Glycome dynamics in T and B cell development: basic immunological mechanisms and clinical applications

Glycans cover the surfaces of all mammalian cells through a process called glycosylation. Nearly all proteins and receptors that integrate the intricate series of co-stimulatory/inhibitory pathways of the immune system are glycosylated. Growing evidence indicates that the development of the immune system at the origins of T and B cell development is tightly regulated by glycosylation. In this opinion, we hypothesize that the glycome composition of developing T and B cells is developmentally regulated. We discuss how glycans play fundamental roles in lymphocyte development and how glycans early define T and B cell functionality in multiple aspects of adaptive immunity. These advances can provide opportunities for the discovery of novel disease factors and more effective candidate treatments for various conditions.This work was funded by the EU (ERC, GlycanSwitch, Grant Agreement N° 101071386). Views and opinions expressed are, however, those of the author(s) only and do not necessarily reflect those of the European Union or the European Research Council Executive Agency. Funded by the European Union (GlycanTrigger, Grant Agreement N°: 101093997. Views and opinions expressed are, however, those of the author(s) only and do not necessarily reflect those of the European Union or European Health and Digital Executive Agency. Neither the European Union nor the granting authority can be held responsible for them. M.M.V. and E.L-G. received funding from the Portuguese Foundation for Science and Technology (FCT) of the Portuguese Ministry of Science, Technology and Higher Education (M.M.V.: PD/BD/135452/2017, COVID/BD/152488/2022; E.L-G.: UI/BD/152866/2022)

Glycans in Trained Immunity: Educators of innate immune memory in homeostasis and disease

Trained Immunity is defined as a biological process normally induced by exogenous or endogenous insults that triggers epigenetic and metabolic reprogramming events associated with long-term adaptation of innate immune cells. This trained phenotype confers enhanced responsiveness to subsequent triggers, resulting in an innate immune "memory" effect. Trained Immunity, in the past decade, has revealed important benefits for host defense and homeostasis, but can also induce potentially harmful outcomes associated with chronic inflammatory disorders or autoimmune diseases. Interestingly, evidence suggest that the "trainers" prompting trained immunity are frequently glycans structures. In fact, the exposure of different types of glycans at the surface of pathogens is a key driver of the training phenotype, leading to the reprogramming of innate immune cells through the recognition of those glycan-triggers by a variety of glycan-binding proteins (GBPs) expressed by the immune cells. β-glucan or mannose-enriched structures in Candida albicans are some of the examples that highlight the potential of glycans in trained immunity, both in homeostasis and in disease. In this review, we will discuss the relevance of glycans exposed by pathogens in establishing key immunological hubs with glycan-recognizing receptors expressed in immune cells, highlighting how this glycan-GBP network can impact trained immunity. Finally, we discuss the power of glycans and GBPs as potential targets in trained immunity, envisioning potential therapeutic applications.This work was supported by grants from SSP: co-funded by the European Union (ERC, GlycanSwitch, 101071386). Views and opinions expressed are however those of the author(s) only and do not necessarily reflect those of the European Union or the European Research Council Executive Agency. Neither the European Union nor the granting authority can be held responsible for them. The work was also co-funded by EU GlycanTrigger-grant Agreement No: 101093997. Views and opinions expressed are however those of the author(s) only and do not necessarily reflect those of the European Union or European Health and Digital Executive Agency. Neither the European Union nor the granting authority can be held responsible for them. SSP also acknowledges funding by “2022 LRA Lupus Innovation Award” and by “European Crohn’s and Colitis Organisation (ECCO) Pioneer Award 2021”. SSP also acknowledges the US Department of Defense, US Army Medical Research Acquisition Activity, FY18 Peer Reviewed Medical Research Program Investigator-Initiated Research Award (award number W81XWH1920053). IA acknowledges the Portuguese Foundation for Science and Technology (FCT) for funding (2022.00337. CEECIND), from BIAL Foundation and Portuguese Medical Association (Maria de Sousa Award 2023), from Núcleo de Estudos de Doenças Autoimunes and Portuguese Society of Internal Medicine (NEDAI-SPMI) and from European Society for Clinical Investigation (ESCI Exploratory Research Grant 2024). ÂF acknowledges FCT for funding (2022.01422. PTDC)

