Generation of Monocyte-Derived Dendritic Cells with Differing Sialylated Phenotypes

The scope of the Glycoimmunology Group research is studying the role of glycans in immunology in human health and disease. This research aims to disentangle biological mechanisms moderated by glycosylations and to discover novel glycan-based therapies for cancer and congenital disorders of glycosylation. Our focus has been on sialylated glycans that affect the function of relevant cell surface molecules, particularly during response.

We developed technologies to alter sialic acid contents and a multimodal platform for high-throughput development of glycan binding proteins with potential clinical applications. To advance the research in the field, we use several technologies such as glycan profiling, selecting or antibody staining, mass spectrometry and immunohistochemistry, in vitro assays with cell lines as disease models, sialic acid content manipulations through enzymatic and metabolic engineering. The current experimental challenges relate to understanding the complex interactions between sialoglycans and lectins.

They are mechanisms and functional consequences in several immune responses. Sialic acid modulates the potency of dendritic cells and MHC-I turnover. This discovery pinpointed new biological and pathological mechanisms and developed innovative therapies.

It also identified novel immune pathways and approaches for their therapeutic modulation. One of the issues in ex vivo production of human monocyte-derived dendritic cells for therapeutic uses is their inability to mature fully. This protocol shows that disease maturation can be achieved by enzymatically treating the cell surface with sialidases while maintaining cell viability.

This method is cost-effective and time-saving. Compared to sialic acid metabolic inhibitors, sialidase treatment offers a rapid, effective, and resilient method of removing cell surface sialic acids, while maintaining cell viability. Understanding the role of sialic acid-containing glycans generates unprecedented awareness the importance of this glycan in cancer, and will foster the identification of new disease mechanisms and new therapeutic strategies.

Begin isolating peripheral blood mononuclear cells by accessing the human buffy coat and transferring seven milliliters into a sterile 15-milliliters conical tube. Add six milliliters of PBS solution for a preliminary wash. Centrifuge the tube for 10 minutes at 1, 100 G in a centrifuge with a swing rotor and brake off at room temperature.

Collect the leukocyte suspension using a Pasteur pipette and transfer it to a new sterile 15-milliliter conical tube. Add PBS solution to the leukocyte suspension until it reaches 10 milliliters and mix it gently by pipetting up and down. Next, prepare the density gradient solution by placing three milliliters of density gradient medium into a new sterile 15-milliliters conical tube.

Allow it to reach room temperature. Slowly add five milliliters of diluted leukocyte suspension on the conical tube walls containing the density gradient medium to create a 5:3 density gradient separation. Avoid disturbing the medium.

Proceed to gradient separation by centrifuging the suspension present in the density gradient medium using a centrifuge with a swing rotor and the brake off. Next, carefully transfer up to 25 milliliters of the thin layer of PBMCs to a new 50-milliliters conical tube filled with PBS using a Pasteur pipette and mix it well. To remove residual cells and debris, centrifuge the samples at room temperature for 10 minutes at 600 G with a normal brake.

Discard the supernatant and fill the sample to 10 milliliters using PBS. Take an aliquot to count the cells. To remove the platelets, centrifuge them with a normal brake and carefully invert the tube to discard the supernatant.

For monocyte CD14+isolation using magnetic activated cell sorting, resuspend the cell pellet in the microbeads buffer and CD14 immunomagnetic beads. Incubate the cell suspension for 15 minutes at four degrees Celsius. To remove the unbound beads, add one to two milliliters of microbeads buffer per one times 10 to the seventh cells before centrifuging suspension at room temperature for 10 minutes at 600 G.Then, carefully invert the tube to discard the supernatant.

Prepare the LS column and place it on the magnet before use. Rinse it with three milliliters of microbeads buffer and immediately resuspend the cell pellet in 500 microliters of microbeads buffer per one times 10 to the eighth cells. Add the cell suspension to the LS column inlet and collect the negative cell fraction in a 15-milliliter conical tube below the column outlet.

