maturation profiling of human monocyte-derived dendritic cells (mo-dcs)

De-Sialylated Monocyte-Derived-DCs from Isolated PBMCs Using Sialidase Treatment

Sialic acid-carrying glycans (sialoglycans) have gained significant interest due to their immunomodulatory role. The monosaccharide sialic acid, which is most prevalent in humans in the form of N-acetylneuraminic acid, presents fundamental ligands for lectins with a recognized role in immunology, such as Selectins and Siglecs. These lectins recognize sialoglycans either on the same cell (cis) or on different cells (trans) and play a significant role in host-pathogen interactions and various physiological and pathological cellular activities1,2,3. Furthermore, since sialic acid occupies the terminal positions of cell surface glycoconjugates, it can conceal the underlying structures, thus inhibiting cell-to-cell contact via non-specific repulsive effects or by obstructing detection by other lectins4. The activity of a variety of sialyltransferases (that transfer sialic acids) and of sialidases (that cleave sialic acid bonds) within the cell determines the amount of sialic acid present at the surface. In addition, soluble sialyltransferases and sialidases expressed by the host or pathogens can extrinsically modify the amount of sialic acid on the cell surface5,6.
Aberrant sialylation is a feature of several pathological conditions. In autoimmune diseases, hyposialylation can contribute to unrestrained immune activation and organ damage, since sialic acid helps to discriminate self-antigens and regulate inflammatory responses7. Conversely, hypersialylation results in the over-expression of sialoglycans, such as sialyl-Tn, sialyl-Lewis antigens, polysialic acid, and gangliosides, which constitutes a hallmark of some cancers8,9. Hypersialylation also depends on increased expression of specific enzymes such as N-acetylglucosaminyltransferase (GNT-V), which generates hypersialylated tri- and/or tetra-antennary N-linked glycans, which have been associated with cancer growth and metastasis10. The sialic acid content also regulates protein stability and function, which are key for the role of relevant oncogenic players11. Therefore, increased sialylation can facilitate tumor development, metastasis, drug resistance, and immune evasion. Moreover, the upregulation of sialoglycans enables tumors to interact with inhibitory Siglec receptors on immune cells and avoid immune surveillance. For that reason, sialoglycans are now considered to be glyco-immune checkpoints and attractive therapeutic targets. For instance, inhibitors of the Siglec-immune axis are already in early clinical trials, since the immune cell receptor Siglec (sialic-acid-binding ImmunoGlobulin-like LECtin) plays an immune-inhibitory role12.
Enzymes have been used to modulate the glycan profile as tools for study or for therapeutic strategies13,14. Sialidase has been employed to alter cancer cell malignancy since sialylated glycans such as sialyl Lewis X are critical for cell migration and cancer metastasis15. At the same time, sialidase inhibitors, which impede sialic acid cleavage, have reached clinics for treating sialic acid-dependent viral infections16. Recently, sialic acid modulation has gained further interest due to the critical role of sialic acids as ligands in the Siglec-immune axis, meaning they offer novel means to reduce cancer escape from immune responses. This interest was further strengthened by the 2022 Nobel laureate Bertozzi and her team&#39;s contribution of several strategies that selectively cleave diverse sialoglycans and improve anti-cancer immune responses17. Thus, sialidase-based strategies represent a promising modality for glyco-immune checkpoint therapy. The glycophenotype of cells of the immune system is dependent on the type of cell and their activation status. Regarding T-cells, glycans have a key role in the pathophysiological steps of T-cell development and thymocyte selection, T-cell activity, differentiation, and proliferation18. For instance, polylactosamine on glycoproteins influences basal levels of B lymphocytes and T lymphocytes and macrophage activation19. In macrophages, distinct glycan expression patterns have an important role in macrophage recruitment to the tumor microenvironment (TME)20. Hence, the expression of O-linked and N-linked glycans by immune cells could be used as potential glycobiomarkers for therapeutic approaches in the treatment of cancer and autoimmune diseases.
Dendritic cells (DCs) are specific antigen-presenting cells with a unique capacity to trigger immune responses, such as anti-cancer immunity21. DCs must undergo an upregulation of their antigen-presenting MHC molecules to present antigens to T-cells (signal 1), co-stimulatory molecules to activate T-cells (signal 2) and pro-inflammatory cytokines, such as IL-12, to trigger type 1 helper T-cell proliferation (signal 3)22. The resulting immune profile is tightly regulated, and checkpoints are essential for preventing healthy cells from being attacked. Since DCs can stimulate various immune responses against tumor cells, they are used as cell-based vaccines, and a sizable number of clinical studies have demonstrated their potential benefits23,24. After the FDA approved the first DC-based vaccine in 201025,26, many other DC-based vaccines have been developed. DC-based vaccines are mainly produced ex vivo and administered to patients to elicit immune responses against tumors. However, insufficient or brief maturation is currently one of the factors limiting the clinical efficacy of DCs and means expensive cytokine cocktails must be used. Without adequate maturation, DCs cannot activate T-cells in clinical circumstances. Instead, the DCs express immune checkpoints and trigger a tolerogenic immune response that prevents cytotoxic T-cells from acting against tumor cells.
Human DCs have heavily sialylated surfaces, and this sialylation decreases upon maturation and during an overall immune response27. The maturation of DCs can be induced by eliminating these sialic acids with sialidase. Desialylation greatly upregulates various cytokines, including IL-12, due to the translocation of the NF-kB transcription factor to the nucleus6,28. In addition, desialylation improves antigen cross-presentation through MHC-I and anti-tumour immune responses29,30. Accordingly, the knockout of the sialyltransferases ST3Gal.l and ST6Gal.l, which have a major role in DC sialylation, generates a more mature phenotype in murine DCs31.
Sialidase treatment provides a method for stimulating all aspects of DC maturation, including increased antigen presentation, increased expression of co-stimulatory molecules, and increased cytokine production, to address the shortcomings mentioned above and enable DCs to elicit effective responses. This article presents a procedure to obtain viable desialylated human DCs through the use of a bacterial sialidase. De-sialylated DCs show an improved maturation profile and can be used as cell models to boost anti-tumour immune responses in vitro. The DCs are obtained from blood monocytes, which are then differentiated in vitro in the presence of the cytokine interleukin-4 (IL-4) and granulocyte macrophage colony-stimulating factor (GM-CSF). This work also describes lectin-based methods to analyze sialic acid at the cell surface and methods to immunophenotype the DC maturation level. The procedure described here can be used to desialylate other cell types, thus providing an approach to investigate the role of sialoglycans, which are vital glyco-immune checkpoints and relevant in immunomodulation.

