The authors acknowledge funding from the European Commission GLYCOTwinning GA 101079417 and EJPRD/0001/2020 EU 825575; the Funda&#231;&#227;o para a Ci&#234;ncia e Tecnologia (FCT) Portugal, under&#160;grants FCT 2022.04607.PTDC, UIDP/04378/2020, UIDB/04378/2020 (UCIBIO), and LA/P/0140/2020 (i4HB). FCT-NOVA. and Stemmatters were also funded by the Fundo Europeu de Desenvolvimento Regional (FEDER), through the Programa Operacional Regional do Norte (Norte 2020) for the SI I&#38;amp;DT DCMatters project (NORTE-01-0247-FEDER-047212). We acknowledge Biolabs facility at FCT-NOVA and GLYCOVID NOVA Saude.

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

The authors declare no competing financial interests or other conflicts of interest.

Monocyte isolation 
This manuscript describes a protocol to generate mo-DCs from human-isolated monocytes CD14+ (Figure 1A), followed by performing a sialidase treatment to reduce the sialic acid content on the surface of these cells.
There are different ways to obtain human DCs, such as directly from peripheral blood or tissues or through differentiation from precursors such as stem cells or monocytes. Obtaining DCs differentiated from monocytes isolated from peripheral blood is far more straightforward due to the ease of obtaining high quantities of monocytes compared to other DC sources41. Still, to obtain a high percentage of isolated monocytes, all the protocol steps must be carefully followed. For instance, the density gradient medium may be toxic to the cells, and to prevent cell death, one must avoid prolonged cell contact with the density gradient medium and wash the cells thoroughly. Cell manipulation must be done as quickly as possible to avoid the loss of cell viability. From PBMCs, monocytes can be isolated through positive selection using the magnetic-activated cell sorting (MACS) method, which is a suitable technology for yielding a high number of monocytes. In addition, compared with other monocyte selection methods, mo-DCs derived from MACS-isolated monocytes possess a greater ability to stimulate anti-tumour T-cell activity42. In this protocol, once isolated, the monocytes were incubated with IL-4 and GM-CSF for a period of 5-6 days to achieve the differentiation into immature mo-DCs (Figure 1). The results showed that morphologically (Figure 1A) and phenotypically (Figure 1B), the isolated monocytes differentiated into immature mo-DCs. Moreover, throughout the differentiation, the mo-DCs lost the expression of CD14 markers and gained the expression of CD1a and MHC-II (Figure 1B), which are required for antigen presentation to T-cells.
This isolation and differentiation of monocytes into mo-DCs are limitations to this protocol. The isolation process is a sensitive step that must be carefully and swiftly executed to avoid cell death, and this step must also be done every time mo-DCs are needed for a new experiment. The differentiation process takes 5-6 days which poses a difficulty in terms of employing this method for high-throughput analyses. Nonetheless, the isolation method and using cytokines to differentiate mo-DCs are useful for generating a high number of functional mo-DCs in vitro for experimentation purposes. The mo-DCs generated in this protocol are able to undergo sialidase treatment, flow cytometry, ELISA, confocal microscopy, and so on, thus emphasizing the importance and usefulness of this method30.
Immature mo-DCs and sialidase treatment 
Sialidases are essential in sialylation regulation and are responsible for removing sialic acids from the cell surface glycans. In mo-DCs, sialic acid removal by sialidase leads to the maturation of these cells, which increases antigen-cross presentation and subsequent T-cell activation and anti-tumor activity30.
Immature human mo-DCs display a high content of cell surface &#945;(2,6)- and &#945;(2,3)-linked sialic acids27 compared to mature mo-DCs31,43. Furthermore, removing sialic acids by treating mo-DCs with sialidase improves the maturation of the DCs28,30,31. The sialidase selected for this experiment was from the bacterium Clostridium perfringens. Still, other organisms also produce sialidases, such as the bacteria Streptococcus pneumoniae, Vibrio cholerae, or Salmonella typhimurium44, the leech Macrobdella decora45, and even Homo sapiens46, and sialidases from these organisms are also used experimentally. However, each sialidase has different substrate specificities. Additionally, using the sialidase enzyme can have its limitations; for example, the manipulation of mo-DCs during the treatment can further stimulate these cells. Furthermore, the amount of sialidase and the incubation times must be optimized based on the type of cells being used and their sialic acid composition. The sialic acid removal is not a permanent effect but rather a transient phenomenon, because the cell will restore its cell surface sialic acid content. Besides sialidase, there are other methods to reduce the sialic acid molecules at the surface of cells, such as using sialyltransferase inhibitors, gene knockouts of sialyltransferase genes, or metabolic blockade of sialic acid using sialic acid mimetics47,48,49. Nonetheless, these methods may present distinct effects on cells, and besides desialylation, the cell viability must be considered. The sialidase enzyme treatment is a practical method for effectively and transiently removing cell surface sialic acids while maintaining the cell viability.
In this work, sialidase was added to the immature mo-DCs at the concentration of 500 mU/5 x 106 cells/mL, and the cells were incubated at 37 &#176;C for 60 min. The treatment was performed using RPMI-1640 without serum to preserve the cell viability and avoid any interaction between the sialylated molecules present in the serum30. Sialidase treatment can be performed with other buffers besides RPMI, such as 50 mM sodium acetate, pH 5.1 (in the case of C. perfingens sialidase), or PBS50,51,52. Nonetheless, RPMI-1640 is the most common culture medium for DCs as it maintains constant experimental conditions during the procedure, avoids inducing maturation, and reduces any stress that may be caused by sialidase buffers or PBS53,54,55,56. After incubation with sialidase, it is critical to wash the cells thoroughly with a serum-supplemented medium to guarantee that the enzyme reaction has stopped. The presence of sialylated molecules in the serum will compete as substrates for sialidase, thus assuring a rapid reaction stop.
Surface marker characterization by flow cytometry and confocal microscopy 
For the determination of the sialic acid profile, in protocol section 3, we utilized lectin staining followed by flow cytometry and confocal laser scanning microscopy. For the cell staining procedure, in both cases, the lectin concentrations and incubation conditions were optimized to avoid cell agglutination and death. It is critical to perform the incubation at 4 &#176;C in buffers containing at least 2% of either FBS or BSA to avoid non-specific binding of the lectins. In this protocol, RPMI-1640 containing 10% FBS was used to maintain constant experimental conditions and avoid cell stress. Regarding confocal microscopy, fixation of the cells prior to staining is essential to preserve the morphology, prevent autolysis, and maintain antigenicity.
The analysis of the mo-DC phenotype by flow cytometry showed that sialidase-treated mo-DCs had a significantly higher amount of PNA lectin bound to the cell surface compared to MMA and SNA lectins, which decreased after the sialidase treatment (Figure 2A). As expected, PNA staining increased, since PNA recognizes non-sialylated antigens, in contrast to MAA and SNA, which bind directly to &#945;2,3- and &#945;2,6-sialic acids, respectively30. This staining confirms the effective removal of sialic acids from the cell surface using this protocol. Another method that can be used to validate the treatment and analyze the cell surface sialic acid content is lectin staining followed by confocal microscopy, as exemplified in Figure 2B.
Besides the former examples, alternative approaches exist to evaluate and characterize sialic acid content, such as lectin probing by western blotting. Alternative sialic acid-specific lectins are also available, such as Siglecs, a group of lectins that have a distinct preference for sialic acid types and linkages57. Besides using lectins in either technique (flow cytometry, microscopy, or western blot), it is also possible to characterize the sialic acid content using antibodies; for instance, &#945;2,8-sialic acids can be assessed by antibodies such as clone 735, which is specific for polysialic acid58. In addition, after sialidase treatment, cells can be functionally tested for their biological or therapeutic efficiency by evaluating their phenotype and ability to activate T-cells40. In fact, as shown in the examples provided, sialidase-treated mo-DCs showed higher maturation phenotype, as well as an elevated expression of antigen-presenting and co-stimulatory molecules.
Furthermore, sialidase-treated mo-DCs can be loaded with antigens and co-cultured with T-cells or other cells and then can be studied regarding the phenotype, cytokine secretion profile, or other features. In the example provided, the data show that sialidase-treated mo-DCs can be loaded with tumor antigens and then used to activate T-cells. In fact, the resulting T-cells showed increased IFN-&#947; secretion, which is in agreement with previous reports on the effect of sialic acid shortage on boosting the capacity of mo-DCs to activate T-cells27,28,29,30,31.
In conclusion, this protocol shows a feasible, viable, and practical method to generate mo-DCs for sialic acid content manipulation by treatment with sialidase. This protocol presents a methodology that can serve different purposes and applications. This method can not only have a crucial role in understanding the role of sialic acids in the maturation and response of immune cells but can also be used as an immunomodulatory tool.

