A mutational analysis of the Aβ^z/Aα^d major histocompatibility complex class II molecule that restricts autoreactive T cells in (NZB×NZW)F1 mice. The critical influence of alanine at position 69 in the Aα^d chain

Abstract

Autoimmune symptoms of (NZB×NZW)F1 (H-2^d/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 Aβ^z/Aα^d 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 Aβ^z/Aα^d molecules for T-cell activation, we generated a panel of transfectant cell lines. These transfectant cell lines express the Aβ^z/Aα^d MHC molecules with a mutation at each residue α11, α28, α57, α69, α70, α76 of Aα^d chain and β86 of Aβ^z chain. Replacing α69 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 β86 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 Aα^d chain is critical for the activation of autoreactive Aβ^z/Aα^d-restricted T-cell clones. Possible explanations for this are discussed.

INTRODUCTION

(NZB×NZW)F1 (B/WF1) mice have been studied as an animal model for human systemic lupus erythematosus (SLE) (reviewed in 1–3). At around 6 months of age, these mice start to show elevated serum immunoglobulin G (IgG) anti-DNA antibodies and severe immune-complex glomerulonephritis. Female mice are more susceptible to autoimmune symptoms than are male mice. In addition to the abnormality in both B and T lymphocytes,^4–7 classic genetic studies revealed that the heterozygosity at the H-2 complex of the murine major histocompatibility complex (MHC) is critically involved in the development of B/WF1 autoimmunity.^8,9

Our previous studies demonstrated the existence of mixed haplotype Aβ^z/Aα^d major histocompatibility complex (MHC) class II molecules on B/WF1 spleen cells and the existence of autoreactive and foreign antigen-reactive T-cell clones restricted by such Aβ^z/Aα^d molecules.¹⁰ Further studies demonstrated that some of these autoreactive T-cell clones induced anti-DNA antibody of IgG class in cell transfer experiments.¹¹ These studies suggested that the recognition of self-peptide(s) in association with Aβ^z/Aα^d class II molecules by pathogenic autoreactive T cells is important for the onset and/or progression of B/WF1 autoimmunity. In an accompanying paper, we examined the characteristics of peptides that bind to Aβ^z/Aα^d class II molecules¹² and suggested that charged residues in the peptide sequence affect the binding to the Aβ^z/Aα^d molecules.

In this study, we attempted to clarify whether unique amino acid residue(s) in the Aβ^z/Aα^d class II molecules might exist which are crucial for the activation of autoreactive T cells. We generated and analysed a panel of transfectant cell lines that express Aβ^z/Aα^d molecules with single amino acid substitutions at several polymorphic sites on the α or β chain. Our results suggest that alanine at position 69 of the Aα^d chain is critical for the activation of autoreactive Aβ^z/Aα^d-restricted T-cell clones.

MATERIALS AND METHODS

Mice

New Zealand Black (NZB, H-2^d) and New Zealand White (NZW, H-2^z) mice were purchased from Japan SLC Inc. (Shizuoka, Japan). (NZB×NZW)F1 (B/WF1) mice were generated by mating female NZB with male NZW mice in our animal breeding facilities. Animals in this study were used in accordance with the Saga Medical School Guidelines for Animal Experimentation.

Antigens, peptides and monoclonal antibodies (mAbs)

Keyhole limpet haemocyanin (KLH) was purchased from Calbiochem-Behring Corp. (La Jolla, CA). Stock solutions (2 mg/ml) were made in Hanks’ balanced salt solution (HBSS) (Gibco BRL, Grand Island, NY) passed through Millipore filters (0·45 μm) and stored at 4°. l-plastin 588–605 peptide (SMARKIGARVYALPEDLV, as expressed by the single letter code of amino acid) that was shown to bind Aβ^z/Aα^dMHC class II molecules¹² was synthesized using the Multipin Peptide Synthesis System (Chiron Mimotopes, Emeryville, CA) according to the manufacturer’s protocol. The sequence was confirmed by automated Edman degradation using a pulsed liquid protein sequencer 477A equipped with an on-line phenylthiohydantoin (PTH) amino acid analyser 120A (Applied Biosystem, Foster City, OR), and the purity was more than 95% by reversed phase high pressure liquid chromatography (HPLC) analysis. The origins and specificities of anti-class II mAb hybridoma cell lines 10.2.16 (anti-Aβ^k, cross-react to Aβ^z),¹³ K24–199 (anti-Aα^d)¹⁴ and BW/9 (rat anti-mouse monomorphic I-A mAb)¹⁵ are described in each reference. Culture supernatants of hybridoma cell lines and appropriately diluted ascitic fluids were used for immunofluorescence staining.

