All animals used in experiments were accommodated in an accredited animal housing facility under a license granted by the Department of Health, Ireland, and were approved by the Animals Ethics Committee of the National University of Ireland, Galway. Bone marrow used in the generation of BMDCs was isolated from male Dark Agouti (DA, RT-1^avl) rats at 8–14 weeks of age. For the allogeneic MLRs, male Lewis (LEW, RT-1^l) rats served as a source of lymphocytes, isolated from both the cervical and mesenteric lymph nodes and spleen. DA and LEW rats were obtained from Harlan Laboratories (Bicester, UK).

Immature DCs were generated using an adapted version of the protocol, which has been previously described (13) (Figure S1 in Supplementary Material). Briefly, on day 0, male DA rats of the specified age were sacrificed and the tibia and femur were surgically removed postmortem. The epiphyses were cut and the bone marrow was flushed from the long bones with a syringe/needle combination. The erythrocytes were removed from the suspension by lysis using a standard red blood cell lysis buffer (Sigma-Aldrich, Dublin, Ireland). After erythrocyte lysis, the cells were washed in RPMI-1640 (Gibco, Grand Island, NY, USA) medium supplemented with 10% heat-inactivated fetal bovine serum (FBS), 2 mmol/L l-glutamine, 100 mmol/L nonessential amino acids, 1 mmol/L sodium pyruvate, 100 U/mL penicillin, 100 µg/mL streptomycin, and 55 µmol/L 2-β-mercaptoethanol (2β-ME) (Gibco). Cells were resuspended at a concentration of 1.5 × 10⁶/mL and plated at a concentration of 4.5 × 10⁶ per well of a 6-well plate. The culture medium was supplemented with 5 ng/mL rat granulocyte-macrophage colony-stimulating factor (GM-CSF) (Invitrogen, Paisley, UK) and 5 ng/mL rat IL-4 (Peprotech EC, London, UK). Cells were incubated under standard cell culture conditions (37°C at 5% CO₂) and, on the third day of culture, half of the medium from each well was harvested and cells were resuspended in fresh medium supplemented with rat GM-CSF and IL-4 and added back into the culture. On the fifth day, the supernatant was exchanged with fresh supplemented growth medium to remove dead granulocytes and lymphocytes. In experiments requiring tDCs, Dexa (Sigma-Aldrich) was added to the culture at 10⁻⁶ mol/L at this point. On the seventh day of culture, half of the medium was again removed and replaced with fresh supplemented medium (Dexa was added as required). To generate mDCs, LPS (1 µg/mL; Sigma-Aldrich) was added 24 h before the cells were cultured. Cultures were maintained until day 10 and then gently pipetted off the bottom of the wells for the in vitro assays.

To produce neuraminidase-treated iDCs and tDCs (niDCs and ntDCs), BMDCs were harvested on day 10 of culture and 2 × 10⁵/mL were treated with 400 U/mL of recombinant Clostridium perfringens neuraminidase (P0720S, New England Biolabs, Ipswitch, MA, USA) in phosphate buffered saline (PBS) supplemented with 1 mM MgCl₂ (Sigma-Aldrich), 1 mM CaCl₂ (Sigma-Aldrich), and 1% bovine serum albumin (Sigma) for 90 min at 37°C.

RNA was exacted from iDCs, tDCs, mDCs, niDCs, and ntDCs on day 10 using Bioline Isolate II RNA mini kits according to manufacturer’s protocols. All cDNA was produced using RevertAid^TM H Minus Reverse Transcriptase (Thermo Fisher Scientific, MA, USA) with random primers. For primer sequences of GAPDH, TNF-α, IL-12p40, inducible nitric oxide synthase (iNOS) IL-10, IDO, IL-6, and IL-1β, see Table S1 in Supplementary Material. All samples were normalized to expression of the house-keeping gene GAPDH and made relative to iDCs. All quantitative real-time PCR was performed according to the standard program using a real-time PCR system (StepOne Plus, Applied Biosystems, Thermo Fisher Scientific).

