The immune system is highly regulated by a series of stimulatory and inhibitory pathways that are crucial to maintain a healthy and balanced system. Disruption of the control of this immunological balance can result in abnormal stimulatory signals associated with the loss of immune tolerance in autoimmunity or in the creation of aberrant immunosuppressive networks that occur in cancer. Accumulating evidences have been demonstrating the importance of glycans and glycans binding proteins [including galectins (1, 2), C-type lectins (3), and sialic acid-binding immunoglobulin-type lectins (siglecs) (4, 5)] in the regulation of both innate and adaptive immune responses. In fact, all cells are covered with a dense coat of glycans that constitute a major molecular interface between cells and their environment. The diversity of glycans presentation at cell surface is enormous, encoding a myriad of important biological information that remains to be fully characterized. Glycosylation is the enzymatic process responsible for the attachment of glycans (carbohydrates) to proteins or lipids (predominantly via nitrogen (N) and oxygen (O) linkages), a process that occurs in the Endoplasmic Reticulum/Golgi compartment of essentially all cells being mediated by the coordinated action of a portfolio of different glycosyltransferases and glycosidases enzymes (6). The proper development and function of the immune system relies both on the dynamic regulation of the expression of glycan-structures and glycan-binding proteins, and the interactions between them (7). This review discusses the role of glycans (with a focus on N-linked glycans) on T cells biology and function, including T cell development, activation, differentiation, and signaling. This dynamic interplay between glycans and T cells activity controlling both auto-reactivity and self-tolerance will be presented and discussed (Figure 1).

T cells are developed in the thymus where a microenvironment is set, which enables the selection of T cell receptors (TCRs) to generate a diverse repertoire of potential antigen recognition (8). Lymphoid progenitors from the bone marrow enter into the cortical tissue of the thymus, where they start to expand and develop (9, 10). Despite the fact that the role of glycosylation in T cell development and thymus selections still remains to be fully understood, some important findings highlight the relevance of glycans in this process (Figure 2).

The proper function of T lymphocytes is highly dependent on their surface receptors, which in turn are highly mediated by glycosylation. Although O-glycan structures have been shown to play important roles on immune-associated molecules (37), the prominent role of N-linked glycans is emphasized in this section (Figure 2).

As previously mentioned, MHC I is expressed by almost all nucleated cells and interacts with TCRs on CD8⁺ T cells; in turn, MHC II is expressed by professional antigen presenting cells (APCs) (dendritic cells - DC, macrophages, B cells and TECs) and is recognized by CD4⁺ T cells (7, 38). More than 3 decades ago, it was demonstrated that blocking MHC1a N-glycosylation, through acceptor site mutation, results in significant increases in intracellular misfolded protein along with decreases in cell surface expression (39). MHC II is assembled by two glycoproteins, α and β chains. The α chain contains N-linked high-mannose and complex glycans whereas the β chain is only constituted by complex N-glycans (40). In contrast to the role of MHC I, MHC II glycosylation was shown to have a particular impact on the effective antigen binding, as well as in the presentation of microbial carbohydrate antigens, which consequently influences downstream T cell responses. This was demonstrated by the depletion of the Mgat2 gene, which compromises N-glycan branching, decreasing carbohydrate antigen presentation by MHC class II and leading to loss of T cell stimulatory activity (41).

During TCR signal transduction, glycans play a key role in stabilizing individual molecules in the complexes at the immunological synapse and by protecting them from the action of proteases during T cell engagement (31). Additionally, glycans can also restrict nonspecific protein-protein interactions, like aggregation of TCRs on the membrane, helping to orient the interactions of the proteins in the central clusters (31). Demetriou et al. demonstrated that β1,6-GlcNAc branched N-glycans structures (catalyzed by GnT-V) regulate T cell activity, namely in CD4⁺ T cells by increasing the threshold of T cell activation, suppressing T cell growth and signaling (30, 42). Moreover, core-fucosylation, which refers to fucose attached to the innermost N-acetylglucosamine of N-linked glycans, catalyzed by α1-6 fucosyltransferase (FUT8), was also shown to affect T cell activity in immune mediated disorders (42, 43).

