Dauer et al. (1) presented a method to differentiate CD14+ cells into mature dendritic cells (DC) within 48 h (FastDC). FastDC displayed a DC-like morphology, down-regulated CD14, and induced proliferation of autologous T cells against soluble Ags as efficiently as standard monocyte-derived DC (Mo-DC). Given the implications of these data for the generation of DC as anti-tumor vaccine, we tried to confirm the results. Monocytes were purified with MACS CD14 isolation kit (Miltenyi Biotec, Germany) and were cultured (1 × 106 cells/ml) for 24 h in complete medium supplemented with GM-CSF (50 ng/ml; Schering Plough, Kenilworth, NJ) and IL-4 (800 IU/ml; Endogen, Woburn, MA). Then, cells were cultured for an additional 24 h with the following maturation stimuli: TNF-α (25 ng/ml; CellGenix, Freiburg, Germany), IL-1β (10 ng/ml; CellGenix), IL-6 (1000 IU/ml; CellGenix), and PGE2 (1 μg/ml; CellGenix). The methods used are the same as those used by the authors. Standard mature Mo-DC were generated from monocytes as previously described (2, 3). In our hands, monocytes cultured for 48 h in presence of the above cytokines displayed a monocytic morphology and expressed CD14, CD86, and HLA-DR. As compared with standard mature Mo-DC, FastDC showed a blunted capacity to induce proliferation of allogeneic T cells and to activate KLH-specific proliferative response of autologous T cells. In conclusion, our results indicate FastDC as activated monocytes. These cells are likely to be good APCs but do not fit the main features of mature DC. Their use as anti-tumor vaccine should be further investigated.

FIGURE 1.

A, The phenotypic profile of Mo-DC and FastDC at the end of culture. Overlay diagrams show the expression of the relevant Ags vs isotype-matched Ab (negative controls). The figure shows both the percentage of positive cells and the mean fluorescence intensity (MFI) value. The results are representative of three independent experiments. B, T cells (1 × 105) were incubated with 198 irradiated DC in presence of 50 μg/ml KLH. Autologous DC alone represented the negative control and were used to calculate the stimulation index as follows: SI = cpm of T cell responders plus KLH-pulsed stimulators KLH, divided by cpm of T cell responders plus stimulators. The results are representative of three independent experiments. C, Increasing numbers of irradiated DC were used to stimulate 1 × 105 allogeneic mononuclear cells. The results are representative of three independent experiments.

FIGURE 1.

A, The phenotypic profile of Mo-DC and FastDC at the end of culture. Overlay diagrams show the expression of the relevant Ags vs isotype-matched Ab (negative controls). The figure shows both the percentage of positive cells and the mean fluorescence intensity (MFI) value. The results are representative of three independent experiments. B, T cells (1 × 105) were incubated with 198 irradiated DC in presence of 50 μg/ml KLH. Autologous DC alone represented the negative control and were used to calculate the stimulation index as follows: SI = cpm of T cell responders plus KLH-pulsed stimulators KLH, divided by cpm of T cell responders plus stimulators. The results are representative of three independent experiments. C, Increasing numbers of irradiated DC were used to stimulate 1 × 105 allogeneic mononuclear cells. The results are representative of three independent experiments.

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We have recently shown that dendritic cells (DCs) generated from monocytes by a novel 2-day strategy possess equal capacity to induce Ag-specific Th1-type immune responses compared with standard monocyte-derived DCs (R1 ). Curti et al. now report that monocytes treated with this 2-day protocol maintained their original phenotype and failed to induce allogeneic or KLH-specific autologous T cell proliferation. Specifically, they observed maintained expression of CD14 and lack of CD83 expression in monocytes that were cultured for 24 h with GM-CSF and IL-4 followed by 24-h stimulation with a combination of proinflammatory mediators (TNF-α, IL-1β, IL-6, and PGE2). We have shown that monocytes isolated from PBMC either by plastic adherence or CD14 positive selection using MACS display an immature DC immunophenotype after 24 h of culture with GM-CSF and IL-4 (R1 ). Unfortunately, Curti et al. do not provide any information on the immunophenotype observed in their cultures after the first 24 h. Our own unpublished observations indicate that the 2-day differentiation and activation protocol fails to yield fully mature DCs if CD14 expression is preserved after the first 24 h of the culture process. In this scenario, proinflammatory activation will not induce maturation of DCs but rather an activated monocytic phenotype as described by Curti et al. Prolonged CD14 expression may be due to early activation of monocytes during the isolation process. Curti et al. also state that FastDC may only represent activated monocytes and thus have a blunted capacity to stimulate proliferative T cell responses. In fact, we have already demonstrated that FastDC display a fully mature DC phenotype, express equal levels of costimulatory molecules as well as MHC molecules and secrete equal levels of IL-12 compared with standard monocyte-derived DCs (R1 ). If phenotypically mature DCs are generated, these cells also possess equal capacity to induce Ag-specific autologous T cell proliferation (R1 ). Consistent with these results, we were able to show that our 2-day protocol does not necessarily require the use of proinflammatory mediators, but also yields fully mature DCs if other stimuli known to activate standard monocyte-derived DCs are used; ATP—which has been identified previously by our group as a potent activator of standard monocyte-derived DCs (R2 )—also induces maturation of FastDC. The same synergistic effect of ATP and TNF that has been observed in activation of standard monocyte-derived DCs can be seen in FastDC (Fig. 1,A). In contrast to the results presented by Curti et al., FastDC are also potent stimulators of allogeneic T cell proliferation in our hands (Fig. 1,B). Moreover, if FastDC are extensively washed after maturation is completed and subsequently cultured in medium alone without addition of any cytokines or growth factors, they maintain their mature DC phenotype for at least 48 h, indicating that these cells represent terminally differentiated DCs rather than activated monocytes (Fig. 1 C). However, we agree with Curti et al. that further studies are required to test whether FastDC loaded with tumor antigen can be used for anti-tumor vaccination.

