Teis Jensen

Processing of glycans on glycoprotein and glycopeptide antigens in antigen-presenting cells

Until about 12 years ago, almost all experimental work with antigens capable of stimulating T cells had been performed with proteins and peptides, as well as haptenated proteins and peptides. By far most of the work had been performed with mice that had been immunized and examined for responses in assays for T cell proliferation. Pure carbohydrates were found to be incapable of major histocompatibility complex (MHC) binding and T cell stimulation (1, 2). However, within the last 10 years it has become evident that both CD4+ and CD8+ T cells can recognize glycopeptides carrying mono- and disaccharides in a MHC-restricted manner provided the glycan group is attached to the peptide at suitable positions. In such glycopeptides, the primed T cells recognize the glycan structure with high fidelity (see below). The question of T cell recognition of glycopeptides may be important in the immune defense against microorganisms, because many microbial antigens are in fact glycosylated. T cell recognition of glycans may also play an important role in the immune defence against tumors, because one of the most consistent traits of a cancer cell is an abnormal glycosylation of the proteins of the malignant cell (3). In this issue of PNAS, Backlund et al. (4) provide evidence that T cell recognition of protein glycans may be crucial also for T cell responses to autoantigens in the course of autoimmune diseases. Below we will describe and discuss the general rules for MHC class II restricted T cell recognition of glycans, the fate of glycoprotein glycans during antigen processing, and the role of antigen glycosylation in tolerance to autoantigens and tumor antigens. T cell recognition of glycans may be crucial for T cell responses to autoantigens.

MUC1-derived glycopeptide libraries with improved MHC anchors are strong antigens and prime mouse T cells for proliferative responses to lysates of human breast cancer tissue

Multi‐component glycopeptide libraries and single glycopeptides were used for immunization of mice with the aim of inducing strong T helper cell responses to the repetitive sequence of MUC1 expressed by human tumor cells. The glycopeptides and glycopeptide libraries were modeled upon the native human MUC1 amino acid variable number of tandem repeats sequence by introduction of modifications in the MHC anchor positions to optimally fulfil the binding requirements of the Ad MHC class II molecule in the BALB/c mouse. The immunogenicity of the MUC1 glycopeptides in BALB/c micewas determined by immunization in complete Freunds adjuvant and assaying lymph node T cells for a proliferative response to the glycopeptide used. Strong proliferative responses with stimulation indices over 50 were obtained with anchor‐improved glycopeptide libraries as well as with single glycopeptides. Immunization with one of the glycopeptide libraries primed T cells for a proliferative cross‐response to the native MUC1 glycopeptide, which by itself was nonimmunogenic. In addition, immunization with the same glycopeptide library primed T cells for a strong response to lysate of a MUC1‐expressing human breast cancer, and immunization with the tumor lysate primed T cells for a response to the glycopeptide library. The T cells responding in the assay for proliferation were restricted to the Ad MHC class II molecule. The results indicate that immunization with MHC anchor‐improved MUC1 glycopeptide libraries can effectively prime T helper cells and may induce long‐term memory. The approach may be useful in the design of preventive cancer vaccines for use in humans.

Synthesis of tumor associated sialyl-T-glycopeptides and their immunogenicity.

Sialyl‐T‐glycopeptides were synthesized by solid‐phase techniques, using a PEGA resin as the solid support. An appropriately protected building block containing α‐Neu5Ac‐(2→3)‐β‐Gal‐(1→3)‐α‐GalN3‐(1→) attached to Fmoc‐Thr/Ser‐OPfp was employed in a solid‐phase glycopeptide assembly of a 10‐mer glycopeptide, using a general Fmoc/OPfp‐ester strategy. Reduction of the azido group of the GalN3 residue was effected on solid‐phase, using DTT and DBU. After acidolytic cleavage from the resin, the methyl ester of the sialic acid residue and acetyl groups were removed with 30% NaOMe/MeOH in MeOH and water pH 14, at −30°C for 2 h. At this low temperature, the highly basic conditions did not result in any detectable β‐elimination. However, one O‐acetyl group, located at the 2‐position of the Gal was resistant to hydrolysis. To remove this remaining acetyl group, reaction with hydrazine hydrate in CHCl3 and MeOH at room temperature for 2.5 h was successful. The two target sequences of sialyl‐T‐glycopeptides were obtained in good yield. In contrast to the the analogs carrying the T‐antigen, the Sial‐T‐glycopeptides were non‐immunogenic, supporting the idea that the sialylation is a method of circumventing the recognition by the immune system. Copyright