The T cell activity is also dependent on glycosylation of co-receptors, such as the complex formation between TCR and CD45. Galectin-3 is a key mediator of this complex, by establishing a molecular lattice through binding to polylactosamine structures in branched N-glycans. Consequently, CD45 phosphatase activity induces downregulation of T cell signaling, preventing T cell activation (44). Furthermore, CD45 is alternatively spliced into five different isoforms on human leukocytes (CD45ABC, CD45AB, CD45BC, CD45B, and CD45RO) (45–47), all decorated with up to 11 N-glycans in the membrane proximal region. Importantly, all isoforms present different glycosylation profiles (48, 49), that change during T cell differentiation and activation (50, 51), as reviewed in (36). CD28 is another T cell surface glycoprotein acting as a secondary signaling molecule of T cell activation. Interestingly, nearly 50% of the molecular mass of CD28 is constituted by N-glycans (52). Previous studies reported that N-glycosylation of human CD28 can negatively regulate CD28-mediated T cell adhesion and co-stimulation, namely the interaction between CD28/CD80. Mutation of all potential N-linked glycosylation sites of CD28 as well as treatment of Jurkat cells with inhibitors of N-glycosylation pathway resulted in a defective CD28 glycosylation with enhancement of the binding to CD80 expressed on APCs (52). The branching N-glycosylation of CD25 receptor also modulates its cell surface retention controlling T differentiation with impact in immune tolerance. Recently, it was demonstrated that a decreased UDP-GlcNAc and complex branching N-glycosylation induces a decreased cell surface retention of CD25 and IL-2 signaling, promoting a T helper (T_H) 17 over induced regulatory T cell (iTreg) differentiation (53) (Figure 2).

Importantly, the co-inhibitory receptors are likewise modulated by N-glycosylation. One of the major negative regulators of T cell response is the cytotoxic T-lymphocyte protein 4 (CTLA-4), that comprises two N-glycosylation sites described to modulate its cell surface retention on T cells and thereby its affinity for CD80/CD28 on APCs (54–56). The impact of N-glycosylation in the modulation of the inhibitory functions of CTLA-4 and programmed cell death protein-1 (PD-1) is discussed in more detail in section “Glycans in tolerogenic/immunosuppressive responses”. Nonetheless, other co-inhibitory receptors like Lymphocyte-activation gene 3 (Lag-3), mucin-domain-containing molecule-3 (Tim-3), and T cell immunoreceptor with Ig and ITIM domains (TIGIT) may also undergo glycans-mediated regulation, as they exhibit N-glycan-binding sites, however the role of glycans on these molecules remains to be explored (57).

Taken together, N-glycosylation plays an instrumental role in the regulation of T cell activation and functions by targeting not only TCR but also its co- receptors (Figure 2).

Autoimmunity is characterized by the loss of self-tolerance and development of an autoreactive immune response toward the individual's own organism. Glycan motifs play a crucial role in the determination of self/non-self antigens. Specific glycan structures, expressed by microbial pathogens, are commonly responsible for the primary activation of the innate immune system; however, the mechanisms involved in the self/non-self discrimination, mediated by glycans are far from being fully elucidated. Abnormal levels of branched N-glycans have been associated with exacerbated immune responses in murine models (58). Particularly, the dysregulation of the N-glycosylation pathway has been associated with autoimmune-like phenotypes. The inability to synthetize β1,6-GlcNAc antennae, in Mgat5^−/− mice has been associated with an increased susceptibility to immune-mediated disorders such as an increased delayed-type hypersensitivity responses, as well as increased susceptibility to develop experimental autoimmune encephalomyelitis (EAE) (30, 59) and severe forms of colitis (60). The lack of β1,6 branching N-glycans favors TCR clustering, leading to a decrease of the TCR threshold and consequently increased T cell activation (30) associated with the hyperimmune response observed in these mice (Figure 2). This hyperimmune phenotype is also due to an abnormal formation of lattices between TCR-branched glycosylation and galectins (61, 62). Accordingly, β3 GnT2-deficient mice show T cell hypersensitivity due to the reduction of polylactosamine on the N-glycans (ligands of galectins), similarly to what is observed in Mgat5 deficient mice (30, 61). Furthermore, absence of α-mannosidase II (which catalyses the last hydrolysis of the α-mannose), was shown to result in signs of glomerulonephritis, deposits of glomerular IgM immunocomplexes and complement component 3 as well as high levels of anti-nuclear antibodies (63, 64), which is consistent with a Lupus-like syndrome (Figure 2). Taken together, these evidences support the role of N-glycosylation in the perspective of T cell biology.

