Classical DCs Generated in Plates

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Dendritic Cells Generated in Clinical Grade Bags Strongly Differ in Immune Functionality When Compared With

Classical DCs Generated in Plates

Re´douane Rouas, Haidar Akl, Hussein Fayyad-Kazan, Nabil El Zein, Bassam Badran, Be´range`re Nowak, Hugues Duvillier, Philippe Martiat, and Philippe Lewalle

Summary:Mature dendritic cells (DCs) represent, by far, the most potent antigen-presenting cells. The development of clinical grade techniques to produce them in large numbers has rendered possible their use in clinical trials. It is therefore crucial to assess the DCs characteristics according to the methodology used to generate them, to improve the comparison and standardization of these trials. We thus compared DCs generated and matured in culture plates (pla-DCs) or in clinical grade bags (bag-DCs) by analyzing, their secretion of bioactive interleukin (IL)-12 and their capacity to induce in-vitro primary responses. We also used several molecular techniques to better characterize the functional differences between the 2 type of DCs. Mature bag-DCs displayed a mature phenotype, but did not secrete significant amounts of IL-12 and failed to initiate primary immune responses. Molecular analyses performed on immature bag-DCs showed them already engaged in a particular maturation process (early activation of nuclear factor k B and b-catenin). Using microarrays, we found underexpression of receptors for the maturation cocktail in bag-DCs. In mature bag-DCs, we found crucial genes (IL-12, chemokines, and costimulatory and adhesion molecules) down-regulated. Electro- phoertic mobility shift assay and Western blots showed a normal activation profile in mature pla-DCs, but not in bag-DCs where the Mek/Erk pathway was still activated. Our results strongly suggest that differentiation of monocytes into DCs in bags generates immature DCs already engaged in an inefficient type of activation, with down-regulation of genes involved in response to the maturation cocktail. This results in mature DCs unable to induce TH1-type responses.

Key Words:dendritic cells generation and maturation, bags or plates, functionality

(J Immunother2010;33:352–363)

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or dendritic cells (DCs) to be used as a cancer vaccine, it is critical that they can be efficiently loaded with tumor- associated antigens and can elicit specific T_H1 and Tc1 lymphocytes response against these antigens.^1–7 Tumor- associated antigens-specific T cells can be detected in patients, and, albeit unable to properly function in the tumoral microenvironment, are capable to lyse tumor cells in vitro.^8–10 Adoptive cell therapy, by ex-vivo stimulated tumor infiltrating lymphocytes is already efficient in some patients,¹¹and its combination with DCs vaccine could be a promising new approach to cancer treatment. Importantly, in the context of allogeneic stem cell transplantation, antitumor specific donor T cells can be ex-vivo expanded to enrich T lymphocytes infusion to increase the graft- versus-tumor effect¹²; tumor specific T cells can be also detected in normal donors.¹³ Nevertheless, most of the lymphocytes that we try to expand ex vivo for cellular immunotherapy, are naive and/or have a low avidity for their cognate antigen, and therefore are expected to be critically dependent on the high levels of costimulatory molecules and cytokines production associated with mature DCs for their expansion.^14–19For lymphocytes priming, the obtention of mature DCs expressing appropriate maturation markers but also capable of secreting large amount of interleukin (IL)-12p70 upon restimulation by CD40L, mimicking the in vivo T cells DCs interaction, is critical.^20–29 Therefore, characterizing the optimal way to generate mature DCs ex vivo, for clinical application is of utmost importance.^30,31We focused on the comparison of monocytes-derived DCs generated and matured in closed bags (bag-DCs) with DCs generated and matured in plates (pla-DCs). One characteristic of mature DCs is a propensity to adhere to plastic surfaces. Recovery of these cells requires a time-consuming procedure that may compromise cell viability and sterility. To facilitate efficient recovery of DCs and maintain a closed system less vulnerable to microbial contamination, several groups have developed methods for generating and maturing monocytes-derived DCs in gas permeable cell culture bags. The theoretical advantage is that, in contrast to cells cultured in conventional polystyrene flasks or plates (pla-DCs), those cultured in bags bind less firmly to container surfaces and therefore can be harvested more easily while being perfectly adequate for clinical grade applications. The ideal way to process these cells in a clinical grade environment is to differentiate monocytes into immature DCs in bags and to perform the maturation and the antigen-loading steps in bags. This results in a completely closed-system procedure, avoiding the risk of patho- gens contamination as much as possible. In phenotypic Copyright^r2010 by Lippincott Williams & Wilkins

Received for publication June 13, 2009; accepted October 29, 2009.

From the Laboratory of Experimental Hematology, Department of Hematology, Jules Bordet Institute, ULB, Brussels, Belgium.

All authors have declared that there are no ﬁnancial conﬂicts of interest in regards to this study.

Re´douane Rouas and Haidar Akl equally contributed to this study.

Philippe Martiat and Philippe Lewalle are the senior co-authors.

This study has been rendered possible thanks to the support of the Medic Foundation, Les Amis de l’Institut Bordet, and of the FRSM and Te´le´vie.

Reprints: Philippe Martiat, Laboratory of Experimental Hematology, Jules Bordet Institute, ULB, 121, Boulevard de Waterloo, 1000, Brussels, Belgium (e-mail: pmartiat@ulb.ac.be).

Supplemental Digital Content is available for this article. Direct URL citations appear in the printed text and are provided in the HTML and PDF versions of this article on the journal’s website, www.immunotherapy.journal.com.

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studies, bags-cultured DCs (bag-DCs) usually express high levels of CD1a, CD1c, class I and II major histocompatibility molecules, CD80, CD86, and CD83 comparable with the levels reported for pla-DCs. Given these results, bag-DCs have been viewed as functionally equivalent to pla-DCs.^32–49

We set up to thoroughly investigate the functional and molecular properties of bag-DCs compared with pla-DCs, using phenotype, primary immune response initiation capacity as functional assays, Electrophoertic mobility shift assay (EMSA), Western blot, and the micro-arrays technology. To better understand the role of contaminating peripheral blood mononuclear cells (PBMCs), we performed experiments using CD14-positive (CD14+) puriﬁed populations in the 2 settings, as it had been suggested that CD14 selection could have a deleterious role in generating fully functional mature DCs⁴¹and to evaluate the role of contaminating nonmonocyte PBMCs on DCs generation.