Cell lines and T-cell clones

An M12.C3 cell line, an immunoselected class II negative mutant of BALB/c (H-2^d)-derived B lymphoma M12.4.1, was established by Dr L. Glimcher.¹⁶ A transfectant antigen-presenting cell (APC) line, TAβ^z, that expresses Aβ^z/Aα^d class II molecules was made by introducing the Aβ^z gene into the M12.C3 B lymphoma cell line as described.¹⁰ Mutant APC line 2C3, obtained by chemical mutagenesis and functional selection from TAβ^z cells,¹⁵ expresses intact Aβ^z chain but has the mutation at residue 13 of the Aα^d chain from valine to asparagine. This mutation resulted in the loss of MHC class II expression probably because of a defect in the association of the α and β chains of class II molecules. The establishment of T-cell lines and clones has been previously described.^10,11 Clones used in this paper were derived from B/WF1 mice. KGU140 and AU3-16 T-cell clones are autoreactive. KLH58 is a KLH-specific T-cell clone. LP207, LP824 and LP10-3 T-cell clones are specific for l-plastin 588–605 peptide.¹⁷ All of the clones have Aβ^z/Aα^d-restriction specificity. cDNA sequencing of the α and β chain of the TCR of these T-cell clones was performed^18,19 and the deduced amino acid sequences are described in Table 1.

Table 1.

Amino acid sequences in single-letter codes of the CDR3 region of TCR α and β chains in Aβ^z/Aα^d-restricted T-cell clones from B/WF1 mice

KGU140 and AU3-16 T-cell clones are autoreactive T-cell clones. KLH58 is a keyhole limpet haemocyanin-specific T-cell clone. LP207, LP824 and LP10-3 are L-plastin 588–605 peptide-specific T-cell clones. The Vα/Jα and Vβ/Jβ gene usages are indicated on the left side of the sequences. The definition of the CDR3 region is according to Rock et al²⁰ These sequence data are available from EMBL/GenBank/DDBJ under accession numbers B015835-AB015845

*ND, not determined.

Site-directed mutagenesis

Site-directed mutagenesis of Aα^d or Aβ^z cDNA was performed using an Altered Sites II in vitro Mutagenesis System (Promega, Madison, WI) or using a polymerase chain reaction (PCR) mutagenesis.²¹ Mutagenic oligonucleotides and PCR primers were purchased from Bio-Synthesis, Inc. (Lewisville, TX) and are listed in Table 2. These mutagenic oligonucleotides or primers were named according to their target positions and the site of the wildtype amino acid to the expected mutant amino acid residues by single-letter codes of amino acid. Most of these mutagenic oligonucleotides or PCR primers were designed to introduce or destroy a restriction enzyme site in their sequences, as described in Table 2, to diagnose the expected mutation for screening. The wildtype Aα^d cDNA was obtained from Dr K. Miyazaki (Osaka University) and cloned into pALTER-1 vector (Promega). Mutagenesis was performed according to the manufacturer’s instructions. The wildtype Aβ^z cDNA was obtained by reverse transcription (RT)–PCR from RNA of the NZW mouse spleen. Mutagenesis was performed by PCR mutagenesis.²¹ After confirming the mutagenesis and the absence of unintentional mutations by DNA sequencing (ALFexpress DNA sequencer, Pharmacia Biotech), mutant Aα^d or Aβ^z cDNA was subcloned into pCEXV vector.²²

Table 2.