The role of N-glycans in antigen presentation and recognition is still elusive, and in fact abnormal glycoantigen presentation might also impact T cell activity. Abnormal accumulation of high-mannose, paucimannose, and agalactosyl bi-antennary glycans, have been detected in kidney tissue from MRL-lpr mouse (a well-stablished murine model of SLE) (65). Moreover, evidences have been showing that Mgat1^f/fSyn1-Cre mice, with Mgat1 deletion at the Synapsin I-expressing cells (abundant in neural tissues), presented neurological defects, with high levels of neuronal apoptosis and caspase 3 activation (66). These high levels of apoptosis are observed in several autoimmune diseases, which results in activation of immune system (67) (Figure 2). Although highly unexplored, rare autoimmune diseases are also associated with N-glycosylation dysfunctions. As example, idiopathic inflammatory myopathies (IIM) are a group of rare diseases of autoimmune nature, whose etiopathogenesis is far from being totally understood (68). Muscle cells surface is enriched with glycoproteins and several lines of evidence provide support for a fundamental role of glycosylation in muscle homeostasis and function (69, 70). Glucosamine (UDP-N-Acetyl)-2-Epimerase/N-Acetylmannosamine Kinase (GNE) genetic mutations (a gene that encodes N-acetylmannosamine (ManNAc) kinase enzyme, responsible for the biosynthesis of N-acetylneuraminic acid) results in hypo-sialylation of muscle glycoproteins; the prophylactic supplementation with sialic acid precursor (ManNAc) was shown to prevent the muscle phenotype in mice with gene mutations that cause hereditary inclusion-body myositis (hIBM), a muscle phenotype that resembles one type of IIM (71). Altogether, these findings highlight the importance of further studies addressing the role of N-glycosylation in the perspective of neoautoantigens, since autoantigens contain a significant amount of glycoantigens due to the increased number of N-glycosylation sites comparing with other proteins (72).

The Glycan binding proteins (GBPs) are expressed in the APCs being characterized by a carbohydrate recognition domain which specifically recognizes glycan structures present at the cell surface receptors. This glycan-GBPs engagement results in either an anti- or pro-inflammatory response (73). C-type lectins, siglecs, and galectins are examples of GBPs, that are instructors of immune responses (5, 73). As example, SIGN1R (expressed by APCs and the analogous of the human dendritic cell-specific ICAM-grabbing non-integrin - DC-SIGN) signaling was shown to result in the expansion of IL-10-secreting Treg cells, preventing the development of autoimmune diseases such as EAE and type 1 diabetes (T1D) (74). Galectin-1 also plays an important immune-regulatory role in EAE (75) as mice deficient in galectin-1 (Lgals1^−/−) have increased T_H1 and T_H17 responses being more susceptible to EAE when compared with wild type mice (76). More recently, Galectin-1 was shown to modulate the cytolytic activity of CD8⁺ T cell. The interaction of Galectin-1 and Fas ligand seems to be responsible for the retention of this glycoprotein at the surface of cytotoxic T lymphocytes hampering the cytolytic ability of these cells (77). Overall, GBPs-glycoprotein interaction is essential to instruct a T cell—mediated immune response.

