Loss of effector function of human cytolytic T lymphocytes is accompanied by major alterations in N- and O-glycosylation

Abstract

Most human tumors are not eliminated by the immune system, and therapeutic vaccination shows poor results, a fact that can be explained at least partially by an immunosuppressive tumor microenvironment that is abundant in galectin-3. On cytolytic T lymphocyte (CTL) clones, maintained in culture by regular stimulation, recently activated CTLs present low effector functions. However, these functions are restored after a short treatment with LacNAc. The latter, which is in agreement with the glycoprotein-galectin lattice concept involving reduced motility, poses the question why galectin-3 ligands improve effector functions. We employed ultrasensitive MALDI-TOF-MS on resting and recently activated CTL clones combined with various glycosidase digestions and GC-MS linkage analyses. Our results showed that compared with the resting CTLs, the N-glycans of the recently activated CTLs consisted of (i) larger LacNAc oligomers of which a significant portion was longer than four-units and (ii) more multi-antennary structures. Interestingly, our results showed that the poly-LacNAc appeared to be equally distributed on all available N-glycan branches and not selectively enriched on a specific branch. The above structural alterations in the recently activated CTLs are expected to increase the galectin-3-LacNAc lattices and multivalent interactions and, therefore, reduce the motility of surface glycoproteins, such as the T-cell receptor. These findings suggest that the loss of effector functions on CTLs may be linked to reduced motility of surface glycoproteins. In addition, our results showed that recently activated CTLs had a reduced abundance of NeuAcα2,6-linked N-glycans and an increased abundance of disialylated core 1 and monosialylated core 2 O-glycan structures.

FIGURE 1.

LacNAc treatment of human CTL can restore their ability to secrete IFN-γ. Resting CTL clones were expanded by stimulation every 2 weeks with peptide-pulsed Epstein-Barr virus immortalized B cells in the presence of feeder cells and IL-2. To assess effector functions, 10,000 CTL were collected either at the resting state (±2 weeks after stimulation) or in an activated state (4 days after stimulation). CTL were incubated (or not) for 2 h with LacNAc (2 mm) and subsequently stimulated with peptide-pulsed HLA-A1 cells. IFN-γ was measured by ELISA in the supernatant of overnight cocultures.

FIGURE 2.

MALDI-TOF mass spectra of permethylated N-glycans of CTL clone A10 derived from resting and recently activated state. N-Glycomic profiles of resting (A) and recently activated (B) CTL clone A10 were obtained from the 50% MeCN fraction from a C₁₈ Sep-Pak column (“Experimental Procedures”). Annotated structures are according to the Consortium for Functional Glycomics guidelines. All molecular ions are [M+Na]⁺. Putative structures are based on composition, tandem MS, and the biosynthetic knowledge. Structures that show sugars outside a bracket have not been unequivocally defined.

FIGURE 3.

MALDI-TOF mass spectra of permethylated O-glycans of CTL clone A10 derived from resting and recently activated state. O-Glycomic profiles of resting (A) and recently activated (B) CTL clone A10 were obtained from the 35% MeCN fraction from a C₁₈ Sep-Pak column (“Experimental Procedures”). Annotated structures are according to the Consortium for Functional Glycomics guidelines. All molecular ions are [M+Na]⁺. Putative structures based on composition, tandem MS, and the biosynthetic knowledge.

FIGURE 4.

Low mass MALDI-TOF spectra of permethylated N-glycans derived from endo-β-galactosidase digestion of resting and recently activated CTL clone A10. N-Glycomic profiles of CTL clone A10 in resting (A) and recently activated (B) states are shown. Profiles of N-glycans are from the 35% MeCN fraction from a C₁₈ Sep-Pak (“Experimental Procedures”). All molecular ions are [M+Na]⁺. Putative structures are based on composition, tandem mass spectrometry, and the literature shown. Annotated structures are according to the Consortium for Functional Glycomics guidelines. Not annotated peaks correspond to no N-glycan structures or to matrix peaks.

FIGURE 5.

Middle mass MALDI-TOF spectra of permethylated N-glycans derived from endo-β-galactosidase digestion of resting and recently activated CTL clone A10. N-Glycomic profiles of CTL clone A10 in resting (A) and recently activated (B) states are shown. Profiles of N-glycans are from the 35% MeCN fraction from a C₁₈ Sep-Pak (“Experimental Procedures”). All molecular ions are [M+Na]⁺. Putative structures are based on composition, tandem mass spectrometry, and the literature shown. Annotated structures are according to the Consortium for Functional Glycomics guidelines.

FIGURE 6.

Long polylactosamine units are more abundant on recently activated CTL clone F3.2 N-glycans. A, shown are the expected resulting N-glycans of resting and recently activated CTLs after HF and sialidase treatments (upper panel structure) and additionally after two consecutive cycles of galactosidase (G) and β-N-acetylhexosaminidase (H) treatments and one endo-β-galactosidase (endo) treatment (lower panel structure). B, shown is the N-glycomic profile of CTL clone F3.2 of resting (upper panel) and recently activated states (lower panel). Profiles of N-glycans are from the 50% MeCN fraction from a C₁₈ Sep-Pak. All molecular ions are [M+Na]⁺. Putative structures based on composition, tandem mass spectrometry, and the literature shown. Annotated structures are according to the Consortium for Functional Glycomics guidelines. Note that the most abundant long polylactosamine units (higher than 4 LacNAc units long) are found at m/z 2081 that corresponds to a tri-antennary N-glycan structure.

FIGURE 7.

Recently activated CTL clone A10 contains less α2,6-linked NeuAc residues than the resting CTL. Partial MALDI-TOF mass spectra of permethylated N-glycans derived from A, resting; and B, recently activated CTL clone A10 after treatment with sialidase-S (α2-3 specific). N-glycomic profiles were obtained from the 50% MeCN fraction from a C₁₈ Sep-Pak column (“Experimental Procedures”). All molecular ions are [M+Na]⁺. Putative structures based on composition, tandem MS and the literature shown. Annotated structures are according to the Consortium for Functional Glycomics (http://www.functionalglycomics.org) guidelines.