FIGURE R1.

A, ATP and TNF-α synergistically induce maturation of FastDC. Monocytes were either cultured for 48 h with GM-CSF (1000 U/ml) and IL-4 (500 U/ml) alone or additionally stimulated with proinflammatory mediators (PIM: 10 ng/ml IL-1β, 1000 U/ml TNF-α, 1 μM PGE2), ATP (100 μM) or ATP plus TNF-α for the last 24 h. Expression of CD83 and CD14 was determined by FACS analysis. Data represent means ± SEM of four experiments with different donors. B, FastDC are potent stimulators of allogeneic T cell proliferation. FastDC or immature DCs were cocultured with 2 × 105/200 μl allogeneic nonadherent PBMC (NAC) in different ratios. On day 5 of coculture, proliferation was determined by standard [3H]thymidine incorporation assay. One representative experiment of three is shown. C, FastDC are terminally differentiated. FastDC were extensively washed and resuspended in medium without addition of cytokines or growth factors. Immediately after completion of the 48-h culture period (0 h) and at different time points of the wash-out phase, CD83 and CD14 expression was determined by FACS analysis. Data represent means ± SEM of three experiments with different donors.

FIGURE R1.

A, ATP and TNF-α synergistically induce maturation of FastDC. Monocytes were either cultured for 48 h with GM-CSF (1000 U/ml) and IL-4 (500 U/ml) alone or additionally stimulated with proinflammatory mediators (PIM: 10 ng/ml IL-1β, 1000 U/ml TNF-α, 1 μM PGE2), ATP (100 μM) or ATP plus TNF-α for the last 24 h. Expression of CD83 and CD14 was determined by FACS analysis. Data represent means ± SEM of four experiments with different donors. B, FastDC are potent stimulators of allogeneic T cell proliferation. FastDC or immature DCs were cocultured with 2 × 105/200 μl allogeneic nonadherent PBMC (NAC) in different ratios. On day 5 of coculture, proliferation was determined by standard [3H]thymidine incorporation assay. One representative experiment of three is shown. C, FastDC are terminally differentiated. FastDC were extensively washed and resuspended in medium without addition of cytokines or growth factors. Immediately after completion of the 48-h culture period (0 h) and at different time points of the wash-out phase, CD83 and CD14 expression was determined by FACS analysis. Data represent means ± SEM of three experiments with different donors.

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R1
Dauer, M., B. Obermaier, J. Herten, C. Haerle, K. Pohl, S. Rothenfusser, M. Schnurr, S. Endres, A. Eigler.
2003
. Mature dendritic cells derived from human monocytes within 48 hours: a novel strategy for dendritic cell differentiation from blood precursors.
J. Immunol.
170
:
4069
.
R2
Schnurr, M., F. Then, P. Galambos, C. Scholz, B. Siegmund, S. Endres, A. Eigler.
2003
. Extracellular ATP and TNF-α synergize in the activation and maturation of human dendritic cells.
J. Immunol.
165
:
4704
.
1
Dauer, M., B Obermaier, J. Herren, C. Haerle, K. Pohl, S. Rothenfuser, M. Schnurr, S. Endres, A Eigler.
2003
. Mature dendritic cells derived from human monocytes within 48 hours: a novel strategy for dendritic cell differentiation from blood precursors.
J. Immunol.
170
:
4069
.
2
Motta, M. R., S. Castellani, S. Rizzi, A. Curti, F. Gubinelli, M. Fogli, E. Ferri, C. Cellini, M. Baccarani, R. M. Lemoli.
2003
. Generation of dendritic cells from CD14+ monocytes positively selected by immunomagnetic adsorption for multiple myeloma patients enrolled in a clinical trial of anti-idiotype vaccination.
Br. J. Haematol.
121
:
240
.
3
Ratta, M., F. Fagnoni, A. Curti, R. Vescovini, P. Sansoni, B. Oliviero, M. Fogli, E. Ferri, G. R. Della Cuna, S. Tura, et al
2002
. Dendritic cells are functionally defective in multiple myeloma: the role of interleukin-6.
Blood
100
:
230
.