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International Immunology 2007 19(9):1103-1113; doi:10.1093/intimm/dxm081
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© The Japanese Society for Immunology. 2007. All rights reserved. For permissions, please e-mail: journals.permissions@oxfordjournals.org

Conformation of MHC class II I-Ag7 is sensitive to the P9 anchor amino acid in bound peptide

Amy Gardiner1, Katherine A. Richards2, Andrea J. Sant2 and Lynne S. Arneson1

1 Department of Biology, American University, 4400 Massachusetts Avenue NW Washington, DC 20016, USA
2 David H. Smith Center for Vaccine Biology and Immunology, University of Rochester, 601 Elmwood Avenue, Box 609, Rochester, NY 14642-8609, USA

Correspondence to: L. S. Arneson; E-mail: larneso{at}american.edu


   Abstract
Top
 Abstract
Introduction
 Methods
 Results
 Discussion
 References
 
Type I diabetes is a chronic autoimmune disease resulting in the destruction of insulin-producing ß cells in the pancreas. In humans, disease incidence is linked to expression of specific MHC class II alleles and in mice type I diabetes is associated with the class II allele I-Ag7. I-Ag7 contains a polymorphism that is shared by human class II alleles associated with the disease, at position 57 in the ß chain, in which aspartic acid is changed to a serine. The P9 pocket in the peptide-binding groove is in part shaped by ß57, and therefore the structure of this pocket is modified in I-Ag7. Using mAbs, we have previously determined that alternative conformations of I-Ag7 form in response to peptide binding. In this study, we have extended these findings by examining how peptides induce I-Ag7 molecules to adopt different conformations. By mutating the amino acid in the P9 position of either class II-associated invariant chain peptide (CLIP) or glutamic acid decarboxylase (GAD) 65 (207–220), we have determined that the chemical nature of the P9 anchor amino acid, either acidic or small hydrophobic, affects the overall conformation of the I-Ag7 class II molecule. T cell hybridomas specific for GAD 65 (207–220) in the context of I-Ag7 were also examined for recognition of I-Ag7 bound to GAD 65 (207–220), in which Glu217 in the P9 position was changed to alanine. We found that although some TCRs were able to recognize both peptides in the context of I-Ag7, and thus both class II conformations, approximately one-third of the T cells tested were not able to recognize the alternate class II conformation formed with the mutated peptide. These results indicate that the I-Ag7 conformations may affect functional activation of T cells, and thus may play a role in autoimmunity.

Keywords: autoimmunity, MHC, T cells


   Introduction
Top
 Abstract
 Introduction
Methods
 Results
 Discussion
 References
 
Type I diabetes is an autoimmune disease in which the insulin-producing ß cells of the pancreas are selectively destroyed. This autoimmune disorder stems from the loss of self-tolerance to ß cell auto-antigens, such as insulin, glutamic acid decarboxylase (GAD) 65 and heat shock protein (HSP) 60. Autoreactive T cells become activated when they encounter these auto-antigens in conjunction with MHC class II molecules. Lymphocytic infiltration into the pancreatic islets eventually occurs, followed by destructive insulitis, which culminates in ß cell elimination and cessation of insulin production.

The MHC accounts for ~50% of the familial risk for type I diabetes (1). Allelic sequencing studies demonstrated that structural polymorphisms in the HLA class II DQß gene are related to type I diabetes (2). The identity of the amino acid at position 57 of the ß chain is important in both disease susceptibility and protection; Ala, Ser or Val at ß57, found in DQ2 and DQ8 as well as the DQ8 murine homolog I-Ag7, characterizes predisposing alleles, while Asp, found in DQ6, characterizes protective alleles (2). Non-obese diabetic (NOD) mice spontaneously develop an autoimmune diabetes that parallels the progression of human type I diabetes. These mice express one type of MHC class II molecule, I-Ag7, and development of diabetes is dependent on homozygosity of that molecule (3). I-Ag7 has a conserved {alpha} chain found in I-Ad haplotypes of non-diabetic mice, but it has a unique ß chain, containing polymorphisms at ß56, histidine instead of proline, and ß57, serine instead of aspartic acid (4). Crystal structures of I-Ak and I-Ad indicate that ß57Asp participates in a salt bridge with {alpha}76Arg, likely stabilizing the {alpha}–ß chain interaction, contributing to the integrity of the class II peptide-binding groove (5–7).

