Mice
Female C57BL/6 J wildtype (WT) mice (7–14 weeks at the beginning of the experiments) were purchased from Charles River (Sulzfeld, Germany). OT-I mice (8–14 weeks at the beginning of the experiments) producing CD8+ T cells that express a transgenic T cell receptor specific for the chicken ovalbumin (OVA) epitope SIINFEKL (OVA257-264) presented on MHC class I H–2 Kb and the congenic marker CD90.1 were bred and held in house. All mice were kept under specific pathogen-free conditions at the Central Animal Laboratory (Nijmegen, The Netherlands). Drinking water and standard laboratory food pellets were provided ad libitum.
Generation of BMDCs and OT-I T cell isolation
Bone marrow (BM) was harvested from the femurs and tibia of female C57BL/6 J mice. The femurs and tibia were soaked in 70% ethanol for 2 minutes (min) and washed in 1 × PBS. BM from the femurs and tibia was flushed out with RPMI 1640 medium (Gibco, Thermo Fisher Scientific) supplemented with 10% fetal calf serum (FCS; GE healthcare, Little Chalfont, UK), 1% UltraGlutamine (Lonza, Bazel, Switzerland), 0.1% β-mercapto-ethanol (Gibco) and 100 U/m penicillin G sodium and 100 μg/mL streptomycin (Pen/Strep) (Gibco). Single cell suspensions were obtained by passing the isolated BM through 100 µm cell strainers (Falcon, Corning Life Sciences, Tewksbury, MA, USA) followed by erythrocyte lysis for 2 min with cold ACK lysis buffer (150 mM NH4Cl, 10 mM KHCO3 and 0.1 mM EDTA). 4*106 cells were cultured for 7 days in 13 ml medium containing 20 ng/ml granulocyte–macrophage colony stimulating factor (GM-CSF; Peprotech, London, UK) in 10 cm petri dishes (VWR, Radnor, PA, USA) at 37 °C and 5% CO2 under humidified conditions. On day 3, the medium was replenished by adding 4 ml medium containing 37.2 ng/ml GM-CSF and on day 6 by adding 1 ml medium containing 158 ng/ml GM-CSF. BMDCs were harvested on day 7 for experiments. Single cells suspensions were prepared from spleens of OT-I mice through mechanical dissociation using 100 µm cell strainers (Falcon). Splenocytes were resuspended in 1 × PBS containing 1 mM EDTA and 2% FBS. CD8+ T cells were negatively isolated using the EasySep™ Mouse CD8+ T cell Isolation Kit (Stemcell Technologies) following the manufacturer’s protocol.
Reagents and antibodies
The sialic acid mimetic Ac53FaxNeu5Ac was synthesized as described previously [21, 23]. Carbo‐free blocking solution and biotinylated lectins MALII, SNA‐I, and PNA were purchased from Vector Laboratories, Inc. (Burlingame, CA, USA). Streptavidin‐PE was purchased from BD Pharmingen (Franklin Lakes, NJ, USA), eFluor 780 and 450 viability dyes from eBioscience, Inc. (San Diego, CA, USA), CellTrace™ Violet (PSBE) and CFSE Cell Proliferation Kits from Thermo Fisher Scientific. CpG ODN 1668 (‘5-TCCATGACGTTCCTGATGCT-3’) was purchased from Sigma Genosys (Haverhill, UK), lipopolysaccharide (LPS) (Escherichia coli O111:B4) from Sigma-Aldrich, and murine recombinant interleukin (IL)-2 from Immunotools. For flow cytometry experiments, the following antibodies were used: BioLegend: anti-I-A/I-E-BV510 (M5/114.15.2), anti-H-2 Kb/H-2Db-PE (28–8-6), anti-CD86-APC/Cy7 (GL-1), Anti-CD90.1-APC/Cy7 (OX-7). Antibodychain: anti-CD80-A488 (16-10A1), anti-CD40-PE (23/3), anti-CD11c-APC (N418), anti-CD11b-PerCP (M1/70). eBioscience: anti-CD274-PE/Cy7 (MIH5). BD: anti-CD8a-V450 (53–6.7).