Cells were characterized by flow cytometry using the monoclonal antibodies (mAbs) CD11b/c-APC, CD80-PE, CD86-PE, MHCI-FITC, and MHCII-PE (BioLegend, San Diego, CA, USA). For analysis of the glycocalyx, lectins from Maackia amurensis (MAL II, indicating α2-3 Sia) and Sambucus nigra (SNA-I, indicating α2-6 Sia) were used (Vector Labs). Lectins were biotin conjugated. PE-streptavidin was used for detection. Negative controls for non-specific fluorescence were used, these consisted of PE-streptavidin staining solutions in the absence of the lectin conjugated to biotin. Lectins were prepared in lectin staining buffer (PBS containing 1% FBS, 1 mmol/L CaCl₂, and 2 mmol/L MgCl₂) and resuspended in FACS buffer (PBS containing 2% fetal calf serum and 0.01% NaN₃, all from Sigma-Aldrich) before analysis using a FACS Canto II (BD Biosciences, Oxford, UK).

For analysis of the assays involving lymphocytes from the lymph nodes and spleen, the following mAbs were used CD3/PE, CD8/PE-Cy7, CD4/APC (BioLegend), and CD25/FITC (eBioscience, San Diego, CA, USA). Prior to staining, cells were washed with FACS buffer. mAbs were diluted in 50 µL FACS buffer, added to the cells, and incubated for 15 min at 4°C. To remove any unbound antibodies, the cells were washed three times with FACS buffer. The cells were then filtered through a nylon mesh (40 µm) before analysis in the cytometer.

Lymphocytes were isolated from the spleen and lymph nodes of LEW rats. T cells were washed with phosphate-buffered saline and stained in prewarmed (37°C) CellTrace™ Violet (CTV) phosphate-buffered saline staining solution (Invitrogen, Carlsbad, CA, USA) as per manufacturer’s instructions. 2 × 10⁵ CTV-stained T cells were stimulated at a 1:1 ratio with anti-rCD3/anti-rCD28-labeled beads in supplemented RPMI 1640 media. Assays were incubated at various BMDC: T-cell ratios in a humidified incubator for 4/5 days at 37°C following which T-cell proliferation and CD4 and CD8 expression were assayed by flow cytometry (mAbs CD4-APC and CD8α-PE-Cy7; Biolegend). T-cell proliferation, activation, and differentiation were analyzed using a FACS Canto II.

Membrane proteins were extracted from iDCs, tDCs, niDCs, and ntDCs using a commercial protein extraction kit (Mem-Per^®, Thermo Fisher Scientific). Proteins recovered from 10⁶ cells were labeled with 100 µg (10 mg/mL in DMSO) Alexa Fluor^® succinimidyl ester 555 dye (Thermo Fisher Scientific) as per the manufacturer’s instructions. Labeled protein was separated from unconjugated dye with Bio-Gel^® P6 (Bio-Rad Laboratories, Dublin, Ireland).

Lectin microarrays were constructed essentially as described previously in Ref. (30). Forty-four lectins (Table S2 in Supplementary Material) sourced from multiple vendors were diluted to 0.5 mg/mL in PBS supplemented with 1 mM of respective haptenic sugar to maintain binding site integrity (see Table S2 in Supplementary Material) and printed on Nexterion^® H (Schott, Mainz, Germany) functionalized glass substrates using a sciFLEXARRAYER S3 non-contact spotter (Scienion, Berlin, Germany). During printing, relative humidity and temperature were maintained at 62% (±2%) and 20°C, respectively. Following printing, slides were incubated in a humidity chamber overnight at 20°C to ensure completion of covalent conjugation. Unoccupied functional groups were deactivated by 1 h incubation with 100 mM ethanolamine in 50 mM sodium borate, pH 8. Finished slides were washed with PBS with 0.05% Tween-20 (PBS-T) three times for 3 min and once with PBS for 3 min, centrifuged dry (450 × g, 5 min), and stored at 4°C with desiccant until use.