The ß56 and ß57 polymorphisms in I-Ag7 occur in the P9 pocket, resulting in the loss of the ß57–{alpha}76 salt bridge. These polymorphisms also cause rearrangement of the hydrogen bond network. The arginine at {alpha}76 shifts slightly to form a hydrogen bond with ß53Leu and ß57Ser via a buried water molecule, resulting in a wider, shallower P9 pocket that allows the side chain of peptide residues to have greater lateral freedom. Additional compensation for the loss of ß57Asp may be provided by electrostatic interaction between {alpha}76Arg and an acidic residue in P9. Also, the substitution at ß56 from Pro to His exposes more surface area of the {alpha}76Arg side chain making the P9 pocket more accessible and more attractive to acidic residues (8, 9). Interestingly, a single amino acid change from ß56His to Pro in the P9 pocket results in protection from type I diabetes (10), suggesting the importance of this pocket in disease susceptibility.

The crystal structure of DQ8 complexed with murine insulin (9–23) has confirmed the structural similarities between DQ8 and I-Ag7 (11, 12). Binding studies with soluble DQ8 and I-Ag7 have demonstrated that these class II molecules bind islet peptides from insulin, GAD 65 and HSP 60 with similar specificity (13). Analyses of transgenic mice that express DQ8 and develop diabetes have further demonstrated the functional similarities between DQ8 and I-Ag7 (14). These studies suggest that the human and murine class II molecules possess common structural features that predispose to lack of effective self-tolerance induction and progressive autoimmune diseases.

Several studies, including those utilizing mAbs, T cell reactivity and protein crystallography, have shown that the sequence of the peptides occupying the peptide-binding pocket of MHC molecules can induce conformational changes in both MHC class I and class II molecules (15–18) Crystal structure and peptide-binding studies indicate that I-Ag7 can accommodate a wide variety of peptides with Gly/Ser/Ala or an acidic amino acid in the P9 pocket. In this study, we have determined that the amino acid in the P9 pocket affects the conformation of the I-Ag7 class II molecule. Based on our mAb analyses, the amino acid in the P9 pocket appears to exert a dominant effect on the class II conformation. Furthermore, TCRs in T cell hybridomas raised against GAD 65 (207–220) in the context of I-Ag7 differ in their ability to recognize peptide-induced conformations.


   Methods
Top
 Abstract
 Introduction
 Methods
Results
 Discussion
 References
 
Reagents and cell lines
B cell hybridomas secreting the mAb 10.2-16 (19), which binds the ß chain of I-Ag7, were obtained from the American Type Culture Collection (Manassas, VA, USA). B cell hybridomas secreting 40 M, which recognizes the ß chain of I-Ag7 (20), K24.199, which recognizes the {alpha} chain of I-Ag7 (21) and In-1, which recognizes an epitope on the cytosolic domain of invariant chain (22), were generously supplied by Jim Miller (University of Rochester, Rochester, NY, USA). The mAb 39E also recognizes an epitope on the ß chain of I-Ag7 (18, 20), and cells secreting this antibody were purchased from the American Type Tissue Collection. Ltk– cells stably transfected with cDNA encoding the {alpha} chain of I-Ad and the ß chain of I-Ag7 were generated as previously described (23). The expression level of class II was verified by staining with the anti-class II mAb 10.2-16 binding using a secondary FITC-labeled goat anti-mouse antibody (ICN Biomedicals) with a FACS at the National Institutes of Health (NIH) (FACS Calibur cytometer).

T cell hybridomas specific for GAD 65 (207–220) were generated as follows. NOD mice were immunized subcutaneously with 50 µl of 10 µM GAD 65 (207–220) peptide in CFA. Nine days later the mice were euthanized and the draining popliteal lymph nodes were collected. CD4+ T cells were isolated by negative selection with anti-class II (10.2-16), anti-class I (3.155) and anti-B cell (B220) antibodies followed by complement lysis. Live cells were isolated by Lympholyte-M (Cedarlane Laboratories) centrifugation. Peptide-specific T cells were activated and expanded by re-stimulation with irradiated NOD spleen cells and GAD 65 (207–220) peptide. After 3 days, T cell blasts were harvested and isolated using Lympholyte-M (Cedarlane Laboratories) and fused with the TCR-negative variant of BW5147 lymphoma cells. After washing, cells were cultured overnight and cloned by limiting dilution into 96-well plates in the presence of HAT to generate clonal T cell hybridoma lines.

Generation and expression of invariant chain constructs
Invariant chain cDNA constructs in which the class II-associated invariant chain peptide (CLIP) region was removed and replaced with heterologous sequences were generated by the ligation of annealed complementary oligonucleotides encoding the desired sequence into an invariant chain construct in which unique restriction sites had been engineered into each side of the CLIP region (18). The engineered invariant chain cDNA was digested with KpnI and XbaI to remove the CLIP region and treated with calf alkaline phosphatase. Oligonucleotides (Integrated Life Technologies) were pre-treated with polynucleotide kinase for 1 h at 37°C to phosphorylate the 5' ends then heated to 65°C to inactivate the kinase. Pre-treated 5' and 3' oligonucleotides were then mixed and placed in a beaker of 95°C water and left to cool to room temperature to allow annealing. Annealed oligonucleotides were then ligated into the digested invariant chain cDNA and plasmid clones were sequenced (Davis Sequencing) to verify replacement of CLIP sequence with that of the heterologous peptide. Single amino acids in either the CLIP region or a heterologous peptide were mutated through site-directed mutagenesis (Stratagene). Briefly, 5' and 3' oligonucleotides containing the mutated sequence were used in a PCR and parental DNA was digested by DpnI. Undigested PCR product was then used to transform competent bacteria and plasmid clones were sequenced to verify the sequence of the entire invariant chain cDNA, including the nucleotides mutated. The constructs containing invariant chain cDNA with CLIP, another peptide or mutated peptide were then transiently transfected into Ltk– cells stably expressing I-Ag7 as previously described (18).