Immune regulatory networks coordinated by glycans and glycan-binding proteins in autoimmunity and infection

The immune system is coordinated by an intricate network of stimulatory and inhibitory circuits that regulate host responses against endogenous and exogenous insults. Disruption of these safeguard and homeostatic mechanisms can lead to unpredictable inflammatory and autoimmune responses, whereas deficiency of immune stimulatory pathways may orchestrate immunosuppressive programs that contribute to perpetuate chronic infections, but also influence cancer development and progression. Glycans have emerged as essential components of homeostatic circuits, acting as fine-tuners of immunological responses and potential molecular targets for manipulation of immune tolerance and activation in a wide range of pathologic settings. Cell surface glycans, present in cells, tissues and the extracellular matrix, have been proposed to serve as “self-associated molecular patterns” that store structurally relevant biological data. The responsibility of deciphering this information relies on different families of glycan-binding proteins (including galectins, siglecs and C-type lectins) which, upon recognition of specific carbohydrate structures, can recalibrate the magnitude, nature and fate of immune responses. This process is tightly regulated by the diversity of glycan structures and the establishment of multivalent interactions on cell surface receptors and the extracellular matrix. Here we review the spatiotemporal regulation of selected glycan-modifying processes including mannosylation, complex Nglycan branching, core 2 O-glycan elongation, LacNAc extension, as well as terminal sialylation and fucosylation. Moreover, we illustrate examples that highlight the contribution of these processes to the control of immune responses and their integration with canonical tolerogenic pathways. Finally, we discuss the power of glycans and glycan-binding proteins as a source of immunomodulatory signals that could be leveraged for the treatment of autoimmune inflammation and chronic infection.This work was supported by grants from SSP: co-funded by the European Union (ERC, GlycanSwitch, 101071386). Views and opinions expressed are however those of the author(s) only and do not necessarily reflect those of the European Union or the European Research Council Executive Agency. Neither the European Union nor the granting authority can be held responsible for them. The work was also co-funded by EU GlycanTrigger-grant Agreement No: 101093997. Views and opinions expressed are however those of the author(s) only and do not necessarily reflect those of the European Union or European Health and Digital Executive Agency. Neither the European Union nor the granting authority can be held responsible for them. SSP also acknowledges funding by “2022 LRA Lupus Innovation Award” and by “European Crohn’s and Colitis Organisation (ECCO) Pioneer Award 2021”. SSP also acknowledges the US Department of Defense, US Army Medical Research Acquisition Activity, FY18 Peer Reviewed Medical Research Program Investigator-Initiated Research Award (award number W81XWH1920053) as well as grant funded by the Portuguese Foundation for Science and Technology – FCT (EXPL/MED-ONC/0496/2021). IA acknowledges FCT for funding (2022.00337.CEECIND). JG acknowledges funding from ESCMID (ESCMID Research Grant 2022), ECCO (ECCO Grant 2023) and FCT (2020.00088.CEECIND). G.A.R acknowledges grants from the Argentinean Agency for Promotion of Science, Technology and Innovation (PICT 2017-0494, PICT-FBB 620 and PICT 2020-01552). The authors are also thankful for generous support from Sales (Argentina), Bunge & Born (Argentina), Baron (Argentina), Williams (Argentina) and Richard Lounsbery (USA) Foundations, as well as donations from Ferioli-Ostry and Caraballo families to GAR

