Peripheral blood from healthy donors was obtained from buffy coats provided by the Red Cross donor center (Red Cross-Flanders, Mechelen, Belgium). Peripheral blood mononuclear cells were isolated by density gradient centrifugation (Ficoll Pacque PLUS, GE Healthcare, Amsterdam, the Netherlands). From the peripheral blood mononuclear cell fraction, monocytes were purified by CD14⁺ immunomagnetic selection (CD14 Reagent, Miltenyi Biotec, Bergisch Gladbach, Germany), according to the manufacturer’s instructions. The CD14-depleted cell fraction [i.e., peripheral blood lymphocytes (PBLs)] was cryopreserved in fetal bovine serum (Thermo Fisher Scientific, Erembodegem, Belgium) supplemented with 10% dimethyl sulfoxide (Sigma-Aldrich, Bornem, Belgium) and stored at −80°C for later use in an allogeneic mixed leukocyte reaction. CD14⁺ monocytes were cultured in vitro at a density of 1–1.2 × 10⁶/ml and differentiated into DCs in culture medium consisting of Iscove’s modified Dulbecco’s medium (IMDM) with l-glutamine (Thermo Fisher Scientific), supplemented with 200 IU/ml of granulocyte-macrophage colony-stimulating factor (Gentaur, Brussels, Belgium), 250 IU/ml of IL-4 (Miltenyi Biotec), 2% human AB (hAB) serum (Thermo Fisher Scientific), 10 µg/ml of gentamicin (Thermo Fisher Scientific), and 1 µg/ml of amphotericin B (Thermo Fisher Scientific). TolDCs were differentiated under the same conditions, except for the addition of 2 nM 1,25(OH)₂-vitamin D₃ (vitD₃, Calcijex, Abbott Laboratories, IL, USA) to the culture medium. On day 4 of culture, DCs were subjected to a proinflammatory cytokine cocktail by the addition of 1,000 IU/ml of IL-1β (Miltenyi Biotec), 1,000 IU/ml of tumor necrosis factor-α (Miltenyi Biotec), and 2.5 µg/ml of prostaglandin E₂ (Pfizer, Elsene, Belgium) to obtain mature control and tolDCs. For tolDC cultures, vitD₃ was replenished on day 4. The cells were cultured in a humidified atmosphere with 5% CO₂ at 37°C. On day 6, DCs were harvested for use in further experiments.

The study was approved by the ethics committee of Antwerp University Hospital and the University of Antwerp (15/50/543) and followed the tenets of the Declaration of Helsinki.

The complementary DNA sequence of human CCR5 (accession number U54994) was modified for optimal codon use (Figure S1 in Supplementary Material) and subcloned into a pST1-plasmid vector under the control of a T7 promotor and with the addition of a poly(A)tail (GeneArt, Thermo Fisher Scientific). After transformation in Escherichia coli and linearization of the circular DNA plasmid, mRNA transcripts were generated using a T7 in vitro transcription kit (mMessage mMachine T7 kit, Ambion, Life Technologies), according to the manufacturer’s protocol. mRNA was resuspended at a concentration of 1 µg/µl, aliquoted, and stored at −20°C.

Messenger RNA EP of DCs was performed as previously described (31, 32). In brief, the cells were resuspended in Opti-MEM (Thermo Fisher Scientific), and a 200 µL aliquot of this cell suspension containing 2–10 × 10⁶ cells was transferred into a 0.4-cm cuvette (Immunosource, Schilde, Belgium). Next, 10 µg of mRNA were added. EP was performed using a Gene Pulser Xcell™ electroporation system (Bio-Rad, Temse, Belgium) with a time constant protocol at 300 V for 7 ms. EP of cells without the addition of mRNA (mock EP) was performed as a control. Immediately after EP, the cells were transferred into fresh DC culture medium. For tolDCs, 2 nM vitD₃ was added to the cell culture medium. After a 30-min resting phase, the cells were washed and resuspended in warm IMDM supplemented with 5% hAB serum. Following an additional resting period of 90 min, the cells were washed again, resuspended in IMDM supplemented with 1% hAB serum, and used in further experiments.

Flow cytometric analysis of the expression of CCR5 by DCs was performed 2, 4, 24, 48, and 72 h after EP. CCR5 mRNA-electroporated, mock-electroporated, and nonelectroporated DCs were stained with a phycoerythrin-cyanin 7-labeled anti-CCR5-antibody (BD Pharmingen, Erembodegem, Belgium) or an isotype-matched control antibody (BD Pharmingen). LIVE/DEAD^® Fixable Violet Dead Cell Stain (Thermo Fisher Scientific) was added to assess cell viability. The indicated percentages of CCR5-positive cells were within the living DC population (i.e., gated for DCs based on light scatter properties and negative for LIVE/DEAD^® Fixable Violet Dead Cell staining). Flow cytometric measurements were performed using a Cyflow ML flow cytometer (Partec, Münster, Germany). The results were analyzed using FlowJo software (Tree Star, Ashland, OR, USA).