Sialic acid blockade, sialidase treatment and TLR stimulation
On day 0, day 3, and day 6 of the BMDC culture, 250 µM Ac53FaxNeu5Ac or DMSO was added to the medium, except for the dose titration experiment, where a concentration between 0 and 500 µM of Ac53FaxNeu5Ac was used. Sialidase treatment was performed with differentiated BMDCs on day 7 by adding 250 mU/ml sialidase from Clostridium perfringens (Sigma-Aldrich) for 1 hour (h) at 37 °C. After incubation, the cells were washed thoroughly and used for experiments. For the TLR stimulation, 1*105 day 7 BMDCs were plated into 96-well round bottom plates (Corning) and stimulated with CpG (200 and 400 ng/ml) or LPS (1 and 10 ng/ml) for 18 h at 37 °C. After the stimulation, the cells were harvested and stained for flow cytometry analysis.
RNA sequencing and bioinformatics analysis
BMDCs differentiated for 7 days in the absence or presence of Ac53FaxNeu5Ac (250 µM) were subjected to sorting for CD11c+ cells (MACS, Miltenyi) according to the manufacturer’s instructions. RNA from CD11c+ cells was isolated using TRIzol (Thermo Fisher Scientific) following the manufacturer’s instructions. RNA seq was performed by BGI Genomics (Hong Kong). The number of aligned reads in the bam files provided by BGI were counted using feature count in the subread package (v. 1.5.3) and the reference genome Gencode GRCm38 (v.M15). All subsequent analysis was conducted in R (v. 3.6.1). After normalization (TMM: trimmed mean of M values), a differential gene expression analysis was performed using edgeR (v. 3.18.1). Significant differently expressed genes (DEGs) were distinguished by a false discovery rate (FDR) under 0.05. Gene ontology analysis was performed with clusterProfiler (v. 3.12.0) and org.Mm.eg.db (v. 3.8.2). In addition, the following dependent package versions were installed: DOSE (v. 3.10.2), AnnotationDbi (v. 1.46.1), IRanges (v. 2.18.3), S4Vectors (v. 0.22.1), BiocGenerics (v. 0.30.0), and Biobase (v. 2.44.0). Results were visualized using ggplot2 (v. 3.2.1).
Flow cytometry analysis
For lectin staining, the cells were first washed with 1 × carbo-free blocking solution. Next, the cells were incubated for 45 min at 4 °C with biotinylated MALII (5 µg/ml), SNA-I (1 µg/ml), or PNA (5 µg/ml) in 1 × carbo-free blocking solution supplemented with 1 mM MgCl2 and 1 mM CaCl2 (both from Merck). After incubation, the cells were washed with PBA (1 × PBS, 1% bovine serum albumin, 0.02% sodium azide), and incubated with Streptavidin-PE for 20 min at 4 °C. For antibody staining, the cells are harvested, washed in 1 × PBS and stained with eFluor 780 or 450 viability dye according to the manufacturer’s instructions. Fc receptors were blocked with anti-CD16/CD32 (2.4G2, BD) in PBA for 10 min at 4 °C. Afterwards, the cells were stained with fluorescent antibodies in PBA at 4 °C for 20 min, and washed three times with PBA. Samples were acquired with a CytoFLEX LX Flow Cytometer (Beckman Coulter), BD FACSVerse™ flow cytometer (BD Bioscience), Gallios (Beckman Coulter) or FACS Cyan (Beckman Coulter), respectively. Data analysis was performed using FlowJo software (Tree Star, Ashland, OR, USA).
