Human T cell glycosylation and implications on immune therapy for cancer

Figures & data

Figure 1. Overview of human N- and O-glycosylation in the Golgi apparatus. On the left side, the synthesis of a human glycoprotein with several relevant complex-type N-glycans is shown. In the cis Golgi, mannosidase I (ManI) activity leads to a Man₅GlcNAc₂ that can be further modified in the medial Golgi. N-acetylglucosaminyltransferase I (GnTI) activity commits the glycan to the complex or hybrid type. Mannosidase II (ManII) activity, followed by several N-acetylglucosaminyltransferases then further commits the glycan to the complex type. If only N-acetylglucosaminyltransferases II (GnTII) acts on it, the result is a biantennary complex type N-glycan. GnTIV and/or GnTV activity then generates different triantennary or a tetraantennary complex type glycan. Fucosyltransferase VIII (FucTVIII) can act on any complex or hybrid type glycan to add a core α-1,6-fucose in the medial Golgi. Afterward, in the trans Golgi, galactosyltransferases (GalT), fucosyltransferases (FucT), sialyltransferases (SiaT) or a combination of GnTs and GalTs synthesize different capping moieties (sialylation, poly-LacNAc repeats, Lewis antigens) on N-glycans

The right side of the figure shows mucin-type O-glycosylation biosynthesis. Polypeptide-GalNAc-transferases (ppGalNAcTs) initiate O-glycosylation in the cis Golgi, which is followed by the action of one or two core synthesizing enzymes: core 1 galactosyltransferase (C1GalT), core 2 N-acetylglucosaminidase I, II or III (C2GnTI/II/III) and core 3 N-acetylglucosaminidase (C3GnT), resulting in the synthesis of four so-called core structures. Core 1 and 2 are common, while core 3 and 4 have a restricted expression pattern. Two SiaT activities in particular (ST6GalNAc and ST3Gal) can add N-Acetylneuraminic acid to the Tn antigen or core 1 (T antigen) to generate the sialyl-Tn or sialyl-T antigen (STn or ST antigen). Several GalTs, GnTs, FucTs and SiaTs can act on the core structures to introduce further branching and terminating structures (not shown). Note that four more core structures (core 5–8) are known, but since their occurrence is extremely rare, they are not shown here. This figure has been adapted from De Wachter et al. with permission.⁷

Table 1. Role of T cell glycosylation during T cell development, activation and signaling

	Glycosylation	Effect
T-CELL DEVELOPMENT	α-(1,3) fucosylation of PSGL-1	Homing of T cell precursors to the thymus (P-Selectin binding)^Citation18,Citation19
	O-GlcNAcylation by OGT	β-selection, maturation of single positive CD4^+ and CD8^+ T cells^Citation21,Citation22
	α-(2,3) and α-(2,6) sialylation of cell surface glycoproteins	Maturation of single positive CD4^+ and CD8^+ T cells^Citation23-25 Regulation of TCR affinity-dependent negative selection^Citation16,Citation26-28
	Core 2 O-glycosylation	Extravasation across activated vascular endothelium and entry in non-lymphoid tissue^Citation29,Citation30
T CELL ACTIVATION AND SIGNALING	N-glycan branching by MGAT5	Increased N-acetyllactosamine modifications (ligand of Galectins) Binding by Galectins holds CD45 and TCR signaling complex in close proximity to prevent low-avidity T cell activation^Citation31,Citation32 Promotion of Th2 development over Th1 responses^Citation33 N-glycan branching on CTLA-4 enhances its retention at the T cell surface, suppressing T cell activation and promoting immune tolerance^Citation34-36
	N-Glycosylation of MHCI	Important for protein folding, trafficking and peptide loading^Citation37-40
	N-Glycosylation of MHCII	Modulation of glycopeptide binding and presentation^Citation41-44
	α-(2,6) sialylation of CD45	Capping of the terminal Gal-β-1,4-GlcNAc-binding sites, used as a ligand by Galectin-1, with consequent reduced Galectin-1 induced cell death Inhibition of CD45 clustering, leading to diminished signaling^Citation45
	N-Glycan branching of CD25	Increased surface expression and retention, thereby controlling T cell differentiation^Citation46
	O-GlcNAcylation of transcription factors involved in IL2-signaling	Regulation of IL-2 production upon TCR stimulation^Citation21
	Sialylation of CCR7 receptor	Modulation of T cell homing to the lymph nodes. Sialylation provides steric hindrance for ligand binding, diminishing receptor potency and decreasing signaling ability; reflected by low CCR7 sialylation on recirculating T cells and high CCR7 sialylation on activated T cells^Citation47