The phenotype of DCs was characterized using the following fluorochrome-labeled mouse antihuman monoclonal antibodies: anti-CD83-fluorescein isothiocyanate (Life Technologies), anti-CD80-phycoerythrin (BD Pharmingen), antihuman leukocyte antigen (HLA)-DR-peridinin chlorophyll (BD Biosciences), anti-CD86-fluorescein isothiocyanate (BD Pharmingen), and anti-CCR5-phycoerythrin-cyanin 7. Isotype-matched control monoclonal antibodies were used to determine nonspecific background staining. For analytical flow cytometry, at least 10⁴ events were acquired using a FACScan flow cytometer (BD). The indicated percentages were within the DC population based on light scatter properties. All the results were analyzed using FlowJo software.

The in vitro BBB model was constructed as described previously (30). In brief, human primary astrocytes (Sanbio, Uden, the Netherlands) were seeded at a density of 15,000 cells/cm² on the poly-l-lysine-coated underside of a transwell (24-well format) with 3.0-µm pore size (Greiner Bio-one, Vilvoorde, Belgium) and allowed to adhere for 2 h. Subsequently, the inserts were transferred into a well filled with EGM-2-MV medium (Lonza, Verviers, Belgium) with 2.5% fetal bovine serum. hCMEC/D3 endothelial cells (Tébu-bio, Le Perray-en-Yvelines, France) were seeded onto the insert’s collagen-coated upper side at a density of 25,000 cells/cm². Cultures were maintained in EGM-2-MV medium in 5% CO₂ at 37°C. Three days after initiating the coculture, the growth medium was replaced by EBM-2-plus medium, consisting of EBM-2 medium (Lonza), supplemented with 1.4 µM hydrocortisone (Pfizer), 1 ng/ml of basic fibroblast growth factor (Thermo Fisher Scientific), 10 µg/ml of gentamicin, 1 µg/ml of amphotericin-B, and 2.5% fetal bovine serum. EBM-2-plus medium was replenished every other day. Migration assays were performed between days 10 and 13 of culture.

Chemotaxis of DCs was studied 2 h after EP or at an equivalent time point for nonelectroporated DCs using 3.0-μm-sized pore transwells and an in vitro BBB model. DCs (2 × 10⁵) were added to the upper compartment of both the transwell and in vitro BBB model. The basolateral compartment contained 25 ng/ml of CCL4 and 25 ng/ml of CCL5 in IMDM, supplemented with 1% hAB serum. DCs were subsequently allowed to migrate for 4 h in the transwell assays or for 24 h in assays using the in vitro BBB model. The negative control consisted of 2 × 10⁵ DCs added to the upper compartment, while no chemokines were added to the basolateral compartment. As a positive control, 2 × 10⁵ DCs were added directly to the basolateral compartment. At the indicated time points, DCs were collected from the basolateral compartment. After resuspension in a fixed volume of 200 µl, they were counted using a BD FACScan flow cytometer. Events were acquired at a fixed flow rate for exactly 120 s. The results were analyzed using FlowJo software. The percentage migration was calculated as follows: [(# migrated cells in the experimental sample−# migrated cells in the negative control)/# cells in the positive control]*100%.

For analysis of the gene expression profile of nonelectroporated and electroporated tolDCs and control DCs, total RNA was isolated. The cells were disrupted and homogenized using guanidine-thiocyanate-containing lysis buffer. Total RNA was isolated using an RNeasy microkit (Qiagen, Antwerp, Belgium). The RNA concentration was determined by measuring absorbance at 260 nm using a Nanodrop spectrophotometer (Wilmington, DE, USA). Reverse transcription of the obtained RNA into cDNA was performed using an iScript™ Advanced cDNA Synthesis Kit (Bio-Rad). Subsequently, SYBR^® Green technology was used for relative mRNA quantification by qPCR in a CFX96 C1000 thermal cycler (Bio-Rad). qPCR reactions were conducted at 95°C for 30 s, followed by 40 cycles at 95°C for 5 s, and at 60°C for 30 s. All primer sets were obtained from Bio-Rad; validation data are shown in Table S1 in Supplementary Material. qPCR was performed in triplicate, and resulting mRNA levels were normalized to levels of the reference genes beta-actin and phosphoglycerate kinase 1. Melt curve analysis was performed to confirm the specificity of the amplified product. Bio-Rad CFX manager v3.1 was used for data processing and analysis.

To assess the allogeneic T-cell stimulatory capacity of DCs, the cells were cocultured with allogeneic PBLs in a 1:10 ratio. Nonstimulated responder PBLs served as a negative control, and allogeneic PBLs stimulated with 1 µg/ml of phytoheamagglutinin (Sigma-Aldrich) were used as a positive control. Cocultures were performed in IMDM supplemented with 5% hAB serum at 37°C. After 6 days in coculture, the secreted level of interferon-γ (IFN-γ) in the cell culture supernatant was determined in duplicate as a measure of allo-stimulatory capacity using a commercially available enzyme-linked immunosorbent assay (ELISA) kit (PeproTech, NJ, USA). In addition, IL-10 secretion was measured in the supernatant using a U-PLEX assay (Meso Scale Discovery, MD, USA), according to the manufacturer’s instructions.

