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Functional inhibition related to structure of a highly potent insulin-specific CD8 T cell clone using altered peptide ligands.

Petrich de Marquesini LG, Moustakas AK, Thomas IJ, Wen L, Papadopoulos GK, Wong FS - Eur. J. Immunol. (2008)

Bottom Line: When tested for antagonist activity with APL differing from the native peptide at either of these positions, the peptide variants, G6H and R8L showed the capacity to reduce the agonist response to the native peptide.We conclude that p6 and p8 of the insulin B15-23 peptide are very important for TCR stimulation of this clone and no substitutions are tolerated at these positions in the peptide.This is important in considering the therapeutic use of peptides as APL that encompass both CD4 and CD8 epitopes of insulin.

View Article: PubMed Central - PubMed

Affiliation: Department of Cellular and Molecular Medicine, School of Medical Sciences, University of Bristol, Bristol, UK.

ABSTRACT
Insulin-reactive CD8 T cells are amongst the earliest islet-infiltrating CD8 T cells in NOD mice. Cloned insulin B15-23-reactive cells (designated G9C8), restricted by H-2K(d), are highly diabetogenic. We used altered peptide ligands (APL) substituted at TCR contact sites, positions (p)6 and 8, to investigate G9C8 T cell function and correlated this with structure. Cytotoxicity and IFN-gamma production assays revealed that p6G and p8R could not be replaced by any naturally occurring amino acid without abrogating recognition and functional response by the G9C8 clone. When tested for antagonist activity with APL differing from the native peptide at either of these positions, the peptide variants, G6H and R8L showed the capacity to reduce the agonist response to the native peptide. The antagonist activity in cytotoxicity and IFN-gamma production assays can be correlated with conformational changes induced by different structures of the MHC-peptide complexes, shown by molecular modeling. We conclude that p6 and p8 of the insulin B15-23 peptide are very important for TCR stimulation of this clone and no substitutions are tolerated at these positions in the peptide. This is important in considering the therapeutic use of peptides as APL that encompass both CD4 and CD8 epitopes of insulin.