Labeled cellular proteins were incubated with finished microarrays following extensive optimization as described in Ref. (30). All processes were carried out with limited light exposure. Samples were applied to microarrays using an 8-well gasket slide and incubation cassette system (Agilent Technologies, Cork, Ireland). 70 µL of each labeled glycoprotein at 0.5 mg/mL, in incubation buffer [TBS-T; Tris-buffered saline (TBS; 20 mM Tris–HCl, 100 mM NaCl, pH 7.2, supplemented with 1 mM CaCl₂, and 1 mM MgCl₂) with 0.05% Tween^®-20], was applied to each well of the gasket. A total of 18 technical replicates were carried out for iDC and tDC profiling (encompassing samples of five biological replicates). Each microarray slide was loaded into a cassette with an accompanying gasket slide and placed in a rotating incubation oven (23°C, approximately 4 rpm) for 1 h. Incubation cassettes were disassembled under TBS-T, and microarrays were washed in a Coplin jar twice in TBS-T for 2 min each and once with TBS for 2 min. Microarrays were dried by centrifugation (450 × g) and imaged immediately using an Agilent G2505B microarray scanner at 5 µm resolution (532 nm laser, 100% laser power, 90% PMT).

Data extraction and analysis was performed essentially as previously described (30, 31). In brief, raw intensity values were extracted from high-resolution *.tif files using GenePix Pro v6.1.0.4 (Molecular Devices, Berkshire, UK) and a proprietary *.gal file (containing feature spot addresses and identities) using adaptive diameter (70–130%) circular alignment based on 230 mm features. Numerical data were exported as text to Excel (Version 2010, Microsoft, Dublin, Ireland). Local background-corrected median feature intensity data (F543median-B543) was analyzed. The median value, derived from data from six replicate spots per subarray, was handled as a single data point for graphical and statistical analyses.

Lectin microarray intensity values were normalized to the median total intensity value for all features across all subarrays. The significance of difference between relative intensity data (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001) was evaluated for each set of replicates on a lectin-by-lectin basis using a standard Student’s t-test (two-tailed, two sample unequal variance). Unsupervised, hierarchical clustering of lectin-binding data was performed with Hierarchical Clustering Explorer v3.0 (http://www.cs.umd.edu/hcil/hce/hce3.html). For clustering analysis, previously, normalized data were imported directly and clustered with the following parameters: no pre-filtering, complete linkage, Euclidean distance. Principal component analysis (PrCA) of previously normalized and pre-filtered data (those lectins which demonstrated p < 0.01 or better in the above t-tests, 15 in total) was performed using Minitab version 16.1.1 (Minitab, Inc., State College, PA, USA).

Data were analyzed using the software package FlowJo v10 (Tree Star, Ashland, OR, USA). All data were analyzed with Graphpad Prism V6 software (Graphpad Software, CA, USA) and are expressed as mean ± SEM unless otherwise indicated. Comparisons among multiple groups were made with one-way ANOVAs followed by Tukey’s multiple comparisons test. Data sets with two groups were analyzed using an unpaired t-test. Differences were considered statistically significant when p-value was <0.05.

Dexamethasone treatment of DCs has been reported to generate tolerogenic DCs (tDCs) (32). To generate iDCs, bone marrow was flushed from the long bones of the tibia and femur of DA rats and cultured in medium supplemented with GM-CSF, IL-4, and Dexa (for tDCs) as required (Figure S1A in Supplementary Material). Following isolation, cell surface characterization was performed using flow cytometry by gating on the CD11b/c population (Figure 1A). tDC generation did not result in any significant changes in cell size (Figure 1B, i) but the number of cells harvested from wells that were treated with Dexa was significantly lower than that of wells that were Dexa-free (Figure 1B, ii). This may be due to Dexa-induced apoptosis of the DCs, which has been reported by other groups (33). While lower numbers of cells were obtained from tDC wells, after harvesting and washing of the cells, no significant changes in viability was noted (Figure 1B, iii). We also analyzed the expression levels of the costimulatory molecules CD80/CD86 and the major histocompatibility complex class I and II molecules (MHCI/II) as an indicator of the maturation status of generated iDCs and tDCs (Figure 1C). The expression levels of CD80, CD86, MHC I, and MHC II indicate that the iDCs display a semi-mature phenotype. However, when the cells were treated with Dexa, a significant reduction in the expression level of MHC II was observed with no changes in MHC I (Figure 1C). To mature iDCs or tDC in vitro, LPS was added to the cultures (1 µg/mL) for 24 h. A significant increase in both CD80/CD86, MHC I and MHC II was noted. However, tDCs following LPS treatment showed significantly reduced expression levels of CD80/CD86 and MHC I/II molecules compared to stimulated iDCs indicating a phenotype that is maturation resistant. iDC and tDC populations were also assessed for expression of pro- and anti-inflammatory markers with and without Dexa-treatment by qRT-PCR (Figure 1D). Results indicate that LPS stimulation of iDCs leads to an increase in mRNA expression of pro-inflammatory molecules such IL-6, IL-12p40, and iNOS. In contrast, tDCs are less sensitive to TLR4 stimulation compared to mDCs, indicated by no observed increases in IL-6, IL-12-p40, and iNOS after LPS treatment. Higher levels of IDO mRNA, which is known as a marker in tolerogenic cells, is present in LPS-treated tDCs when compared to mDCs. Interestingly, IL-1β mRNA expression does not seem to be regulated by Dexa, as LPS stimulation leads to a profound increase, which cannot be blocked by Dexa. All together these data indicate that Dexa treatment of iDCs leads to the generation of a tolerogenic DC phenotype with reduced expression of markers of immunogenicity and reduced expression of pro-inflammatory molecules but increases in immunoregulatory molecules.

