Functional characterization of monocyte-derived dendritic cells generated under serumfree culture conditions

Immunology Letters 99 (2005) 209–216

Functional characterization of monocyte-derived dendritic cells generated under serumfree culture conditions Manfred Lehner 1 , Patrick Morhart 1 , Andrea Stilper, Wolfgang Holter ∗ Department of Cellular Therapy, Children’s University Hospital, Loschgestr. 15, D-91054 Erlangen, Germany Received 14 December 2004; received in revised form 25 February 2005; accepted 27 February 2005 Available online 2 April 2005

Abstract The culture of human monocyte-derived dendritic cells (DCs) is typically performed in media containing human or fetal calf serum, supplements with the potential to influence the cells phenotype and their functional properties. Published clinical trails based on serumfree cultured DCs reported the use of the commercially available medium AIMV. In this study, we directly compared DCs generated in AIMV medium (“AIMV/sf-DCs”) with DCs generated in RPMI supplemented with 2% human serum (“RPMI/HS-DCs”) in functional assays of potential relevance for vaccine application. Using TNF-␣/PGE2 /IL-1␤/IL-6 as maturation stimulus, AIMV/sf-DCs revealed to be comparable with RPMI/HS-DCs with regard to phenotypic expression of maturation markers, survival in vitro, migratory capacity and stimulation of lymphocyte proliferation except for CD1a which was expressed on a fraction of DCs only when cultured in serumfree AIMV medium. However, IL-12p70 production in response to Toll-like receptor (TLR) stimulating agents plus IFN-␥ was consistently lower in AIMV medium although also under serumfree culture conditions, nanogram quantities of IL-12 were produced. Together, DCs with functional characteristics important for in vivo application can be generated under defined serumfree conditions; however, medium and/or serum conditions appear to have strong influence on the production of relevant T cell differentiating cytokines. © 2005 Elsevier B.V. All rights reserved. Keywords: Serumfree; Dendritic cells; IL-12; CD1a

1. Introduction Dendritic cells (DC) have gathered enormous interest because of their potential role for tumor-specific immune therapy. With regard to such an application, many parameters of DC culture are relevant because of their influence on subsequent cell function [1]. Among the basic variables influencing DC function, the appropriate choice of culture medium as well as potential serum supplements remain an important issue. From the first description of a clinical grade protocol for the generation of mature monocyte-derived DCs [2] up to now, most DCs used for clinical vaccination were generated in media containing serum or plasma. Both autologous as ∗ 1

Corresponding author. Tel.: +49 9131 85 34759; fax: +49 9131 85 33113. E-mail address: [email protected] (W. Holter). Contributed equally.

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well as allogeneic serum derived from the patients or healthy donors, respectively, potentially introduce undefined ingredients with variable effects on DC function together with a lot to lot variability to the culture system. Serumfree culture of monocyte-derived DCs has been reported with media such as XVivo15, XVivo20 and AIMV [2–10]. From these reports, however, we could not deduce whether DCs cultured under such conditions are functionally equivalent to DCs cultured in the presence of human serum. In this study, we, therefore, compared monocyte-derived DCs differentiated in AIMV with cells cultured in RPMI 1640 plus human serum. Since immature DCs are no longer considered as good candidates for vaccination trials [11–14], we compared our DCs primarily following maturation with a combination of TNF-␣, PGE2 , IL-1␤ and IL-6. This stimulus represents a mimic of monocyte conditioned medium (“MCM-mimic” or “cytokine cocktail”) and functions currently as a standard

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maturation stimulus for clinically applied DCs [1]. Because IL-12p70 production, which is not typically observed following this maturation stimulus, may be of central importance for cytotoxic T lymphocyte (CTL) induction [10,15–18], DC cytokine production was evaluated using various Toll-like receptor (TLR) stimuli in the presence of IFN-␥. With this experimental setup, we show that DCs cultured serumfree in AIMV (“AIMV/sf-DC”) appear to be very similar in vitro compared to DCs cultured in RPMI supplemented with 2% human serum (“RPMI/HS-DCs”) with regard to cell yield, expression of maturation markers, migratory capacity, stimulation of lymphocyte proliferation and cell survival. Of possible importance, however, IL-12p70 production was consistently lower under synthetic serumfree culture conditions. Serumfree culture, on the other hand, allowed expression of the lipid antigen presenting molecule CD1a on a fraction of immature and mature DCs.