Radically altered T cell receptor signaling in glycopeptide‐specific T cell hybridoma induced by antigen with minimal differences in the glycan group

A T cell hybridoma raised against the synthetic glycopeptide T72(Tn) was used to study whether the initial TCR signaling events are markedly different when the hybridoma is stimulatedwith glycopeptides closely related to the cognate glycopeptide antigen. T72(Tn) has an α‐D‐GalNAc group O‐linked to the central threonine in the decapeptide VITAFTEGLK, and the hybridoma is known to be highly specific for this carbohydrate group. T72(Tn)‐pulsed APC induced tyrosine phosphorylation of the TCR‐ζ 21‐ and 23‐kDa proteins and the downstream p42/44 MAP kinase and strong IL‐2 secretion. APC pulsed with T72(α‐D‐GlcNAc), which differs from T72(Tn) solely by the orientation of a hydroxy group in the carbohydrate structure, completely failed to induce detectable tyrosine phosphorylation and IL‐2 secretion. APC pulsed with S72(Tn), which differs from T72(Tn) by not having a methyl group in theserine amino acid side chain to which the glycan is attached, induced partial tyrosine phosphorylation of the TCR‐ζ 21‐kDa protein, no tyrosine phosphorylation of the MAP kinases and no IL‐2 production. Molecular modeling of the MHC/glycopeptide complex revealed that the dramatic difference between the stimulatory power of T72(Tn) and T72(α‐D‐GlcNAc) is mainly due to very small differences in the TCR exposed carbohydrate structure.

Shared structural motifs in TCR of glycopeptide‐recognizing T cell hybridomas

The TCR structure of T cell hybridomas recognizing a tumor glycan‐defined epitope has been studied using reverse transcriptase‐PCR and gene sequencing. The hybridomas had been raised against a glycopeptide, T72(Tn), consisting of the mouse hemoglobin‐derived decapeptide Hb(67 – 76), O‐glycoslated in position 72 with α‐D‐GalNAc. The glycan‐specific hybridomas varied widely in their use of Vα genes although Vα4 was predominant, being present in one third of them. The Vβ gene usage was more restricted and dominated by Vβ1 and Vβ15. There was no correlation between Vα and Vβ usage and antigen fine specificity of the hybridomas. The overall amino acid composition of the complementarity‐determining region (CDR) 3 of the hybridomas was dominated by small polar residues such as Gly, Asn, Ser, Glu and Ala, amino acids reported in the literature to be frequent in glycan‐recognizing proteins. Furthermore, the CDR3 of most hybridomas also contained an aromatic residue with preference for Tyr. A few of the hybridomas raised against the T72(Tn) glycopeptide were peptide specific, i.  e. they responded to the unglycosylated peptide only. The amino acid usage of their CDR3 regions was not radically different from that of the glycopeptide specific hybridomas. They also preferentially used Vα4. However, Vβ4 and Vβ8 were the dominating β chains.

Versatile solid-phase thiolytic reduction of azido and N-Dts groups in the synthesis of haemoglobin (67–76) O-glycopeptides and photoaffinity labelled analogues to study glycan T-cell specificity