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Related in: MedlinePlus

Molecular modeling of H-2Kd−Ins B15–23 and mutated B15–23 peptides. (A) TCR view of the H-2Kd−Ins B15–23 complex, and side view of the peptide chain as bound in the groove of H-2Kd. This simulated structure is based on the crystal structure of H-2Kd in complex with an influenza nucleoprotein peptide 29. Several modes of structural rendering are shown simultaneously in order to appreciate how the peptide fits into the groove. The antigenic peptide is in space-filling form with its carbon atoms shown in green, nitrogen in blue, oxygen in red, hydrogen in white, and sulfur in orange. The α1α2 domain of the molecule is depicted according to its secondary structure in different regions: α-helix in red, β-pleated sheet in turquoise and random coil in grey. The solvent-accessible surface of the α1α2 domain is shown in grey with colorings according to the electrostatic surface potential (blue for positive, red for negative and intermediate hues for neutral). The surface of the heavy chain is made transparent so that peptide residue p2Y that is buried in pocket B, as well as the heavy chain residues making contact with the insulin peptide can be seen, albeit in a lighter color. These residues are shown with their carbon atoms in orange, while the color convention for the other atoms is identical to that for the antigenic peptide. (B) Modeled structure of the H-2Kd molecule with the Ins B15–23 p6H variant peptide, in the same orientation as the figure for the corresponding complex of the native peptide (A). Rendering and color conventions as in (A). (C) Molecular environment in p6 of the modeled complex with view of p6 containing the insulin peptide B15–23/B20G (p6G) residue and surrounding MHC heavy chain amino acids, in the complex of H-2Kd and Ins B15–23, as seen from above (TCR view). Color, surface electrostatic, and transparency conventions are as in (A). Some of the surrounding residues are at a distance longer than 4 Å, but are shown for comparison with (D). (D) View of p6 of the complex of H-2Kd with Ins B15–23/B20H (p6H), in the same orientation and conventions as in (C). All the surrounding residues from the heavy chain are at a distance of less than 4 Å. (E) View of p8 containing the Ins B15–23/B22R (p8R) residue and surrounding heavy chain amino acids, in the complex of H-2Kd and Ins B15–23, as seen from above (TCR view). Representations, color, surface electrostatic, and transparency conventions are as in (A). All the surrounding residues from the heavy chain are at a distance of less than 4 Å. (F) View of p8 of the complex of Kd with Ins B15–23/B22Leu (p8L), in the same orientation and conventions as in (E).
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fig05: Molecular modeling of H-2Kd−Ins B15–23 and mutated B15–23 peptides. (A) TCR view of the H-2Kd−Ins B15–23 complex, and side view of the peptide chain as bound in the groove of H-2Kd. This simulated structure is based on the crystal structure of H-2Kd in complex with an influenza nucleoprotein peptide 29. Several modes of structural rendering are shown simultaneously in order to appreciate how the peptide fits into the groove. The antigenic peptide is in space-filling form with its carbon atoms shown in green, nitrogen in blue, oxygen in red, hydrogen in white, and sulfur in orange. The α1α2 domain of the molecule is depicted according to its secondary structure in different regions: α-helix in red, β-pleated sheet in turquoise and random coil in grey. The solvent-accessible surface of the α1α2 domain is shown in grey with colorings according to the electrostatic surface potential (blue for positive, red for negative and intermediate hues for neutral). The surface of the heavy chain is made transparent so that peptide residue p2Y that is buried in pocket B, as well as the heavy chain residues making contact with the insulin peptide can be seen, albeit in a lighter color. These residues are shown with their carbon atoms in orange, while the color convention for the other atoms is identical to that for the antigenic peptide. (B) Modeled structure of the H-2Kd molecule with the Ins B15–23 p6H variant peptide, in the same orientation as the figure for the corresponding complex of the native peptide (A). Rendering and color conventions as in (A). (C) Molecular environment in p6 of the modeled complex with view of p6 containing the insulin peptide B15–23/B20G (p6G) residue and surrounding MHC heavy chain amino acids, in the complex of H-2Kd and Ins B15–23, as seen from above (TCR view). Color, surface electrostatic, and transparency conventions are as in (A). Some of the surrounding residues are at a distance longer than 4 Å, but are shown for comparison with (D). (D) View of p6 of the complex of H-2Kd with Ins B15–23/B20H (p6H), in the same orientation and conventions as in (C). All the surrounding residues from the heavy chain are at a distance of less than 4 Å. (E) View of p8 containing the Ins B15–23/B22R (p8R) residue and surrounding heavy chain amino acids, in the complex of H-2Kd and Ins B15–23, as seen from above (TCR view). Representations, color, surface electrostatic, and transparency conventions are as in (A). All the surrounding residues from the heavy chain are at a distance of less than 4 Å. (F) View of p8 of the complex of Kd with Ins B15–23/B22Leu (p8L), in the same orientation and conventions as in (E).

Mentions: The results of molecular structural simulation, based on the crystal structure of a H2-Kd nucleoprotein peptide complex, show that the orientation of the Ins peptide in the groove is mostly the same (with the exception of residues p5C and p7E) as that obtained by modeling based on the HLA-A2-Tax peptide complex 21 (Fig. 5A). Remarkably, the crucial TCR contact residues p6G/p8R remain in nearly identical orientations as in the previous simulation (see below). There is a minor shift in the anchoring of the peptide, as it is now shown in this work, compared to previously 21, that the p5 residue in this peptide points into the groove (pocket C, 22). Furthermore, p7 is shown to be pointing towards the α2 helix and away from the groove, hence just outside the canonical “footprint” of the cognate T cell receptor 23. Assuming that the T cell receptor docks onto this complex in a diagonal manner as suggested by nearly all structures of such complexes, we conclude that p5C and p7E participate only slightly in TCR recognition 24–28. The position of p8R is nearly on the same level and in front of the α1 helix of H2-Kd. We find it significant that in both molecular simulations, based on HLA-A2 21, and H2-Kd crystal structures (this work), the relative orientations of p6G and p8R are nearly identical. The residues p6G and p8R form a special pair such that any canonical TCR contact will necessarily involve both of them, as well as the peptide backbone of p7E (Fig. 5A).