2.2. Flow cytometric analysis

2. Materials and methods

For immunophenotypic analysis of the DCs, the following monoclonal antibodies were used: anti-CD1a-FITC (clone HI149; Becton Dickinson Co., Mountain View, CA), antiCD14-APC (clone RMO52; Immunotech Marseille, France), anti-CD80-PE (clone MAB104; Immunotech), anti-CD83PE (clone HB15A; Immunotech), anti-CD83-APC (clone HB15e; BD), anti-CD86-FITC (clone 2331; BD), anti-CCR7 (clone 2H4; BD), biotinylated rat-anti-mouse IgM (clone R660.2; BD), streptavidin-PE (BD) and isotype controls: murine IgM (clone G155-228; BD), IgG1-FITC (clone DAK-GO1; Dako), IgG1-PE (clone DAK-GO1; Dako), IgG1-APC (clone DAK-GO1; Dako), IgG2a-APC (clone X39; BD). Cells were analyzed on a FACSCalibur flow cytometer (BD) and data analysis was performed with CellQuest software (BD). For the determination of viable cell numbers, the cells were counted after the addition of 5 mM EDTA and propidiumiodide by FACS analysis using Counting Beads (Caltag, Burlingame, CA).

2.1. Monocyte isolation and DC culture

2.3. Cytokine measurements

Human mononuclear cells (MNCs) were obtained from the blood of healthy donors by density gradient centrifugation using endotoxin-free Ficoll-Paque-PLUS® (Amersham Pharmacia Biotech AB, Uppsala, Sweden). Monocytes were isolated from these MNCs by a second density gradient centrifugation using Ficoll-Paque-PLUS® adjusted to 1.068 g/ml by dilution with PBS [19]. The remaining cells, containing 70–90% monocytes as assessed by counting on a Cell-Dyn 3500R hematocytometer, were frozen in RPMI 1640 (Invitrogen Corporation, Bethesda, MD) supplemented with 10% DMSO (Sigma, St. Louis, MO) and 20% human serum (HS) (BioWhittaker, Walkersville, MD) containing less than 100 pg/ml endotoxin. For DC differentiation, monocytes were cultured and at a density of 0.5 × 106 ml–1 in RPMI 1640 or AIMV medium (Invitrogen Corporation) supplemented with 2 mM l-glutamine, 1000 U/ml GM-CSF (Leukine® , Schering AG, Berlin, Germany) and 1000 U/ml recombinant human IL-4 (Strathmann Biotec, Hamburg, Germany). Two percent HS was added to RPMI 1640 medium and to some of the AIMV cultures as indicated. Half of the medium was exchanged every 2 or 3 days. Immature DCs were matured on day 5 by a previously described cytokine cocktail (“MCM-mimic”) [20] containing 1000 U/ml recombinant human TNF-␣ (Bender Med Systems, Vienna, Austria), 1 ␮g/ml PGE2 (Sigma), 10 ng/ml IL-1␤ (RnD Systems, Minneapolis, MN) and 1000 U/ml IL-6 (Strathmann Biotec) or where indicated by the addition of 1000 U/ml IFN-␥ (Bender Med Systems) combined with 10 ␮g/ml suspended peptidoglycan (PGN) (Staphylococcus aureus; Sigma), 1 ␮g/ml lipopolysaccharide (LPS) (Escherichia coli 055:B5; Sigma), 20 ␮g/ml poly(I:C) (Amersham, Freiburg, Germany) or 10 ␮g/ml R848 (Pharmatech, Shanghai, China), respectively.