MATERIALS AND METHODS

After informed consent, healthy donors underwent mononuclear cell leukaphereses on a Cobe Spectra Apher- esis cell separator with V6-Auto PBSC software (Gambro BCT, Lakewood, CO). Leukaphereses were performed according to standard clinical routines. The leukaphereses products were centrifuged at 540gfor 10 minutes to remove the platelet-rich plasma, diluted in Hank buﬀered salt solution (HBSS; Lonza, Basel, Switzerland) and 10%

anticoagulant citrate dextrose, solution-A (Baxter Health- care Corp, Deerﬁeld, IL). PBMCs were isolated by dilution with HBSS and centrifugation at 400g for 20 minutes over lymphocyte separation medium (PAA Laboratories GmbH, Austria). The interface cells were collected pooled and washed twice. After washing, the cells were pelleted and resuspended in endotoxin ‘‘free’’ Roswell Park Memorial Institute (RPMI) 1640 medium, with 2% human AB serum (PAA Laboratories, Linz, Austria).

DC Generation in Plates

Plate DCs were generated according to earlier described methods.⁵⁰PBMCs obtained from leukaphereses products were plated in 6-wells culture plates, at a density of 20 to 2510⁶ cells/well. After 2 hours, the wells were washed 3 times to remove nonadherent cells. Then adherent cells were cultured for 6 days in RPMI 1640 supplemented with 2 mM L-glutamine, penicillin 50 U/mL, streptomycin 50mg/mL (all from Lonza), 2% AB serum (PAA Labora- tories), granulocyte macrophage colony-stimulating factor (GM-CSF) 800 U/mL (Leucomax, Novartis Pharmaceuti- cal), and IL-4 1000 U/mL (RyD Systems, Abingdon, UK) at 371C in a humidiﬁed incubator. Cultures were fed every other day. On day 6 cells were harvested, counted, and resuspended at a concentration of 0.510⁶ cells/mL, 1.510⁶cells per well in a 6 well plate, in fresh maturation or control media, for 6 to 48 hours. Maturation cocktail used in this study included tumor necrosis factor (TNF)-a 50 ng/mL, IL-1b 25 ng/mL (Peprotech, Rocky Hill, NJ), interferon (IFN)-g 1000 U/mL (R&D systems Europe), IFN-a3000 U/mL (Intron A, Schering-Plough Kenilworth, NJ), and Poly(I:C) 20mg (Sigma Aldrich, Saint-Louis, MO). After maturation, cells were harvested and the plates vigorously washed 3 times, with additional media to collect residual cells. Cells were counted using a Bu¨rker hemocytometer and viability determined by Trypan blue dye

exclusion. Cells were mostly used fresh excepted for mixed leukocytes reaction (MLR) stimulation. For MLRs stimulation, immature cells were frozen in aliquot of 5 to 1010⁶ DCs and thawed every week for Ag loading and maturation at the same cells concentration as fresh cells. DCs were frozen in RPMI, 50% human stable plasmatic proteins solution (4% m/V; DCF Red Cross, Belgium) and 10%

dimethylsulfoxide.

DC Generation in Bags

PBMCs from the same healthy volunteers were seeded at 510⁶ cells/mL in Cell Differentiation Bags (Miltenyi Biotec, Utrecht, The Netherlands) in RPMI, 2% AB serum, 800 U/mL GM-CSF, and 1000 U/mL IL-4, at 371C in a humidified incubator. Bags were placed flat in the incubator. Cultures were fed on days 2 and 4 by adding 25%

fresh culture medium including 4 times concentrated cytokines. DCs were puriﬁed by counterﬂow centrifugal elutriation on day 6 and resuspended in the same maturation media as pla-DCs at a concentration of 0.5 10⁶cells/mL.

Elutriation at Day 6

Elutriation was performed using a Beckman-Coulter (Miami, FL) J6-MC centrifuge with a rotor type JE-5-0 and 5 mL volume chamber. Cell fractions were collected in bags (Baxter, Chicago) at different flow rate using a peristaltic pump (Masterflex 7016 to 20). Elutriation of the different fractions was performed by changing the rotor speed as follows: fraction 1: 65 mL/min during 8 minutes to load the cells, fraction 2: 70 mL/min during 7 min, fraction 3: 75 mL/

min during 7 min, fraction 4: 77 mL/min during 4 minutes, fraction 5: 80 mL/min during 2 minutes, and fraction 6:

83 mL/min during 2 minutes, then the centrifugation is stopped to wash the chamber and to collect the DCs. This last elutriation fraction was centrifuged and the cells counted then transferred into cell culture bags, in maturation media. After maturation, bag-DCs were harvested after gentle massage and washed with additional media to collect residual cells. Cells were counted using Bu¨rker hemocytometer and viability determined by Trypan blue dye exclusion. Cells were mostly used fresh excepted for MLR stimulation. For MLRs stimulation, immature cells were also frozen in aliquot of 5 to 1010⁶DCs, following the same procedure as for pla-DCs and thawed weekly for antigen loading and maturation.

DC Generation From Immunomagnetic Selected CD14+ Monocytes

Monocytes selection was performed with the Clini- MACS cell selection system (Miltenyi, Bergisch-Gladbach, Germany) using anti-CD14-coated magnetic beads to retain monocytes in a clinical scale magnetic column. The semi- automated monocyte enrichment procedures were made on half of full fresh leukapheresis product to design direct comparison with the same collected cells diﬀerently processed.

CD14+ selected monocytes were cultivated in plates at the concentration of 10⁶/mL with a total of 310⁶/well or in bags at the same cell concentration. The media, the cytokines, the freezing procedure, and the timing were similar those used for DCs generation from PBMCs either by adherence or by day 6 elutriation.

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Cell Surface Immunophenotyping

Cells were analyzed on a Facscalibur ﬂow cytometer (Becton Dickinson) after acquisition of 10,000-gated events.

