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)

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

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)

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

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)

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)

Loss and Recovery of Mgat3 and GnT-III Mediated E-cadherin N-glycosylation Is a Mechanism Involved in Epithelial-Mesenchymal-Epithelial Transitions

BACKGROUND: N-acetylglucosaminyltransferase-III (GnT-III) is a glycosyltransferase encoded by Mgat3 that catalyzes the addition of β1,4-bisecting-N-acetylglucosamine on N-glycans. GnT-III has been pointed as a metastases suppressor having varying effects on cell adhesion and migration. We have previously described the existence of a functional feedback loop between E-cadherin expression and GnT-III-mediated glycosylation. The effects of GnT-III-mediated glycosylation on E-cadherin expression and cellular phenotype lead us to evaluate Mgat3 and GnT-III-glycosylation role during Epithelial-Mesenchymal-Transition (EMT) and the reverted process, Mesenchymal-Epithelial-Transition (MET). METHODOLOGY/PRINCIPAL FINDINGS: We analyzed the expression profile and genetic mechanism controlling Mgat3 expression as well as GnT-III-mediated glycosylation, in general and specifically on E-cadherin, during EMT/MET. We found that during EMT, Mgat3 expression was dramatically decreased and later recovered when cells returned to an epithelial-like phenotype. We further identified that Mgat3 promoter methylation/demethylation is involved in this expression regulation. The impact of Mgat3 expression variation, along EMT/MET, leads to a variation in the expression levels of the enzymatic product of GnT-III (bisecting GlcNAc structures), and more importantly, to the specific modification of E-cadherin glycosylation with bisecting GlcNAc structures. CONCLUSIONS/SIGNIFICANCE: Altogether, this work identifies for the first time Mgat3 glycogene expression and GnT-III-mediated glycosylation, specifically on E-cadherin, as a novel and major component of the EMT/MET mechanism signature, supporting its role during EMT/MET

Glycans as regulatory elements of the insulin/IGF system: Impact in cancer progression

The insulin/insulin-like growth factor (IGF) system in mammals comprises a dynamic network of proteins that modulate several biological processes such as development, cell growth, metabolism, and aging. Dysregulation of the insulin/IGF system has major implications for several pathological conditions such as diabetes and cancer. Metabolic changes also culminate in aberrant glycosylation, which has been highlighted as a hallmark of cancer. Changes in glycosylation regulate every pathophysiological step of cancer progression including tumour cell-cell dissociation, cell migration, cell signaling and metastasis. This review discusses how the insulin/IGF system integrates with glycosylation alterations and impacts on cell behaviour, metabolism and drug resistance in cancer.Financial support from Portugal: Institute of Molecular Pathology and Immunology of University of Porto (IPATIMUP) integrates the i3S research unit, which is partially supported by the Portuguese Foundation for Science and Technology (FCT). This article is a result of the project NORTE-01-0145-FEDER-000029, supported by the Norte Portugal Regional Programme (NORTE 2020) under the PORTUGAL 2020 Partnership Agreement through the European Regional Development Fund (ERDF). This work was also funded by FEDER—Fundo Europeu de Desenvolvimento Regional funds through the COMPETE 2020—Operacional Programme for Competitiveness and Internationalisation (POCI), Portugal 2020, and by Portuguese funds through FCT—Fundação para a Ciência e a Tecnologia/ Ministério da Ciência, Tecnologia e Inovação in the framework of the project (POCI-01/0145-FEDER-016601). Financial support from Brazil: Ministério da Saúde and Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES), under the project 336/13

Dysregulation of T cell receptor N-glycosylation: A molecular mechanism involved in ulcerative colitis

The incidence of inflammatory bowel disease is increasing worldwide and the underlying molecular mechanisms are far from being fully elucidated. Herein, we evaluated the role of N-glycosylation dysregulation in T cells as a key mechanism in the ulcerative colitis (UC) pathogenesis. The evaluation of the branched N-glycosylation levelsandprofile of intestinalTcell receptor (TCR)wereassessedin colonic biopsies fromUCpatientsand healthy controls. Expression alterations of the glycosyltransferase gene MGAT5 were also evaluated. We demonstrated thatUCpatients exhibit a dysregulation ofTCRbranchedN-glycosylationonlamina propriaTlymphocytes. Patients with severe UC showed the most pronounced defect on N-glycan branching in T cells. Moreover, UC patients showed a significant reduction of MGAT5 gene transcription in T lymphocytes. In this study, we disclose for the first time that a deficiency in branched N-glycosylation on TCR due to a reduced MGAT5 gene expression is a new molecular mechanism underlying UC pathogenesis, being a potential novel biomarker with promising clinical and therapeutic applications.This work was supported by grants from the Portuguese Foundation for Science and Technology (FCT), project grants (PTDC/ CVT/111358/2009; PTDC/BBB-EBI/0786/2012; EXPL/ BIM-MEC/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 Nacio-nal QREN. This work was further supported by a portuguese grant from ‘Grupo de Estudo da Doenc¸a Inflamatória Intestinal’ (GEDII). S.S.P. (SFRH/BPD/63094/2009); S.C. (SFRH/BD/ 77386/2011) also acknowledge FCT. IPATIMUP is an Associate Laboratory of the Portuguese Ministry of Science, Technology and Higher Education, and is partially supported by FCT

Mannosylated glycans impair normal T-cell development by reprogramming commitment and repertoire diversity

T-cell development ensures the formation of diverse repertoires of T-cell receptors (TCRs) that recognize a variety of antigens. Glycosylation is a major posttranslational modification present in virtually all cells, including T-lymphocytes, that regulates activity/functions. Although these structures are known to be involved in TCR-selection in DP thymocytes, it is unclear how glycans regulate other thymic development processes and how they influence susceptibility to disease. Here, we discovered stage-specific glycome compositions during T-cell development in human and murine thymocytes, as well as dynamic alterations. After restricting the N-glycosylation profile of thymocytes to high-mannose structures, using specific glycoengineered mice (Rag1CreMgat1fl/fl), we showed remarkable defects in key developmental checkpoints, including ß-selection, regulatory T-cell generation and γδT-cell development, associated with increased susceptibility to colon and kidney inflammation and infection. We further demonstrated that a single N-glycan antenna (modeled in Rag1CreMgat2fl/fl mice) is the sine-qua-non condition to ensure normal development. In conclusion, we revealed that mannosylated thymocytes lead to a dysregulation in T-cell development that is associated with inflammation susceptibility.Funded by the “2022 Lupus Research Alliance (LRA) Lupus Innovation Award”. Institutional funding from the Portuguese Foundation for Science and Technology (FCT): projects NORTE-01-0145-FEDER-000029, POCI-01/0145-FEDER-016601, POCI-01-0145-FEDER-028772, and PTDC/MEC-REU/28772/2017 (SSP). This study was co-funded by the European Union (ERC Synergy, 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. The study was also co-funded by the European Union, GlycanTrigger project, 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. A grant was received from the Portuguese group of study in autoimmune diseases (NEDAI) to SSP. MMV (PD/BD/135452/2017; COVID/BD/152488/2022) received funding from the FCT