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.

<table><tbody><tr><td>15 mL conical tube</td><td>AstiK&amp;rsquo;s</td><td>CTGP-E15-050</td><td>Obtaining Monocyte-derived Dendritic Cells; Treatment of Cells with Sialidase</td></tr><tr><td>24-well plate</td><td>Greiner Bio-one</td><td>662 160</td><td>Obtaining Monocyte-derived Dendritic Cells; Treatment of Cells with Sialidase</td></tr><tr><td>50 mL conical tube</td><td>AstiK&amp;rsquo;s</td><td>CTGP-E50-050</td><td>Obtaining Monocyte-derived Dendritic Cells</td></tr><tr><td>7-Aminoactinomycin D (7-AAD)</td><td>BioLegend</td><td>420404</td><td>Obtaining Monocyte-derived Dendritic Cells</td></tr><tr><td>Annexin V</td><td>Immunotools</td><td>31490013</td><td>Obtaining Monocyte-derived Dendritic Cells</td></tr><tr><td>Attune Acoustic Focusing Flow Cytometer</td><td>Thermo Fisher Scientific&amp;nbsp;</td><td></td><td>Determination of Sialic Acid Profile; Maturation Profiling of mo-DCs</td></tr><tr><td>BSA</td><td>Sigma - Aldrich</td><td>A3294-100G</td><td>Obtaining Monocyte-derived Dendritic Cells; Determination of Sialic Acid Profile</td></tr><tr><td>CD14 (Monoclonal T&amp;Uuml;K4)</td><td>Miltenyi Biotec</td><td>130-080-701</td><td>Obtaining Monocyte-derived Dendritic Cells</td></tr><tr><td>CD80</td><td>Immunotools</td><td>21270803</td><td>Maturation Profiling of mo-DCs</td></tr><tr><td>CD86</td><td>Immunotools</td><td>21480863</td><td>Maturation Profiling of mo-DCs</td></tr><tr><td>Cell counting slides and trypan blue</td><td>EVE</td><td>EVS-050</td><td>Obtaining Monocyte-derived Dendritic Cells</td></tr><tr><td>Centrifuge</td><td>Eppendorf</td><td>5430 R</td><td>Obtaining Monocyte-derived Dendritic Cells; Treatment of Cells with Sialidase; Determination of Sialic Acid Profile; Maturation Profiling of mo-DCs</td></tr><tr><td>Density gradient medium (Histopaque)</td><td>Sigma - Aldrich</td><td>10771-100ML</td><td>Obtaining Monocyte-derived Dendritic Cells</td></tr><tr><td>EDTA</td><td>Gibco, ThermoFisher</td><td>15400054</td><td>Obtaining Monocyte-derived Dendritic Cells</td></tr><tr><td>Elisa kit (IFN-&amp;gamma;)</td><td>Immunotools</td><td>31673539</td><td>Maturation Profiling of mo-DCs</td></tr><tr><td>EVE automated cell count</td><td>NanoEntek</td><td>10027-452</td><td>Obtaining Monocyte-derived Dendritic Cells</td></tr><tr><td>Fetal bovine serum (FBS)</td><td>Gibco</td><td>10500064</td><td>Obtaining Monocyte-derived Dendritic Cells; Treatment of Cells with Sialidase; Determination of Sialic Acid Profile</td></tr><tr><td>Granulocyte-macrophage colony-stimulating factor (GM-CSF)</td><td>Miltenyi Biotec</td><td>&amp;nbsp; 130-093-864</td><td>Obtaining Monocyte-derived Dendritic Cells</td></tr><tr><td>Human CD14 microbeads (Immunomagnetic beads)</td><td>Miltenyi Biotec</td><td>130-050-201</td><td>Obtaining Monocyte-derived Dendritic Cells</td></tr><tr><td>Interleukin (IL)-1&amp;beta;</td><td>Sigma - Aldrich</td><td>I9401</td><td>Obtaining Monocyte-derived Dendritic Cells</td></tr><tr><td>Interleukin (IL)-4</td><td>Miltenyi Biotec</td><td>130-093-919</td><td>Obtaining Monocyte-derived Dendritic Cells</td></tr><tr><td>Interleukin (IL)-6</td><td>Sigma - Aldrich</td><td>SRP3096</td><td>Obtaining Monocyte-derived Dendritic Cells</td></tr><tr><td>L-glutamine</td><td>Gibco</td><td>A2916801</td><td>Obtaining Monocyte-derived Dendritic Cells</td></tr><tr><td>LS column and plunger</td><td>Miltenyi Biotec</td><td>130-042-401</td><td>Obtaining Monocyte-derived Dendritic