DCs were harvested, washed, and labeled (30 min at 41C) using the following ﬂuorescein isothiocyanate (FITC) or phycoerythrin (PE)-conjugated monoclonal antibodies. Iso- typic controls FITC/PE: IgG2a/IgG1 (Becton Dickinson), IgG2a/IgG2a, and IgG1/IgG1 (Immunotech). Speciﬁc markers: CD1a-FITC (IgG2a; Dako), CD1a-PE (IgG1;

Coulter), CD14-FITC (IgG2b; Becton Dickinson), CD80- PE (IgG1; Becton Dickinson), CD83-PE (IgG2b; Immu- notech), CD86-FITC (IgG1; Pharmingen), human leukocyte antigen (HLA)-DR–FITC (IgG2a; Pharmingen), CD19-PE (IgG2a; Probio), CD40-PE (IgG1; Immunotech), CD3-FITC (IgG1; Pharmingen), CD54-PE (IgG1; Becton Dickinson), and CD58-FITC (IgG2a; Immunotech). Dead cells and debris were gated out on the basis of their light scatter properties. Data were analyzed for significant differences using Student paired t test. P value less than 0.05 was considered statistically significant.

Wash Out Test and CD40L Rechallenge

To analyze the irreversibility of the maturation process after its initiation, DCs were collected, washed at different time point after the beginning of the maturation step, and divided in 3 populations one third of the cells were kept in control media with GM-CSF and IL-4, another third was resuspended in new fresh maturation media, and the remaining cells were cultivated on CD40L-expressing cell line for a total of 48 hours. Cells were analyzed at the different time points by flow cytometry for maturation markers and supernatants were assessed for IL-12p70 secretion.

IL-12p70 Secretion

Culture supernatants from maturating DC cultures were kept at 201C. Bioactive IL-12p70 heterodimer concentration was determined using an enzyme-linked immunosorbent assay (Pierce Endogen, Rockford, IL) according to manufacturer’s instructions.

DC-mediated Specific T-cell Response

DCs were loaded with the recombinant human immunodeﬁciency virus (HIV) gp41protein (Prospec-Tany, Technogene, Rehovot, Israel), at a concentration of 25 mg/mL, by 4 hours incubation and then washed twice, irradiated (15 Gy). PBMCs were used as responders in a 1/10 DC/T cells ratio in 3 weeks primary MLR.

MLR were performed in RPMI, 10% inactivated AB serum, supplemented during the first week with IL-6 1000 U/mL and with or without IL-12 40 U/mL (both of R&D Systems, Europe). Then, the cells were restimulated at days 7 and 14, and cultured with IL-2 20 and IL-7 40 U/mL from day 8. Cultures were fed every other day by changing one third of the culture media; and transferred to larger wells depending on lymphocyte density. On day 21, lymphocytes were used, in an intracytoplasmic IFN-g flow cytometry detection assay, to test their capacity to specifically recognize HIV protein loaded DCs. Controls consisted of T cells alone, DCs alone, and stimulation by unloaded DCs. For detection of antigen-specific intracytoplasmic IFN-g production, lymphocytes were harvested and cocultured with targets at a 10:1 ratio in the presence of Golgi stop (BD). After 4 hours, cells were harvested,

stained with anti-CD4 or anti-CD8, permeabilized and stained for intracellular IFN-g(all from BD), and subjected to fluorescence-activated cell sorting analysis. Data were analyzed for significant differences using Student paired t test. P value less than 0.05 was considered statistically significant.

Western Blot Assays

Cell lysates were subjected to sodium dodecyl sulfate- polyacrylamide gel electrophoresis using 10% polyacrylamide gels and transferred to polyvinylidene ﬂuoride membranes (Amersham Biosciences, Uppsala, Sweden) using a semidry electroblot chamber. Membranes were blocked with tris buﬀered saline containing 0.1% Tween 20 containing 5% bovine serum albumin overnight at 41C.

The blots were incubated with primary antibodies diluted in tris buﬀered saline containing 0.1% Tween 20 for 1 hour at 251C. After 1 hour of incubation with goat anti-rabbit peroxidase-conjugated antibody (Sigma Aldrich, St Louis, MO) at room temperature, proteins were detected by the electrogenerated chemiluminescence method (Amersham Biosciences) according to the manufacturer’s instructions.

Detection of p38, phospho-ERK 1/2 (Tyr204) and phospho-p38 (Tyr182) was carried out using speciﬁc monoclonal antibodies (Santa Cruz Biotechnology, Santa Cruz, CA).

Detections of phospho-b-catenin, phospho-Akt (Ser473), and total AKT were performed using standard immuno- blotting procedures. Antibodies were from Abcam (Abcam, Cambridge, UK).

Nuclear Extracts and Electrophoretic Mobility Shift Assay

Nuclear extracts were prepared from 410⁶ cells as follows: all buffers contained a mixture of protease inhibitors (Complete, Roche Diagnostics, Brussels, Bel- gium) to minimize proteolysis. The cellular pellet was washed with ice-cold phosphate-buffered saline and then resuspended twice with 1 mL of ice-cold buffer A [10 mM N-2-hydroxyl piperazine-N⁰-2-ethane sulfonic acid (HEPES) buffer, pH 7.9, 1.5 mM MgCl2, and 10 mM KCl]. Cells were collected by centrifugation (600gfor 10 min), resuspended, and incubated for 10 minutes with 40mL of ice-cold lysis buffer A containing 0.2% Nonidet P-40 (this step was repeated twice). The pellet (nuclear fraction) was incubated with 30mL of ice-cold extraction buffer C [20 mM HEPES buffer, pH 7.9, 25% glycerol, 1.5 mM MgCl2, 420 mM NaCl, and 0.2 mM ethylenediaminetetraacetic acid (EDTA)] for 20 minutes at 41C and then centrifuged at 20,800g for 10 minutes at 41C. The nuclear supernatants were diluted with 150mL of buffer D (20 mM HEPES buffer, pH 7.9, 20% glycerol, 50 mM KCl, and 0.2 mM EDTA) and stored frozen at –801C. Protein concentrations were determined by the Bradford method.

EMSA experiments were performed as described earlier⁵¹ using the following radiolabeled oligonucleotide probe encoding wild-type nuclear factor-k B (NFkB) binding site from the IL-12 promoter: 5⁰-AGTCCCGG GAAAGTCCTGCC-3⁰. The oligonucleotide bound complexes were separated on a 6% Tris-glycine-EDTA polyacrylamide gel migrated overnight at 50 V, and the radiolabeled protein complexes were detected by auto- radiography.