Mutagenic oligonucleotides and PCR primers

Diagnostic restriction enzyme sites are underlined. Mismatches with the template are indicated by lowercase letters. The names of the oligonucleotides or primers indicate the target position and the wildtype amino acid to the expected mutant amino acid residues by single-letter codes

*(+) Introduced enzyme site

†(−) Destroyed enzyme site

DNA transfection

For Aα^d cDNA transfection, 2×10⁷ class II negative 2C3 cells¹⁵ were cotransfected with 20 μg of pCEXVAα^d cDNA and 1 μg of pSV2-hyg plasmid (kindly provided by Dr Itoh, Saga Medical School, Japan) by electroporation. The electroporation was performed three times at 1·0 kV/cm with a capacitance of 25 μF using a Gene-Pulser (Bio-Rad Laboratories, Richmond, CA). 48 hr after transfection, a selection medium containing 300 μg/ml hygromycin (Wako Chemical, Osaka, Japan) was added to the culture. After 2–3 weeks, hygromycin resistant clones were stained with anti-class II mAb (BW/9). Positive clones were then subcloned by the limiting dilution and stable transfectant cell lines were established. In some experiments, a cell-sorting technique by EPICS ELITE (Coulter Cytometry, Hialeah, FL) was used to enrich the positive clones before the limiting dilution. For Aβ^z cDNA transfection, M12.C3 cells were cotransfected with 20 μg of pCEXVAβ^z cDNA and 1 μg of the pSV2-neo plasmid by electroporation. Class II expression by G418 (Gibco, BRL) resistant clones was screened using anti-class II mAb (BW/9), and stable transfectant cell lines were established as above.

Flow cytometry analysis and quantitative immunofluorescence analysis

One million cells were first incubated with 100 μl of appropriately diluted mAbs for 30 min at 4°. Cells were washed three times with HBSS containing 1% fetal calf serum (FCS) and 0·1% NaN₃ (staining buffer). Then, 20 μl of appropriately diluted fluoroscein isothiocyanate (FITC)–protein A (Zymed Lab. Inc., San Francisco, CA) or FITC–Mar 18.5 (mouse anti-rat κ chain mAb)²³ was added and the mixture was incubated for 30 min at 4°. This was followed by addition of 10 μl of 50 μg/ml propidium iodide (Sigma, St. Louis, MO) for the final 5 min to gate out dead cells. Cells were then washed three times with cold staining buffer and analysed by fluorescence-activated cell sorting (FACScan®; Becton-Dickinson Immunocytometry Systems, Mountain View, CA). The quantitative immunofluorescence analysis was performed as described by Braunstein and Germain²⁴ with slight modifications. Briefly, 3×10⁵ transfectant cells in 100 μl staining buffer were stained with 100 μl of a serial dilution of the K24-199 mAb followed by FITC–protein A. Cells were analysed on a FACScan and the mean fluorescence intensity (MFI) was calculated.

Peptide binding assay

This was performed according to Malcherek et al.²⁵ with slight modification.¹² Briefly, a saturating concentration of 100 μm N-terminally biotinylated l-plastin 588–605 peptide was incubated with 5×10⁴ wildtype or mutant transfectant cell lines in 200 μl culture medium and the mixture was incubated at 37° for 20 hr in a microtitre plate (Falcon no. 3075, Becton-Dickinson, Lincoln, NJ). The cells were then washed and incubated with FITC–streptavidin (Gibco BRL, Rockville, MD) at 4°. Propidium iodide was added for the final 5 min to gate out dead cells. Stained cells were analysed on a FACScan. The peptide binding assay was repeated three times with reproducible results.

T-cell stimulation assay

Complete medium consists of RPMI-1640 culture medium (Gibco BRL) containing 10% heat-inactivated fetal calf serum (Intergen, Purchase, NY), 5×10⁻⁵ m 2-mercaptoethanol, 10 mm HEPES (Gibco BRL), 100 U/ml penicillin and 100 μg/ml streptomycin. l-glutamine was added at a final concentration of 2×10⁻³ m before use. Cytokine release of T-cell clones was assayed as described.^11,15 Thus, 1×10⁴ T-cell clones and the indicated number of transfectant cells were cultured with or without the appropriate antigens in 0·2 ml complete medium in a 96-well culture plate (Falcon no. 3075). After 24 hr, 0·1 ml supernatant was transferred to wells containing 1×10⁴ CT-6 cells (an IL-2 and IL-4 cytokine dependent cell line)²⁶ in 0·1 ml complete medium. CT-6 cells were cultured for 48 hr and proliferative responses were assayed by the pulse of 0·5 μCi [³H]-TdR (ICN pharmaceuticals Inc., CA) for the final 16 hr, and the uptake of [³H]-TdR was measured on a Betaplate flat-bed scintillation counter (Pharmacia-Wallac, Gaithersburg, MD). Experiments were performed in triplicate cultures and repeated at least twice, but usually three times, with similar results.