Western blot analysis
Cells were lysed on ice in a buffer containing 150 mM NaCl, 50 mM Tris (pH 7.6), 5 mM EDTA, aprotinin and phenylmethylsulfonylfluoride as protease inhibitors and 0.5% Igepal 40 for 15 min (23). Post-nuclear supernatants were then aliquoted into four equal volumes and a sample was removed from each aliquot to determine invariant chain levels in each sample. Class II molecules were then isolated by incubating with mAbs pre-bound to Protein-A Sepharose (Amersham) for 2 h at 4°C. Immunoprecipitated proteins were eluted by addition of 2% SDS, 0.0625 M Tris (pH 6.8) and 10% glycerol at room temperature, and then boiled for 2 min. Samples were electrophoresed on SDS–10% PAGE and transferred to nitrocellulose as described (24). The nitrocellulose membrane was blocked in TBST [10 mM Tris (pH 7.6), 150 mM NaCl and 1% Tween 20] containing 2% dry milk for 1 h, and then incubated in TBST/2% milk containing the anti-invariant antibody In-1 overnight. After washing in TBST, the blot was probed with goat anti-rat antibody conjugated to HRP (Kierkegaard and Perry Laboratories, KPL) and developed by chemiluminescence (Pierce). Blots were quantified using Image J (NIH), and presented as ratios of 40 M, K24.199 or 39E compared with 10.2-16. Antibody ratios were determined for each experiment, and ratios for individual experiments were averaged for each peptide. Average antibody ratios and standard deviations are shown.

T cell assays
Briefly, 5 x 104 L cell transfectants and 5 x 104 T cell hybridomas were incubated with various concentrations of either wild-type (WT) GAD 65 (207–220) or the mutated P9 E to A GAD 65 (207–220) peptide in 96-well flat-bottom plates in culture media as previously described (23). After overnight culture, 50 µl of supernatant was removed, frozen, thawed and then co-cultured with CTLLs, an IL-2-dependent cell line. After overnight culture, CTLL viability was determined by 3-(4,5-dimethyl thiazon-2-yl)-2,5-diphenyl tetrazolium bromide assay as previously described (23). In antibody blocking experiments, various dilutions of the appropriate antibody were added to each well and incubated overnight with L cell transfectants, T cell hybridomas and either WT or the mutated GAD 65 (207–220) peptide prior to freezing.


   Results
Top
 Abstract
 Introduction
 Methods
 Results
Discussion
 References
 
I-Ag7 conformation is peptide dependent
The murine fibroblast Ltk– cell line had previously been transfected with cDNAs encoding the {alpha} chain of I-Ad and the ß chain of I-Ag7 (23). FACS analysis indicates that this transfected cell line stably expresses a uniform level of I-Ag7 class II at the cell surface. This level of class II expression remained stable throughout experimentation (data not shown).

We previously showed that the conformation of I-Ag7 is dependent on the peptide bound (18). In this study, we began by examining the conformation of I-Ag7 induced by peptides associated with diabetes, GAD 65 (524–543), GAD 65 (207–220) and murine insulin B (9–23) (Fig. 1). A major problem encountered when examining MHC class II molecules bound to soluble peptide is ensuring that all the molecules surveyed are in fact bound to the peptide of choice. Previous studies have solved this problem by tethering the peptide to the ß chain of the class II molecule (25). However, in this study, we have developed a different technique utilizing the chaperone molecule invariant chain to ensure high peptide occupancy and exclusion from our analyses of those class II molecules that have never bound or that have released the peptide of choice. A region of invariant chain, the CLIP, naturally associates with the peptide-binding groove of invariant chain during class II synthesis and transport (26). In this study, we have replaced the CLIP region of invariant chain with a heterologous peptide (GAD 65 aa207–220, GAD 65 aa524–543 or mInsB aa9–23). These modified invariant chain constructs were then expressed in cells stably expressing I-Ag7.