Based on standard sandwich ELISA methodology, commercially available pairs of monoclonal antibodies (BD) were used to quantify human IL-12p70. 2.4. Cell proliferation assay The DCs (10,000, 2000 or 400) were cocultured in triplicates with 100,000 allogeneic naive MNCs in 200 ␮l XVIVO 15 medium (BioWhittaker) supplemented with 1% HS in round bottom wells of a 96-well plate (Nunc, Roskilde, Denmark). The allogeneic responder MNCs were depleted of CD45RO and CD56 positive cells using the MACS isolation kit for CD45RO and CD56 (Miltenyi Biotec, Bergisch Gladbach, Germany). For a positive reference, 100,000 MNCs were stimulated in 200 ␮l medium with 1.2 ␮g/ml PHAL (Biochrom AG, Berlin, Germany) plus 1 × 10–7 M PMA (Sigma). On day 4, the cultures were labeled with 1 ␮Ci/well 3 H-thymidine (NEN, Boston, MA). Eighteen hours later, the cells were harvested onto glass fiber filters with a Skatron harvesting device (Skatron, Lier, Norway) and the incorporated tritium thymidine was measured on a Trilux ␤-plate reader (Wallac Oy, Turku, Finland) using the Wallac Microbeta Meltilex® scintillation system (Perkin-Elmer, Boston, MA). 2.5. Migration assay The DCs were washed twice and tested for migration toward chemokines using the transwell assay similarly as described by Luft et al. [21]. The lower chambers of 24well transwell plates (8.0 ␮m pore size; Costar, Corning, NY) were filled with 600 ␮l IMDM (Invitrogen Corporation) supplemented with 5% HS with or without the

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chemokines CCL19 (300 ng/ml; PeproTech, Rocky Hill, NJ) and CXCL12 (300 ng/ml; PeproTech). Thereafter, the plates were incubated 1 h at 37 ◦ C before the DCs were added. 20,000–50,000 DCs were then seeded in 100 ␮l IMDM/5% HS into the upper chamber and were incubated at 37 ◦ C for 1.5 h. Cells migrated in the lower chamber and adherent on the bottom side of the porous insert of the transwell were harvested by the addition of 5 mM EDTA and counted by flow cytometry using beads (Caltag Laboratories, Burlingame, CA).

3. Results 3.1. Phenotype of DCs matured under serumfree conditions Mature DCs are characterized by surface expression of CD83, the chemokine receptor CCR7 and an enhanced expression of the costimulatory molecules CD80 and CD86. Therefore, we were interested if stimulated DCs cultured in the two different media, i.e. RPMI 1640 supplemented with 2% HS (RPMI/HS-DCs) and AIMV without serum (AIMV/sf-DCs), would show comparable expression of these maturation markers. Maturation was induced with the cytokine cocktail TNF-␣/PGE2 /IL-1␤/IL-6 on day 5. As shown in Fig. 1, DCs cultured in RPMI/HS and AIMV/sf both upregulated the maturation markers after stimulation with a tendency of AIMV/sf cultured cells for a more complete maturation compared to RPMI/HS culture condition. Interestingly, expression of CD1a, a protein which presents lipid antigens to T cells [22], was observed in our experiments on a fraction of the cell population cultured in AIMV/sf only. The percentage of positive cells ranged from 27.87 ± 4.80% (AIMV/sf) versus 0.48 ± 0.15% (RPMI/2% HS) in DCs matured by the cytokine cocktail to 22.93 ± 6.49% (AIMV/sf) versus 1.16 ± 0.93% (RPMI/2% HS) after maturation via Toll-like receptors by PGN/IFN-␥, poly(I:C)/IFN-␥, LPS/IFN-␥ and R848/IFN-␥ (n = 3 for each stimulus; data not shown). Apart from CD1a expression, the observed similarity in the FACS phenotype was paralleled by a similar morphological appearance after maturation by the cytokine cocktail (Fig. 2). In both the cultures, the mature DCs displayed a typical dendritic morphology and could easily be suspended.

Fig. 1. Phenotype of AIMV/sf-DCs and RPMI/HS-DCs. Immature DCs cultured in RPMI/HS and serumfree AIMV were matured by the cytokine cocktail on day 5 of culture. The dotplots show the expression of the indicated surface markers analyzed by FACS on day 7.

remained highly viable under both media conditions for up to 11 days of total culture (Fig. 3).

3.2. Recovery and longevity of mature DCs in serumfree culture

3.3. Migratory and T cell stimulatory capacity of mature DCs generated in serumfree culture

After having confirmed that differentiation of monocytes under both the culture conditions allows the generation of homogeneous populations of mature DCs, we compared both culture conditions for their yield of mature DCs. For all the experiments, we used frozen aliquots of monocytes and the number of seeded viable monocytes after thawing was set to 100%. Similar yields of immature and mature DCs were obtained in medium without and with serum. Furthermore, the experiments confirmed that cytokine cocktail matured DCs

To functionally compare mature DCs generated in RPMI/HS and AIMV/sf, we addressed first their capability to migrate towards a concentration gradient of the chemokines CXCL12 and CCL19. CXCL12 binds to the receptor CXCR4 expressed on immature and mature DCs and might be important for the initial phase of DC homing into inflamed lymph nodes as speculated for plasmacytoid DCs [23]. CCL19 is produced in the T cell zones of lymph nodes and binds to CCR7, the chemokine receptor which is thought to mediate

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Fig. 2. Morphological appearance of mature DCs cultured in different media. The DC cultures were photographed on day 7, i.e. 48 h after induction of maturation by the cytokine cocktail.