RESULTS

Expression of mutant Aβ^z/Aα^d class II molecules in B lymphoma cells

We selected polymorphic sites in the Aα and Aβ chain of class II molecules for the target sites of mutagenesis because polymorphic sites in the MHC molecules were shown to be critical for the binding of antigenic peptides (reviewed in 27). Almost all the mutageneses were arbitrarily designed to change from the wildtype to one of the parental (d or z haplotype) amino acid residues. For residue 69 in the Aα^d chain, the mutagenesis was also designed to change to representative amino acids in the hydrophobic or hydrophilic side chain group. Each mutated Aα^d or Aβ^z cDNA was transfected together with a hygromycin-resistance gene or a neomycin-resistance gene into the class II mutant B lymphoma line 2C3 cells or M12.C3 cells, respectively. After selection, stable transfectant cell lines were analysed for their expression of class II molecules by staining with monomorphic anti-class II mAb (BW/9). We selected transfectant cell lines of the highest class II molecule expression out of several independent clones of each mutant for analysis in this study. As shown in Fig. 1, most of the transfectant cell lines expressed considerable amounts of class II molecules on the cell surface, although there was variability among the lines. A transfectant cell line that expressed β86 V-D mutant Aβ^z/Aα^d showed low expression of class II molecules probably due to decreased efficiency in the association of the α and β chains (see Discussion). Haplotype-specific mAbs (10.2.16 as anti-Aβ^z and K24-199 as anti-Aα^d) also showed positive staining (data not shown), indicating that the expressed class II molecules originated from Aβ^z/Aα^d. We also created several transfectant cell lines that express various amounts of wildtype Aβ^z/Aα^d. These TAβ^z high (TAβ^z-H), TAβ^z intermediate (TAβ^z-I) and TAβ^z low (TAβ^z-L) expressing cells were used for the positive control of T-cell stimulation (see below). None of the transfectant cell lines showed positive staining with anti-I-E (13/4)²⁸ mAb (data not shown), indicating that mixed isotype Aβ/Eα class II molecules are not expressed. They were also negative for anti-Aβ^d (MKD6)²⁹ and anti-Aα^z (4D5–12)³⁰ mAb staining, indicating that only wildtype or mutant Aβ^z/Aα^d molecules are expressed on the cell surface.

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.

In spite of multiple transfection experiments, we were not able to obtain transfectant cell lines with mutations at residues β11 and β13 of the Aβ^z chain. This was not due to inefficient transfection because we were able to demonstrate the existence of β11F-L and β13P-M mutant transcripts in transfectant cell lines by RT–PCR. We speculate that a reason for the lack of expression could be that the mutation at β11 or β13 results in defects in pairing ability to the α chain. The importance for the N-terminal residues in pairing α and β chains of class II molecules was already suggested.²⁴

Stimulation of Aβ^z/Aα^d-restricted T-cell clones by transfectant cell lines

We examined the ability of these transfectant cell lines to stimulate Aβ^z/Aα^d-restricted autoreactive and antigen-reactive T-cell clones. Proliferative responses of T-cell clones against each transfectant APC line were performed in one set of experiments to compare the stimulatory activity of each mutation. Because the amounts of Aβ^z/Aα^d class II molecules on transfectant cells vary for each line, we first assessed whether the amounts of class II molecules affect the stimulatory activity on T-cell clones. As shown in Fig. 2, wildtype transfectant cell lines that express various amounts of Aβ^z/Aα^d (TAβ^z-H, TAβ^z-I, TAβ^z-L) showed a similar degree of stimulation against all the T-cell clones. This indicates that the amounts of Aβ^z/Aα^d molecules do not significantly affect this assay within the range of amounts that are expressed in wildtype TAβ^z-H to TAβ^z-L cells.

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.

As shown in Fig. 2, the substitution of wildtype alanine at position 69 of the Aα^d chain into threonine (z haplotype) resulted in the complete loss of stimulatory activity to two different autoreactive T-cell clones. However, this substitution did not affect the stimulatory activity against any of the antigen-specific T-cell clones examined. Furthermore, substitution of alanine at α69 into valine (hydrophobic) or serine (hydrophilic) also eliminated the stimulatory activity against autoreactive T-cell clones. Surprisingly, these mutations did not affect the stimulatory activities against all four independent antigen-reactive T-cell clones examined. Because the mutation at α69 affected only the autoreactive but not the antigen-reactive T-cell clones, alanine at this position in the Aβ^z/Aα^d molecules may have critical influences on the stimulation of autoreactive T-cell clones. It should be noted that these two autoreactive T-cell clones have different T-cell receptor (TCR) α and β chains (Table 1).