Cells</td></tr><tr><td>&lt;em&gt;Maackia amurensis&lt;/em&gt;&amp;nbsp;(MAA) lectin (MAA lectin - Biotinylated)</td><td>Vector labs</td><td>B-1265-1</td><td>Determination of Sialic Acid Profile</td></tr><tr><td>MHC-I (HLA-ABC)</td><td>Immunotools</td><td>21159033</td><td>Maturation Profiling of mo-DCs</td></tr><tr><td>MHC-II (HLA-DR)</td><td>Immunostep</td><td>HLADRA-100T</td><td>Maturation Profiling of mo-DCs</td></tr><tr><td>Microtubes</td><td>AstiK&amp;rsquo;s</td><td>PCRP-E015-500</td><td>Obtaining Monocyte-derived Dendritic Cells; Treatment of Cells with Sialidase; Determination of Sialic Acid Profile; Maturation Profiling of mo-DCs</td></tr><tr><td>Neuraminidase (Sialidase)</td><td>Roche</td><td>11585886001</td><td>Treatment of Cells with Sialidase</td></tr><tr><td>Non-essential amino acids (NEAA)</td><td>Gibco</td><td>11140-050</td><td>Obtaining Monocyte-derived Dendritic Cells</td></tr><tr><td>Paraformaldehyde (PFA 2%)</td><td>Polysciences Europe</td><td>25085-1</td><td>Determination of Sialic Acid Profile; Maturation Profiling of mo-DCs</td></tr><tr><td>Paraformaldehyde (PFA 4%)</td><td>Biotium</td><td>22023</td><td>Determination of Sialic Acid Profile</td></tr><tr><td>Pasteur pipettes</td><td>Labbox</td><td>PIPP-003-500</td><td>Obtaining Monocyte-derived Dendritic Cells</td></tr><tr><td>&lt;em&gt;Peanut (Arachis hypogaea) Agglutinin&lt;/em&gt;&amp;nbsp;(PNA) lectin (PNA lectin - FITC)</td><td>Vector labs</td><td>FL-1071</td><td>Determination of Sialic Acid Profile</td></tr><tr><td>Penicillin/streptomycin</td><td>Gibco</td><td>15140163</td><td>Obtaining Monocyte-derived Dendritic Cells</td></tr><tr><td>Phosphate Buffered Saline (PBS)</td><td>NZYTech</td><td>&amp;nbsp; MB18201</td><td>Obtaining Monocyte-derived Dendritic Cells; Treatment of Cells with Sialidase; Determination of Sialic Acid Profile; Maturation Profiling of mo-DCs</td></tr><tr><td>Prostaglandin E2 (PGE2)</td><td>Sigma - Aldrich</td><td>P0409</td><td>Obtaining Monocyte-derived Dendritic Cells</td></tr><tr><td>RBC lysis buffer</td><td>BioLegend</td><td>420302</td><td>Obtaining Monocyte-derived Dendritic Cells</td></tr><tr><td>RPMI-1640 medium&amp;nbsp;(containing 11.1 mM glucose)</td><td>Gibco</td><td>31870074</td><td>Obtaining Monocyte-derived Dendritic Cells; Treatment of Cells with Sialidase; Determination of Sialic Acid Profile</td></tr><tr><td>&lt;em&gt;Sambucus nigra &lt;/em&gt;lectin (SNA lectin - Biotinylated)</td><td>Vector labs</td><td>&amp;nbsp; B-1305-2</td><td>Determination of Sialic Acid Profile</td></tr><tr><td>&lt;em&gt;Sambucus nigra &lt;/em&gt;lectin (SNA lectin - FITC)</td><td>Vector labs</td><td>FL-1301-2</td><td>Determination of Sialic Acid Profile</td></tr><tr><td>Sodium pyruvate</td><td>Thermofisher</td><td>11360-070</td><td>Obtaining Monocyte-derived Dendritic Cells</td></tr><tr><td>SpectroMax190</td><td>Molecular Devices</td><td></td><td>Maturation Profiling of mo-DCs</td></tr><tr><td>Streptavidin-PE</td><td>BioLegend</td><td>&amp;nbsp; 405203</td><td>Determination of Sialic Acid Profile; Maturation Profiling of mo-DCs</td></tr><tr><td>Tetramethylbenzidine (TMB)</td><td>Sigma - Aldrich</td><td>T0440</td><td>Maturation Profiling of mo-DCs</td></tr><tr><td>Tumour necrosis factor-&amp;alpha; (TNF-&amp;alpha;)</td><td>Sigma - Aldrich</td><td>H8916</td><td>Obtaining Monocyte-derived Dendritic Cells</td></tr><tr><td>Zeiss LSM710 confocal microscope</td><td>Zeiss</td><td></td><td>Determination of Sialic Acid Profile</td></tr></tbody></table>