Figure 1
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  		Fig. 1. Sequences of peptides inserted into the CLIP region of invariant chain. The sequence of WT murine invariant chain amino acids 87–99 or the heterologous peptides replacing CLIP are shown. The nucleotide sequence is given with the corresponding amino acid beneath the appropriate codon. The four codons containing the unique restriction sites KpnI and XbaI inserted into the p31 invariant chain cDNA are indicated.

 
To assess the conformational status of the class II molecules occupied with heterologous peptides replacing the invariant chain CLIP region, an immunowestern strategy was employed. Class II molecules were immunoprecipitated from total cell lysate using different anti-I-Ag7 mAbs, and invariant chain was detected in the immunoprecipitate by immunowestern. I-Ag7 molecules bound to invariant chain retain the heterologous peptide in the peptide-binding groove, and thus exhibit the conformation induced by this peptide. Only class II molecules both bound to intact invariant chain and thus bound to the heterologous peptide and exhibiting a conformation recognized by the mAb could be detected by this assay. Therefore, the amount of invariant chain detected on the immunowestern would indicate the relative reactivity of the mAb with the peptide-induced class II conformation.

To examine the conformation of I-Ag7 induced by peptides associated with diabetes, GAD 65 (524–543), GAD 65 (207–220) and murine insulin B (9–23), invariant chain constructs in which CLIP had been replaced by these peptides were transiently transfected into L cells stable expression I-Ag7. After equally dividing cell lysates from the cell lines expressing the test invariant chain constructs, a small portion of each was analyzed by western blot for intact invariant chain to verify that equal amounts of lysate were used for each immunoprecipitation and that the relative levels of invariant chain expression for each construct were similar (Fig. 2B). Cells were also checked for expression of endogenous invariant chain (Fig. 2A, vector-only lane).


Figure 2
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  		Fig. 2. Bound peptides affect I-Ag7 conformation as determined by antibody recognition. L cell fibroblasts stably expressing I-Ag7 were transiently transfected with either vector alone or the invariant chain constructs shown in Fig. 1. Cells were lysed 3 days after transfection, and the lysate was split into four equal portions. Thirty microliter was removed from each portion and the class II and associated invariant chain molecules were immunoprecipitated from the remaining lystate with either 10.2-16, 40 M, K24.199 or 39E (panel A). The blots were exposed for 15 min. Each experiment was performed three times. The triangles indicate molecular weight markers to the left of each gel. Bands on blots were quantified using Image J and compared with 10.2-16 (panel B). Ratios of 40 M/10.2-16 (white), K24.199/10.2-16 (black) and 39E/10.2-16 (gray) are shown. Standard deviations are indicated by error bars.

 
mAb recognition of I-Ag7 bound to CLIP was identical to that seen previously (18). Specifically, I-Ag7 bound to CLIP was recognized well by 10.2-16, considerably less well by both 40 M and K24.199 and not at all by 39E (Fig. 2A). Westerns were quantitated by densitometry and averages of three experiments are shown. Amounts of I-Ag7:peptide:invariant chain complex precipitated by each mAb are shown relative to amounts precipitated by 10.2-16 (Fig. 2C). When the CLIP region of invariant chain was replaced with auto-immunogenic peptides and, therefore these alternate peptides occupied the binding pocket of I-Ag7, different conformations of I-Ag7 were formed, as determined by different patterns of antibody recognition of the class II–peptide complex. I-Ag7 bound to GAD 65 (524–543) was recognized well by 10.2-16, to a low level by 40 M (~8% of 10.2-16 levels), slightly better by K24.199 (~12% of 10.2-16 levels) and undetectably by 39E. In contrast, I-Ag7 bound to GAD 65 (207–220) was recognized equally well by 10.2-16 and 40 M, ~60% as well by 39E, but not at all by K24.199. I-Ag7 bound to Ii in which CLIP had been replaced by murine insulin B (9–23) was recognized by the mAb panel in a pattern similar to that seen with GAD 65 (207–220), although 10.2-16 recognized the class II–peptide complex better than either 40 M or 39E (Fig. 2A and C). These data reconfirm that the conformation of I-Ag7 is dependent on the bound peptide and that these different auto-antigens lead to different conformational states of the I-Ag7 class II molecule.

P9 anchor amino acids affect I-Ag7 conformation
Structural analyses of peptide:MHC class II co-crystals have indicated that some amino acids in the bound peptide interact directly with the class II molecule, fitting into pockets in the floor of the peptide-binding groove. Other peptide residues project out of the groove and have the potential to interact more directly with the TCR, thus affecting T cell recognition and activation (5–7). We hypothesized that anchor amino acids in the pockets would most likely affect the class II conformation, and the amino acid in the P9 pocket would have the greatest effect on I-Ag7, as the polymorphisms in I-Ag7 are located near the P9 pocket and affect its structure (8, 9, 27).