Substitution of valine at position 86 of the Aβ^z chain into aspartic acid resulted in a decrease of stimulatory activity against almost all the antigen- and autoreactive T-cell clones. This would be caused by the very low expression of class II molecules on β86V-D mutant cell lines (Fig. 1), possibly because this position is a critical site for the association of α and β chains as well as peptide binding, as reported.^31–34 Mutations at other positions in Aβ^z/Aα^d molecules did not show significant effects for the stimulation of auto- and antigen-reactive T-cell clones.

Conformation of mutant Aβ^z/Aα^d molecules on transfectant cell lines examined by mAbs

One explanation for the loss of or decreased stimulatory activity of α69 and β86 mutant APC lines is that these mutations resulted in gross conformational changes in the structure of Aβ^z/Aα^d class II molecules. To examine this, we performed quantitative immunofluorescence analysis that allows evaluation of relative affinity of the antibody binding²⁴ and thus reflects the conformational change of class II molecules by mutagenesis. As shown in Fig. 3(a), α69A-T and α69A-S mutant APC lines showed decreased affinity to anti-Aα^d (K24-199) mAb compared to the wildtype TAβ^z cells. The data of α69A-V mutant APC lines was difficult to interpret because of the weak intensity of staining. These results indicate that the mutation at position 69 of the Aα chain induced slight conformational changes in the Aβ^z/Aα^d molecules. Although weak in staining intensity of the β86V-D mutant cell line, it showed increased affinity to anti-Aα^d mAb staining, indicating that this mutation also resulted in a slight conformational change of Aβ^z/Aα^d molecules. Mutations at other positions did not show significant changes in affinity to anti-Aα^d (K24–199) mAb (Fig. 3b) and may not have caused conformational changes in these class II molecules.

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.

Peptide binding assay of mutant Aβ^z/Aα^d class II molecules

We further examined the conformational alterations of mutant class II molecules by peptide binding assay. Our previous study revealed that l-plastin 588–601 peptide as a naturally eluted peptide from purified Aβ^z/Aα^d class II molecules and synthetic l-plastin 588–605 peptide (SMARKIGARVYALPEDLV) bind to Aβ^z/Aα^d.¹² Transfectant cell lines with wildtype or mutant Aβ^z/Aα^d molecules were incubated with biotinylated l-plastin 588–605 peptide followed by FITC–streptavidin and analysed by FACScan. Because each transfectant cell line expressed different amounts of Aβ^z/Aα^d molecules, the efficiency of peptide binding was expressed as the percentage relative peptide binding as described in the legend to Fig. 4. The peptide binding was specific for all the transfectant cell lines because the Aβ^z/Aα^d negative parental APC line (M12.C3) did not bind the peptide.¹² Also the anti-Aβ^z mAb (10.2.16) partially blocked the peptide binding to each transfectant (data not shown). As shown in Fig. 4, almost all transfectant cell lines with wildtype or mutant Aβ^z/Aα^d molecules showed similar binding ability to this peptide except α69 A-T mutant cell lines. The α69A-T mutation appears to affect the peptide binding to Aβ^z/Aα^d molecules. Although the results show some variability, especially when class II expression is low, other mutant APC lines in this study may not have altered Aβ^z/Aα^d conformation for peptide binding.

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.

DISCUSSION

The most interesting finding in this study is that substitution of alanine to threonine, valine or serine at position 69 in the Aα^d chain of Aβ^z/Aα^d class II molecules resulted in the complete loss of stimulatory activity against Aβ^z/Aα^d-restricted autoreactive T-cell clones. The mutations affected two independent autoreactive T-cell clones with different TCR α and β chains. The α69 mutations, however, did not show a significant effect on the stimulatory activity against antigen-reactive Aβ^z/Aα^d-restricted T-cell clones. The reason for the complete loss of stimulatory activity of α69 mutant APCs was probably not a result of the gross conformational alterations of the Aβ^z/Aα^d molecules by the mutagenesis, as (i) immunofluorescence staining by haplotype specific anti-class II mAbs did not show loss of staining and, (ii) with one exception (the α69A-T mutant), l-plastin 588–605 peptide binding studies did not show a substantial difference between mutant and wildtype Aβ^z/Aα^d lines (Fig. 4). Also, the observation that antigen-reactive T-cell clones showed a similar degree of proliferation against α69 mutant and wildtype APCs supports the idea that gross conformational changes are unlikely.

