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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.

Show MeSH
MHC-APL-binding assays of the APL with substitutions at p6 and p8 of the Ins B15–23 peptide. The binding of the APL at p6 and p8 to the MHC molecule is shown at a peptide concentration of 6.25 μg/mL. All the APL, for p6 or p8, fall below the baseline (given by the MHC binding of RMAS-Kd cells stained with the FITC-conjugated anti-Kd mAb but without the addition of any peptide), and the binding of the native peptide (NAT) is just above baseline levels. By contrast, good binding to the H-2Kd molecule is seen for the peptides LLO, HA and G9V (NAT with the residue valine substituting glycine at p9, previously shown to bind well to the MHC 21).
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fig04: MHC-APL-binding assays of the APL with substitutions at p6 and p8 of the Ins B15–23 peptide. The binding of the APL at p6 and p8 to the MHC molecule is shown at a peptide concentration of 6.25 μg/mL. All the APL, for p6 or p8, fall below the baseline (given by the MHC binding of RMAS-Kd cells stained with the FITC-conjugated anti-Kd mAb but without the addition of any peptide), and the binding of the native peptide (NAT) is just above baseline levels. By contrast, good binding to the H-2Kd molecule is seen for the peptides LLO, HA and G9V (NAT with the residue valine substituting glycine at p9, previously shown to bind well to the MHC 21).

Mentions: To evaluate the binding of the APL to the MHC, binding assays for each altered peptide at both p6 and p8 were performed and results are shown in Fig. 4. The binding of the APL at p6 and p8 of the Ins B15–23 peptide is extremely weak in this binding assay and no substituted peptide showed MHC-binding capacity greater than the native peptide. Thus, the ability of the peptides to antagonize the T cell functions is not related to competition for MHC binding with the native peptide as the altered peptides bound to the MHC more weakly compared to the native peptide.


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)

MHC-APL-binding assays of the APL with substitutions at p6 and p8 of the Ins B15–23 peptide. The binding of the APL at p6 and p8 to the MHC molecule is shown at a peptide concentration of 6.25 μg/mL. All the APL, for p6 or p8, fall below the baseline (given by the MHC binding of RMAS-Kd cells stained with the FITC-conjugated anti-Kd mAb but without the addition of any peptide), and the binding of the native peptide (NAT) is just above baseline levels. By contrast, good binding to the H-2Kd molecule is seen for the peptides LLO, HA and G9V (NAT with the residue valine substituting glycine at p9, previously shown to bind well to the MHC 21).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2901522&req=5

fig04: MHC-APL-binding assays of the APL with substitutions at p6 and p8 of the Ins B15–23 peptide. The binding of the APL at p6 and p8 to the MHC molecule is shown at a peptide concentration of 6.25 μg/mL. All the APL, for p6 or p8, fall below the baseline (given by the MHC binding of RMAS-Kd cells stained with the FITC-conjugated anti-Kd mAb but without the addition of any peptide), and the binding of the native peptide (NAT) is just above baseline levels. By contrast, good binding to the H-2Kd molecule is seen for the peptides LLO, HA and G9V (NAT with the residue valine substituting glycine at p9, previously shown to bind well to the MHC 21).
Mentions: To evaluate the binding of the APL to the MHC, binding assays for each altered peptide at both p6 and p8 were performed and results are shown in Fig. 4. The binding of the APL at p6 and p8 of the Ins B15–23 peptide is extremely weak in this binding assay and no substituted peptide showed MHC-binding capacity greater than the native peptide. Thus, the ability of the peptides to antagonize the T cell functions is not related to competition for MHC binding with the native peptide as the altered peptides bound to the MHC more weakly compared to the native peptide.

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