Data were analyzed using Graphpad Prism software version 5.01 (Graphpad, San Diego, CA, USA), except for qPCR data, which were analyzed using CFX Manager software, version 3.1 (Bio-Rad). Comparison of nonelectroporated, mock-electroporated, and CCR5 mRNA-electroporated tolDCs was performed by a repeated measures one-way ANOVA, followed by Tukey’s multiple comparisons test. For data that were not normally distributed according to the Kolmogorov–Smirnov test, the Friedman test, with Dunn’s multiple comparison test was performed. Comparison of CCR5 expression levels at several time points after EP in mock-electroporated, CCR5 mRNA-electroporated, and nonelectroporated tolDCs was performed using a two-way repeated measures ANOVA, with post hoc Bonferroni tests. For qPCR results, differences were considered significant when p < 0.01. For other data, statistical significance was considered at the 5% level. Data are shown as mean ± SEM. The number of biological replicates is indicated in the figure or table legend.

Only a minority of in vitro generated vitD₃-treated tolDCs expressed CCR5 (i.e., 12.96 ± 2.02% on average). This translated into marginal chemotaxis of tolDCs. Only 0.10 ± 0.05% of tolDCs migrated in response to the chemokines CCL4 and CCL5.

To increase CCR5 protein expression, tolDCs were electroporated with mRNA encoding CCR5. Using flow cytometric analysis of CCR5 protein expression at consecutive time points after EP, an incremental increase was detected in CCR5 expression levels from 2 to 48 h following EP, after which expression decreased again (Figure 1). CCR5 expression levels of CCR5 mRNA-electroporated DCs were significantly higher as compared with those of both nonelectroporated and mock-electroporated DCs 4 h (36.84 ± 5.89 vs. 2.87 ± 0.60 and 4.75 ± 0.79%, respectively; p < 0.05), 24 h (55.42 ± 10.09 vs. 7.09 ± 3.04 and 5.04 ± 1.74%, respectively; p < 0.001), and 48 h (59.52 ± 9.59 vs. 14.44 ± 9.48 and 14.08 ± 7.86%, respectively; p < 0.001) after EP.

To investigate whether elevated CCR5 expression translated into a higher capacity to migrate in vitro, chemotaxis of tolDCs across transwells in response to the CCR5 ligands CCL4 and CCL5 was studied. Although only 0.22 ± 0.11% of nonelectroporated tolDCs and 0.11 ± 0.10% of mock-electroporated tolDCs migrated toward a CCL4 and CCL5 gradient, 2.59 ± 0.37% of CCR5 mRNA-electroporated tolDCs showed chemokine-mediated migration (p < 0.05 and p < 0.01, respectively) (Figures 2A,B). Likewise, there was an 18-fold increase in CCR5-driven transmigration of tolDCs across an in vitro BBB model following CCR5 mRNA EP of tolDCs. Only 0.22 ± 0.16% of nonelectroporated tolDCs and 0.35 ± 0.35% of mock-electroporated tolDCs succeeded in transmigrating across the in vitro BBB model. In contrast, 4.98 ± 1.24% of tolDCs electroporated with CCR5 mRNA crossed the BBB in response to CCL4 and CCL5 (p < 0.05) (Figures 2C,D).

To ensure that the semi-mature phenotype of tolDCs was unaffected by mRNA EP, the expression of DC maturation markers, as well as that of molecules involved in antigen presentation, was investigated (Table 1). Mock or mRNA EP did not affect the proportion of control DCs or that of tolDCs expressing CD80, CD83, CD86, and HLA-DR. In addition, the level of protein expression per cell, as assessed by mean fluorescence intensity, was not significantly affected for the membrane molecules expressed by tolDCs following mRNA EP. In control DCs, a modest but significant decrease in protein expression levels was observed after EP for all molecules tested. However, expression levels of CD80, CD83, CD86, and HLA-DR were still significantly higher in CCR5 mRNA-electroporated control DCs as compared to tolDCs. Interestingly, neither mock nor mRNA EP affected mRNA expression levels of LILRB4 and TLR2, two established regulators of tolerogenicity, in vitD₃-treated tolDCs (33). Normalized expression levels of both markers remained significantly higher in tolDCs as compared with those of control DCs (Table 1).

Functionally, tolDCs maintained their capacity to induce T-cell hyporesponsiveness following mRNA EP (Figure 3A). No differences were observed in the level of secreted IFN-γ in the supernatant of PBLs stimulated with nonelectroporated tolDCs as compared with that of PBLs stimulated with either mock- or CCR5 mRNA-electroporated tolDCs. In contrast, the levels of IL-10 secreted in the coculture supernatant were significantly higher when mock- or CCR5 mRNA-electroporated tolDCs were cocultured with PBLs as compared with cocultures of PBLs with corresponding control DCs (p < 0.05 and p < 0.001, respectively) (Figure 3B).