Notably, one of the first evidences addressing the relationship between the dysregulation of N-glycosylation and human autoimmunity was observed in multiple sclerosis (MS) patients. During active, relapse or in very early stages of remission, peripheral blood mononuclear cells from MS patients display a significant decrease of the enzymatic activity of Golgi β1,6 N-acetylglucosaminyltransferase (core 2 GlcNAc-T), compared to healthy subjects (78). Moreover, MGAT5 polymorphisms were associated with MS severity (79) together with MGAT1, IL2R, and IL7R Single Nucleotide Polymorphisms (80–82). Additionally, in Inflammatory Bowel Disease (IBD), it was also demonstrated that lamina propria T lymphocytes from ulcerative colitis (UC) patients exhibited a deficiency in β1,6-GlcNAc branching N-glycans due to decreased levels of MGAT5 gene expression (83). Importantly, low levels of branched N-glycans in lamina propria early at diagnosis were shown to predict UC patients that will fail the response to standard therapy, thus displaying a bad disease course (84). The supplementation of intestinal T cells from UC patients and mouse models with colitis with GlcNAc promoted the enhancement of β1,6 branching N-glycans on T cells, suppressing TCR signaling and reducing the production of pro-inflammatory cytokines such as tumor necrosis factor alpha (TNFα) and interferon gamma (IFNγ). Pre-clinical studies both in IBD and MS demonstrated the immunomodulatory properties of N-glycans in the control of T cell-mediated immune response (60, 85), paving the way for the development of human clinical trials, that are currently on going (53, 60). Less explored but of utmost importance is the study of N-glycosylation profile in rare autoimmune disorders, since its etiopathogenesis is still very elusive. Glycosylation changes in muscle-associated human disease have focused in muscular dystrophies (86) and congenital disorders of glycosylation (87). Recent studies have shown that muscle cell surface glycosylation is finely regulated and subjected to alterations under inflammatory conditions (88), pointing to a possible interaction between muscle glycocalyx and the extracellular milieu, which is particularly enriched in immune cells and antibodies in IIM patients (89).

Overall, glycans are critical determinants in autoreactive responses both by directly regulating T cell activity and also through the creation of abnormal glycoantigens that may unleash an autoreactive immune response.

Recent studies have been highlighted that alterations on the glycosylation pattern of T cells' receptors, as well as the alterations of the glycosylation profile of tumor cells (tumor glyco-code), are implicated in the modulation of the immune response leading to immunosuppressive pathways, known to occur in the tumor microenvironment associated with tumor immunoescape (90).

The impact of glycosylation on T cell development and functions is enormous, as revealed by the critical roles of glycans in the development and progression of major diseases such as auto-immunity and cancer, as described herein. In order to accompany the bioenergetic and biosynthetic demands required for T cell proliferation and activation, a shift in the T cell metabolism is required. While naïve T cells are in a metabolic quiescent state, mainly using oxidative phosphorylation to maximize ATP production, T cells under clonal expansion or under differentiation, reprogram their metabolic status to aerobic glycolysis and glutaminolysis in order to increase the availability of glycolytic precursors for the biosynthesis of nucleotides, amino acids and lipids (125–127). During T cell activation, the hexosamine biosynthetic pathway (HBP—a branch of the glucose metabolism) is upregulated in order to generate the nucleotide sugar-donor substrate UDP-GlcNAc, required for N-glycosylation, O-GlcNAcylation, and glycosaminoglycans production that are needed for a proper T cell function (128).

Mediators from the glycolytic pathway such as glucose (Glc), glutamine (Gln), acetyl CoA are known to interfere with the availability of the UDP-GlcNAc in the cell (129–131). Together Glc and Gln were shown to increase UDP-GlcNAc in nutrient-starved T cells. In the same setup, the supplementation of both Glc and glucosamine (GlcN–a metabolite of the HBP) further increased the UDP-GlcNAc cellular content, demonstrating the sensitivity of the HBP to nutrients that enter directly (GlcN) or through a precursor pathway (Glc in glycolysis) (130). Despite the general use of the UDP-GlcNAc as a substrate donor of HBP, there are some glycosyltransferases that are more susceptible to nutrient changing than others, such as the case of OGT (132). In fact, the supply with Glc and Gln are crucial for protein O-GlcNAcylation, that is important during T cell development, being associated with T cell malignant transformation (23). Among the N-acetylglucosaminyltransferases (GnTs) that participate in the HBP, the less sensitive to nutrient changing (and thus substrate availability) are GnT1, GnT2, and GnT3, due to lower Michaelis Constant (K_m) levels, meaning that these enzymes require low levels of the substrate to synthetize the specific glycans. In contrast, GnT4 and GnT5 present higher K_m and therefore their activity is highly dependent on the availability of the UDP-GlcNAc substrate (119, 133). Therefore, these two enzymes are sensitive to alterations in glucose and HBP metabolism (as the GlcN or N-acetyl glucosamine–GlcNAc) (62), which ultimately will interfere in the N-glycan branching biosynthesis on T cells with impact in their activity, as detailed in section “Glycans in the regulation of T cell activity and functions” (60). In fact, supplementation with Glc, Gln, and GlcNAc increases branching N-glycans on Jurkat cells and resting T cells from mice (85, 119, 130). Moreover, CD4⁺ T cells from MGAT5^+/+ or MGAT5^+/−− mice supplemented with oral GlcNAc also results in up to 40% increase of branching N-glycans, detected by L-PHA (130). This enhancement of branching N-glycosylation upon GlcNAc supplementation was shown to functionally impact on T cells activity by reducing T cell activation, decreasing T_H1 differentiation, and increasing retention of the growth inhibitory receptor CTLA-4 at T cell surface (85, 130).

