An example of different responses to glycosylated CII between DR4 and huCII/DR4 mice. T cell hybridomas clones mDR-16.2 and hDR-9.1 were obtained from DR4 and huCII/DR4 mice, respectively, after immunization with the Gal264/270-peptide. Cells were stimulated with titrated amounts of huCII or CII-peptides (see Fig. 1) and investigated for production of IL-2.

Specific recognition of CII-glycopeptides by human T cells. The two-color flow-cytometry of in vitro-stimulated T cells from an RA-patient shows fluorescence intensities for surface-binding of a FITC-labeled anti-CD4 antibody on the x axis whereas the y axis represents signal intensities for intracellular staining with a phycoerythrin-labeled anti-IL-2 antibody. PBMCs were cultured overnight without antigen, with tetanus toxoid, the K264/270-peptide, or the Gal264-peptide. The figures in the upper right quadrant of the different panels represent the percentage of double-positive cells.

Comparative analysis of the individual response to the nonmodified and the galactosylated T cell epitope in DR4 and huCII/DR4 mice. Recall in vitro response of lymph node cells from DR4 and huCII/DR4 mice immunized 10 days earlier with human CII. Cells were restimulated with either 10 or 50 μg/ml of the K264/270- or the Gal264-peptide. A ratio above 1 indicates a stronger response against the K264/270-peptide whereas a ratio below 1 indicates a stronger response to the Gal264-peptide. The response of individual DR4 mice was stronger to the K264-peptide whereas the responses of huCII/DR mice were stronger to the Gal264-peptide (Wilcoxon signed rank test).

Strong but incomplete tolerance to glycosylated CII in humanized mice. Recall in vitro response of lymph node cells from DR4 and huCII/DR4 mice immunized 10 days earlier with human CII (huCII). Cells were restimulated with huCII and the following CII-peptides: nonglycosylated (K264/270), glycosylated at hydroxylysine 264 (Gal264), glycosylated at hydroxylysine 270 (Gal270), or glycosylated at both hydroxylysine residues (Gal264/270) with β-d-galactopyranosyl residues. Ten animals (5 males and 5 females) of each mouse line were investigated. As a positive control, the recall response to mycobacteria antigen PPD (purified protein derivate, present in complete Freund's adjuvant) was measured. The response in huCII/DR4 mice was significantly reduced against all CII antigens (P < 0.05) as compared with DR4 mice, except for Gal264/270-specific IFN-γ production. *, Significant responses above background levels for huCII/DR4 mice. *, P < 0.05, Mann–Whitney U test; bars represent mean ± SEM. Antigen-specific proliferation and IFN-γ production: Δcpm and ΔU/ml respectively (response with antigen—response in the absence of antigen).