generation of monocyte-derived dendritic cells with differing sialylated phenotypes

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.

Monocyte isolation and monocyte differentiation into mo-DCs 
In accordance with the protocol, human PBMCs were isolated from the buffy coat using density gradient separation with density gradient medium and thoroughly washed. Trypan blue was used to perform viable cell counting on the day of isolation, as described previously in step 1.4.1. Subsequently, CD14+ monocyte isolation was carried out through positive selection. To achieve this, PBMCs were incubated with magnetic beads containing an antibody that recognizes the CD14 antigen. The selected CD14+ monocytes were cultured in a medium supplemented with GM-CSF and IL-4 for 5-6 days27 to differentiate into immature mo-DCs (Figure 1A). The maturation of mo-DCs can be obtained by applying a cocktail of cytokines, including IL-6, IL-1&#946;, TNF-&#945;, and PGE235 (Figure 1A).
During the differentiation process, as a result of IL-4 and GMCSF stimulation, the cell phenotype is expected to change. Data shows that mo-DCs lose expression of surface marker CD14, mainly expressed by monocytes (Figure 1B), and gain significant expression of CD1a, a marker expressed by human DCs36,37. mo-DCs also obtain higher MHC-II (HLA-DR) expression, an antigen-presenting molecule expressed by human DCs and other antigen-presenting cells38 (Figure 1B).
Cell surface sialic acid content 
The treatment of mo-DCs with sialidase reduces the sialic acid content on the surface of mo-DCs, which can be confirmed by staining with lectins, which are proteins capable of binding to carbohydrates39. Since the enzyme used removes both &#945;2,3 and &#945;2,6-linked sialic acids from the cell surface, mo-DCs were stained with PNA, which recognizes T antigen-Gal&#946;1-3GalNAc&#945;1-Ser/Thr, as well as MAA and SNA lectins, which bind to &#945;2,3- and &#945;2,6-sialic, respectively. The effectiveness of the sialidase treatment was evaluated by flow cytometry and by confocal microscopy (Figure 2). As shown in Figure 2A, sialidase treatment significantly decreased MAA and SNA binding while increasing PNA staining. The decrease in SNA staining after sialidase treatment was further confirmed by confocal microscopy analysis showing a significantly reduced SNA staining at the cell surface (Figure 2B).
Functional characterization of sialidase-treated mo-DCs 
To evaluate how sialidase treatment affects mo-DC functions, the maturation level of mo-DCs was assessed after sialidase treatment. As shown in Figure 3A, the sialidase treatment leads to a significant increase in the expression of the antigen-presenting molecules MHC I and MHC II and the expression of the CD80 and CD86 co-stimulatory molecules. To assess the effect of sialic acid removal on the ability of mo-DCs to induce T-cell responses, sialidase-treated mo-DCs loaded with tumor cell lysates were used to prime autologous T-cells (Figure 3). Next, the profile of the resulting T-cells was characterized based on their capacity to secrete the Th1 cytokine IFN-&#947;. As shown in Figure 3B, when compared with T-cells primed by fully sialylated mo-DCs, the T-cells primed by sialidase-treated mo-DCs secreted significantly higher levels of IFN-&#947;. These results suggest that sialidase-treated mo-DCs have improved capacity to prime autologous T-cells.
Cell viability 
After sialidase treatment, a viability assay was performed to ensure the treatment was not cytotoxic to the cells. Following treatment, mo-DCs were stained with 7-AAD and Annexin V, to detect nonviable and apoptotic cells, and analyzed by flow cytometry (Figure 4). Data shows no significant difference in cell viability between untreated (Figure 4, left panel) and sialidase-treated cells (Figure 4, right panel).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/65525/65525fig01.jpg" /> 
Figure 1: Differentiation of isolated monocytes into mo-DCs. (A) CD14+ monocytes were isolated from buffy coats and cultured at a concentration of 1.3 x 106 cells/mL at 37 &#176;C. The monocytes were differentiated in medium supplemented with 750 U/mL of IL-4 and 1,000 U/mL of GM-CSF. Microscopic analysis of the morphology of monocytes isolated from human buffy coat at day 0 (top image). Immature mo-DCs; cells were differentiated during a period of 5 days using IL-4 and GM-CSF (middle image). Matured mo-DCs were obtained by using IL-6, IL-1&#946;, TNF-&#945;, and PGE2 cytokines for 24 h (bottom image). Scale bars: 100 &#181;m. (B) The cells were analyzed at day 0, day 2, and day 5 throughout the differentiation period using flow cytometry. The following antibodies were used to characterize cell surface markers: (a-c) CD14; (d-f) CD1a, and (g-i) HLA-DR (MHC class II). The figure shows representative histograms of at least three independent assays. Panel (B) has been modified from Videira et al.40, patent WO2017002045A1. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/65525/65525fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/65525/65525fig02.jpg" /> 
