Limits...
Prevention of cytotoxic T cell escape using a heteroclitic subdominant viral T cell determinant.

Butler NS, Theodossis A, Webb AI, Nastovska R, Ramarathinam SH, Dunstone MA, Rossjohn J, Purcell AW, Perlman S - PLoS Pathog. (2008)

Bottom Line: We demonstrate that a single amino acid substitution at a secondary anchor residue (Q to Y at position 3) increased the stability of the engineered determinant in complex with H-2K(b).The structural basis for this enhanced stability was associated with local alterations in the pMHC conformation as a result of the Q to Y substitution.Recombinant viruses encoding this engineered determinant primed CTL responses that also reacted to the wildtype epitope with significantly higher functional avidity, and protected against selection of virus mutated at a second CTL determinant and consequent disease progression in persistently infected mice.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology, University of Iowa, Iowa City, Iowa, United States of America.

ABSTRACT
High affinity antigen-specific T cells play a critical role during protective immune responses. Epitope enhancement can elicit more potent T cell responses and can subsequently lead to a stronger memory pool; however, the molecular basis of such enhancement is unclear. We used the consensus peptide-binding motif for the Major Histocompatibility Complex molecule H-2K(b) to design a heteroclitic version of the mouse hepatitis virus-specific subdominant S598 determinant. We demonstrate that a single amino acid substitution at a secondary anchor residue (Q to Y at position 3) increased the stability of the engineered determinant in complex with H-2K(b). The structural basis for this enhanced stability was associated with local alterations in the pMHC conformation as a result of the Q to Y substitution. Recombinant viruses encoding this engineered determinant primed CTL responses that also reacted to the wildtype epitope with significantly higher functional avidity, and protected against selection of virus mutated at a second CTL determinant and consequent disease progression in persistently infected mice. Collectively, our findings provide a basis for the enhanced immunogenicity of an engineered determinant that will serve as a template for guiding the development of heteroclitic T cell determinants with applications in prevention of CTL escape in chronic viral infections as well as in tumor immunity.

Show MeSH

Related in: MedlinePlus

Refined structures of WT and Q600Y S598-Aba bound to H-2Kb.(A) View of the H-2Kb antigen binding cleft from above. The HC is shown as a cartoon representation and coloured slate. The peptide is in stick format with carbon atoms coloured yellow. The final 2Fo-Fc map density for the peptide contoured at 1.0 σ is shown as a magenta mesh. (B) Detail of the antigen binding cleft displaying key interactions (dashed lines) between H-2Kb and S598-Aba in the region surrounding position 3 of the peptide. Selected residues of the HC are drawn in stick format (slate carbon atoms). Peptide residues are labelled in italics. (C) Surface representation of the H-2Kb/S598-Aba complex as seen from above. Peptide residues Arg-1, Ile-4 and Asn-7 are coloured wheat. Position 3 of the peptide (Gln) is coloured red and the HC residue E-152 is purple. D, E and F, Equivalencies to A, B and C, respectively, for the H-2Kb/S598Q600Y-Aba structure. In these panels the HC is drawn in green and the peptide in cyan.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2563037&req=5

ppat-1000186-g005: Refined structures of WT and Q600Y S598-Aba bound to H-2Kb.(A) View of the H-2Kb antigen binding cleft from above. The HC is shown as a cartoon representation and coloured slate. The peptide is in stick format with carbon atoms coloured yellow. The final 2Fo-Fc map density for the peptide contoured at 1.0 σ is shown as a magenta mesh. (B) Detail of the antigen binding cleft displaying key interactions (dashed lines) between H-2Kb and S598-Aba in the region surrounding position 3 of the peptide. Selected residues of the HC are drawn in stick format (slate carbon atoms). Peptide residues are labelled in italics. (C) Surface representation of the H-2Kb/S598-Aba complex as seen from above. Peptide residues Arg-1, Ile-4 and Asn-7 are coloured wheat. Position 3 of the peptide (Gln) is coloured red and the HC residue E-152 is purple. D, E and F, Equivalencies to A, B and C, respectively, for the H-2Kb/S598Q600Y-Aba structure. In these panels the HC is drawn in green and the peptide in cyan.

Mentions: While these studies clearly demonstrated that S598Q600Y is heteroclitic, they did not provide a mechanism for the immune enhancement. To address this, we determined the crystal structures of the H-2Kb/S598 (PDBid 2ZSV, Protein Data Bank Japan (http://www.pdbj.org/)) and H-2Kb/S598Q600Y (PDBid 2ZSW) complexes to 1.8 Å and 2.8 Å resolution respectively. The structure of H-2Kb/S598 consists of two heterodimers in the asymmetric unit (r.m.s.d. of 0.18 Å for Cα atoms), with the S598 peptide clearly bound in the antigen binding cleft of the heavy chains (HC, Figure S3A). The two peptide copies display a virtually identical configuration with root mean square deviation (rmsd) values of only 0.09 Å for all peptide atoms (0.05 Å for Cα atoms). The mode of S598 and S598Q600Y binding within the Ag-binding cleft is unambiguous, with the exception of Arg-1, whose side chain is partially disordered (Figure 5A,D; Figure S3C). The S598 peptide adopts an extended conformation, with the side chains of Arg-1, Ile-4 and Asn-7 extending prominently out of the cleft (Figure 5C). Ala-6 is also largely solvent exposed with its side chain pointing towards the α2 helix, while the side chains of Cys (Aba)-2, Gln-3, Phe-5 and Ile-8 are buried within the cleft. While the cysteine analogue's side chain is not involved in any hydrophilic interactions, there are a number of suitably positioned hydrogen bonding partners (Glu-24, Tyr-45 and Asn-70), with which the original thiol side chain could potentially interact (Figure S3B; Table S2).


