Anti cd86

BMDCs were stimulated with Rv2005c (10 μg/mL) for 24 h. The stimulated cells were harvested and washed with PBS. The BMDCs were stained with PE-conjugated anti-CD40, anti-CD80, anti-CD86, anti-MHC class I and anti-MHC class II and with FITC-conjugated CD11c (eBioscience) for 30 min at 4 °C. The cells were washed with PBS and suspended in 250 μL PBS. The fluorescence was measured by flow cytometry, and the data were processed using FlowJo software.

M1 macrophages (3 × 10⁵) were incubated with 7.5 × 10⁹ SF-derived exosomes for 24 hours or stimulated with 150 UI/ml IFNγ (Peprotech) for 12 hours and then with 10 ng/ml LPS (Sigma-Aldrich) for 24 hours. The stimulation with IFNγ/LPS was used as a positive control for these experiments. The expression level of the costimulatory molecules CD80 and CD86 was evaluated by flow cytometry using the anti-CD80 (eBiosciences) and anti-CD86 (eBiosciences) antibodies. Flow cytometry was carried out on the FACSCalibur (Becton Dickson) and data analysed using Flowing software. Supernatants were also harvested, centrifuged for 10 minutes at 14,000g, and cytokine/chemokine and MMP concentrations were quantified with a magnetic bead-based multiplex assay (Bio-plex Assay, Bio-Rad Laboratories). To ensure that cytokines were not previously present in our exosome preparation as contaminants, their presence was investigated directly in SF-derived isolated exosomes by magnetic bead-based multiplex assay.

Primary human B cells were purchased from BioSpecialty and were used to study CD40 signaling. Cells were cultured in RPMI with 10% heat-inactivated serum, 2 mM L-glutamine, 1 mM sodium pyruvate and 100μg/ml Pen Strep. To measure agonist activity, the cells were plated at 0.1 million per well of a 96-well plate and dilutions of CD40 antibodies were incubated with the cells for 2 days. Cells were then harvested and CD86 upregulation was measured by FACS. To assess antagonist activity, B cells were incubated with CD40L-expressing Jurkat cells (ATCC CRL-10915) at 2:1 ratio per well of 96-well plate in the presence of CD40 antibodies. Cells were harvested 2 days later and stained for CD19, CD20 (B cell marker) and CD86. Briefly, cells were washed by PBS and incubated with antibodies in PBS with 2% serum for 20 min on ice. The cells were then washed by PBS and analyzed by a BD LSRFortessa™ cell analyzer. Anti-CD19 and anti-CD20 were purchased from BD bioscience and anti-CD86 was from eBiosciences. The ability of the anti-CD40 antibodies to inhibit the interaction between CD40 and its receptor CD40L was compared to an IgG control.

Tumor tissues were minced and incubated with collagenase type II (1 mg/ml, Thermo Fisher) at 37°C for 40 min to obtain a single-cell suspension. For the staining of extracellular target proteins, 1 × 10⁶ cells were first incubated in a mixture of PBS, 1% FBS, and anti-CD16/32 antibody (BioLegend, #101301) to block non-specific binding and then labeled with the indicated antibodies at room temperature for 30 min. Fluorescence-activated cell sorting (FACS) was performed using a Beckman Coulter Gallios flow cytometer (USA), and the results were analyzed using FlowJo software version 10.0.7 (TreeStar). The following fluorochrome-coupled antibodies were used in the experiment: anti-CD45 (#103115), anti-CD206 (#141711), anti-CD3 (#100235), anti-CD4 (#100203), anti-CD8 (#100733), anti-NK1.1 (#108709), and anti-Gr1 (#108425) (all from BioLegend); and anti-CD11b (#12-0112), anti-F4/80 (#11-4801), anti-CD11c (25–0114), anti-MHCII (#17-5321), and anti-CD86 (#17-0862) (all from eBioscience) antibodies; the fixable viability dye eFluor 506 was from eBioscience (#65-0866).

Expression of CD90, CD105, CD45, and CD11b/c (eBioscience, USA) on MSCs surface was assessed by flow cytometry (Becton Dickinson, USA). The expression of CD11b/c, CD80, CD83, CD86, and MHC II (eBioscience, USA) on DCs surface was identified using flow cytometry. Each group of DCs were collected on the sixth day. After washing with PBS, the cells were immunolabeled with monoclonal anti-CD80, anti-CD83, anti-CD86, and anti-MHC II (eBioscience, USA) antibodies, as well as their isotype control antibodies. After that they were incubated in darkness at 4°C for 30 min and were detected using FACSCalibur flow cytometer.

Immature DCs (10⁷) were stimulated for 6 h with LPS (1 µg/ml E. coli strain O111:B4, Calbiotech Merck) or LPS in combination with IFN-γ (0.02 µg/ml, BD Pharmingen) with or without Dex (10⁻⁸ M, Sigma-Aldrich) for 20 min before adding other reagents. To monitor maturation, DCs (10⁵) were stained with 5 µl of mix including anti-MHC-I, anti-MHC-II, anti-CD11b, anti-CD11c, anti-CD80, anti-CD83 and anti-CD86 (all from eBioscience, Austria).
T-cells were stained with anti-CD3, anti-CD4, anti-CD8, anti-CD25 and anti-Vα2 TCR for OT-I and OT-II mice (eBioscience). Cell viability was analysed with DAPI (Sigma-Aldrich). Apoptosis was measured with Annexin V (BD Pharmingen). CFSE (7 µM, Invitrogen) or Cell Proliferation Dye eFluor 670 (CPD, 5 µM, eBioscience) were used to detect proliferation. Flow cytometry was done on an LSR II (BD Pharmingen). Data were analysed by FlowJo (Version 9.6.2 Treestar). The difference in apoptosis induction was calculated using absolute cell number, determined with BD Trucount tubes.
This method is based on lyophilized pellet, containing a known number of fluorescent beads, which dissolves once the monoclonal antibody reagent is added. Absolute numbers (cells/µl) of positive cells in the sample are calculated following the equation: number of cell events/number of bead events x Trucount bead concentration.

Single cells prepared from the uterus were treated
with antibody against CD16/CD32 (anti-Fcγ receptor
III/ II antibody) to avoid non-specific antibody binding
through Fc receptors blockage. Cells were then washed
twice with ice-cold PBS (pH=7.2) and stained with PEconjugated
hamster anti-mouse CD11c and one of the
APC-conjugated monoclonal antibodies (anti-MHCII,
anti-CD86, anti-CD11b, and anti-CD40) and APCCy7-
conjugated antibody (anti-CD45) (all antibodies
obtained from eBioscience, San Diego, USA). Cells were
subsequently analyzed by flow cytometry (FACSCanto
II, BD, San Jose, CA, USA) and the obtained data were
analyzed using the FlowJo software (version 6.07). The
uterine cells were selected on dot plots of side and forward
scatters. CD45-positive cells as uterine leukocytes were
gated and the frequency of CD11c-positive cells (mouse
uDCs) was evaluated in uterine leukocyte population
(Fig .1). The expression of the DC lineage marker (CD11b)
and co-stimulatory molecules (CD40, CD86, and MHCII)
were assessed on CD11c⁺ cells.