Glycosylation in cancer: Mechanisms and clinical implications

Despite recent progress in understanding the cancer genome, there is still a relative delay in understanding the full aspects of the glycome and glycoproteome of cancer. Glycobiology has been instrumental in relevant discoveries in various biological and medical fields, and has contributed to the deciphering of several human diseases. Glycans are involved in fundamental molecular and cell biology processes occurring in cancer, such as cell signalling and communication, tumour cell dissociation and invasion, cell-matrix interactions, tumour angiogenesis, immune modulation and metastasis formation. The roles of glycans in cancer have been highlighted by the fact that alterations in glycosylation regulate the development and progression of cancer, serving as important biomarkers and providing a set of specific targets for therapeutic intervention. This Review discusses the role of glycans in fundamental mechanisms controlling cancer development and progression, and their applications in oncology.The Institute of Molecular Pathology and Immunology of the University of Porto integrates the Institute for Research and Innovation in Health, which is partially supported by the Portuguese Foundation for Science and Technology (FCT). This work is funded by the European Regional Development Fund (FEDER) through the Operational Programme for Competitiveness Factors (COMPETE) and by national funds through the FCT, under the projects PEst‑C/SAU/ LA0003/2013, PTDC/BBB-EBI/0786/2012 and EXPL/BIM-MEC/0149/2012. S.S.P. acknowledges a grant from the FCT (number SFRH/BPD/63094/2009). C.A.R. acknowledges sup­port from the European Union Seventh Framework Programme GastricGlycoExplorer (grant number 316929). The authors apologize that they cannot include all the relevant studies on glycosylation in cancer in this article owing to limitation of space. The authors thank Tiago Fontes- Oliveira for support in figures preparations

The role of N-glycans in regulatory T cells in autoimmunity

Regulatory T cells (Tregs) have a key role in the maintenance of immune tolerance and in the prevention of autoimmunity. Recent studies have shown an association between decreased Treg frequency and a deficient suppressive activity with the development of many autoimmune diseases. Although glycosylation, which consists in the addition of glycans to proteins and lipids on the cell surface, is recognized as a critical modification for T cell development and function, the relevance of glycans in Treg biology and activity, as well as their impact in the immunopathogenesis of autoimmune diseases, deserves more attention, as it is far from being fully understood. This review discusses the biological impact of N-glycans in Treg biology, highlighting their potential to uncover novel pathogenic mechanisms in autoimmunity and new targets for promising therapeutic approaches with clinical applications in autoimmune disease patients.ÂF acknowledges FCT (Portuguese Foundation for Science and Technology) for funding (Grant: 2022.01422.PTDC); Portuguese Society of Neurology (SPN) – (Grant: Bolsa Pereira Monteiro); and PARSUK/ FCT/British Embassy Lisbon - UK Science and Innovation Network (Grant: PARSUK Bilateral Research Fund). SSP also acknowledges funding by “2022 LRA Lupus Innovation Award”, and by “European Crohn's and Colitis Organisation (ECCO) Pioneer Award 2021”. Co- funded by the European Union (ERC, GlycanSwitch, 101071386). Views and opinions expressed are however those of the author(s) only and do not necessarily reflect those of the European Union or the European Research Council Executive Agency. Neither the European Union nor the granting authority can be held responsible for them

Cadherins Glycans in Cancer: Sweet Players in a Bitter Process

Cadherins are key components in tissue morphogenesis and architecture, contributing to the establishment of cohesive cell adhesion. Reduced cellular adhesiveness as a result of cadherin dysfunction is a defining feature of cancer. During tumor development and progression, major changes in the glycan repertoire of cancer cells take place, affecting the stability, trafficking, and cell-adhesion properties of cadherins. Importantly, the different glycoforms of cadherins are promising biomarkers, with potential clinical application to improve the management of patients, and constitute targets for the development of new therapies. This review discusses the most recent insights on the impact of glycan structure on the regulation of cadherin function in cancer, and provides a perspective on how cadherin glycans constitute tumor biomarkers and potential therapeutic targets.IPATIMUP integrates the I3S Research Unit, which is partially supported by FCT, the Portuguese Foundation for Science and Technology (Fundação para a Ciência e a Tecnologia/Ministério da Ciência, Tecnologia e Inovação). This work was financed by FEDER – Fundo Europeu de Desenvolvimento Regional funds through the COMPETE 2020 – Operational Program for Competitiveness and Internationalization (POCI), Portugal 2020, and by Portuguese funds through the FCT in the framework of the project ‘Institute for Research and Innovation in Health Sciences’ (POCI-01-0145-FEDER-007274), PTDC/DTP-PIC/0560/2014, and PTDC/BBB-EBI/0567/2014. S.C. also acknowledges funding from the FCT (SFRH/BD/77386/2011)

The role of glycans in health and disease: Regulators of the interaction between gut microbiota and host immune system