Table 2. Role of glycan binding proteins in T cell cancer immunity

	GBP	EFFECT		PRO OR ANTI-TUMORIGENIC
GALECTINS	Galectin-1	Induction of tolerogenic signaling and immune suppression^Citation68,Citation69		PRO
	Galectin-3
	Intracellular (T-cell)  Extracellular	Blocking T cell apoptosis^Citation70 Negative regulation of T cell activation by destabilizing the immunological synapse and by promoting downregulation of the TCR. Binding to CTLA-4 and Lag3 leads to inhibition and cell death of activated T cells^Citation71,Citation72		ANTI PRO
	Galectin-9	Negatively regulates T cell immunity by binding to Tim-3^Citation67		PRO
SELECTINS	L-Selectin
	On T cells  On leukemia cells	Acquisition of T cell effector functions^Citation73 Improved trafficking and distribution abilities (dissemination) (CLL patients)^Citation74		ANTI PRO
SIGLECS	Siglec-9
	On TILs	Effector memory T cells with higher functional responses after stimulation^Citation75,Citation76		ANTI
	Siglec-15
	On tumor cells and TIMs	Suppression of antigen-specific T cell responses^Citation77		PRO

Table 3. Examples of glycan-engineering and its implications in immune therapy

	Intervention	Effect
GENETIC ENGINEERING	Deletion of N-glycosylation sites in TCR chains	Increased functional avidity by improved TCR multimerization and decreased TCR-MHC dissociation^Citation108
	Overexpression of ST6GalI (increased α-(2,6) sialylation)	Shielding LacNac residues from Galectin-1 binding, reducing Galectin-1 induced cell death of T cells^Citation45
	Genetic ablation of FUT8	Reduced cell surface expression of PD-1 and enhanced T cell activation^Citation109
METABOLIC GLYCOENGINEERING	Inhibition of LacNAc synthesis Addition of Galectin-binding variant of lactose	Increased number of infiltrating tumor-specific CD8^+ T cells and intra-tumoral IFN-γ expression^Citation110 Increased TIL numbers and reduced tumor growth^Citation111
	Inhibition of fucosylation and sialylation Sialic acid biosynthesis blockade	Decreased Selectin ligand synthesis and impaired cancer cell adhesion and migration in vitro and prevention of metastasis in vivo^Citation112 Enhanced T cell killing capacity^Citation113
	Enhanced N-glycan branching	Control of the T cell immune response^Citation114
CHEMOENZYMATIC GLYCOENGINEERING	LacNAc labeling LacNAc modification with antibodies to generate antibody-T cell conjugate	Biophysical probe for imaging or glycomics analysis^Citation115 Increased tumor targeting capacity and resistance to inhibitory signals^Citation116
	Fucosyltransferase treatment of regulatory T cells	Increased expression of sLe^x antigens leading to improved trafficking, homing and engraftment^Citation117
	PNGaseF treatment of regulatory T cells	Increased proliferation of naïve and memory T cells^Citation118
	Sialidase treatment of cytotoxic T cells	Increased T cell activation and cytolytic activity^Citation119

Figure 2. In this review, we discuss the role of different types of glycans decorating the surface of T cells, and their binding to GBPs and resulting functions. Central in the figure, several mammalian glycoforms that can occur on the T cell surface are shown. Above them, the GBPs that bind to them and the effects they have on T cells are outlined and below them we elaborate on the effects of therapeutic interventions on the glycan composition. The color of the arrows relates to the glycoforms, as indicated centrally in the figure, and the direction indicates decreased or increased expression of those glycoforms. The text next to the arrow then explains the effects on the T cell, resulting from the indicated changes in the glycoforms

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