Functional inhibition related to structure of a highly potent insulin-specific CD8 T cell clone using altered peptide ligands.

Petrich de Marquesini LG, Moustakas AK, Thomas IJ, Wen L, Papadopoulos GK, Wong FS - Eur. J. Immunol. (2008)

Molecular modeling of H-2Kd−Ins B15–23 and mutated B15–23 peptides. (A) TCR view of the H-2Kd−Ins B15–23 complex, and side view of the peptide chain as bound in the groove of H-2Kd. This simulated structure is based on the crystal structure of H-2Kd in complex with an influenza nucleoprotein peptide 29. Several modes of structural rendering are shown simultaneously in order to appreciate how the peptide fits into the groove. The antigenic peptide is in space-filling form with its carbon atoms shown in green, nitrogen in blue, oxygen in red, hydrogen in white, and sulfur in orange. The α1α2 domain of the molecule is depicted according to its secondary structure in different regions: α-helix in red, β-pleated sheet in turquoise and random coil in grey. The solvent-accessible surface of the α1α2 domain is shown in grey with colorings according to the electrostatic surface potential (blue for positive, red for negative and intermediate hues for neutral). The surface of the heavy chain is made transparent so that peptide residue p2Y that is buried in pocket B, as well as the heavy chain residues making contact with the insulin peptide can be seen, albeit in a lighter color. These residues are shown with their carbon atoms in orange, while the color convention for the other atoms is identical to that for the antigenic peptide. (B) Modeled structure of the H-2Kd molecule with the Ins B15–23 p6H variant peptide, in the same orientation as the figure for the corresponding complex of the native peptide (A). Rendering and color conventions as in (A). (C) Molecular environment in p6 of the modeled complex with view of p6 containing the insulin peptide B15–23/B20G (p6G) residue and surrounding MHC heavy chain amino acids, in the complex of H-2Kd and Ins B15–23, as seen from above (TCR view). Color, surface electrostatic, and transparency conventions are as in (A). Some of the surrounding residues are at a distance longer than 4 Å, but are shown for comparison with (D). (D) View of p6 of the complex of H-2Kd with Ins B15–23/B20H (p6H), in the same orientation and conventions as in (C). All the surrounding residues from the heavy chain are at a distance of less than 4 Å. (E) View of p8 containing the Ins B15–23/B22R (p8R) residue and surrounding heavy chain amino acids, in the complex of H-2Kd and Ins B15–23, as seen from above (TCR view). Representations, color, surface electrostatic, and transparency conventions are as in (A). All the surrounding residues from the heavy chain are at a distance of less than 4 Å. (F) View of p8 of the complex of Kd with Ins B15–23/B22Leu (p8L), in the same orientation and conventions as in (E).
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Related In: Results  -  Collection