Fig. 3. Longevity of mature DCs cultured under serumfree conditions. Cytokine cocktail was added to the cultures on day 5. On days 7 and 11, respectively, the viable cells were harvested by adding 5 mM EDTA and their number was determined by FACS analysis using reference beads. The yields are given as percentage of the number of seeded monocytes on day 0 (mean ± S.D.; n = 4).

homing of mature DCs and T cells to lymph nodes [24]. Fig. 4 shows the similarity of RPMI/HS-DCs and AIMV/sf-DCs in their response to CCL19 and CXCL12. After maturation with the cytokine cocktail, both the cell populations could migrate well towards CCL19 and also towards CXCL12. Immature DCs showed low migration towards both chemokines. The T cell stimulatory capacity was tested in an allogeneic MLR using CD45RO and CD56 depleted MNCs as responders (Fig. 5). Mature DCs generated under both the culture conditions similarly stimulated potent proliferation of allogeneic naive T cells whereas immature DCs were weak stimulators in comparison. 3.4. Capacity of DCs cultured in AIMV/sf-DCs for production of IL-12p70 In our comparison of AIMV/sf-DCs and RPMI/HS-DCs, we finally focused on their capacity of IL-12p70 production

Fig. 4. Migratory capacity of mature DCs towards CXCL12 and CCL19. The migratory capacity of DCs was determined 48 h after induction of maturation by the cytokine cocktail using 8 ␮m pore size transwell plates. The fraction of DCs, which migrated within 1.5 h to the bottom well containing medium with or without chemokine, is given as percentage of the number of cells seeded into the upper well (mean ± S.D. of three independent experiments).

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Fig. 5. Capacity of mature DCs generated in serumfree AIMV medium to stimulate allogeneic naive T cells. Two days after induction of maturation by the cytokine cocktail, the DCs were harvested, washed and graded doses of DCs (indicated as ratios DCs/lymphocytes) were added to allogeneic CD45RO-negative lymphocytes. Immature DCs harvested on day 7 were used as reference. Proliferation of lymphocytes was determined by 3H-tymidine incorporation from days 4 to 5 of coculture. The results of three experiments using different donors are separately shown (displayed as the mean of triplicates ± S.D. of each experiment).

because the presence of this cytokine may be of central importance for the differentiation of CTLs [10,15–18,25]. Since the PGE2 containing cytokine cocktail does not induce IL-12 release at least in vitro [26], we used different stimuli previously reported to induce maturation of DCs in conjunction with IL-12 secretion [27–34]. Fig. 6 shows the production of IL-12p70 after stimulation with peptidoglycan plus IFN-␥, poly(I:C) plus IFN-␥, LPS plus IFN-␥ and R848 plus IFN-␥. When DCs were stimulated in serumfree AIMV medium, they produced consistently less IL-12p70 in response to all four stimuli than DCs stimulated in RPMI medium supplemented with HS (p < 0.001; Wilcoxon-rank-sum test); however, it should be noted that nanogram amounts of IL-12p70 per millilitre of culture medium were still secreted. In the absence of IFN-␥, IL-12 production was reduced, in particular to stimulation with LPS and PGN (data not shown).

Since at least LPS signaling is partially amplified by serumproteins [35], we performed additional stimulation experiments with LPS/IFN-␥ in AIMV medium supplemented with 2% HS; however, no increase in IL-12 production was observed (not shown). Finally, DCs stimulated with the cytokine cocktail (containing PGE2 ) did not produce IL-12p70, regardless of the culture medium applied (not shown), in accordance with previously published data [26]. 3.5. Phenotype of serumfree cultured DCs upon stimulation with TLR ligands In view of the somehow reduced IL-12p70 production under serumfree condition, it was important to document the efficient maturation of TLR stimulated DCs. Fig. 7 illustrates that indeed all maturation markers investigated were upregulated in response to PGN, poly(I:C), LPS and R848 in

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Fig. 6. Cytokine production of DCs cultured in different media. Immature DCs were stimulated on day 5 with the indicated stimuli. IL-12p70 was assayed in the supernatants 48 h later by sandwich ELISA. The result of each single experiment comparing RPMI/HS with AIMV/sf is indicated by a pair of linked data points (for each stimulus n = 8, obtained with four different donors).