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Microarrays Analyses and Statistical Methodology

Oligonucleotide microarray total RNA (2 to 3 mg) was labeled using the BioArray High-Yield RNA Transcript Labeling Kit (Enzo Biochem, New York, NY) following the manufacturer’s standard procedures (Affymetrix, Santa Clara, CA). The labeled cRNA were hybridized on test-3 arrays (Affymetrix) to ensure the quality of the probes. The probes were recovered and hybridized on Human Genome U133 Plus 2.0 Array Genechips. The hybridization, washing, staining, and scanning of the array slides were performed according to standard protocols (Affymetrix).

Gene expression values from the cell intensity files were normalized using the Robust Multiarray Analysis which processes a group of cell intensity files simultaneously.⁵² Gene expression profile was determined in 6 immature DCs (3 bag-DCs and 3 pla-DCs) and 6 mature ones (same repartition). We identified significant differences between sample groups using the BRB array tools (available without charge for noncommercial applications at http://linus.nci.

nih.gov/BRB-ArrayTools.html). The class comparison tool of BRBArray Tools provides powerful methods for finding differentially expressed genes while controlling either the number or proportion of false discoveries.⁵³ Only genes defined as ‘‘present’’ by the Affymetrix algorithm in at least 25% of all experiments were considered for further analysis, and we calculated unpaired (as microarrays analyses performed on immature and mature DCs were carried out using the same donors, but in distinct experiments) 2-samplettests (with a random variance model) of the 2 groups (immature and mature DCs) for each gene and addressed the multiple comparison problems by estimating the false discovery rate.

The genes used for random variance estimation had to have passed the ﬁltering criteria. The nominal signiﬁcance level of each univariate test was 0.05. The genes were sorted by Pvalue of the univariate test.

Bags Conditioned Media (CM) for the Differentiation of Monocytes in Plates

To get more insights into the mechanisms leading to the difference between immature bag-DCs and pla-DCs, we investigated the role of bag conditioned media (CM) to differentiate monocytes into DCs in plates. To evaluate whether physical contact or soluble ‘‘factors’’ in bags are responsible for bag-DC IL-12p70 secretion deficiency, we prepared different bag-CM and used plate-CM as a control.

CMs were generated in bags and in plates using monocytes, PBMCs, or medium alone. The culture media were renewed

every other day, as described above. At day 5, CM were harvested and frozen for further experimentation.

Monocytes were diﬀerentiated for 6 days in plates using CMs that had been conditioned in bags with or without cells. Plate-CM was used as control. After washing, fresh unconditioned medium containing the cocktail of maturating agents was used for 24 hours, as described above, and secretion of IL-12p70 by matured DCs was chosen as a single informative assay.

Comparison of 2 Different Bag Brands With Plates for the Generation of Mature DCs

Monocytes were diﬀerentiated and matured as described above in plates and bags: Cell Diﬀerentiation Bags (Miltenyi Biotec, Utrecht, The Netherlands) and PermaLife bags (OriGen Biomedical, Inc, Austin, TX). This was carried out to avoid drawing conclusions from a single bag brand and to generalize the observations regarding the properties of DCs generated and matured in cell culture treated bags. Again, we chose the secretion of mature DCs after 24 hours of maturation as a single informative assay.

RESULTS DCs Phenotyping

Immature DCs generated in plates or in bags from leukapheresis products of healthy donors showed a typical DCs immunophenotype: high expression of HLA-DR and CD40, costimulatory molecule (CD86), adhesion molecules (CD54, CD58, CD11b, and CD11c), a variable CD1a expression, and a lack of lineage specific markers expression (CD14, CD3, and CD19). Kinetics of surface-antigen expression during 48 hours of maturation, with inflammatory cytokines and a toll-like receptor (TLR) 3 agonist were analyzed. Upon maturation, cell surface expression of CD80 costimulatory molecules and CD83 maturation marker appear on both cell types. DCs up-regulated the costimulatory molecules CD86, major histocompatibility complex class II molecules (HLA-DR), and CD40 molecules expression. For DCs matured in plate, the percentage of CD80-positive cells was higher and also its mean fluorescence intensity. Moreover CD86, HLA-DR, and CD40 cell surface density were also higher in mature DCs generated in plates compared with bag-DCs, as shown in Table 1.

As far as the origin of cells used to generate DCs was concerned (puriﬁed monocytes or PBMCs), there was neither substantial diﬀerence with respect to cell surface markers expression among DCs generated in plates from

TABLE 1. Flow Cytometric Measurements on the Different Types of Mature Dendritic Cells

Median Fluorescence Intensities Percentage of Positive Cells

CD83 CD80 CD86 CD40 HLA-DR CD83 CD80 CD86 CD40 HLA-DR

Plates 49.51 74.55 231.52 372.36 217.44 49.31 71.97 96.07 99.65 99.56

Bags 47.21 48.64 154.55 165.64 101.64 51.21 51.15 98.98 99.30 97.94

Pvalue NS 0.0269 0.0003 0.04 0.0081 NS 0.0189 NS NS NS

Bags matured in plates 38.69 60.65 217.90 249.80 184.26 55.25 60.21 98.35 99.78 99.56 Plates matured in bags 39.94 47.49 148.60 106.28 128.25 56.23 53.97 98.28 99.03 99.16 Puriﬁed CD14+ plates 45.13 61.28 201.32 653.55 285.39 51.923 61.11 97.79 99.93 99.92

Puriﬁed CD14+ bags 37.94 33.69 95.88 233.8 139.56 60.54 46.84 98.77 95.95 99.35

Pvalue NS 0.0095 0.0104 0.0493 0.0057 NS 0.0025 NS NS NS

HLA indicates human leukocyte antigen; NS, not signiﬁcant.

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adherent monocytes or from immunomagnetically sorted monocytes nor was there any diﬀerence between DCs generated in bags, either using day 6 elutriation of mononuclear cells or monocytes puriﬁed by CD14 immunomagnetic selection.

To analyze whether the impact of the bag or plate was more important during the generation or the maturation, we matured immature bag-generated DCs in plates, and vice-versa. The mean ﬂuorescence intensities obtained in these 2 types of mature ‘‘hybrid’’ DCs were intermediate between the phenotypes obtained by the 2 types of DCs, generated and matured in plates or bags.

