CD40L-expressing fibroblasts, mimicking an encounter with T cells. DCs matured with our new cocktails containing TLR8 ligand, with or without poly (I:C), induced alloresponses and stimulated virus-specific T cells after peptide-pulsing. DCs matured in cocktails containing TLR8 ligand without poly (I:C) could also be loaded with RNA as a source of antigen, whereas DCs matured in cocktails containing poly (I:C) were unable to express proteins following RNA transfer by electroporation.

Conclusion: Our new maturation cocktails allowed easy DC harvesting, stable maturation and substantial recoveries of mature DCs after cryopreservation. Our procedure for generating DCs is easily adaptable for GMP-compliance and yields IL-12p70-secreting DCs suitable for development of cancer vaccines using peptides or RNA as sources of immunizing antigens.

Figures

Figure 1

Time-line of experimental setup using monocytes derived from one representative donor. Monocytes were prepared from a leukapheresis by elutriation on day 0 and cultured for 6 days with GM-CSF and IL-4 to produce iDCs, which were then incubated with different maturation cocktails. After 24 h, mDCs were harvested and washed twice, phenotypes determined by FACS and aliquots were cryopreserved. The primary culture supernatants were collected to assess IL-12p70 and IL-10 by ELISA. Samples of the different DC populations were cocultured with fibroblast L-cells (signal-3 assay) for an additional 24 h and supernatants collected once again for IL-12p70 and IL-10 measurements. Mixed lymphocyte cultures were established using autologous and allogeneic lymphocytes (fraction 3 of elutriated leukaphesis cells, cryopreserved on day 0) as responding cells and DC1–DC5 cells as stimulating cells. Tritiated-thymidine incorporation into dividing cells was measured during the final 24 h of a 7-day coculture. Mature DC1–DC5 cells that were harvested and washed on day 7 were loaded with CEF peptides and used as stimulating cells for autologous lymphocytes (fraction 3 cells after elutriation, cryopreserved on day 0). Lymphocytes and the various DC populations were cocultured for 7 days, washed and restimulated with autologous monocytes (fraction 5 cells, cryopreserved on day 0), with or without CEF peptides. IFNγ secretion was assessed in a standard ELISPOT analysis 24 h later. DCs cryopreserved on day 7 were thawed and reassessed for phenotype after storage in liquid nitrogen. Cryopreserved monocytes (fraction 5 from day 0) were thawed and used to generate new mDCs which were loaded with EGFP RNA by electroporation on day 7. Flow cytometry to detect percentages of positive cells and intensity of fluorescence was performed at 24 h.

Figure 2

Generation of stable, mature DCs using different maturation cocktails. (A) Percentages of DCs harvested after primary cell culture (6 d differentiation + 24 h maturation) calculated on total seeded cells (mononuclear cells) or CD14-positive monocytes detected by FACS and manual counting. DC populations DC1–DC5 were matured in different cocktails as listed in Table 1. Viability was detected by 7AAD exclusion measured by flow cytometry in FL-3 of the FACS-Calibur and viable cells are expressed as percentages of total cells. Percentages of cells expressing various surface markers were determined by flow cytometry using the antibodies specified in "Methods", including CD14 (a monocyte marker), CD83 (a marker of mDCs), CD80 and CD86 (costimulatory molecules) and chemokine receptor 7 (CCR7 = CD197) as an indicator for DC migratory potential into lymph nodes. Data are expressed as percentages of total cells with acquisition of 1 × 104 events. CCR7 values presented here (Fig. 2A) represent the percentages in histograms overlayed by IgG2a isotype control, although generated from the same FACS stain they are slightly different to the CCR7 values shown as dot plots in Figure 2B. Broken lines indicate marker levels for DCs matured with DC1 (Jonuleit) cocktail. (B) Representative dot plots of DC1–DC5 populations showing percentages of cells positive for CD83 versus CCR7 and CD80 versus CD86. (C) DCs were washed free of maturation cytokines and cultured an additional 44 h in culture medium without cytokines. Viability was determined by 7AAD incorporation. Percentages of CD14, CD83, CD80, CD86 and CCR7 were determined as described above. Broken lines indicate marker levels for DCs matured with DC1 (Jonuleit) cocktail.

Figure 3

Production of IL-12p70 and IL-10 by DCs matured using different cocktails. Immature DCs were cultured with different maturation cocktails and the amounts of IL-12p70 and IL-10 were determined by standard ELISA. Filled bars indicate IL-12p70 and empty bars IL-10, respectively. Cytokine content was measured (A) in supernatant medium of primary maturation cultures after 7 d and (B) in supernatant medium of cultures containing washed mDCs and CD40L-transfected fibroblasts following coculture for 24 h, representing a signal-3 assay as described in "Methods". (C) The quotients of IL-12p70/IL-10 were determined for the DC populations matured in different cocktails, based on the pg/ml values of the signal-3 assay. For calculation of quotients, it was assumed that IL-12p70 and IL-10 have theoretically equal biological potential. Filled circles indicate quotients ranging from 0 (DC1 cells in Jonuleit cocktail) to 3.2 (DC5 cells in cocktail 5). The values of DCs matured in DC1 (Jonuleit) versus DC2 (Kalinski) cocktails are indicated by broken lines.

Figure 4

Analysis of allostimulatory capacity of different DCs in mixed lymphocyte reactions. (A) Negative controls of proliferation of irradiated (40 Gy) DCs alone. (B) Proliferation of autologous T cells stimulated by DC1–DC5 populations. (C) Proliferation of one representative allogeneic T cell responder stimulated by DC1–DC5 cells. Note that the y-axis is different for autologous and allogeneic T cell responses. (D) Summary of proliferation data of three independent allogeneic T cell responders in comparison to autologous T cells stimulated by DC1–DC5 cells.

Figure 5

Response of autologous lymphocytes from an HLA-A*0201-positive donor responding to virus-peptide pulsed DCs. T cell responses were assessed in an IFNγ-ELISPOT experiment using peripheral blood lymphocytes (PBLs: T cell enriched ELUTRA fraction 3 = 54.8% CD3 positive cells) that were first activated for 7 d with mature peptide-pulsed DCs and then restimulated for 24 h with monocytes plus CEF peptides. For the ELISPOT analyses, 4 × 103 autologous in vitro activated lymphocytes were stimulated with 2 × 103 monocytes together with the CEF peptide pool. The mean ± S.D. was calculated for triplicate wells. Note: Due to insufficient recoveries, lymphocytes activated by DC2 cells were not included in the assay. Because of limitations in HLA-A2 subtyped donors available for leukapheresis, this experiment was only included once in the full DC evaluation protocol.

Figure 6

Expression of EGFP in DCs transfected with EGFP-encoding in vitro transcribed RNA. Flow cytometry histogram overlays show EGFP expression following RNA transfer into mDCs on day 7 (filled curves) 24 h after electroporation and corresponding untransfected DCs (empty curves) as negative controls. DCs were matured in the four cocktails indicated, RNA was introduced by electroporation, the DCs were returned to their corresponding media containing maturation cocktails and harvested for flow cytometry 24 h later (day 8). Numbers indicate the percentages of EGFP-positive DCs and their mean fluorescence intensities. These data are representative of two independent experiments with measurements at 24 and 48 h.