One explanation for the effect of α69 mutations would be that the peptides recognized by autoreactive T-cell clones have specific affinity to the structure created by alanine at α69. A conservative change of alanine to hydrophobic residue valine or a non-conservative changes to hydrophilic residue serine or threonine resulted in the complete loss of stimulation against autoreactive T-cell clones. This may indicate that alanine, by itself or in combination with other surrounding residues around α69, is critical for binding to the target peptides. The small side chain of alanine could exert this effect. Attempts to substitute alanine by the smaller glycine at α69 were unsuccessful in achieving its expression, probably due to the conformational unstability of the mutated polypeptide chain.

Because two independent T-cell clones with different TCR sequences and, therefore, two different target peptides, were affected in a similar way, these peptides may have common features for the binding to Aβ^z/Aα^d and, specifically, to the structure around position α69. It is interesting to note here that our accompanying paper¹² showed that the side chain of binding peptide at relative position 6 (p6) influenced the peptide binding and that residues with large and negatively charged side chains are not tolerated at p6. The position of α69 corresponds to pocket 6 in the class II molecule that accommodates the side chain of residue at relative position 6 of the binding peptide, as speculated by the crystallographic analysis of class II molecules.^35,36

Another explanation for the loss of stimulatory activity of α69 mutant APCs against auto- but not antigen-reactive T-cell clones is that the responsiveness of these autoreactive T-cell clones, and potentially the autoreactive T-cell clones in general, is sensitive to the slight structural change of class II molecules. As shown in the quantitative immunofluorescence experiments (Fig. 3), substitution of alanine at α69 to other residues resulted in a slight conformational change of the structure of Aβ^z/Aα^d class II molecules. Although crystallographic analysis^35,36 revealed that position 69 in the Aα chain resides deep in the peptide binding groove and is not exposed to the surface of the class II molecule, a mutation at this residue would induce a slight conformational change on the surface structure of class II molecules. Autoreactive but not antigen-reactive T-cell clones were sensitive against such slight conformational change and lost the responsiveness to the mutated class II molecule. Such a difference in sensitivity against conformational changes of class II molecules between autoreactive and antigen-reactive T cells could be derived from positive and negative selection during the maturation of T cells in the thymus. Autoreactive T cells that exit from the thymus may have relatively high affinity TCR to class II molecules (reviewed in 37, 38), and thus may be more sensitive to the class II structure compared to foreign antigen-reactive T cells.

The mutation of β86V-D resulted in a decrease in stimulation for almost all the T-cell clones examined irrespective of their antigen-specificity. The reason for this could be a marked decrease in the expression of MHC molecules on the cell surface (Fig. 1). Quantitative immunofluorescence analysis indicated a mutation-induced change of mAb binding affinity, also suggesting the conformational change of Aβ^z/Aα^d molecules. Although we were not able to demonstrate a change in peptide binding ability, these findings are essentially consistent with the results reported by several investigators. Substitution of the residue at β86 resulted in very low levels of cell surface expression of class II molecules,³¹ decreased peptide binding^33,34 and weakened T-cell recognition.^32–34

Our previous report suggested that mixed haplotype Aβ^z/Aα^d class II molecules play important roles in the pathogenicity of autoimmunity in B/WF1 mice.¹¹ It would be interesting to examine whether in vivo expression of mutated Aβ^z/Aα^d class II molecules at position 69 in the α chain can prevent the onset and/or progression of autoimmune symptoms of B/WF1 mice. It is also interesting that autoreactive T cells in general are sensitive to mutation at position 69 in the α chain, irrespective of the haplotype specificity.

Glossary

KLH

keyhole limpet haemocyanin

MHC

major histocompatibility complex

NZB

New Zealand black mice

NZW

New Zealand white mice

B/WF1

(NZB×NZW)F1

SLE

systemic lupus erythematosus

APC

antigen-presenting cell