Author Spotlight: Dendritic Cells Maturation Using Sialidases-Based Enzymatic Treatment of the Cell Surface 

Generation of Monocyte-Derived Dendritic Cells with Differing Sialylated Phenotypes

The scope of the glycoimmunology group research is studying the role of glycan immunology in human health and disease. This research aims to disentangle biological mechanisms modulated 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 molecules, particularly during response.We developed technologies to alter sialic acid contents and a multimodal platform for high-throughput dissolvement 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 sialylated glycans and lectins.There are mechanisms and functional consequences in several immune responses. The sialic acid modulates the potency of dendritic cells and MHC1 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 DCs 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 method of removing cell surface sialic acids while maintaining cell viability. Understanding the role of sialic acid content in glycans generates some precedent awareness of the importance of this glycan in cancer and will foster the identification of new disease mechanisms and neuropath strategies.

Watch this Scientific Journal Video about Maturation Profiling of Human Monocyte-Derived Dendritic Cells Mo-DCs at JoVE.com

Maturation Profiling of Human Monocyte-Derived Dendritic Cells (Mo-DCs)  

Maturation Profiling of Human Monocyte-Derived Dendritic Cells (mo-DCs)

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 break. Distribute the cells into micro tubes. 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 micro tubes. 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.

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Research

JoVE Journal

Immunology and Infection

411 Views.  Universidade NOVA de Lisboa. Iniziare a raccogliere un nuovo campione delle cellule di interesse per la colorazione degli anticorpi lavando le cellule a temperatura ambiente per cinque minuti a 300 G con una pausa normale. Distribuire le cellule in microtubi. Per eseguire la colorazione utilizzando gli anticorpi desiderati, incubare gli anticorpi coniugati a fluorescenza al buio per 15 minuti a temperatura ambiente.Lavare le cellule con un millilitro di PBS prima di centrifugarle per cinque minuti a 300 G a temper...