Figure 2: Sialidase treatment of human mo-DCs to remove &#945;2,6- and &#945;2,3-linked sialic acids from the cell surface. (A) Analysis of mo-DCs by flow cytometry using lectin staining to test the efficacy of sialidase treatment. Human mo-DCs were treated with sialidase (grey bars) or left untreated (white bars) and stained with SNA lectin (recognizing [2,6]-sialic acids), MAA lectin (recognizing [2,3]-sialic acids), and PNA lectin (recognizing T antigen-Gal&#946;1-3GalNAc&#945;1-Ser/Thr). The values represent the mean fluorescence intensity (MFI) of at least three independent assays. Statistical significance was determined using a two-tailed paired t-test (*P &#60; 0.05 or ***P &#60; 0.0001), referring to the difference between the untreated and sialidase-treated DCs. Sialidase treatment decreased MAA binding and increased PNA staining in human mo-DCs, resulting from the removal of &#945;(2,3)-linked sialic acids; the removal of &#945;(2,6)-linked sialic acids after sialidase treatment was detected by a decrease in SNA staining. (B) Confocal microscopy of mo-DCs treated with sialidase and prepared on coverslips for observation. A range of z-stack images was collected from different cells and processed to include the mean staining intensity. Scale bars: 20 &#181;m. Panel (A) has been modified from Silva et al.30; panel (B) has been modified from Silva et al.29. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/65525/65525fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/65525/65525fig03.jpg" /> 
Figure 3: Sialidase treatment of mo-DCs inducing a higher expression of maturation markers. (A) mo-DCs treated with sialidase showed a higher maturation phenotype than fully sialylated mo-DCs. Flow cytometry was used to evaluate the expression of several maturation markers. The mo-DCs were treated with sialidase for 1 h at 37 &#176;C; the graph values represent the mean fluorescence intensity (MFI) (average &#177; SEM) of at least three independent assays. Statistically significant differences were calculated using a t-test (*P &#60; 0.05, **P &#60; 0.01), referring to the difference between the untreated and sialidase-treated conditions. (B) Desialylated human mo-DCs loaded with whole tumor antigens induced specific T-cell responses. The mo-DCs were treated with sialidase for 1 h at 37 &#176;C or left untreated, followed by loading with MCF-7 lysates (TL) as a source of whole tumor cell antigens. Co-culture between mo-DCs and autologous T-cells was performed for 4-8 days in the presence of IL-2 (10 U/mL). T-cells primed with desialylated mo-DCs showed significantly higher secretion of the Th1 cytokine, IFN-&#947;. Following T-cell stimulation with mo-DCs, the cytokines secreted into the co-culture supernatants were measured by ELISA (n = 7). The graph values represent the concentration (pg/mL) (average &#177; SEM). Statistically significant differences were calculated using a t-test (*P &#60; 0.05). The figure has been modified from Silva et al.30. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/65525/65525fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/65525/65525fig04.jpg" /> 
Figure 4: Lack of impact of sialidase treatment on the viability of human mo-DCs. Untreated mo-DCs (left panel) and sialidase-treated mo-DCs (right panel) were subjected to dual staining with annexin V and 7-AAD, and the staining was analyzed by flow cytometry. The data showed no significant difference in cell viability between the untreated and sialidase-treated cells, suggesting that mo-DCs can tolerate sialidase treatment and remain viable to exert their immunologic function. The figure has been modified from Silva et al.30. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/65525/65525fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Dendritic Cells Maturation Using Sialidases-Based Enzymatic Treatment of the Cell Surface at JoVE.com

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

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.

Generation of Monocyte-Derived Dendritic Cells with Differing Sialylated Phenotypes

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

generation-monocyte-derived-dendritic-cells-with-differing-sialylated

Nanyang Technological University

Research

JoVE Journal

Immunology and Infection

3.4K Views.  Universidade NOVA de Lisboa. De reikwijdte van het onderzoek van de glyco-immunologiegroep is het bestuderen van de rol van glycaanimmunologie in de gezondheid en ziekte van de mens. Dit onderzoek heeft tot doel biologische mechanismen te ontrafelen die worden gemoduleerd door glycosylaties, en nieuwe op glycaan gebaseerde therapieën te ontdekken voor kanker en aangeboren glycosyleringsstoornissen. Onze focus lag op gesiaalyleerde glycanen die de functie van relevante moleculen beïnvloeden, met name tijdens respons.