Changes in DC glycocalyx after induction of tolerogenic phenotype have not been investigated. To address this knowledge gap, lectin microarray profiling of proteins extracted from the membranes of iDCs and tDCs and lectin-coupled flow cytometry of intact iDCs and tDCs was undertaken.

Comparisons of all lectin microarray replicate profiles were made by unsupervised hierarchical clustering. This clustering approach revealed two major clusters with separation at 53% minimum similarity (Figure 2A). With the complete linkage method employed, two untreated iDC replicates were placed into the tDC group while only three of the iDC replicates, two from biological set 2 and one from set 5 (Figure 2A), showed outlier behavior and were excluded from the major cluster containing the balance of the iDC replicate data. However, the well-defined separation of the vast majority of the iDC and tDC replicates into two groups (Figure 2A, Group 1 and 2) supports the solidity of the subtle profile differences and also the high level of reproducibility for the lectin profiling method in distinguishing membrane glycoprotein samples from iDCs and tDCs.

Median values obtained from normalized lectin microarray profile data (n = 18) for iDCs and tDCs were broadly similar with only small, but significant, differences in intensities noted at a subset of the lectin panel (Figure 2B). The general profiles of tDC glycoproteins remained similar to those of iDCs across lectin features. Furthermore, the lectin profiles displayed no obvious signs of cell stress as evidenced by a lack of elevation of signals suggesting increased endoplasmic reticulum- and proximal Golgi-associated glycan structures (i.e., increased evidence of high mannose structures). However, SNA-I showed a consistent intensity increase with tDC surface glycoproteins (p = 2 × 10⁻¹⁰) relative to iDCs, which is in line with previous findings from our group (13). PrCA performed using the 15 lectins, which demonstrated p < 0.01 (SNA-II, BPA, PNA, DSA, LEL, SNA-I, RCA-I, CPA, ECA, LTA, UEA-I, EEA, GS-I-B4, MPA, and VRA) revealed a division of replicate lectin profiles dominated by distinct groups containing iDCs or tDCs with minimal overlap and further reinforced the ability of these lectins to distinguish untreated iDCs from tDCs (Figure 2C). In short, these lectin microarray profiles demonstrate that the glycocalyxes of the iDC and tDCs are distinct. These changes were validated using lectin-coupled flow cytometry. The increase in SNA-I binding suggests an increase in quantity or better accessibility to α2-6-linked with no significant change suggested for α2-3-linked Sia (MAL-II) confirmed lectin microarray findings (Figure 2D).