The human gut microbiota is home to a diverse collection of microorganisms that has co-evolved with the host immune system in which host-microbiota interactions are essential to preserve health and homeostasis. Evidence suggests that the perturbation of this symbiotic host-microbiome relationship contributes to the onset of major diseases such as chronic inflammatory diseases including Inflammatory Bowel Disease. The host glycocalyx (repertoire of glycans/sugar-chains at the surface of gut mucosa) constitutes a major biological and physical interface between the intestinal mucosa and microorganisms, as well as with the host immune system. Glycans are an essential niche for microbiota colonization and thus an important modulator of host-microorganism interactions both in homeostasis and in disease. In this review, we discuss the role of gut mucosa glycome as an instrumental pathway that regulates host-microbiome interactions in homeostasis but also in health to inflammation transition. We also discuss the power of mucosa glycosylation remodelling as an attractive preventive and therapeutic strategy to preserve gut homeostasis.Co-funded by the European Union (GlycanTrigger, Grant Agreement No: 101093997). Views and opinions expressed are however those of the author(s) only and do not necessarily reflect those of the European Union or the European Research Council Executive Agency. Neither the European Union nor the granting authority can be held responsible for them. Salomé S. Pinho acknowledges funding from European Crohn’s and Colitis Organisation (ECCO) Pioneer Award 2021. Salomé S. Pinho also acknowledges the International Organization for the study of Inflammatory Bowel Disease (IOIBD) and the Portuguese Foundation for Science and Technology (FCT; EXPL/MED-ONC/0496/2021). Cláudia Rodrigues thanks FCT for funding (2020.08422.BD). Joana Gaifem acknowledges funding from European Crohn’s and Colitis Organisation (ECCO Grant) and FCT (DOI 10.54499/2020.00088.CEECIND/CP1608/ CT0001). The Crouch Lab is funded by The Academy of Medical Sciences (SBF0061175) and the Wellcome Trust/Royal Society Henry Dale Fellowship (224240/Z/21/Z)

Studying T Cells N-Glycosylation by Imaging Flow Cytometry

Imaging flow cytometry is an emerging imaging technology that combines features of both conventional flow cytometry and fluorescence microscopy allowing quantification of the imaging parameters. The analysis of protein posttranslational modifications by glycosylation using imaging flow cytometry constitutes an important bioimaging tool in the glycobiology field. This technique allows quantification of the glycan fluorescence intensity, co-localization with proteins, and evaluation of the membrane/cytoplasmic expression. In this chapter we provide the guidelines to analyze glycan expression, particularly the ß1,6 GlcNAc branched N-glycans, on the membrane of intestinal T cells from inflammatory bowel disease patients.This work was supported by grants from the Portuguese Foundation for Science and Technology (FCT), project grants (PTDC/DTPPIC/0560/2014; PTDC/BBB-EBI/0786/2012; EXPL/BIMMEC/0149/2012), “financiados no âmbito do Programa Operacional Temático Factores de Competitividade (COMPETE) e comparticipado pelo fundo Comunitário Europeu FEDER,” e do Quadro de Referência Estratégia Nacional QREN. This work was further supported by a Portuguese grant from “Grupo de Estudo da Doença Infl amatória Intestinal” (GEDII). This work had also the fi nantial support of FCT/MEC through National Funds and, when applicable, co-fi nanced by the FEDER via the PT2020 Partnership Agreement under the 4293 Unit I&D. S.S.P. (SFRH/BPD/63094/2009) also acknowledges FCT. A.M.D. PD/BD/105982/2014 also acknowledges FCT and BiotechHealth Doctoral Programme. The Institute of Molecular Pathology and Immunology of the University of Porto (IPATIMUP) integrates the Institute for Research and Innovation in Health (I3S), which is partially supported by the Portuguese Foundation for Science and Technology (FCT). Data was acquired at the Bioimaging Center for Biomaterials and Regenerative Therapies (b.IMAGE, INEB, Porto, Portugal)

"Mannose glycans as key players in trained immunity: A novel anti-tumoral catalyst"