We also observed that when the maturation process was started during 12 hours, the DCs continued their maturation, even if they were washed and kept in culture media without the maturation cocktail (data not shown).

Mature DC Cytokine Release

We performed a kinetic analysis of bioactive IL-12 secretion by pla-DCs or bag-DCs upon maturation. Further- more, to investigate whether matured DCs kept their ability to secrete bioactive IL-12 after subsequent CD40-CD40L interaction (mimicking T-cell dependent DC activation), we transferred both type of matured DCs, after extensive washing, to new culture plate for IL-12p70 production assessment after CD40 triggering in the absence of IFN-a˜

compared with medium alone (Fig. 1, panels A and B) Mature pla-DCs released high amount of IL-12p70.

IL-12p70 secretion peaked between 6 and 18 hours after the addition of the maturation stimuli. DCs rechallenged by CD40 ligation showed a capacity to release new IL-12p70 up to 12 hours after initiation of maturation, but signiﬁcantly less after 18 or 24 hours. In contrast, DCs generated in bags showed no signiﬁcant IL-12p70 secretion at any time of the kinetics or when rechallenged by CD40 ligation.

To assess whether bags exerted their inhibitory effect on mature DCs IL-12 production by affecting them at the differentiation stage, during the maturation or at both stages, we compared IL-12p70 production by DCs generated in bags but matured in plates, and DCs generated in plates but matured in bags within the same condition throughout the whole culture (Fig. 1, panel C). Our results demonstrate that the culture in bags affect their capacity of secreting IL-12p70 during the 2 consecutive steps (differentiation and maturation). Importantly, it also shows that changes induced by the generation in bags are irreversible as of the stage of immature DCs.

We also assessed the impact of the lymphocyte population on DCs generated in bags and elutriated at day 6 by comparing them with DCs generated in bags from immunomagnetically puriﬁed monocytes before culture and found no diﬀerences regarding IL-12p70 production.

This eﬀect was not speciﬁc to a particular brand of bags as demonstrated by a new set of experiments comparing 2 brands of bags with plates (depicted in Fig. 2, panel A) using IL-12p70 secretion as a single informative assay.

Finally, the use of CM to diﬀerentiate monocytes into immature DCs in plates revealed that the use of bag CMs, irrespective of the presence of cells during the medium conditioning, had the same eﬀect on DCs generated in plates, as their direct generation in bags. This is to say immature DCs unable to secrete IL-12p70 upon maturation in plate using fresh unconditioned medium containing the maturation cocktail (Fig. 2, panel B).

Induction of IFN-c Secretion by T Cells After coculture With Ag-loaded DCs

To assess another critical characteristic of functional mature DC more speciﬁcally, we performed MLR using FIGURE 1. A, IL-12p70 secretion kinetics. Immature DCs were plated at 510⁵ cells/mL for 6 to 48 hours in DCs culture medium with the maturation cocktail (Poly:IC 20mg/mL, IL-1b 25 ng/mL, IFN-a3000 U/mL, tumor necrosis factora50 ng/mL, and IFN-g1000 U/mL). Data represent the mean±SD of experiments performed on 8 healthy volunteers. B, IL-12p70 secretion after CD40L rechallenge. DCs at different maturation time were split in 3, and plated at a concentration of 0.510⁶cells/mL for further culture to a total of 48 hours with either the maturation cocktail, or CD40L-expressing cell line, or medium alone. C, IL- 12p70 secretion by DCs generated in bags but matured in plates, and DCs generated in plates but matured in bags. IL-12p70 secretion by DCs generated from day 0 purified CD14+

monocytes in plates or bags. Day 6 immature DCs were plated at 0.510⁶cells/mL for 48 hours with the maturation cocktail.

Generated and matured pla-DCs, after monocytes adherence and day 6-elutriated bag-DCs were used as control. Data represent the mean ± SD of experiments performed on 4 healthy volunteers. DC indicates dendritic cell; IFN, interferon; IL, interleukin.

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recombinant gp41 HIV protein loaded DCs with lymphocytes from HIV-seronegative donors. Those MLRs allowed us to evaluate the ability of generated DCs to initiate primary immune cellular response. Conventional immature DCs generated in plates are very poor stimulator and often no response was observed, the situation was slightly improved when exogenous IL-12 was added to the culture.

In contrast, 12 or 48 hours mature pla-DCs showed strong T_H1 stimulatory capacity, independent from exogenous IL-12 addition, especially DCs matured for 12 hours before their use as stimulators. By comparison mature bag-DCs showed very weak stimulatory capacity, not better than immature DCs, and addition of exogenous IL-12p70, only partially restored the stimulatory capacity. (Fig. 3, panels A and B).

The same comparison, performed using puriﬁed CD8 T-cell as responders, showed that only mature pla-DCs

were able to generate in vitro Ag-speciﬁc IFN-g-secreting CD8 T lymphocytes in 7 out of 9 volunteers (range, 2.3% to 20.6% speciﬁc CD8+ T cells). In this case, the results were superior with the addition of exogenous IL-12p70. (Fig. 3, panels C and D).

NFjB Nuclear Translocation

Given the lack of IL-12p70 expression in mature bag- DCs, we asked whether there could be any differences in the NFkB activation pathway, after differentiation and maturation. NFkB is known to play a critical role in DCs maturation. Using an oligonucleotide probe extending from 70 to 49, a sequence known to bind NFkB in the IL-12p35 promoter and nuclear extracts from immature and mature bag-DCs and pla-DCs, we demonstrated an early activation of NFkB in immature bag-DCs (Fig. 4, panel A) in contrast with immature pla-DCs, where the NFkB binding site was vacant. The differential binding observed between nuclear extracts from immature bag-DCs and pla-DCs was specific and reproducible among different preparations of nuclear extracts. Indeed, binding of the constitutively expressed Oct-1 transcription factor to its consensus sequence did not vary (Fig. 4, panel B). This showed that some of the mechanisms involved in mature DCs were already active in immature elutriated bag-DCs before the addition of the maturation stimuli.

Western Blot Assays

These assays investigated the status of signaling proteins involved both in the diﬀerentiation and maturation processes of GM-CSF/IL4 monocyte-derived DCs (b-catenin and PI3K-Akt) and in the control of IL-12 expression in mature DCs (Erk and MAPPK-p38). We ﬁrst found that the PI3K-Akt pathway was activated in immature bag-DCs, but not in immature pla-DCs (Fig. 5).