Video: Maturation Profiling of Human Monocyte-Derived Dendritic Cells (Mo-DCs)  

Sialic acids are negatively charged monosaccharides typically found at the termini of cell surface glycans. Due to their hydrophilicity and biophysical characteristics, they are involved in numerous biological processes, such as modulation of the immune response, recognition of self and non-self antigens, carbohydrate-protein interactions, etc. The cellular content of sialic acid is regulated by sialidase, which catalyzes the removal of sialic acid residues. Several studies have shown that sialo-glycans are critical in monitoring immune surveillance by engaging with cis and trans inhibitory Siglec receptors on immune cells. Likewise, glyco-immune checkpoints in cancer are becoming crucial targets for developing immunotherapies. Additionally, dendritic cells (DCs) are envisioned as an important component in immunotherapies, especially in cancer research, due to their unique role as professional antigen-presenting cells (APC) and their capacity to trigger adaptive immune responses and generate immunologic memory. Nevertheless, the function of DCs is dependent on their full maturation. Immature DCs have an opposing function to mature DCs and a high sialic acid content, which further dampens their maturation level. This downregulates the ability of immature DCs to activate T-cells, leading to a compromised immune response. Consequently, removing sialic acid from the cell surface of human DCs induces their maturation, thus increasing the expression of MHC molecules and antigen presentation. In addition, it can restore the expression of co-stimulatory molecules and IL-12, resulting in DCs having a higher ability to polarize T-cells toward a Th1 phenotype and specifically activate cytotoxic T-cells to kill tumor cells. Therefore, sialic acid has emerged as a key modulator of DCs and is being used as a novel target to advance their therapeutic use. This study provides a unique approach to treat in vitro monocyte-derived DCs with sialidase, aimed at generating DC populations with different cell surface sialic acid phenotypes and tailored maturation and co-stimulatory profiles.

A unique, comprehensive protocol to generate de-sialylated human monocyte-derived dendritic cells (mo-DCs) from isolated peripheral blood mononuclear cells (PBMCs) using a sialidase treatment is presented. Further, methods to assess the phenotypic and functional characterization of mo-DCs and evaluate how sialidase treatment improves the maturation level of mo-DCs are described.

Sialic acids are negatively charged monosaccharides typically found at the termini of cell surface glycans. Due to their hydrophilicity and biophysical ...

Monocyte Isolation and Monocyte Differentiation into Monocyte-Derived Dendritic Cells

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 Pasture 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 five to three 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 Pasture 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 positive isolation using magnetic activated cell sorting, resuspend the cell pellet in the microbeads buffer and CD14 immuno magnetic 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 the 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 negative and CD14 positive 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 positive cells. Resuspend the monocytes by pipetting with a Pasture 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 micro pipette 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 media or buffer for the experimental setup. During the monocyte differentiation, interleukin four and granulocyte macrophage colony stimulating factor stimulation changed the cell phenotype. Data showed that human monocyte derived dendritic cells lost the expression 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 MHC2 HLA-DR, an antigen presenting molecule expressed by human dendritic cells and other antigen presenting cells.

Treatment of the Monocyte-Derived Dendritic Cells (mo-DCs) with Sialidase

Treatment of the Monocyte-Derived Dendritic Cells (Mo-DCs) with Sialidase  

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 300G for five to seven minutes at room temperature, using the normal break, 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 300G, using the normal break, 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 micro tubes labeled as number one and number two.To micro tube 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 micro tubes 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 300G using the normal break and add five milliliters of complete RPMI 1640 medium to the pellet. Plate one milliliter cells per well.The effectiveness of Sailidase treatment for reducing the sialic acid content down the surface of human monocyte derived dendritic cells was evaluated by flow cytometry and confocal microscopy. Sailidase 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 Sailidase treatment showed a significantly reduced Sambucus nigra lectin staining at the cell's surface.