Sia has long been reported to be important in DC biology (28). Considering the dramatic increase observed after Dexa treatment confirmed by both flow cytometry and lectin microarray (Figures 2B–D), we cleaved Sia using neuraminidase to study phenotypical and functional changes upon removal. iDCs and tDCs were treated with neuraminidase (designated niDC and ntDC, respectively) and lectin binding profiles for SNA-I and MAL-II were analyzed using flow cytometry. Both niDCs and ntDCs showed a significant reduction in SNA-I binding intensities and trend decreases MAL-II binding intensities suggesting the successful removal of α2-6-linked and α2-3-linked Sia, respectively (Figure 3A, i–iv). Based on these results, we further investigated if the removal of Sia resulted in a detectable increase of the expression of MHC I, MHC II, CD80, and CD86 immunogenicity markers after treatment with neuraminidase. niDCs (Figure 3B, i) had small but significant increases in MHC II and CD86 expression when compared to iDCs. MHC I showed a trend increase in expression on niDCs compared to iDCs, and there was no change in CD80 expression after treatment with neuraminidase. ntDCs (Figure 3B, ii) displayed a significant increase in both MHC I and MHC II with no changes in CD80 and a trend increase in CD86 after neuraminidase treatment. niDC and ntDC populations were also assessed for expression of pro- and anti-inflammatory markers by qRT-PCR (Figure 3C). Although there was some sample-to-sample variation, our data indicate that neuraminidase treatment of iDCs leads to dramatic increases in pro-inflammatory mRNA expression of IL-6, IL-1β, iNOS, TNF-α, and IL-12-p40. However, ntDCs are protected from this strong increase in pro-inflammatory cytokine expression in the case of iNOS and IL-12-p40, but mRNA levels of IL-6, IL-1β, and TNF-α are increased. Interestingly, levels of anti-inflammatory IL-10 are lost after neuraminidase treatment in both iDCs and tDCs. In summary, these results indicate that neuraminidase treatment reduces Sia on the cell surface of both iDCs and tDCs and leads to the stimulation of pro-inflammatory cytokine mRNA expression, which can be largely inhibited by Dexa treatment.

Considering that the removal of Sia altered the immunogenic phenotype of both iDCs and tDCs, we further analyzed the effects of neuraminidase treatment on iDCs and tDCs through in vitro allogeneic coculture experiments. iDCs or tDCs from DA rats were treated with neuraminidase and cocultured with allogeneic lymphocytes. The immunogenic potential or the ability of niDCs and ntDCs to induce the proliferation and/or the activation of allogeneic lymphocytes was analyzed by T-cell proliferation assays (Figure 4A). Responder LEW rat T cells were analyzed based on their co-expression of CD3⁺CD4⁺ or CD3⁺CD8⁺ (Figure 4B). Proliferation of lymphocytes was measured using CellTrace^TM Violet (CTV) and activation of lymphocytes was measured using CD25 as an activation marker. DA iDCs (Figure 4C, i) and tDCs (Figure 4C, ii) did not induce an allogeneic response as indicated by a lack of changes in LEW CD3⁺CD4⁺ or CD3⁺CD8⁺ T cell proliferation when compared to unstimulated lymphocytes alone. Additionally, we observed no significant changes in CD3⁺CD4⁺CD25 or CD3⁺CD8⁺CD25 expression (data not shown) supporting our data on reduced immunogenicity of iDCs and tDCs. However, niDCs (Figure 4C, i) significantly stimulated both CD3⁺CD4⁺ and CD3⁺CD8⁺ T cell proliferation when compared to both unstimulated lymphocyte controls and iDCs. This indicates the importance of Sia in the maintenance of an iDCs phenotype. While ntDCs (Figure 4C, ii) show a trend increase to stimulate CD3⁺CD8⁺ T cells, there were no significant changes noted (Figure 4C). To eliminate the possibility of cell death as a potential cause of this increase in proliferation, we assessed cell death using Sytox Blue. We observed that iDCs have less cell death after neuraminidase treatment than tDCs (Figure S2 in Supplementary Material) enabling us to exclude this possibility. Finally, we investigated if niDCs and ntDCs can regulate the proliferation of stimulated T cells. LEW T cells were labeled with CTV, stimulated with CD3/CD28 labeled beads, and cocultured with niDCs and ntDCs (Figure 5A) and CD3⁺CD4⁺ and CD3⁺CD8⁺ proliferation was measured by flow cytometry. Neuraminidase treatment completely abrogates the T cell inhibitory effect of iDCs leading to full restoration of T cell proliferation (Figure 5A, i). Interestingly, Dexa treatment is not sufficient to enable iDCs to inhibit the proliferation of activated T cells as no differences were observed between tDCs and ntDCs (Figure 5B, ii). In summary, these data indicate that the removal of Sia from iDCs increases the immunogenicity by its ability to stimulate CD4 and CD8 T cell proliferation, which can be prevented by Dexa treatment. In contrast, neuraminidase treatment completely restores the proliferation of polyclonally activated T cells, which cannot be prevented by Dexa treatment.