We next decided to investigate the status ofb-catenin in immature DCs, as it is known, in a mouse model,⁵⁴that the early activation of this pathway leads to a form of mature DCs that lacks the capacity to initiate TH1-type responses.

When we looked atb-catenin, we found it to be in its active phosphorylated form in immature bag-DCs, but not in their pla-DCs counterpart (Fig. 5). Finally, although p38 phosphorylation increased during the maturation process in both DCs types, Erk, which was activated at the immature stage in both, remained activated only in mature bag-DCs (Fig. 5).

Microarrays Analyses Part 1: Immature DCs

In this analysis comparing immature bag-DCs with immature pla-DCs (see Supplemental Data 1, Supplemental Digital Content 1, http://links.lww.com/JIT/A36), we focused on several sets of gene expression, related to their function (costimulatory molecules, adhesion molecules, IL-12 p35 and p40, and IL23 p19), protein processing and the expression of genes involved in the response to the maturating cocktail agents. There were no signiﬁcant diﬀer- ences in the basal level of IL-12p40 and p35 expression.

Regarding the expression of genes involved in protein processing, no differences were found, particularly in the expression of proteosome subunits. In contrast, when looking at the expression of genes related to the response to the cocktail components, important statistically significant differences (Table 2) were found in bag-DCs: a FIGURE 2. A, IL-12p70 secretion by DCs generated in plates or

bags. Immature DCs were seeded at 510⁵cells/mL, either in plates or in 2 commercially available bags, for 24 hours in DCs culture medium containing the maturation cocktail (Poly:IC 20mg/mL, IL-1b 25 ng/mL, IFN-a 3000 U/mL, tumor necrosis factor-a 50 ng/mL, and IFN-g1000 U/mL). Data represent the mean ± SD of experiments performed on 2 healthy volunteers.

B, IL-12p70 secretion by DCs generated in plates using different bag CM. Medium conditioning was carried out in absence or presence of cells (peripheral blood mononuclear cells and CD14+

monocytes) in bags. Monocytes were differentiated into immature DCs in plates using bag CM, then matured using identical fresh unconditioned medium containing the maturation cocktail.

Plate CM was used as control condition. IL-12p70 was assessed after 24 hours in the supernatants. Data represent the mean ± SD of experiments performed on 3 healthy volunteers. CM indicates conditioned media; DC, dendritic cell; IFN, interferon; IL, interleukin.

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decreased expression of IFN-greceptor-1, TNF-areceptor (NFkB activator), TNF receptor associated factor, and of MyD88, known for its role in TLR and IL-1 signaling and in the diﬀerentiation of monocytes into DCs, using GM-CSF.^55,56There was also a lower expression of TLR-1, but not of other TLRs, in immature bag-DCs. Finally, we looked at the expression of the receptors for the HIV gp41 protein, CCR5, and CXCR4, and no diﬀerence was found.

Part 2: Mature DCs

In this analysis, we focused on differences that could be related to a lesser efficiency to induce T_H1-type responses. The whole analysis revealed (see Supplemental data 2, Supple- mental Digital Content 2, http://links.lww.com/JIT/A37) that the 2 types of mature DCs differed by more than 1000 genes in their transcriptome. Among this huge set of differentially expressed mRNAs, we found significant overexpression of some genes related to the induction of T-cell responses in pla-DCs (Table 3), among which IL- 12p35 and p40, CCL24, CD1c, a-Integrins, HLA-DP, HLA-DRB, CD80, CD83, and CD40. It is worth noting that IL-23p19 expression did not differ between bag and pla-DCs.

DISCUSSION

DCs have been incorporated into active immunotherapy treatment strategies for over decade. However,

challenges remain in optimizing DC-based therapy within manufacturing process that permits quality control and scale-up of consistent products.³¹

Bags offer significant practical and regulatory advantage for the generation of clinical-grade DCs in a completely closed system. Most of the earlier studies examining DCs generated in bags found them to be similar in phenotype and function to DCs generated in culture plates.35,36,40,42,44–47). However, our first series of experiments, aiming at comparing the functional characteristics of both type of mature DCs, generated some unexpected results. Although both types of DCs had the classical immunophenotype of mature DCs, with a lower level of expression of CD80, CD40, CD86, and HLA-DR for bag- DCs, the latter failed both to secrete appropriate amounts of IL-12p70 and to induce primary immune response.

These differences remained the same in bag-DCs, differentiated from PBMCs and elutriated after 6 days of culture with GM-CSF and IL-4 before the maturation step, or originating from immunomagnetic purified CD14+ cells.

As the CD14 molecule possesses some important signaling capacities that may disturb the maturation process, we compared day 6 elutriation and day 0 CD14+

immunomagnetic selection. Elkord et al⁵⁷ have suggested that the use of CD14+ selection had a deleterious eﬀect on IL-12 secretion by monocytes-derived DCs, matured using lipopolysaccharide alone. This is not conﬂicting with our results as we use an optimal cocktail including FIGURE 3. Pla-DCs (A and B) or bag-DCs (C and D) were loaded with human immunodeficiency virus gp41 protein (25mg/mL) and then used irradiated, immature, or after 12 and 48 hours maturation. Peripheral blood mononuclear cells were used as responder cells in a 3 weeks primary mixed leukocytes reaction with or without exogenous recombinant interleukin (IL)-12. On day 21, T lymphocytes (CD4-positive on panels A and C, and CD8-positive on panels B and D) were assessed for intracytoplasmic detection of IFN-gby flow cytometry to analyze the capacity of the DCs to prime Ag-specific type-1 T-cell response. The controls consisted of T cells alone, DCs alone, and T cells with unloaded DCs. Data represent percentage of T cells secreting IFN-g. DC indicates dendritic cell; IFN, interferon;

PIC, poly:IC.