A series of O-glycosylated peptides and photoaffinity labelled glycopeptide analogues of the mouse haemoglobin-derived decapeptide Hb (67–76), VITAFNEGLK, which binds well to the MHC class II Ek molecule and is non-immunogenic in CBA/J mice, was synthesized by multiple-column peptide synthesis employing the glycosylated building blocks 1–4 and 7–21. The non-immunogenic peptide VITAFNEGLK was converted into an immunogen by introducing different tumour-associated carbohydrate moieties [β-D-GlcNAc-O -Ser/Thr, α-D-GalNAc-O-Ser/Thr (TN-antigen) core 1 (T-antigen), core 2, core 3 and core 4] to the central position Asn-72 in the decapeptide. Previous studies suggest that T cells may be capable of recognizing epitopes which are partially defined by glycans and may be in direct contact with the T-cell receptor. In order to study the specificity of glycan interactions with the T-cell receptor a series of corresponding glycopeptides labelled with 2-azidobenzamide on the carbohydrate amino function was synthesized. The glycan structure was varied with respect to O-GlcNAc, T and TN-antigen moieties and anomeric configuration. Throughout, efficient reduction of the N-dithiasuccinyl- and azido-functionality-containing building blocks 1, 2, 7, 8, 11, 12, 13, 16, 18 and 20 could be achieved either (i) in solution by utilizing simultaneous in situ reduction with Zn in THF–HOAc–Ac2O or (ii) on solid-phase upon treatment with diisopropylethylamine and an excess of dithiothreitol or α-mercapto-N-methylacetamide. N-Acetylation of the resin-bound glycopeptides furnished the O-glycopeptides 24, 25 and 31–36. No further modification of the carbohydrate moiety on the solid phase was required when utilizing the N-acetylated building blocks 3, 4, 9, 10, 14, 15, 17, 19 and 21. In addition, comparative studies with solid-phase reduction were conducted for the syntheses of the O-linked glycopeptides 24, 25 and 31–36 by employing any of the building blocks 1–4 and 7–21. The photoaffinity labelled glycopeptides 39–45 were synthesized by employing building blocks 1, 2, 7, 8 and 11–13 by reduction of azido or N-Dts functionalities by thiolysis with dithiothreitol and subsequent coupling of the activated photoaffinity label 38 to the glycanamino group of the resin-bound glycopeptides. The synthesized mucin O-glycopeptides 24, 25 and 31–36 and the photoaffinity labelled analogues 39–45 were fully characterized by 1D and 2D 1H NMR spectroscopy and by electrospray mass spectrometry.

Characterization of T cell hybridomas raised against a glycopeptide containing the tumor-associated T antigen, (βGal (1-3) αGalNAc-O/Ser)

T cell hybridomas were raised against the glycopeptide S72 (Core-1) containing the tumor-associated disaccharide βGal (1–3) αGalNAc (Core-1) O-linked to serine at position 72 in the mouse hemoglobin derived decapeptide Hb (67–76). All hybridomas recognized the glycopeptide S72 (Core-1). Two of the selected hybridomas responded, however, much better to the S72 (Tn) glycopeptide containing the monosaccharide αGalNAc O-linked to serine. In addition, one hybridoma cross-responded to the glycopeptide T72 (Core-1) having a threonine at position 72 instead of a serine. No cross-responses were found to other glycopeptides consisting of the same hemoglobin peptide with different glycans attached or to the unglycosylated peptides. The T cell receptor Vα and Vβ usage was clearly diverse. The CDR3α regions demonstrated moreover a predominance of small polar amino acid side chains, and three hybridomas contained a common sequence motif. All the sequenced CDR3β regions contained furthermore a conserved proline-glycine motif. In conclusion, immunization with the disaccharide containing glycopeptides S72 (Core-1) created a heterogeneous population of glycopeptide specific T cells with the ability of cross-responding toward related glycopeptides.

Convenient synthesis of Thr and Ser carrying the tumor associated sialyl-(2→3)-T antigen as building blocks for solid-phase glycopeptide synthesis

Sialyl-T antigens have been synthesized, conveniently and in high yield, by a block condensation approach. Glycosylation of the D-GalN3 derivative with the suitably protected sialyl-α-(2→3)-D-galactopyranosyl trichloroacetimidate by using trimethylsilyl trifluoromethanesulfonate (TMS-OTf) as a promoter in dichloromethane gave the trisaccharide in high yield. Cleavage of the tert-butyldimethylsilyl ether (TBDMS) at the anomeric position of the sialic acid containing trisaccharide derivative, using tetrabutylammonium fluoride (TBAF) in pyridine, occurred in very high yield. After conversion to the trichloroacetimidate coupling of the sialyl-containing trisaccharide derivative with Fmoc-Thr/Ser-OPfp, using silver trifluoromethanesulfonate (AgOTf) as a promoter in dichloromethane, gave the target building blocks in good yields.

Determining the Fate of Glycopeptides During Antigen Processing in Antigen Presenting Cells

Malignant cells can be differentiated from normal cells by the presence of aberrant glycosylation. A large part of anti-tumor immune responses is directed against the tumour cell-associated Tn (α-D-GalNAc) and T (β-D-Gal(l-3)-α-D-GalNAc) carbohydrate antigens [1]. In the present study, the Ek MHC class II molecule found in CBA/J mice was used to evaluate the immunogenicity of synthetic antigens [2]. It has been shown that synthetic glycopeptides can bind to the Ek molecule and produce an immune response provided the glycan points away from the MHC binding cleft and are located towards the central part of the T cell receptor recognition site [3].