Prevention of cytotoxic T cell escape using a heteroclitic subdominant viral T cell determinant.

Butler NS, Theodossis A, Webb AI, Nastovska R, Ramarathinam SH, Dunstone MA, Rossjohn J, Purcell AW, Perlman S - PLoS Pathog. (2008)

Refined structures of WT and Q600Y S598-Aba bound to H-2Kb.(A) View of the H-2Kb antigen binding cleft from above. The HC is shown as a cartoon representation and coloured slate. The peptide is in stick format with carbon atoms coloured yellow. The final 2Fo-Fc map density for the peptide contoured at 1.0 σ is shown as a magenta mesh. (B) Detail of the antigen binding cleft displaying key interactions (dashed lines) between H-2Kb and S598-Aba in the region surrounding position 3 of the peptide. Selected residues of the HC are drawn in stick format (slate carbon atoms). Peptide residues are labelled in italics. (C) Surface representation of the H-2Kb/S598-Aba complex as seen from above. Peptide residues Arg-1, Ile-4 and Asn-7 are coloured wheat. Position 3 of the peptide (Gln) is coloured red and the HC residue E-152 is purple. D, E and F, Equivalencies to A, B and C, respectively, for the H-2Kb/S598Q600Y-Aba structure. In these panels the HC is drawn in green and the peptide in cyan.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2563037&req=5

ppat-1000186-g005: Refined structures of WT and Q600Y S598-Aba bound to H-2Kb.(A) View of the H-2Kb antigen binding cleft from above. The HC is shown as a cartoon representation and coloured slate. The peptide is in stick format with carbon atoms coloured yellow. The final 2Fo-Fc map density for the peptide contoured at 1.0 σ is shown as a magenta mesh. (B) Detail of the antigen binding cleft displaying key interactions (dashed lines) between H-2Kb and S598-Aba in the region surrounding position 3 of the peptide. Selected residues of the HC are drawn in stick format (slate carbon atoms). Peptide residues are labelled in italics. (C) Surface representation of the H-2Kb/S598-Aba complex as seen from above. Peptide residues Arg-1, Ile-4 and Asn-7 are coloured wheat. Position 3 of the peptide (Gln) is coloured red and the HC residue E-152 is purple. D, E and F, Equivalencies to A, B and C, respectively, for the H-2Kb/S598Q600Y-Aba structure. In these panels the HC is drawn in green and the peptide in cyan.
Mentions: While these studies clearly demonstrated that S598Q600Y is heteroclitic, they did not provide a mechanism for the immune enhancement. To address this, we determined the crystal structures of the H-2Kb/S598 (PDBid 2ZSV, Protein Data Bank Japan (http://www.pdbj.org/)) and H-2Kb/S598Q600Y (PDBid 2ZSW) complexes to 1.8 Å and 2.8 Å resolution respectively. The structure of H-2Kb/S598 consists of two heterodimers in the asymmetric unit (r.m.s.d. of 0.18 Å for Cα atoms), with the S598 peptide clearly bound in the antigen binding cleft of the heavy chains (HC, Figure S3A). The two peptide copies display a virtually identical configuration with root mean square deviation (rmsd) values of only 0.09 Å for all peptide atoms (0.05 Å for Cα atoms). The mode of S598 and S598Q600Y binding within the Ag-binding cleft is unambiguous, with the exception of Arg-1, whose side chain is partially disordered (Figure 5A,D; Figure S3C). The S598 peptide adopts an extended conformation, with the side chains of Arg-1, Ile-4 and Asn-7 extending prominently out of the cleft (Figure 5C). Ala-6 is also largely solvent exposed with its side chain pointing towards the α2 helix, while the side chains of Cys (Aba)-2, Gln-3, Phe-5 and Ile-8 are buried within the cleft. While the cysteine analogue's side chain is not involved in any hydrophilic interactions, there are a number of suitably positioned hydrogen bonding partners (Glu-24, Tyr-45 and Asn-70), with which the original thiol side chain could potentially interact (Figure S3B; Table S2).

Bottom Line: We demonstrate that a single amino acid substitution at a secondary anchor residue (Q to Y at position 3) increased the stability of the engineered determinant in complex with H-2K(b).The structural basis for this enhanced stability was associated with local alterations in the pMHC conformation as a result of the Q to Y substitution.Recombinant viruses encoding this engineered determinant primed CTL responses that also reacted to the wildtype epitope with significantly higher functional avidity, and protected against selection of virus mutated at a second CTL determinant and consequent disease progression in persistently infected mice.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology, University of Iowa, Iowa City, Iowa, United States of America.

ABSTRACT
High affinity antigen-specific T cells play a critical role during protective immune responses. Epitope enhancement can elicit more potent T cell responses and can subsequently lead to a stronger memory pool; however, the molecular basis of such enhancement is unclear. We used the consensus peptide-binding motif for the Major Histocompatibility Complex molecule H-2K(b) to design a heteroclitic version of the mouse hepatitis virus-specific subdominant S598 determinant. We demonstrate that a single amino acid substitution at a secondary anchor residue (Q to Y at position 3) increased the stability of the engineered determinant in complex with H-2K(b). The structural basis for this enhanced stability was associated with local alterations in the pMHC conformation as a result of the Q to Y substitution. Recombinant viruses encoding this engineered determinant primed CTL responses that also reacted to the wildtype epitope with significantly higher functional avidity, and protected against selection of virus mutated at a second CTL determinant and consequent disease progression in persistently infected mice. Collectively, our findings provide a basis for the enhanced immunogenicity of an engineered determinant that will serve as a template for guiding the development of heteroclitic T cell determinants with applications in prevention of CTL escape in chronic viral infections as well as in tumor immunity.

Show MeSH
Related in: MedlinePlus