To test this hypothesis, we replaced the CLIP region in invariant chain with GAD 65 (207–220). Crystallization studies of the peptide GAD 65 (207–220) in I-Ag7 have indicated that Glu217 occupies the P9 pocket in the peptide-binding groove (9). To determine the effect of the P9 anchor amino acid on the class II conformation, we used site-directed mutagenesis to change Glu217 to aspartic acid (D), glycine (G), alanine (A) or methionine (M). An invariant chain construct containing a mutated GAD 65 (207–220) peptide in place of CLIP was expressed in cells expressing I-Ag7. After equally dividing cell lysates, a small portion of each was analyzed by western blot for intact invariant chain to verify that equal amounts of lysate were used for each immunoprecipitation and that the relative levels of invariant chain expression for each construct were similar (Fig. 3B). I-Ag7 associated with WT GAD 65 (207–220), with Glu in the P9 pocket, was recognized well by 10.2-16 and 40 M almost equally well and by 39E at ~60% of 10.2-16 reactivity, but not at all by K24.199 (Fig. 3A and C). A very similar pattern was seen when Glu217 was mutated to Asp (Fig. 3A and C), retaining the acidic nature of the P9 anchor residue. However, when Glu217 was mutated to a small hydrophobic amino acid, either glycine or alanine, or methionine, an amino acid not modeled to fit well in the P9 pocket, the I-Ag7–peptide complex was recognized by K24.199 at ~50% (G and M) or 15% (A) of 10.2-16 reactivity, indicating the conformation of the class II–peptide complex had changed (Fig. 3A and C). This high level of recognition of I-Ag7:GAD 65 (P9G) by K24.199 also indicates that the {alpha} chain-specific mAb K24.199 binds class II with high avidity when its favored conformation is present.


Figure 3
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  		Fig. 3. The amino acid in the P9 position of GAD 65 (207–220) affects the conformation of I-Ag7. The invariant chain construct in which CLIP was replaced with GAD 65 (207–220) (Fig. 1) was used for site-directed mutagenesis to replace Glu217 with aspartic acid (D), glycine (G), alanine (A) or methionine (M). These invariant chain constructs were then transiently transfected into L cell fibroblasts stably expressing I-Ag7 and post-nuclear cell lysates were collected 3 days after transfection. The lysates were split into four equal fractions from which 30 µl aliquots were removed, and class II and associated invariant chain molecules were immunoprecipitated with 10.2-16, 40 M, K24.199 or 39E (panel A). The blots were exposed for 15 min, except for GAD 65 (207–220) E217 to A, which was exposed 10 min. Each experiment was performed three times. The triangles indicate molecular weight markers to the left of each gel. Bands on blots were quantified using Image J and compared with 10.2-16 (panel B). Ratios of 40 M/10.2-16 (white), K24.199/10.2-16 (black) and 39E/10.2-16 (gray) are shown. Standard deviations are indicated by error bars.

 
To verify that the P9 anchor residue can independently alter the I-Ag7 conformation, we mutated the amino acid in the P9 position of the CLIP from methionine to either glutamic acid or glycine and transiently transfected cells expressing I-Ag7 with these constructs. I-Ag7 associated with WT CLIP, with Met98 in the P9 position, is recognized well by 10.2-16, to a much lower level by 40 M and K24.199 (~10% and 5% of 10.2-16 reactivity, respectively), but not at all by 39E (Fig. 4A and C), as seen previously. When Met98 is mutated to glutamic acid, the entire pattern of antibody reactivity changes, reflecting a conformational change in the class II structure. I-Ag7 bound to CLIP with Glu98 is recognized equally well by 10.2-16 and 40 M and by 39E at ~30% of 10.2-16 reactivity, but not at all by K24.199 (Fig. 4A and C), a pattern seen when I-Ag7 is bound to WT GAD 65 (207–220), which contains an acidic P9 residue. However, when Met98 is mutated to glycine, mAb reactivity is similar to that seen when I-Ag7 is bound to WT CLIP. Together, these data indicate that the amino acid in the P9 pocket of I-Ag7 affects the conformation of the class II molecule.


Figure 4
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  		Fig. 4. Acidic amino acids in the P9 position of CLIP dominantly affect the conformation of I-Ag7. The WT invariant chain construct was used for site-directed mutagenesis to change Met98 to glutamic acid (E) or glycine (G). These invariant chain constructs were then transiently transfected into L cell fibroblasts stably expressing I-Ag7 and post-nuclear cell lysates were collected 3 days after transfection. The lysates were split into four equal fractions from which 30 µl aliquots were removed, and class II and associated invariant chain molecules were immunoprecipitated with 10.2-16, 40 M, K24.199 or 39E (panel A). The blots were exposed for 15 min. Each experiment was performed three times. The triangles indicate molecular weight markers to the left of each gel. Bands on blots were quantified using Image J and compared with 10.2-16 (panel B). Ratios of 40 M/10.2-16 (white), K24.199/10.2-16 (black) and 39E/10.2-16 (gray) are shown. Standard deviations are indicated by error bars.