OT-I proliferation assay
BMDCs generated in the presence or absence of Ac53FaxNeu5Ac (250 µM) were washed in 1 × PBS and pulsed with OVA protein (Endograde, Hyglos GmbH, Germany) for 3 h or OVA H-2 Kb peptide (257–264, AS-60193, Tebu-bio) for 1 h at 37 °C. After the incubation, the BMDCs were washed three times, and 50.000 cells were seeded per well into a round bottom plate in culture medium. The CD8+ OT-I T cells were labeled with 3 µM CFSE according to the manufacturer’s instructions. 50.000 CFSE-labeled CD8+ OT-I T cells were added to the BMDCs (1:1 ratio) in culture medium. The co-cultures were incubated for 3 days at 37 °C in the presence of 0 or 10 ng/ml IL-2. After 3 days, the cells were analyzed by flow cytometry. Interferon gamma (IFNγ) levels in the supernatants were analyzed using an IFNγ ELISA (Thermo Fisher Scientific) following the manufacturer’s protocol.
OVA uptake and degradation assay
Control and Ac53FaxNeu5Ac (250 µM) treated BMDCs were washed with 1 × PBS and seeded into round bottom plates at a density of 2*105 cells/well in culture medium. The BMDCs were pulsed with 100 ng/ml Alexa Fluor 647-OVA to follow uptake or 1 µg/ml DQ-OVA to follow degradation (both from Thermo Fisher Scientific), respectively, washed and cultured at 37 °C. At different timepoints, the cells were washed three times and the fluorescence was determined by flow cytometry.
BMDC:OT-I T cell clustering assay
Control and Ac53FaxNeu5Ac (250 µM) treated BMDCs were labeled with 12 µM CellTrace Violet dye (Thermo Fisher Scientific) according to the manufacturer’s instructions. Afterwards, the BMDCs were pulsed with 0 or 1 ng/ml OVA peptide for 1 h at 37 °C. The isolated CD8+ OT-I T cells were labeled with 1 µM CFSE according to the manufacturer’s instructions. In total 50.000 BMDCs and 50.000 OT-I T cells were co-cultured in Falcon round-bottom polystyrene test tubes for 45 min at 37 °C. The cells were fixed with 2% PFA for 10 min at RT and subsequently analyzed using a CytoFlex LX flow cytometer.
Cell avidity analysis
Cell–cell interaction strength between BMDCs and CD8+ T cells was analyzed using a z-Movi® Cell Avidity Analyzer (LUMICKS, Amsterdam, The Netherlands). BMDCs were allowed to adhere to a microfluidic z-Movi chip (LUMICKS) for 30 min after which CD8+ T cells were added and co-incubated with the BMDCs for indicated time-periods, before assessing cell avidity using acoustic force. Briefly, the chips were coated with poly-L-lysine (Sigma-Aldrich) for 10 min and air-dried for 60 min at 37 °C. Control and Ac53FaxNeu5Ac treated BMDCs were pulsed with 1 ng/ml OVA H-2 Kb peptide, 1 ng/ml irrelevant murine HPV peptide (RAHYNIVTF, kindly gifted by Thorbald van Hall, Leiden University Medical Center, The Netherlands) or medium for 1 h at 37 °C. After washing thoroughly, 3–5*106 BMDCs were flushed into the poly-L-lysine coated chip and incubated for 30 min at 37 °C. The chips were placed onto the z-Movi Cell Avidity Analyzer, where experiments were performed at 37 °C. To ensure attachment of the BMDC monolayer to the surface of the chip, an initial constant force of 1000 pN (as calibrated on 10 μm polystyrene beads) was applied. CD8+ OT-I T cells were fluorescently labeled with CellTrace Far Red dye (Thermo Fisher Scientific) according to manufacturer’s protocol and 1–3*105 cells were flushed into the z-Movi Chip. CD8+ T cells were allowed to interact with the BMDC monolayer for indicated time-periods. Subsequently, a linear force ramp was applied from 0 to 1500 pN (as calibrated for 10 μm polystyrene beads) for 3 min 45 s (6.7 pN/s). Avidity runs were analyzed using Oceon Software (LUMICKS).
Statistical analysis
Statistical significance was calculated by performing a t test or a one-way analysis of variance (ANOVA) followed by Bonferroni’s correction using Prism 8 software (GraphPad, Inc., La Jolla, CA). P values < 0.05 were considered significant. Significance is shown as: ns. > 0.05, *P < 0.05, **P < 0.01, ***P < 0.001.