Anti cd3 pe

Cells were stained with 7-AAD viability dye, anti-CD3 eFluor450, anti-CD4 PE-eFluor 647 (eBioscience), anti-CD4 electron coupled dye (Beckmann Coulter), anti-CD4 fluorescein isothiocyanate (FITC), anti-CD3 PE, anti-CD3 APC-Cy7, anti-CD8 PE, anti-CD8 APC-Cy7, anti-CD25 PECy7, anti-CD127 PE, and anti-CD27 FITC (BD), anti-CD45RA FITC, FOXP3 Alexa Fluor 647 (BioLegend) specific antibodies. FOXP3 intracellular staining was performed using FOXP3 staining buffers (eBioscience). Data were acquired using a fluorescence activated cell sorting (FACS)CantoII and analysed using FACSDiva software (BD) and Flojo software (Treestar).

FCM assay was conducted as we previously reported with several modifications [2 (link), 15 (link)]. In details, the unstimulated MNCs (day 0) and the MNC-derived cells (day 7, day10, day 14) were harvested by centrifugation at 1200 rpm for 5 min at room temperature (RT) and washed by 1 × PBS for twice. Then, the cells were resuspended in phosphate buffer solution (PBS) contained 2% fetal bovine serum (FBS) and labelled with the fluorescence conjugated antibodies including anti-CD3-PE, anti-CD3-FITC, anti-CD4-PE, anti-CD8-PE-Cy7, anti-CD56-APC, anti-CD16-FITC, anti-NKG2D-Percp5.5, anti-NKp44-APC-Cy7, anti-NKp46-PE-Cy7, anti-NKG2A-PE, anti-CD107a-PE, 7-AAD, PI or Annexin V-FITC (BD Biosci, USA) in dark for 30 min. Finally, the cells were washed by 1 × PBS for twice and turned to FACS Canto II (BD Biosci, USA) flow cytometer for detection and FlowJo 10.0 software (Tree Star, USA) for analysis. The detailed information of the indicated antibodies was listed in Additional file 1: Table S2.

IFN-γ and TNF-α production by NK cells was evaluated in all samples available for phenotypic analysis after overnight stimulation with or without IL-12 and IL-18 (5 ng/mL) in the presence of 10 µg/mL of brefeldin A (BFA) added during the last 3 h of culture. Surface staining with anti-CD3-PE, anti-CD56-PE-CF594, and anti-CD16-FITC (BD Bioscience, Franklin Lakes, NJ, USA) was performed. Then, cells were fixed with medium A reagent and permeabilized with medium B reagent (Nordic Mubio, Lifespan Biosciences, Seattle, WA, USA) in accordance with manufacturer’s instructions. Cytokine determinations were performed by intracellular cytokine staining (ICS) with anti-IFN-γ-PerCp-Cy5.5 (BioLegend, San Diego, CA, USA) and anti-TNF-α-APC (BioLegend, San Diego, CA, USA) monoclonal antibodies and analyzed by flow cytometry. The CD107a degranulation assay was performed to assess the cytotoxic potential. Cells were stimulated overnight with IL-12 and IL-18 (as above), then incubated with K562 target cells for the last 4 h in the presence of BFA and anti-CD107a-PE-Cy7 (BD Bioscience, Franklin Lakes, NJ, USA).
Data are expressed as the difference between the percentage of cytokine or CD107a-positive⁺ NK cells in the stimulated and unstimulated samples.

To stain Ramos B cells for BCR and CD22 expression, 200,000 cells were used for each condition. Cells were washed with washing buffer (PBA/PBS, 0.5% BSA, 0.02% NaN3) and incubated Fixation buffer (BioLegend, Cat. No. 420801) diluted 1:1 with washing buffer for 15–20 min. Cells were washed twice with washing buffer and incubated with a mix of Abs for 30 min in the dark. Cells were washed 3 times with wash buffer and taken up in wash buffer and stored at 4 °C until measuring time. Staining Abs used were anti-IgG Fc PE (Thermo Fisher; Cat. No. 12-4998-82) and anti-CD22 APC (BD Biosciences; Cat. No. 562860); isotype control Abs used were anti-CD3 PE (BD Biosciences; Cat No. 345765) and anti-Ig Kappa LC APC (BioLegend; Cat No. 316509).BCR

B-cell receptor

CDR

Complementarity Determining Region

FR

Framework

GBP

Glycan-Binding Partner

Ig

Immunoglobulin

IP

Immunoprecipitation

Lat-A -Latrunculin-ALC

Liquid chromatography

mAb

Monoclonal antibody

MS

Mass spectrometry

NANA

N-Acetylneuraminic acid

NG

Non-glycosylated

PAL

Photoaffinity labeling

PLA

Proximity Ligation Assay

PV

Pervanadate

RA

Rheumatoid arthritis

SIGLEC

Sialic acid-binding, immunoglobulin-type lectin

SNA

Sambucus Nigra Agglutinin

ST6GAL

β-Galactoside α2–6 Sialyltransferase

UV

Ultraviolet

VDG

Variable domain glycan

WT

Wild type

20 μl of peripheral blood were collected from the tail vein and collected in FACS buffer. Splenocytes were prepared by grinding spleens between frosted-end microscopic slides in petri dishes containing RPMI medium supplemented with 5% FCS. For analysis of pulmonary cells, mouse lungs were perfused through the pulmonary artery with 5 ml PBS. The lungs were excised, cut into small pieces and incubated for 60 min at 37°C in RPMI medium supplemented with 5% FCS and 200 μg/ml Collagenase D (Roche Diagnostics, Risch, Switzerland) and 10 μg/ml DNAse I (Sigma, Deisenhofen, Germany). The suspension was resuspended with a Pasteur pipette every 15 min. The reaction was stopped by 5 min incubation at 37°C with 5 mM EDTA. Lung cells were then mechanically separated in 100 μm mesh sized cell strainers.
Erythrocytes were lysed with Tris-buffered 0.15 M ammonium chloride for 1 min (lung) or 7 min (blood, spleen). Unspecific binding sites were blocked with anti-FcγR at 4°C for 10 min. The cells were extracellularly stained with anti-CD4-FITC, anti-CD4-APC, anti-B220-APC, anti-CD3-PE, anti-CD8-PerCP-Cy5.5, or anti-CD62L-FITC (all from BD, Heidelberg, Germany), respectively, for 1 h at 4°C. Cells were analyzed on an Accuri C6 cytometer or fixed in 1% PFA for 30 min and analyzed on an LSR-II cytometer (both BD). Absolute cell numbers of blood cell populations were measured with the C6 cytometer.