 
TCR recognition of altered class II–peptide conformation
To examine whether the altered class II–peptide conformations detected by mAbs had functional consequences on T cell recognition, we extended our studies to antigen-specific T cells. T cell hybridomas reactive to GAD 65 (207–220) were generated from NOD mice immunized with synthetic peptide in CFA and cloned T cell hybridomas were examined for IL-2 production in response to either WT GAD 65 (207–220) or GAD 65 (207–220) in which the amino acid in P9 has been changed from the charged glutamic acid to the uncharged alanine. The only amino acid changed in this variant should be completely sequestered in the peptide-binding groove (5–7). Therefore, any change in T cell recognition by altering this residue should be induced by a change in the structure of the class II molecule bound to the peptide. All T cell hybridomas were expected to respond to the WT peptide, but if the variant peptide at P9 induces a conformational change, we speculated that some T cells might detect this change and respond less well, or not at all, to the mutated peptide. Four of the six T cell hybridomas examined responded to both the WT peptide, as expected, and to the mutated peptide, although at a higher concentration of peptide (Fig. 5A). The dose shift with the mutated peptide, ~100-fold, is most likely due to the decreased affinity for the peptide with alanine in the P9 pocket. The positive reactivity of these T cells with the variant GAD peptide shows that the mutated GAD 65 (207–220) binds to I-Ag7 when it is offered as free peptide in culture and that it binds in the same register as the WT peptide.


Figure 5
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  		Fig. 5. TCRs may discriminate between class II conformations. T cell hybridomas specific for GAD 65 (207–220) were incubated with L cells stably expressing I-Ag7 plus either WT GAD 65 (207–220) (squares) peptide or peptide in which Glu217 had been changed to alanine (circles). Optical density of 3-(4,5-dimethyl thiazon-2-yl)-2,5-diphenyl tetrazolium bromide taken up by CTLLs incubated with supernatant from the T cell assay is shown. This experiment was repeated three times with similar results.

 
Interestingly, two of the six T cell hybridomas tested responded to the WT GAD 65 (207–220) peptide but did not respond to the mutated peptide (Fig. 5B). This result suggests that the TCRs expressed by these T cells were not able to recognize the class II conformation induced by the peptide altered at its P9 position. These two classes of GAD-specific T cells, those that do recognize the alternate I-Ag7:mutated peptide complex and those that do not, do not appear to differ systematically in affinity for GAD–I-Ag7 because both sets of T cells display a range in sensitivities to antigen when they are tested in dose–response curves with the WT GAD peptide (Fig. 5). Collectively, these data support the hypothesis that changing the P9 residue in the peptide bound to I-Ag7 affects the conformation of this class II molecule, resulting in differential recognition by antigen-specific T cells.

In addition, these T cell assay data (Fig. 5) support the hypothesis that the variant class II conformation is expressed on the cell surface. To verify surface expression of the variant class II conformations, cells expressing I-Ag7 and the invariant chain construct with GAD 65 (207–220) in place of CLIP where incubated with each of the T cell hybridomas used above. The WT GAD 65 (207–220) construct resulted in dose-dependent stimulation of each of the T cells (Fig. 6) indicating that the peptide originating in the invariant chain construct is retained in the class peptide binding groove and binds in the correct register. When CLIP is replaced by the mutated GAD 65 (207–220), with alanine at P9, stimulation is decreased in each of the T cell hybridomas, consistent with the results presented above. The two cell lines that do not recognize the mutated peptide also do not respond to the construct encoding the mutated peptide. These data indicate that the peptides generated by the invariant chain constructs are binding in the correct register and that the class II structural variants are present on the cell surface.


Figure 6
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  		Fig. 6. Peptides encoded in the constructs bind in the correct register. Increasing amounts of L cells stably expressing I-Ag7 and the invariant chain construct encoding either the WT GAD 65 (207–220) peptide or GAD 65(207–220) in which P9 as changed to alanine were incubated with T cell hybridomas. Optical density of 3-(4,5-dimethyl thiazon-2-yl)-2,5-diphenyl tetrazolium bromide taken up by CTLLs incubated with supernatant from the T cell assay is shown. This experiment was repeated twice with similar results.