Fig. 7. Phenotype of AIMV/sf-DCs after stimulation with TLR ligands. Immature DCs cultured in serumfree AIMV medium were stimulated with the cytokine cockail and different TLR ligands plus IFN-␥ on day 5 of culture. The histograms show the expression of DC maturation markers as analyzed by FACS on day 7.

the presence of IFN-␥ to an extent very similar to cytokine cocktail simulated DCs with the exception of CCR7, which is optimally expressed only in PGE2 containing cultures.

4. Discussion Monocyte-derived DCs are typically differentiated in media containing serum or plasma supplements. In view of existing reports describing also the serumfree culture of DCs for clinical application [5,8,9] and because of potential

disadvantages associated with the use of serum such as lot to lot variability and the presence of undefined serum factors, we considered DC culture in serumfree AIMV medium as an attractive alternative. However, from existing literature, we missed a conclusive direct comparison of such differently cultured DCs. Using different kinds of maturation stimuli, we tested RPMI/HS-DCs and AIMV/sf-DCs for the expression of maturation markers, yield and longevity, migratory capacity, stimulation of lymphocyte proliferation and cytokine secretion. We observed that both the culture conditions resulted in equal yields of DCs which were

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comparable in all tested parameters except the production of IL-12p70 and the expression of CD1a. Recovery of DCs was our first decisive issue considering AIMV in our experimental setup. Our previous attempts of serumfree DC culture in XVivo15 or XVivo20 as well as RPMI supplemented with 2% human albumin as suggested by Araki et al. [36] resulted in very low DC yields only (not shown). When changing to AIMV medium, we obtained reasonable recoveries of about 50% of seeded thawed monocytes which were identical to an RPMI/HS culture system. Together with the approximately 60% recovery of monocytes enriched by our previously described density gradient monocyte isolation procedure [19], this amounts to a final recovery of mature DCs generated from frozen monocytes of around 30% of the number of monocytes initially present in the mononuclear cell preparation obtained by a standard Ficoll® density gradient separation. The DCs resulting from serumfree culture in AIMV medium exhibited no functional defect in the MLR, migration assay and also survived in vitro for at least 6 days following maturation. Longevity was considered important since enhanced survival could further promote T cell priming [37] and, as discussed by Schuler et al. [1], reprocessing of excessively dying DCs by viable recipient DCs in the lymph nodes might lead to tolerance [38]. With regard to the capacity of T cell stimulation and migration towards chemokines DCs matured with the cytokine cocktail, in comparison to DCs matured in response to LPS/IFN-␥, behaved in a superior manner, regardless which culture medium for differentiation had been applied (not shown). Such a comparably good performance of DCs matured with the cytokine cocktail with regard to MLR stimulation was also seen in a previous report where this higher stimulatory capacity was correlated with an increased IL-2 secretion by responding T cells [21]. With regard to the question of culture medium, however, no difference between AIMV/sf-DCs and RPMI/HS-DCs matured by the cytokine cocktail was noted in their migratory capacity towards CCL19 and CXCL12. Minor differences between the two culture systems became visible in the FACS phenotype where expression of CD1a was not seen or at least very weak in RPMI/2% HS and clearly visible in AIMV, independent on the maturation state (Figs. 1 and 7 and data not shown). CD1a is a member of the CD1 family involved in lipid antigen presentation [22] and, thus, of potential functional importance. Furthermore, we observed a tendency for a more complete maturation of DCs in AIMV medium following stimulation with cytokine cocktail or TLR ligands as estimated by a variable expression of CD14 on a fraction of cells cultured in RPMI/2% HS (Fig. 1 and data not shown). However, all other phenotypic markers were expressed to a similar extent under both the culture conditions, in particular CD80 and CD86, important for T cell co-stimulation. Accordingly, T cell stimulation in an allogeneic MLR was very similar (Fig. 5). An important issue seemed to us to evaluate the capacity of AIMV/sf-DCs for IL-12p70 production as this was

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