Importantly, evidences suggest that the glycolysis and glutaminolysis compete with HBP pathway for the same metabolites. Recently, Araujo et al showed that, during T_H17 differentiation the existence of common mediators shared between HBP, glycolysis (fructose-6-phosphate) and glutaminolysis (Gln) results in a starvation of the HBP mediators, translated in a reduction of N-glycan branching due to the limitation on the UDP-GlcNAc availability (53). Fueling HBP with GlcNAc switched the cell fate from T_H17 to iTreg differentiation, through stimulation of IL2-Rα signaling (53). This interplay between metabolic pathways was further demonstrated by the increase on Glc, Gln, fatty-acids uptake, and lipid storage upon stimulation of the HBP with GlcNAc supplementation, suggesting a reprogramming of the cellular metabolism upon GlcNAc flux (53, 134).

The impact of glycans as metabolic regulators of T cells is also testified by its effects in ex vivo and in vivo models of autoimmune diseases. The metabolic supplementation with GlcNAc in ex vivo human colonic T cells from IBD patients resulted in an enhancement of the branching N-glycosylation pathway that was accompanied by a significant reduction of T cell proliferation, supression of T_H1/T_H17 immune response (through decreased production of IFN-γ and IL-17A pro-inflammatory cytokines) and decreased TCR signaling (60). Accordingly, the GlcNAc supplementation of mice models with auto-immune diseases such as EAE, TID, and IBD results in inhibition of T_H1, T_H17 immune response concomitantly with a significant improvement of the clinical symptoms (60, 85). Treatment with GlcNAc after disease onset also demonstrate inhibitory effects on the development of the EAE, by reducing the secretion of INF-γ, TNF-α, IL-17, and IL-22 (85). Interestingly, a dual role of GlcN (the precursor of GlcNAc) on the progression of autoimmune disorders was shown, by demonstrating its impact in preventing T_H1-mediated Type I diabetes (through the reduction of IFN-γ producing CD4⁺ T cells), but also the GlcN effects in exacerbating T_H1/T_H17–mediated EAE symptoms (trough stimulation of T_H17 response) (135). In contrast, another study showed that GlcN suppresses acute EAE through the blockage of T_H1 and induction of T_H2 response (136). GlcN supplementation was further shown to mediate T cell activation by decreasing the N-glycosylation of CD25 (IL-2Rα) from CD4⁺ T cell (135). This down-regulation of N-glycosylation might be explained by the competition between GlcN and Glc for the same glucose transporter which might impact in the reduction of the GlcNAc concentration.

Altogether, alterations on the glucose metabolism and partially changes in the metabolic flux of HBP have a direct impact on T cells N-glycosylation profile with major consequences in their function and activity. Ultimately, the modulation of the HBP constitutes an important metabolic target able to control both autoreactive and immunosuppressive responses known to occur, respectively, in autoimmunity and cancer.

The contribution of the glycome as a major regulator of the immune system is clear. Glycans actively participate in the cellular and molecular mechanisms underlying the genesis of the loss of immunological tolerance associated with (auto)immunity, from one hand, participating also in the creation of tolerogenic pathways associated with cancer progression, from the other. The importance of glycans in immune response spans from its role in the modulation the T cell development; their importance as a source of glycoantigens presentation; as well as their role as fine tuners of T cell response. In this context, glycans can exert a dual role, acting either as immune inhibitory checkpoints or as immune stimulatory signals. Understanding in depth the influence of glycans in the immune regulatory circuits that mediate the pathophysiology of autoimmunity and cancer will generate a platform with extraordinary potential to illuminate the identification of novel biomarkers and targets for the development of efficient immunomodulatory strategies with applications in the clinical setting.

All the authors wrote the manuscript. AD and NP created the figures. SP performed the critical review of the manuscript.