 
Finally, T cell assays were done in the presence of either 10.2-16 or K24.199, antibodies that recognize I-Ag7. As shown above, 10.2-16 recognition of I-Ag7 is high when the class II molecule is bound to either WT GAD 65 (207–220) or the mutated GAD 65 (207–220) peptide. However, K24.199 does not bind to I-Ag7–WT GAD 65 (207–220) complexes, but does recognize the variant class II conformation induced by the mutated GAD 65 (207–220) peptide. Therefore, the response of T cells to either the WT or mutated GAD 65 (207–220) peptide in the presence of either 10.2-16 or K24.199 was determined. In all assays, the addition of 10.2-16 blocked T cell reactivity to either of the peptides, resulting in 3–20% of maximum reactivity in the absence of antibody (Fig. 7). However, the addition of K24.199 had minimal effect on T cell reactivity to WT GAD 65 (207–220), but blocked reactivity to the mutated peptide at levels similar to those achieved in the presence of 10.2-16. Data from the two T cell hybridomas that did react with the mutated peptide are not shown, as no change in antibody blocking would be observable. Together, these data indicate that the variant class II conformation induced by the altered residue in the P9 position is expressed at the cell surface.


Figure 7
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  		Fig. 7. Altered I-Ag7 conformations are present on the cell surface. T cell hybridomas specific for GAD 65(207–220) were incubated with L cells stably expressing I-Ag7, either WT GAD 65 (207–220) or peptide in which Glu217 had been changed to alanine, plus either 10.2-16 (black bars) or K24.199 (white bars). The percent of maximal stimulation is shown. This experiment was repeated three times with each T cell hybridoma with similar results.

 

   Discussion
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
References
 
MHC class II genes, specifically HLA-DQ in humans and I-Ag7 in mice, are strongly linked to diabetes susceptibility. Although the ß57Asp to Ser polymorphism shared by these alleles is thought to be important in disease initiation or progression, how this polymorphism affects class II or diabetes susceptibility is not clear. Previous studies have shown that the peptide bound to I-Ag7 can affect the conformation of the class II molecule (18), and studies presented here indicate that the amino acid sequence of the bound peptide, specifically the residue in the P9 pocket of the peptide-binding groove, affects the conformation of the I-Ag7 class II molecule.

Alternate class II conformations documented in this report and in previous studies (15–18, 28) suggest that class II molecules are flexible, and may subtly change shape in response to occupancy with different peptides. However, this flexibility is difficult to assess on a molecular level. In this study, we have used both T cell and mAb recognition to examine conformational changes in the class II molecule, I-Ag7. We found that changing the amino acid in the P9 pocket [glutamic acid in GAD 65 (207–220)] changed antibody recognition of the class II–peptide complex. Antibodies that recognized the class II–peptide complex also blocked reactivity of T cells raised against the WT GAD 65 (207–220) peptide. We further found that these T cells raised against the WT GAD 65 (207–220) peptide varied in their ability to cross-react when a non-acidic amino acid is in the P9 position. Some TCRs were able to recognize the altered conformation of the class II–peptide complex, whereas other TCRs were not. These results suggest that the amino acid in the P9 pocket affects the conformation of the class II–peptide complex, which in turn affects TCR recognition.

In this study, the effect of peptide on class II conformation was assessed both using biochemical assays, in which the CLIP region of invariant chain was replaced with amino acids from a different peptide, and using assays measuring T cell reactivity to peptides. Although it is possible that the altered antibody recognition seen in the biochemical assays could be due to a structural change in the intact invariant chain molecule, which then altered the class II conformation, this interpretation seems unlikely as T cell reactivity assays, specifically the antibody-blocking experiments, suggest similar antibody interaction profiles as those seen in the biochemical assays. In addition, T cell reactivity to cells transfected with the invariant chain construct containing either the WT of mutated GAD 65 (207–220) peptide resulted in very similar results to T cell assays with added peptide, suggesting that the peptide is binding in the correct register. Together, these data argue that the change in the P9 amino acid is responsible for the altered class II structure as determined by antibody reactivity.

The panel of mAbs used in this study recognizes epitopes on both the {alpha} and ß chains along the peptide-binding groove. K24.199 recognizes an epitope on the {alpha} chain located toward the amino terminal end of the peptide-binding groove. The 10.2-16 epitope is located near amino acid 64 on the ß chain, whereas both 40 M and 39E recognition of class II rely on amino acid 70 in the ß chain, a region that contains a kink in the {alpha} helix that may contribute to conformational shifts in class I and class II molecules (15, 17, 29). All mAbs can bind class II with high avidity, although avidity is dependent on the class II:peptide conformation (Figs 2C, 3C and 4CGoGo). Previous studies have shown that the peptide sequence, or an absence of peptide, can affect the conformation of MHC class I and class II molecules and that this conformational change can be detected by differential antibody binding (15–17,29–31). The {alpha} helices of the class II peptide-binding groove have been shown to be specifically affected by peptide sequence resulting in class II conformational change (26, 32). The ß57 polymorphism specific to I-Ag7 has been shown to affect the structure of the carboxy terminal end of the peptide-binding groove (9), potentially allowing increased flexibility to this end of the groove that would be translated through the {alpha} helices to the rest of the molecule.

