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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

Cytotoxicity and IFN-γ production assays using native peptide and APL with substitutions at p6 and p8. (A) 51Cr-release cytotoxicity assay was performed using increasing concentrations of native Ins B15–23 peptide with P815 target cells and G9C8 cloned T cells as effectors at an E:T ratio of 10:1 following 4 h of incubation. IFN-γ production was measured by ELISA following incubation with P815 cells and increasing concentrations of B15–23 peptide after 24 h incubation. Proliferation is shown by [3H]thymidine incorporation in cpm following incubation of G9C8 cloned T cells, in triplicate, with increasing concentrations of Ins B15–23 peptide. (B) P815 target cells were first incubated with 51Cr-sodium chromate and secondly with different concentrations of native peptide (NAT), irrelevant control peptides (LLO and HA) and peptides altered at position 6 and position 8. The G9C8 cloned T cells as effectors were added to the plates at an E:T ratio of 10:1. Data are shown for a peptide concentration of 1 μg/mL. The background when effectors and targets were incubated in the absence of peptide was 8.6% in this assay. The data are presented as percentage of specific lysis. Each value corresponds to the average of duplicate samples. Results shown represent one of at least two independent assays. (C) G9C8 cloned T cells were stimulated by peptide-pulsed P815 cells at five peptide concentrations within the range 0.008–5 μg/mL. IFN-γ production at 1 μg/mL is shown. Supernatants were tested in duplicate taken after 24-h incubation at 37°C. Results correspond to one of two independent experiments. The limit of detection for the assay was 0.27 U/mL.
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fig01: Cytotoxicity and IFN-γ production assays using native peptide and APL with substitutions at p6 and p8. (A) 51Cr-release cytotoxicity assay was performed using increasing concentrations of native Ins B15–23 peptide with P815 target cells and G9C8 cloned T cells as effectors at an E:T ratio of 10:1 following 4 h of incubation. IFN-γ production was measured by ELISA following incubation with P815 cells and increasing concentrations of B15–23 peptide after 24 h incubation. Proliferation is shown by [3H]thymidine incorporation in cpm following incubation of G9C8 cloned T cells, in triplicate, with increasing concentrations of Ins B15–23 peptide. (B) P815 target cells were first incubated with 51Cr-sodium chromate and secondly with different concentrations of native peptide (NAT), irrelevant control peptides (LLO and HA) and peptides altered at position 6 and position 8. The G9C8 cloned T cells as effectors were added to the plates at an E:T ratio of 10:1. Data are shown for a peptide concentration of 1 μg/mL. The background when effectors and targets were incubated in the absence of peptide was 8.6% in this assay. The data are presented as percentage of specific lysis. Each value corresponds to the average of duplicate samples. Results shown represent one of at least two independent assays. (C) G9C8 cloned T cells were stimulated by peptide-pulsed P815 cells at five peptide concentrations within the range 0.008–5 μg/mL. IFN-γ production at 1 μg/mL is shown. Supernatants were tested in duplicate taken after 24-h incubation at 37°C. Results correspond to one of two independent experiments. The limit of detection for the assay was 0.27 U/mL.

Mentions: The G9C8 cloned T cells were used as effectors in a standard 51Cr-release assay against P815 target cells after incubation with six different concentrations of the peptides ranging from 0.0016–5 μg/mL (Fig. 1A). No stimulation was seen above the background (no peptide) for any of the substituted peptides or the two control peptides for any of the six concentrations of peptide tested (Fig. 1B and data not shown).


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)

Cytotoxicity and IFN-γ production assays using native peptide and APL with substitutions at p6 and p8. (A) 51Cr-release cytotoxicity assay was performed using increasing concentrations of native Ins B15–23 peptide with P815 target cells and G9C8 cloned T cells as effectors at an E:T ratio of 10:1 following 4 h of incubation. IFN-γ production was measured by ELISA following incubation with P815 cells and increasing concentrations of B15–23 peptide after 24 h incubation. Proliferation is shown by [3H]thymidine incorporation in cpm following incubation of G9C8 cloned T cells, in triplicate, with increasing concentrations of Ins B15–23 peptide. (B) P815 target cells were first incubated with 51Cr-sodium chromate and secondly with different concentrations of native peptide (NAT), irrelevant control peptides (LLO and HA) and peptides altered at position 6 and position 8. The G9C8 cloned T cells as effectors were added to the plates at an E:T ratio of 10:1. Data are shown for a peptide concentration of 1 μg/mL. The background when effectors and targets were incubated in the absence of peptide was 8.6% in this assay. The data are presented as percentage of specific lysis. Each value corresponds to the average of duplicate samples. Results shown represent one of at least two independent assays. (C) G9C8 cloned T cells were stimulated by peptide-pulsed P815 cells at five peptide concentrations within the range 0.008–5 μg/mL. IFN-γ production at 1 μg/mL is shown. Supernatants were tested in duplicate taken after 24-h incubation at 37°C. Results correspond to one of two independent experiments. The limit of detection for the assay was 0.27 U/mL.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig01: Cytotoxicity and IFN-γ production assays using native peptide and APL with substitutions at p6 and p8. (A) 51Cr-release cytotoxicity assay was performed using increasing concentrations of native Ins B15–23 peptide with P815 target cells and G9C8 cloned T cells as effectors at an E:T ratio of 10:1 following 4 h of incubation. IFN-γ production was measured by ELISA following incubation with P815 cells and increasing concentrations of B15–23 peptide after 24 h incubation. Proliferation is shown by [3H]thymidine incorporation in cpm following incubation of G9C8 cloned T cells, in triplicate, with increasing concentrations of Ins B15–23 peptide. (B) P815 target cells were first incubated with 51Cr-sodium chromate and secondly with different concentrations of native peptide (NAT), irrelevant control peptides (LLO and HA) and peptides altered at position 6 and position 8. The G9C8 cloned T cells as effectors were added to the plates at an E:T ratio of 10:1. Data are shown for a peptide concentration of 1 μg/mL. The background when effectors and targets were incubated in the absence of peptide was 8.6% in this assay. The data are presented as percentage of specific lysis. Each value corresponds to the average of duplicate samples. Results shown represent one of at least two independent assays. (C) G9C8 cloned T cells were stimulated by peptide-pulsed P815 cells at five peptide concentrations within the range 0.008–5 μg/mL. IFN-γ production at 1 μg/mL is shown. Supernatants were tested in duplicate taken after 24-h incubation at 37°C. Results correspond to one of two independent experiments. The limit of detection for the assay was 0.27 U/mL.
Mentions: The G9C8 cloned T cells were used as effectors in a standard 51Cr-release assay against P815 target cells after incubation with six different concentrations of the peptides ranging from 0.0016–5 μg/mL (Fig. 1A). No stimulation was seen above the background (no peptide) for any of the substituted peptides or the two control peptides for any of the six concentrations of peptide tested (Fig. 1B and data not shown).

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