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fig05: Molecular modeling of H-2Kd−Ins B15–23 and mutated B15–23 peptides. (A) TCR view of the H-2Kd−Ins B15–23 complex, and side view of the peptide chain as bound in the groove of H-2Kd. This simulated structure is based on the crystal structure of H-2Kd in complex with an influenza nucleoprotein peptide 29. Several modes of structural rendering are shown simultaneously in order to appreciate how the peptide fits into the groove. The antigenic peptide is in space-filling form with its carbon atoms shown in green, nitrogen in blue, oxygen in red, hydrogen in white, and sulfur in orange. The α1α2 domain of the molecule is depicted according to its secondary structure in different regions: α-helix in red, β-pleated sheet in turquoise and random coil in grey. The solvent-accessible surface of the α1α2 domain is shown in grey with colorings according to the electrostatic surface potential (blue for positive, red for negative and intermediate hues for neutral). The surface of the heavy chain is made transparent so that peptide residue p2Y that is buried in pocket B, as well as the heavy chain residues making contact with the insulin peptide can be seen, albeit in a lighter color. These residues are shown with their carbon atoms in orange, while the color convention for the other atoms is identical to that for the antigenic peptide. (B) Modeled structure of the H-2Kd molecule with the Ins B15–23 p6H variant peptide, in the same orientation as the figure for the corresponding complex of the native peptide (A). Rendering and color conventions as in (A). (C) Molecular environment in p6 of the modeled complex with view of p6 containing the insulin peptide B15–23/B20G (p6G) residue and surrounding MHC heavy chain amino acids, in the complex of H-2Kd and Ins B15–23, as seen from above (TCR view). Color, surface electrostatic, and transparency conventions are as in (A). Some of the surrounding residues are at a distance longer than 4 Å, but are shown for comparison with (D). (D) View of p6 of the complex of H-2Kd with Ins B15–23/B20H (p6H), in the same orientation and conventions as in (C). All the surrounding residues from the heavy chain are at a distance of less than 4 Å. (E) View of p8 containing the Ins B15–23/B22R (p8R) residue and surrounding heavy chain amino acids, in the complex of H-2Kd and Ins B15–23, as seen from above (TCR view). Representations, color, surface electrostatic, and transparency conventions are as in (A). All the surrounding residues from the heavy chain are at a distance of less than 4 Å. (F) View of p8 of the complex of Kd with Ins B15–23/B22Leu (p8L), in the same orientation and conventions as in (E).
Mentions: The results of molecular structural simulation, based on the crystal structure of a H2-Kd nucleoprotein peptide complex, show that the orientation of the Ins peptide in the groove is mostly the same (with the exception of residues p5C and p7E) as that obtained by modeling based on the HLA-A2-Tax peptide complex 21 (Fig. 5A). Remarkably, the crucial TCR contact residues p6G/p8R remain in nearly identical orientations as in the previous simulation (see below). There is a minor shift in the anchoring of the peptide, as it is now shown in this work, compared to previously 21, that the p5 residue in this peptide points into the groove (pocket C, 22). Furthermore, p7 is shown to be pointing towards the α2 helix and away from the groove, hence just outside the canonical “footprint” of the cognate T cell receptor 23. Assuming that the T cell receptor docks onto this complex in a diagonal manner as suggested by nearly all structures of such complexes, we conclude that p5C and p7E participate only slightly in TCR recognition 24–28. The position of p8R is nearly on the same level and in front of the α1 helix of H2-Kd. We find it significant that in both molecular simulations, based on HLA-A2 21, and H2-Kd crystal structures (this work), the relative orientations of p6G and p8R are nearly identical. The residues p6G and p8R form a special pair such that any canonical TCR contact will necessarily involve both of them, as well as the peptide backbone of p7E (Fig. 5A).

Bottom Line: When tested for antagonist activity with APL differing from the native peptide at either of these positions, the peptide variants, G6H and R8L showed the capacity to reduce the agonist response to the native peptide.We conclude that p6 and p8 of the insulin B15-23 peptide are very important for TCR stimulation of this clone and no substitutions are tolerated at these positions in the peptide.This is important in considering the therapeutic use of peptides as APL that encompass both CD4 and CD8 epitopes of insulin.

View Article: PubMed Central - PubMed

Affiliation: Department of Cellular and Molecular Medicine, School of Medical Sciences, University of Bristol, Bristol, UK.

ABSTRACT
Insulin-reactive CD8 T cells are amongst the earliest islet-infiltrating CD8 T cells in NOD mice. Cloned insulin B15-23-reactive cells (designated G9C8), restricted by H-2K(d), are highly diabetogenic. We used altered peptide ligands (APL) substituted at TCR contact sites, positions (p)6 and 8, to investigate G9C8 T cell function and correlated this with structure. Cytotoxicity and IFN-gamma production assays revealed that p6G and p8R could not be replaced by any naturally occurring amino acid without abrogating recognition and functional response by the G9C8 clone. When tested for antagonist activity with APL differing from the native peptide at either of these positions, the peptide variants, G6H and R8L showed the capacity to reduce the agonist response to the native peptide. The antagonist activity in cytotoxicity and IFN-gamma production assays can be correlated with conformational changes induced by different structures of the MHC-peptide complexes, shown by molecular modeling. We conclude that p6 and p8 of the insulin B15-23 peptide are very important for TCR stimulation of this clone and no substitutions are tolerated at these positions in the peptide. This is important in considering the therapeutic use of peptides as APL that encompass both CD4 and CD8 epitopes of insulin.

Show MeSH
Related in: MedlinePlus