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inflammatory cytokines (IFN-g, IFN-a, IL-1b, and TNF-a) and poly(I:C) as TLR agonist^20,22with which we did not see any impact on IL-12p70 secretion when CD14+ selected monocytes were cultivated in plates as large amount of bioactive IL-12 were then produced. The cellular environment in which our bag-DCs elutriated at day 6 were generated could also have impacted on their final functionality. Nevertheless, combining the results from day 6 elutriated DCs and from immunomagnetic CD14+ selected derived DCs, and the results comparing plates and bags DCs, it is very unlikely that the final defects observed in mature bag-DCs originated mainly from their differentiation in bags, rather than from the culture medium, the cellular environment and the way the cells were purified.

Production of bioactive IL-12 can be ampliﬁed in vivo by T cells but must be initiated by a ﬁrst signal, such as TLR triggering.^21,23 It has nevertheless been shown that CD40/CD154 interactions are critical for the priming and expansion of CD4+T_H1 type cells and CD8+cytotoxic T cells in response to protein antigen, and that protective

antitumor immunity is therefore critically dependent of the CD40 maturation signals induced during cognate interaction between CD154 bearing CD4+ T cells and activated matured DC.^24,25 As the timing of the CD40L rechallenge could have an impact on the ability of the cells to produce a second peak of bioactive IL-12, we did a kinetic analysis of DC maturation and IL-12p70 secretion.

Our results in plates showed that IL-12p70 secretion is a rapid event in the maturation process, occurring between 6 and 18 hours after the beginning of the maturation stimuli, and the second peak of IL-12p70 is maximal when the CD40L rechallenge is performed after 12 hours of maturation. These results show that, for clinical use, a short DC maturation step with an association of a TLR agonists and inﬂammatory cytokines allows further in-vivo interaction with speciﬁc T-lymphocytes through CD40/CD40L interactions. The same did not hold true for bag-DCs. As a matter of fact, despite equal level of TLR3 expression, FIGURE 4. Using an oligonucleotide probe extending from70

to 49, a sequence known to bind NFkB in the interleukin-12 p35 promoter and nuclear extracts from immature and mature bag-DCs and pla-DCs, we demonstrated an early activation of NFkB in immature bag-DCs (panel A, lane 2), in contrast with immature pla-DCs (panel A, lane 1). In mature DCs, NFkB was activated, as expected, in both type of DCs (panel A, lanes 3 and 4). As a control, the binding of the constitutively expressed Oct-1 transcription factor to its consensus sequence did not vary (Fig. 3, panel B). DC indicates dendritic cell; IFN, interferon; NFkB, nuclear factorkB.

FIGURE 5. These assays investigated the status of signaling proteins involved both in the differentiation and maturation processes of granulocyte macrophage colony-stimulating factor/

interleukin-4 monocyte-derived DCs. The PI3K-Akt pathway was activated in immature (Im) bag-DCs, but not in immature pla-DCs (panel B). Theb-catenin was found to be in its active phosphorylated form in immature bag-DCs, but not in their pla- DCs counterpart (panel B). Finally, although p38 phosphorylation increased during the maturation (Mat) process in both DCs types, Erk, which was activated at the immature stage in both, remained activated only in mature bag-DCs (panel A). DC indicates dendritic cell; Neg, negative; Pos, positive.

TABLE 2. Differential Expression (Fold-change) of Selected Genes in Immature Dendritic Cells Generated in Plates Versus in Bags

Gene

Diﬀerential

Expression Plates/Bags P

Interferon-g, receptor 1 2.43 0.0002

TNFareceptor, NFkB activator

2.43 0.0002

TLR 1 2.07 0.0012

TNF receptor, associated factor 5

1.95 0.0011

TNF 1.94 0.0080

CD40 1.85 0.0170

MyD88 1.60 0.0159

NFkB indicates nuclear factorkB; TLR, toll-like receptor; TNF, tumor necrosis factor.

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bag-DCs were not only unable to secrete significant amount of IL-12p70 after maturation, but, importantly, failed to secrete IL-12p70 in response to CD40L. It is worth noting that maturating immature bag-DCs in plates did only increase slightly IL-12p70 secretion, far below the results achieved with pla-DCs. It is also worth noting that these results were not dependent on the bag brand, as shown by the last series of experiments comparing IL-12p70 secretion after differentiation and maturation in plates and 2 different brands of culture treated bags. Finally, the fact that bag CM, used for the differentiation experiments in plates, ultimately resulted in mature DCs with a much lower IL- 12p70 secretion, further confirmed that crucial events, leading to the observed differences, occurred during the differentiation phase. The fact that CM made in bags containing cells did not bring significant changes in comparison with the CM made without cells, suggests a major role for potential soluble factors, such as bags polymers. This is further supported by the fact that the use of plate CM to differentiate the monocytes led to an IL- 12p70 secretion by mature DCs identical to what was observed when the whole experiment was performed in plate from the start using fresh medium.

How do our results compare with the literature data?

First of all, some groups used bags to perform a good manufacturing practice enrichment in monocytes, using elutriation for this purpose, but then diﬀerentiated and matured the cells in ﬂasks, which cannot be compared with performing the whole procedure in bags.^39,49 In other articles, the whole procedure was performed in bags, but the capacity of DCs to secrete bioactive IL-12 was not addressed.32,34,36,38,40,42,50

Adamson et al⁴¹ showed that DCs cultured in bags were able to secrete IL-12p70 upon poly(I:C) stimulation.

The amount of IL-12 produced was far below the amount we measured in plate DCs, but nevertheless our bag-DCs are unable to produce IL-12p70 at the same level. There are 2 methodologic diﬀerences that could explain this diﬀer- ence. First, they enriched monocytes by elutriation of

leukapheresis product on day 0 and they used the Cell Gro DC serum-free medium (Cell Genix, Freiburg Germany) and it is known that culture media has an impact on DCs maturation, but it is unlikely that this impact will be diﬀerent on bag and plate DCs.⁵⁸ For bag-DCs, we compared elutriation at day 6 and immunomagnetic CD14+ monocyte selection. The second diﬀerence is that the stimulatory capacity of the DCs was only tested in an allogeneic MLR.

