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

387 Views.  Universidade NOVA de Lisboa. 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 P...