Video: Auteur in de schijnwerpers: Rijping van dendritische cellen met behulp van op sialidasen gebaseerde enzymatische behandeling van het celoppervlak 

Generation and Labeling of Murine Bone Marrow-derived Dendritic Cells with Qdot Nanocrystals for Tracking Studies

Dendritic cells (DCs) are professional antigen presenting cells (APCs) found in peripheral tissues and in immunological organs such as thymus, bone marrow, spleen, lymph nodes and Peyer's patches 1-3. DCs present in peripheral tissues sample the organism for the presence of antigens, which they take up, process and present in their surface in the context of major histocompatibility molecules (MHC). Then, antigen-loaded DCs migrate to immunological organs where they present the processed antigen to T lymphocytes triggering specific immune responses. One way to evaluate the migratory capabilities of DCs is to label them with fluorescent dyes 4.
Herewith we demonstrate the use of Qdot fluorescent nanocrystals to label murine bone marrow-derived DC. The advantage of this labeling is that Qdot nanocrystals possess stable and long lasting fluorescence that make them ideal for detecting labeled cells in recovered tissues. To accomplish this, first cells will be recovered from murine bone marrows and cultured for 8 days in the presence of granulocyte macrophage-colony stimulating factor in order to induce DC differentiation. These cells will be then labeled with fluorescent Qdots by short in vitro incubation. Stained cells can be visualized with a fluorescent microscopy. Cells can be injected into experimental animals at this point or can be into mature cells upon in vitro incubation with inflammatory stimuli. In our hands, DC maturation did not determine loss of fluorescent signal nor does Qdot staining affect the biological properties of DCs. Upon injection, these cells can be identified in immune organs by fluorescent microscopy following typical dissection and fixation procedures.

Dendritic cells uptake antigens and migrate towards immune organs to present processed antigens to T cells. Qdot nanocrystal labeling provides a long-lasting and stable fluorescent signal. This allows tracking of dendritic cells to different organs by fluorescent microscopy.

Dendritic cells (DCs) are professional antigen presenting cells (APCs) found in peripheral tissues and in immunological organs such as thymus, bone ...

Isolation of Mouse Lung Dendritic Cells

Lung dendritic cells (DC) play a fundamental role in sensing invading pathogens 1,2 as well as in the control of tolerogenic responses 3 in the respiratory tract. At least three main subsets of lung dendritic cells have been described in mice: conventional DC (cDC) 4, plasmacytoid DC (pDC) 5 and the IFN-producing killer DC (IKDC) 6,7. The cDC subset is the most prominent DC subset in the lung 8. 
The common marker known to identify DC subsets is CD11c, a type I transmembrane integrin (&beta;2) that is also expressed on monocytes, macrophages, neutrophils and some B cells 9. In some tissues, using CD11c as a marker to identify mouse DC is valid, as in spleen, where most CD11c+ cells represent the cDC subset which expresses high levels of the major histocompatibility complex class II (MHC-II). However, the lung is a more heterogeneous tissue where beside DC subsets, there is a high percentage of a distinct cell population that expresses high levels of CD11c bout low levels of MHC-II. Based on its characterization and mostly on its expression of F4&#47;80, an splenic macrophage marker, the CD11chiMHC-IIlo lung cell population has been identified as pulmonary macrophages 10 and more recently, as a potential DC precursor 11. 
In contrast to mouse pDC, the study of the specific role of cDC in the pulmonary immune response has been limited due to the lack of a specific marker that could help in the isolation of these cells. Therefore, in this work, we describe a procedure to isolate highly purified mouse lung cDC. The isolation of pulmonary DC subsets represents a very useful tool to gain insights into the function of these cells in response to respiratory pathogens as well as environmental factors that can trigger the host immune response in the lung.

A highly purified preparation of mouse lung dendritic cells is described. Specific emphasis is given to the isolation of conventional dendritic cell subset.

Lung dendritic cells (DC) play a fundamental role in sensing invading pathogens 1,2 as well as in the control of tolerogenic responses 3 in the ...

Generation of a Novel Dendritic-cell Vaccine Using Melanoma and Squamous Cancer Stem Cells

We identified cancer stem cell (CSC)-enriched populations from murine melanoma D5 syngeneic to C57BL/6 mice and the squamous cancer SCC7 syngeneic to C3H mice using ALDEFLUOR/ALDH as a marker, and tested their immunogenicity using the cell lysate as a source of antigens to pulse dendritic cells (DCs). DCs pulsed with ALDHhigh CSC lysates induced significantly higher protective antitumor immunity than DCs pulsed with the lysates of unsorted whole tumor cell lysates in both models and in a lung metastasis setting and a s.c.&#160;tumor growth setting, respectively. This phenomenon was due to CSC vaccine-induced humoral as well as cellular anti-CSC responses. In particular, splenocytes isolated from the host subjected to CSC-DC vaccine produced significantly higher amount of IFN&#947; and GM-CSF than splenocytes isolated from the host subjected to unsorted tumor cell lysate pulsed-DC vaccine. These results support the efforts to develop an autologous CSC-based therapeutic vaccine for clinical use in an adjuvant setting.

Evaluated in syngeneic immunocompetent hosts, cancer stem cell (CSC) based dendritic cell (DC) vaccine demonstrated significantly higher antitumor immunity than traditional DC vaccines pulsed with heterogeneous bulk tumor cells.

We identified cancer stem cell (CSC)-enriched populations from murine melanoma D5 syngeneic to C57BL/6 mice and the squamous cancer SCC7 syngeneic to C3H ...

