Epitope glycosylation plays a critical role for T cell recognition of type II collagen in collagen-induced arthritis.

Immunization of mice with type II collagen (CII) leads to collagen-induced arthritis (CIA), a model for rheumatoid arthritis. T cell recognition of CII is believed to be a critical step in CIA development. We have analyzed the T cell determinants on CII and the TCR used for their recognition, using twenty-nine T cell hybridomas derived from C3H.Q and DBA/1 mice immunized with rat CII. All hybridomas were specific for the CII(256-270) segment. However, posttranslational modifications (hydroxylation and variable O-linked glycosylation) of the lysine at position 264 generated five T cell determinants that were specifically recognized by different T cell hybridoma subsets. TCR sequencing indicated that each of the five T cell epitopes selected its own TCR repertoire. The physiological relevance of this observation was shown by in vivo antibody-driven depletion of TCR Valpha2-positive T cells, which resulted in an inhibition of the T cell proliferative response in vitro towards the non-modified CII(256-270), but not towards the glycosylated epitope. Most hybridomas (20/29) specifically recognized CII(256-270) glycosylated with a monosaccharide (beta-D-galactopyranose). We conclude that this glycopeptide is immunodominant in CIA and that posttranslational modifications of CII create new T cell determinants that generate a diverse TCR repertoire.

Convergent synthesis of neoglycopeptides by coupling of 2-bromoethyl glycosides to cysteine and homocysteine residues in T cell stimulating peptides.

The 2-bromoethyl beta-glycosides of the disaccharide galabiose [Gal(alpha1-4)Gal] and the trisaccharides globotriose [Gal(alpha1-4)Gal(beta1-4)Glc] and 3'-sialyllactose [Neu5Ac(alpha2-3)Gal(beta1-4)Glc] have been prepared by improved routes. The 2-bromoethyl glycosides were then used in cesium carbonate promoted alkylations of the sulfhydryl groups of cysteine and homocysteine residues in T cell stimulating peptides. This convergent and general approach was used to prepare 16 neoglycopeptides which were obtained in 52-95% yields after purification by HPLC. 1H NMR spectroscopy revealed that beta-elimination and epimerization of neoglycopeptide stereocentres did not occur during the synthesis.

The structural basis of MHC control of collagen-induced arthritis; binding of the immunodominant type II collagen 256-270 glycopeptide to H-2Aq and H-2Ap molecules.

The Aq major histocompatibility complex (MHC) class II molecule is associated with susceptibility to murine collagen-induced arthritis (CIA), whereas the closely related H-2Ap molecule is not. To understand the molecular basis for this difference, we have analyzed the ability of H-2Aq and H-2Ap molecules (referred to as Aq and Ap) to bind and present collagen type II (CII)-derived glycosylated and non-glycosylated peptides. T cell clones specific for the immunodominant CII 256-270 peptide and restricted to both Aq and Ap molecules were identified. When these clones were incubated with CII protein and either Aq- or Ap-expressing antigen-presenting cells (APC), only Aq-expressing APC were able to induce stimulation. With the use of A(beta) transgenic mice this could be shown to be solely dependent on the MHC class II molecule itself and to be independent of other MHC- or non-MHC genes. Peptide binding studies were performed using affinity-purified MHC class II molecules. The CII 256-270 peptide bound with lower affinity to the Ap molecule than to the Aq molecule. Using a set of alanine-substituted CII 256-270 peptides, MHC class II and T cell receptor (TCR) contacts were identified. Mainly the side chains of isoleucine 260 and phenylalanine 263 were used for binding both the Aq and Ap molecule, i.e. the peptide was orientated similarly in the binding clefts. The major TCR contact amino acids were lysine 264, which can be posttranslationally modified, and glutamic acid 266, which is the only amino acid in the heterologous peptide which differs from the mouse sequence. Glycosylation at positions 264 and 270 of the CII 256-270 peptide did not change the anchor positions used for binding to the Aq or Ap molecules. The autologous form of the peptide (with aspartic acid at position 266) bound with lower affinity to the Aq molecule as compared with the heterologous peptide. The variable affinity displayed by the immunodominant CII 256-270 peptide for different MHC class II molecules, the identification of MHC and TCR contacts and the significance of glycosylation of these have important implications for the understanding of the molecular basis for inherited MHC class II-associated susceptibility to CIA and in turn, for development of novel treatment strategies in this disease.

Antigen processing and presentation of a naturally glycosylated protein elicits major histocompatibility complex class II-restricted, carbohydrate-specific T cells.

It is well known that T cells recognize antigen as processed peptides bound to major histocompatibility complex molecules on the surface of antigen-presenting cells. Recently, it has been shown that T cells can specifically recognize synthetic glycopeptides. However, whether glycopeptides are selected for presentation during antigen processing of glycoproteins and eventually elicit carbohydrate-specific T cells is still an open question. In this study, we utilized synthetic glycopeptides to analyze T cell recognition of the naturally glycosylated immunodominant peptide representing type II collagen (CII) residues 256-270. In this peptide, lysines at positions 264 and 270 may be post-translationally modified by hydroxylation and subsequent O-linked glycosylation with beta-galactosyl or alpha-glucosyl-(1-->2)-beta-galactosyl residues. T cell hybridomas established from type II collagen-immunized mice specifically recognized CII 256-270 with either galactose or glucosyl-galactose at position 264. The T cell hybridoma recognizing glucosyl-galactose displayed no cross-reactivity either to galactose or to the structurally different alpha-galactosyl-(1-->4)-beta-galactose. Furthermore, the T cell hybridoma recognizing galactose did not cross-react to glucosyl-galactose or galactosyl-galactose, indicating that the antigen-presenting cells (bulk spleen cells, lipopolysaccharide-stimulated spleen cells, anti-CD40-stimulated spleen cells, peritoneal exudate cells or CFA-primed lymph node cells) inefficiently processed carbohydrates when the antigen was given as a glycopeptide.

Synthesis of a water-soluble serine-based neoglycolipid which can be covalently linked to solid phases.

N alpha-Fmoc-serine pentafluorophenyl ester was glycosylated with perbenzoylated lactosyl bromide. The resulting product was coupled to a resin functionalized with 6-aminohexanoic acid and then N alpha-acylated to give a serine-based analogue of lactosylceramide. The water-soluble neoglycolipid was covalently linked to microtiter plates via its carboxyl group and was recognized by a lactose-binding lectin in an ELISA.