Cell wall glycans isolated from microorganisms are long known to provoke strong immune responses piloted by innate immune cell populations, including monocytes, in the context of Trained Immunity (TI). However, the contribution of yeast-derived mannan in the reprogramming of monocytes remains ill-defined. Here, we demonstrated that TI is often accompanied by an altered gene expression profile of selected glycan-binding proteins expressed by monocytes, including DC-SIGN and Dectin-2. Additionally, we showed that mannan, a mannose rich glycan, can trigger an enhanced immune phenotype compatible with TI in healthy monocytes, with glycan-primed cells exhibiting enhanced pro-inflammatory cytokine secretion (TNFα and IL-6) and higher activation (CD86) levels. Furthermore, the glycan-mediated priming of monocytes also imposed alterations to the expression of certain Glycan-Binding Proteins, such as DC-SIGN. Importantly, we established that these mannan-trained immune cells displayed an improved capacity to kill tumor cells in vitro. Lastly, we confirmed that monocytes from non-muscle invasive bladder cancer patients treated with BCG instillations presented a TI phenotype, as was revealed by the higher cytokine production and activation. Altogether, this study lays the foundations for exploiting the immunological potential of glycan-derived pathogens in reprogramming innate immune cells towards an effective anti-tumor immune response.This work was supported by grants from SSP: co-funded by the European Union (ERC, GlycanSwitch, 101071386). Views and opinions expressed are however those of the author(s) only and do not necessarily reflect those of the European Union or the European Research Council Executive Agency. Neither the European Union nor the granting authority can be held responsible for them. The work was also co-funded by EU GlycanTrigger-grant Agreement No: 101093997. Views and opinions expressed are however those of the author(s) only and do not necessarily reflect those of the European Union or European Health and Digital Executive Agency. Neither the European Union nor the granting authority can be held responsible for them. SSP also acknowledges funding by “2022 LRA Lupus Innovation Award” and by “European Crohn’s and Colitis Organisation (ECCO) Pioneer Award 2021”. SSP also acknowledges the US Department of Defense, US Army Medical Research Acquisition Activity, FY18 Peer Reviewed Medical Research Program Investigator-Initiated Research Award (award number W81XWH1920053). IA acknowledges the Portuguese Foundation for Science and Technology (FCT) for funding (2022.00337.CEECIND), from BIAL Foundation and Portuguese Medical Association (Maria de Sousa Award 2023), from Núcleo de Estudos de Doenças Autoimunes and Portuguese Society of Internal Medicine (NEDAI-SPMI) and from European Society for Clinical Investigation (ESCI Exploratory Research Grant 2024). ˆAF acknowledges FCT for funding (2022.01422.PTDC) and the Portuguese Society of Neurology (SPN) – Grant: Bolsa Pereira Monteiro. CM-L holds a contract funded by the FCT under the project UCIPredict- TRANSCAN3/0001/2021. The authors thank Prof. Margarida Saraiva from i3S for the contribution to the discussion of this project

Glycan Structures Contain Information for the Spatial Arrangement of Glycoproteins in the Plasma Membrane

Glycoconjugates at the cell surface are crucial for cells to communicate with each other and the extracellular microenvironment. While it is generally accepted that glycans are vectorial biopolymers, their information content is unclear. This report provides evidence that distinct N-glycan structures influence the spatial arrangement of two integral membrane glycoproteins, Kv3.1 and E-cadherin, at the adherent membrane which in turn alter cellular properties. Distinct N-glycan structures were generated by heterologous expression of these glycoproteins in parental and glycosylation mutant Chinese hamster ovary cell lines. Unlike the N-linked glycans, the O-linked glycans of the mutant cell lines are similar to those of the parental cell line. Western and lectin blots of total membranes and GFP immunopurified samples, combined with glycosidase digestion reactions, were employed to verify the glycoproteins had predominantly complex, oligomannose, and bisecting type N-glycans from Pro(-)5, Lec1, and Lec10B cell lines, respectively. Based on total internal reflection fluorescence and differential interference contrast microscopy techniques, and cellular assays of live parental and glycosylation mutant CHO cells, we propose that glycoproteins with complex, oligomannose or bisecting type N-glycans relay information for localization of glycoproteins to various regions of the plasma membrane in both a glycan-specific and protein-specific manner, and furthermore cell-cell interactions are required for deciphering much of this information. These distinct spatial arrangements also impact cell adhesion and migration. Our findings provide direct evidence that N-glycan structures of glycoproteins contribute significantly to the information content of cells