Structural analyses of co-crystals of I-Ag7 and peptide have indicated that a wide variety of peptides may bind in the I-Ag7 peptide-binding groove (8, 9, 33). The P9 pocket of I-Ag7 is most influenced by the ß57 polymorphism resulting in the loss of the salt bridge with {alpha}76. As a result, the P9 pocket can accept two different classes of amino acids. Small hydrophobic amino acids such as glycine, alanine and serine are accepted with their side chains pointing down into the pocket; however, the basic environment of the pocket favors the insertion of acidic amino acids with their side chains pointed sideways (9). Crystallization of I-Ag7 bound to GAD 65 (207–220) has shown that Glu217 resides in the P9 pocket, whereas other studies have shown that Met98 of CLIP (26) and Ala535 of GAD 65 (524–543) (34) reside in the P9 pocket. The difference in the amino acid in the P9 pocket is likely responsible for the difference in the conformation of the class II molecule bound to these peptides. Amino acids in other pockets, such as P1, may also contribute to the class II conformation, as suggested by the differential antibody recognition patterns of I-Ag7 bound to either GAD 65 (524–543) or GAD 65 (207–220) in which Glu217 is mutated to glycine. Both of these class II–peptide complexes have glycine in the P9 pocket, yet the peptides induce different class II conformations. However, the presence of an acidic amino acid in the P9 pocket appears to affect the class II conformation in a dominant fashion, as GAD 65 (207–220) and CLIP have very different peptide-binding motifs, but when Met98 in CLIP is mutated to aspartic acid, these two peptides induce similar conformations when bound to I-Ag7.

The high degree of structural flexibility of I-Ag7, which may be important in diabetogenesis, may contribute to the formation of alternate conformations based on anchor amino acids in peptide binding. Similar results have been documented in another class II molecule, HLA-DRB1*0401 (DR4), associated with rheumatoid arthritis. A study examining peptide–class II affinity noted that two peptides with similar binding affinity and similar TCR contact residues did not activate the same population of T cells (35). T cell activation remained highly specific even when the TCR contact residues in the peptides were changed to replicate each other, although the class II anchor amino acids remained different. These results suggest that a different class II molecule, DR4, can assume different conformations dependent on peptide anchor amino acids and that these alternate conformations are highly T cell specific (35). In our study, the T cells examined were able to cross-react on both the WT and mutated peptide (expressing alanine rather than glutamic acid in the P9 pocket) because the T cells were selected with the WT peptide. Increased levels of mutated peptide were required for T cell activation likely due to decreased peptide-binding avidity and decreased ability of the TCR to recognize the altered class II–peptide conformation.

That two class II molecules shown to assume different peptide-dependent conformations, depending on the particular amino acid residues at anchor residues are both associated with autoimmune disease, either type I diabetes or rheumatoid arthritis is interesting and suggests a potential difference in the way autoimmune T cells interact with class II–peptide complexes. Indeed, crystal studies of autoimmune TCRs bound to class II–peptide suggests that the topology of binding is different than that first found in anti-microbial TCRs (36, 37). Initial crystallization studies of class II-restricted anti-microbial TCRs found a diagonal to orthogonal-binding orientation that allowed the TCR to come into contact with the bound peptide while avoiding the class II helices especially in the region around the amino terminus of the peptide (reviewed in refs 38–40). However, two autoimmune TCRs that recognize peptide from MBP bind to the class II–peptide complex in a more off-centered mode, centered over the P2 residue, with contacts made to the class II helices in the area of the amino terminus of the peptide (36, 37), suggesting that TCR–class II–peptide interaction may vary depending on the functional capabilities of the TCR. For both TCRs mentioned above, as well as an additional TCR from an experimental autoimmune encephalomyelitis model, interaction with the class II-bound self-peptide may be inefficient, as only two or three complementarity-determining regions loops contact the peptide, perhaps permitting survival during thymic selection (39). Analyses such as surface plasmon resonance to compare the affinities of pathogen-specific TCR versus autoreactive TCR is necessary to rigorously assess this possibility.

Together the data presented in this manuscript suggest that T cell recognition is highly dependent on structural features imparted by the bound peptide, and can be modified not only by the TCR contact residues of the peptide, as previously described (5–7), but can also be affected to a significant degree by peptide anchor residues that structurally affect the class II molecule. Although not shown here, it is possible that the peptide-dependent class II conformation could play a role in altering the structure of the TCR–class II interaction, thus potentially affecting T cell survival in the thymus, and consequently playing a role in susceptibility to autoimmunity.


   Abbreviations
 
CLIP, class II-associated invariant chain peptide
GAD, glutamic acid decarboxylase
HSP, heat shock protein
NIH, National Institutes of Health
NOD, non-obese diabetic
WT, wild type

   Notes
 
Transmitting editor: R. A. Flavell

Received 22 August 2006, accepted 19 June 2007.


   References
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 

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