Activation and Measurement of NLRP3 Inflammasome Activity Using IL-1&#946; in Human Monocyte-derived Dendritic Cells

Inflammatory processes resulting from the secretion of Interleukin (IL)-1 family cytokines by immune cells lead to local or systemic inflammation, tissue remodeling and repair, and virologic control1,2 . Interleukin-1&#946; is an essential element of the innate immune response and contributes to eliminate invading pathogens while preventing the establishment of persistent infection1-5.
Inflammasomes are the key signaling platform for the activation of interleukin 1 converting enzyme (ICE or Caspase-1). The NLRP3 inflammasome requires at least two signals in DCs to cause IL-1&#946; secretion6. Pro-IL-1&#946; protein expression is limited in resting cells; therefore a priming signal is required for IL-1&#946; transcription and protein expression. A second signal sensed by NLRP3 results in the formation of the multi-protein NLRP3 inflammasome. The ability of dendritic cells to respond to the signals required for IL-1&#946; secretion can be tested using a synthetic purine, R848, which is sensed by TLR8 in human monocyte derived dendritic cells&#160;(moDCs) to prime cells, followed by activation of the NLRP3 inflammasome with the bacterial toxin and potassium ionophore, nigericin.
Monocyte derived DCs&#160;are easily produced in culture and provide significantly more cells than purified human myeloid DCs. The method presented here differs from other inflammasome assays in that it uses in vitro human, instead of mouse derived, DCs thus allowing for the study of the inflammasome in human disease and infection.

Dendritic cells (DCs)&#160;secrete IL-1&#946; in response to TLR8 recognition of synthetic purine, R848, followed by NLRP3 inflammasome activation with nigericin, therefore, IL-1&#946; can be used to measure NLRP3 inflammasome activity. Intracellular cytokine staining, immunoblotting, and ELISA are used to accurately measure NLRP3 inflammasome priming and activation via IL-1&#946; expression.

Inflammatory processes resulting from the secretion of Interleukin (IL)-1 family cytokines by immune cells lead to local or systemic inflammation, tissue ...

In Vitro Generation of Murine Plasmacytoid Dendritic Cells from Common Lymphoid Progenitors using the AC-6 Feeder System

Plasmacytoid dendritic cells (pDCs) are powerful type I interferon (IFN-I) producing cells that are activated in response to infection or during inflammatory responses. Unfortunately, study of pDC function is hindered by their low frequency in lymphoid organs, and existing methods for in vitro DC generation predominantly favor the production of cDCs over pDCs. Here we present a unique approach to efficiently generate pDCs from common lymphoid progenitors (CLPs) in vitro. Specifically, the protocol described details how to purify CLPs from bone marrow and generate pDCs by coculturing with &#947;-irradiated AC-6 feeder cells in the presence of Flt3 ligand. A unique characteristic of this culture system is that the CLPs migrate underneath the AC-6 cells and become cobblestone area-forming cells, a critical step for expanding pDCs. Morphologically distinct DCs, namely pDCs and cDCs, were generated after approximately 2 weeks with a composition of 70-90% pDCs under optimal conditions. Typically, the number of pDCs generated by this method is roughly 100-fold of the number of CLPs seeded. Therefore, this is a novel system with which to robustly generate the large numbers of pDCs required to facilitate further studies into the development and function of these cells.

In Vitro Generation of Murine Plasmacytoid Dendritic Cells from Common Lymphoid Progenitors using the AC-6 Feeder System

In this study we present an in vitro culture system that can efficiently generate pDCs by co-culturing common lymphoid progenitors with AC-6 feeder cells in the presence of Flt3 ligand.

Plasmacytoid dendritic cells (pDCs) are powerful type I interferon (IFN-I) producing cells that are activated in response to infection or during ...

Generation of Immature, Mature and Tolerogenic Dendritic Cells with Differing Metabolic Phenotypes

Immune response results from a complex interplay between the antigen non-specific innate immune system and the antigen specific adaptive immune system. The immune system is a constant balance in maintaining tolerance to self-molecules and reacting rapidly to pathogens. Dendritic cells (DCs) are powerful professional antigen presenting cells that link the innate immune system to the adaptive immune system and balance the adaptive response between self and non-self. Depending on the maturation signals, immature dendritic cells can be selectively stimulated to differentiate into immunogenic or tolerogenic DCs. Immunogenic dendritic cells provide proliferation signals to antigen-specific T cells for clonal expansion; while tolerogenic dendritic cells regulate tolerance by antigen-specific T-cell deletion or clonal expansion of regulatory T-cells. Due to this unique property, dendritic cells are highly sought after as therapeutic agents for cancer and autoimmune diseases. Dendritic cells can be loaded with specific antigens in vitro and injected into the human body to mount a specific immune response both immunogenic and tolerogenic. This work presents a means to generate in vitro from monocytes, immature monocyte derived dendritic cells (moDCs), tolerogenic and mature moDCs that differ in surface marker expression, function and metabolic phenotypes.

Immature dendritic cells can be selectively differentiated into tolerogenic or mature dendritic cells to regulate the balance between immunity and tolerance. This work presents a means to generate from immature monocyte derived dendritic cells (moDCs), in vitro tolerogenic and mature moDCs that differ in metabolic phenotypes.

Immune response results from a complex interplay between the antigen non-specific innate immune system and the antigen specific adaptive immune system. ...