Wash the column three times with three milliliters of microbead buffer. After the final wash, remove the column from the magnet and place it on a sterile 15-milliliter conical tube. Pipette five milliliters of microbeads buffer into the column inlet.

Immediately insert the syringe plunger filled with the target cells into the column inlet and dispense the cells in the column. Then, centrifuge CD14-and CD14+cell fractions and discard the supernatant. For monocyte differentiation into human monocyte-derived dendritic cells, prepare a cell suspension containing 1.3 times 10 to the sixth cells per milliliter by adding the appropriate volume of differentiation medium to the CD14+cells.

Resuspend the monocytes by pipetting with a Pasteur pipette. After plating the 1.3 times 10 to the sixth cells per milliliter suspension per well of a 24-well plate, incubate in a culture incubator at 37 degrees Celsius with 5%carbon dioxide. Change the culture medium and supplement it with fresh cytokines every two to three days.

To collect the differentiated cells, transfer the entire cell suspension to a sterile conical tube using a micropipette and wash the culture flask twice with PBS. After centrifuging the conical tubes at room temperature for 10 minutes at 180 G, resuspend the pellet in the appropriate medium or buffer for the experimental setup. During the monocyte differentiation, interleukin-4 and granulocyte macrophage colony stimulating factor stimulation changed the cell phenotype.

Data showed that human monocyte-derived dendritic cells lost the of surface marker CD14, mainly expressed by monocytes, and gained significant expression of CD1a, a marker expressed by human dendritic cells. Human monocyte-derived dendritic cells also obtain higher expression of MHC-II, HLA-DR, an antigen-presenting molecule expressed by human dendritic cells and other antigen-presenting cells. Collect approximately 10 times 10 to the sixth human monocyte-derived dendritic cells differentiated from the monocytes from the 10 wells of the 24-well plate, having 1.3 times 10 to the sixth cells per well.

Transfer them to a new sterile 15-milliliter conical tube. After centrifuging the cells at 300 G for five to seven minutes at room temperature using the normal brake, discard the supernatant to remove dead cells and debris. Add 10 milliliters of RPMI 1640 medium containing 11.1-millimolar glucose to the tube.

After centrifuging at room temperature for four minutes at 300 G using the normal brake, discard the supernatant again. Add two milliliters of RPMI 1640 medium to the cell pellet and divide one milliliter of the cell suspension into two new sterile microtubes, labeled as number one and number two. To microtube one, add 500 milli-units of sialidase from Clostridium perfringens.

Incubate both tubes for 60 minutes at 37 degrees Celsius. After incubation, transfer the cells from both microtubes into corresponding new 15-milliliter conical tubes, labeled as number one and number two. Then, add approximately four milliliters of complete RPMI 1640 medium containing 10%FBS to each conical tube.

Centrifuge the tubes at room temperature for four minutes at 300 G using the normal brake and add five milliliters of complete RPMI 1640 medium to the pellet. Plate one milliliter cells per well. The effectiveness of sialidase treatment for reducing the sialic acid content on the surface of human monocyte-derived dendritic cells was evaluated by flow cytometry and confocal microscopy.

Sialidase treatment significantly decreased Maackia amurensis lectin and Sambucus nigra lectin binding, while increasing peanut agglutinin lectin staining. The decrease in Sambucus nigra lectin staining after sialidase treatment showed a significantly reduced Sambucus nigra lectin staining at the cell surface. Begin collecting a new sample of the cells of interest for antibody staining by washing the cells at room temperature for five minutes at 300 G with normal brake.

Distribute the cells into microtubes. To perform the staining using the desired antibodies, incubate the fluorescence conjugated antibodies in the dark for 15 minutes at room temperature. Wash the cells with one milliliter of PBS before centrifuging for five minutes at 300 G at room temperature.

Add up to 100 microliters of PBS to all the microtubes. Then, resuspend the cells in 300 microliters of 2%paraformaldehyde, and store the tubes in the dark at four degrees Celsius until flow cytometry. A significant increase was observed in the expression of the antigen-presenting molecules MHC-I and MHC-II and the expression of the CD80 and CD86 co-stimulatory molecules due to sialidase treatment.