Kurlander et al⁴³ also published results comparing mature DCs prepared in bags or flask, generated after day 0 elutriation of the leukapheresis product and showed that DCs maturation by CD40L and IFN-g in bags markedly reduced, by more than 10 times, the production of IL-12p70, but again bag-DCs-upon poly(I:C) and inflammatory cytokines stimulation or CD40L rechallenge were unable to reach the level of IL-12p70 production that we found in pla-DCs. The amount of IL-12 produced in the Kurlander et al’s experiments was significant both in plates and bags, but the timing and the maturation agents were different. Differences in the DCs production process can also give us possible explanation for this discrepancy. In this article, PBMCs were also elutriated at day 0 before and the culture medium was supplemented by 10% of AB serum. Functional assay to assess DCs stimulation capacity was carried out with a pool of viral or tumor-associated antigen after a short (7 d) MLR. Importantly, they also found a drastic decrease in IL-12 production by bag-DCs in comparison with pla-DCs.

To better understand the global impact of plates and bags generation on DCs, we decided to perform functional tests. Secondary reactions and allogeneic MLR are often used in articles to assess DCs functions and are not perfect tests to address specific DCs function.32,36,38,40–46,50 As already shown by others, memory lymphocytes can be expanded easily. Cytomegalovirus (CMV)-specific helper T cells expansion were easily obtained from all our CMV- seropositive donors with CMV lysate loaded immature DCs, regardless that they were generated in bags or in plates. Moreover, CMV-specific helper T cells expansion from CMV-seropositive donors can easily be achieved using loaded monocytes as antigen-presenting cells.⁵⁹In the data not shown of the article of Macke et al,³⁵DC function was analyzed by the capacity to stimulate a tyrosinase specific cytotoxic T lymphocytes clone. In the same line of reasoning, Pullarkat et al⁴⁷ and Erdmann et al⁴⁸ used gp100 and MelanA melanoma peptide to evaluate DCs stimulation capacity and peptide-specific cytotoxic T-cell responses.

Fully functional DC are not necessary to achieve these above results, although they are required to address the impact of bags and ﬂask containers on the ability of protein loaded DCs to prime naive CD4+ T helper cells.

Kurlander et al did not find any differences in the expansion of specific CD-8 T cells using various peptides. However, these peptides were derived from antigens that can be assumed, considering the short 7 days MLR, to stimulate memory T cells.

To assess the mature DC function more accurately, we performed MLR using HIV gp41protein loaded DCs as antigen stimulation with lymphocytes from HIV- seronegative donors. Those MLRs allow us to study primary reaction with naive lymphocytes. Regarding the functional assay performed with gp41 protein loading, there was no bias, in terms of gp41 receptors. Both types of TABLE 3. Differential Expression (Fold-change) of Selected

Genes in Mature Dendritic Cells Generated in Plates Versus Bags (P< 0.05). Fold change <0.6 or >1.6

Gene

Diﬀerential Expression Plates/Bags

IL-12 p40 29.7

TNFareceptor (NFkB activator) 8.02

CCL24 4.71

CD1c 4.53

IL-12p35 3.58

a-Integrin (9) 3.04

CD40 2.48

BCL-2 2.38

IL-18 receptor 2.22

HLA-DP 2.12

CD80 1.99

HLA-DRB 1, 3, 4, and 5 1.86

a-Integrin (4) 1.84

CD83 1.78

The HLA-DRB found signiﬁcantly diﬀerent were those of the donors used for these experiments.

HLA indicates human leukocyte antigen; IL, interleukin; NFkB, nuclear factorkB; TNF, tumor necrosis factor.

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DCs expressed CCR5 and CXCR4, at the immature and mature stages, which ruled out that the diﬀerence, could be due to a lack of gp41 internalization in bag-DCs. More- over, although very concerning for in-vivo use, the lack of bioactive IL-12 secretion cannot explain by itself the absence of response to gp41, as addition of exogenous bioactive IL-12 in the MLRs, could not restore a satisfac- tory primary response. Other mechanisms had therefore to be contemplated.

As far as EMSA were concerned, we decided to focus on NFkB binding to the IL-12p35 subunit promoter and found that, in immature bag-DCs, NFkB was already activated, at the opposite of immature pla-DCs. However, at the stage of mature DCs, there was no longer a difference regarding NFkB activation. This observation was a hint that some sort of premature activation had already started in bag-DCs at the immature stage. This led us to examine the status of different pathways involved in the function of DCs, at the immature and mature stages, using Western blot techniques. The following conclusion can be drawn: at the immature stage, bag-DCs displayed an unusual activation of b-catenin and an activation of the PI3K-Akt pathway. Early activation ofb-catenin has been associated with the generation of mature DCs unable to induce T_H1-type response,⁵⁴ whereas activation of the PI3K-Akt pathway can negatively regulate IL-12 expression.^60,61At the immature stage, both DCs types did not differ regarding p38 and Erk activation status (inactivated and activated, respectively). When turning on to the mature stage, it was remarkable to observe an activation of p38 in both, but also the persistence of the Erk activated status in bag-DCs, consistent with their low level of IL-12 secretion and lack of response to CD40 rechallenge.^62–66

Finally, our microarrays analyses can be summarized as follows. First of all, the 2 types of DCs already differ by the expression of more than a 1000 genes at the immature stage and cluster apart, confirming that they had started to diverge during the differentiation process. The main characteristics of immature bag-DCs are a lower expression of molecules involved in the response to proinflammatory signals and to TLRs stimulation. With respect to mature DCs, the main observation, apart from the obvious down- regulation of the IL-12p35 and p40 genes, was a significant underexpression, in bag-DCs, of genes related to the induction of T_H1-type response. In conclusion, major molecular events have already taken place during the differentiation process. After the differentiation of monocytes into immature DCs in bags, some form of premature maturation, not effective to lead to fully efficient mature DCs, which in turn results in altered mature bag-DCs, this fact being worsened by the underexpression on immature DCs of several molecules important for a T_H1-type response to the different stimuli they should encounter in vitro or in vivo.

We conclude that monocyte-derived DCs, generated in bags, result eventually in mature DCs unable to generate primary immune response. We speculate that this effect is due partly to the impaired secretion of IL-12, but also to a perturbed expression profile of molecules crucial for their functionality. As maturation of bag-DCs in plates could not restore their functionality, and given the molecular analyses, these defects are already present at the immature stage and have taken place during differentiation. Our experiments suggest that at least part of these observations might be because of the material used, perhaps a release of bags

polymers into the culture medium. Those phenomena result eventually in mature bag-DCs, unable to secrete bioactive IL-12 and with lower expression of molecules involved in the capacity of eliciting primary responses from naive T cells.

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