A mutational analysis of the Abetaz/Aalphad major histocompatibility complex class II molecule that restricts autoreactive T cells in (NZBxNZW)F1 mice. The critical influence of alanine at position 69 in the Aalphad chain

Abstract

Autoimmune symptoms of (NZBxNZW)F1 (H-2d/z) mice are reported to be critically related to the heterozygosity at the H-2 complex of the murine major histocompatibility complex (MHC). We previously showed that several Abetaz/Aalphad MHC class II molecule-restricted autoreactive T-cell clones from B/WF1 mice were pathogenic upon transfer to preautoimmune B/WF1 mice. In this study, to identify the crucial amino acid residues in Abetaz/Aalphad molecules for T-cell activation, we generated a panel of transfectant cell lines. These transfectant cell lines express the Abetaz/Aalphad MHC molecules with a mutation at each residue alpha11, alpha28, alpha57, alpha69, alpha70, alpha76 of Aalphad chain and beta86 of Abetaz chain. Replacing alpha69 alanine with threonine, valine or serine completely eliminated the ability to stimulate autoreactive T-cell clones without affecting the ability to present foreign antigen keyhole limpet haemocyanin (KLH) or L-plastin peptide to specific T-cell clones. Replacing beta86 valine with aspartic acid resulted in a decrease in the stimulation for antigen-reactive as well as autoreactive T-cell clones. Substitutions at other residues had minimal or no effect on the stimulation of either auto- or antigen-reactive T-cell clones. These results suggest that alanine at residue 69 of the Aalphad chain is critical for the activation of autoreactive Abetaz/Aalphad-restricted T-cell clones. Possible explanations for this are discussed.

Figure 1

Flow cytometric analysis of expression of the Aβ^z/Aα^dMHC class II molecules on the surface of transfectants. The cells were stained with BW/9 (anti-I-A) mAb followed by FITC–Mar 1 8.5. Immunofluorescence analysis was performed on FACScan. Transfectant cell lines with mutant Aβ^z/Aα^d (e–m) express various amounts of β^z/Aα^d class II molecules. The expected mutations in β^z/Aα^d molecules are as described in Table 2 and indicated at the right upper corner in each panel. Staining of three independent wildtype transfectant cell lines with high (a, TAβ^z-H), intermediate (b, TAβ^z-I) or low (c, TAβ^z-L) expression of Aβ^z/Aα^d class II molecules and a class II negative 2C3 cell line (d) for transfection are also shown. Mean fluorescence intensity (MFI) of staining with BW/9 mAb for each cell line is indicated at the right side of each panel.

Figure 2

Stimulation of T-cell clones by wildtype or mutant transfectant APC cell lines. 1×10⁴ autoreactive T-cell clones KGU140 (a) or AU3-16 (b) were cultured with 3×10⁴ APC cell lines. 1×10⁴ T-cell clones KLH58 (c) were cultured with 1×10⁴ APCs in the presence of 250 μg/ml of KLH antigen. 1×10⁴ T-cell clones LP207 (d), LP824 (e) and LP10–3 (f) were cultured with 1×10⁴ APCs in the presence of 75 μm l-plastin 588–605 peptide. Cytokines released in culture medium were measured by the proliferative responses of IL-2 and IL-4 dependent CT-6 cells. The results were expressed as a mean c.p.m. of triplicate cultures±SD.

Figure 3

Quantitative immunofluorescence analysis of mutant transfectant cell lines. 3×10⁵ transfectant cell lines were incubated with one batch of serially diluted K24-199 mAb culture supernatants followed by FITC-protein A and analysed on a FACScan. Mean fluorescence intensity (MFI) of each staining is indicated for mutant: α69A-T (×), α69A-V (▵), α69A-S (▪), β86V-D (◊) (a); α11F-S (□), α28H-F (▴), α57L-S (+), α70E-G (○), α76I-V (•) (b) cells. MFI of wildtype TAβ^z-H (···▪···), TAβ^z-I ···•···) and TAβ^z-L (···♦;···) transfectant cell lines is also shown.

Figure 4

Peptide binding assay of transfectant cell lines.Biotinylated l-plastin 588–605 peptide (100 μm) were incubated with each transfectant cell line followed by FITC–streptavidin and analysed by FACScan. The same transfectant cell lines were also stained with monomorphic anti-class II mAb (BW/9) followed by FITC–Mar 18·5. Relative peptide binding to each transfectant cell line was calculated by a correction of the amounts of class II molecules as follows: Relative peptide binding=(MFI of staining with biotinylated-l-plastin 588–605 peptide minus MFI of staining with FITC–streptavidin only)*(MFI of staining with BW/9 mAb minus MFI of staining with FITC-Mar 18·5 only). Representative data of three independent experiments.