Culture of Macrophage Colony-stimulating Factor Differentiated Human Monocyte-derived Macrophages

A protocol is presented for cell culture of macrophage colony-stimulating factor (M-CSF) differentiated human monocyte-derived macrophages. For initiation of experiments, fresh or frozen monocytes are cultured in flasks for 1 week with M-CSF to induce their differentiation into macrophages. Then, the macrophages can be harvested and seeded into culture wells at required cell densities for carrying out experiments. The use of defined numbers of macrophages rather than defined numbers of monocytes to initiate macrophage cultures for experiments yields macrophage cultures in which the desired cell density can be more consistently attained. Use of cryopreserved monocytes reduces dependency on donor availability and produces more homogeneous macrophage cultures.

A protocol is presented for cell culture of macrophage colony-stimulating factor (M-CSF) differentiated human monocyte-derived macrophages. The protocol utilizes cryopreservation of monocytes coupled with their bulk differentiation into macrophages. Then harvested macrophages can then be seeded into culture wells at required cell densities for carrying out experiments.

A protocol is presented for cell culture of macrophage colony-stimulating factor (M-CSF) differentiated human monocyte-derived macrophages. For initiation ...

Characterization of Human Monocyte-derived Dendritic Cells by Imaging Flow Cytometry: A Comparison between Two Monocyte Isolation Protocols

Dendritic cells (DCs) are antigen presenting cells of the immune system that play a crucial role in lymphocyte responses, host defense mechanisms, and pathogenesis of inflammation. Isolation and study of DCs have been important in biological research because of their distinctive features. Although they are essential key mediators of the immune system, DCs are very rare in blood, accounting for approximately 0.1 - 1% of total blood mononuclear cells. Therefore, alternatives for isolation methods rely on the differentiation of DCs from monocytes isolated from peripheral blood mononuclear cells (PBMCs). The utilization of proper isolation techniques that combine simplicity, affordability, high purity, and high yield of cells is imperative to consider. In the current study, two distinct methods for the generation of DCs will be compared. Monocytes were selected by adherence or negatively enriched using magnetic separation procedure followed by differentiation into DCs with IL-4 and GM-CSF. Monocyte and MDDC viability, proliferation, and phenotype were assessed using viability dyes, MTT assay, and CD11c/ CD14 surface marker analysis by imaging flow cytometry. Although the magnetic separation method yielded a significant higher percentage of monocytes with higher proliferative capacity when compared to the adhesion method, the findings have demonstrated the ability of both techniques to simultaneously generate monocytes that are capable of proliferating and differentiating into viable CD11c+ MDDCs after seven days in culture. Both methods yielded &gt; 70% CD11c+ MDDCs. Therefore, our results provide insights that contribute to the development of reliable methods for isolation and characterization of human DCs.

This study compares two different methods of human monocyte isolation for obtaining in vitro dendritic cells (DCs). Monocytes are selected by adherence or negatively enriched by magnetic separation. Monocyte yield and viability along with MDDC viability, proliferation and CD11c/CD14 surface marker expression will be compared between both methods.

Dendritic cells (DCs) are antigen presenting cells of the immune system that play a crucial role in lymphocyte responses, host defense mechanisms, and ...

Generation of Human Monocyte-derived Dendritic Cells from Whole Blood

Dendritic cells (DCs) recognize foreign structures of different pathogens, such as viruses, bacteria, and fungi, via a variety of pattern recognition receptors (PRRs) expressed on their cell surface and thereby activate and regulate immunity. 
The major function of DCs is the induction of adaptive immunity in the lymph nodes by presenting antigens via MHC I and MHC II molecules to na&#239;ve T lymphocytes. Therefore, DCs have to migrate from the periphery to the lymph nodes after the recognition of pathogens at the sites of infection. For in vitro experiments or DC vaccination strategies, monocyte-derived DCs are routinely used. These cells show similarities in physiology, morphology, and function to conventional myeloid dendritic cells. They are generated by interleukin 4 (IL-4) and granulocyte-macrophage colony-stimulating factor (GM-CSF) stimulation of monocytes isolated from healthy donors. Here, we demonstrate how monocytes are isolated and stimulated from anti-coagulated human blood after peripheral blood mononuclear cell (PBMC) enrichment by density gradient centrifugation. Human monocytes are differentiated into immature DCs and are ready for experimental procedures in a non-clinical setting after 5 days of incubation.

Here, we demonstrate how monocytes are isolated by magnetic bead separation from peripheral blood mononuclear cells after density gradient centrifugation of human anti-coagulated blood. Following incubation for 5 days, human monocytes are differentiated into immature dendritic cells and are ready for experimental procedures in a non-clinical setting.

Dendritic cells (DCs) recognize foreign structures of different pathogens, such as viruses, bacteria, and fungi, via a variety of pattern recognition ...

Generation of Immature, Mature, and Tolerogenic Dendritic Cells from Monocytes

Source: Sim, W. J. et al., <a href="https://app-jove-com.remotexs.ntu.edu.sg/v/54128">Generation of Immature, Mature and Tolerogenic Dendritic Cells with Differing Metabolic Phenotypes.</a>&#160;J. Vis. Exp.&#160;(2016).
This video demonstrates the generation of immature, mature, and tolerogenic dendritic cells, or DCs, from human monocytes. The incubation of monocytes with growth factors and cytokines leads to the production of immature DCs. In response to immune regulatory molecules, immature DCs differentiate into tolerogenic and mature DCs.

Generation of Monocyte-Derived Dendritic Cells with Differing Sialylated Phenotypes

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