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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.

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Fitness of and CTL responses elicited by rJ and rJ.SQ600Y.(A) Schematic depictions of recombinant wild type JHMV (rJ), recombinant JHMV encoding the Q600Y substitution (rJ.SQ600Y) and recombinant JHMV encoding the S598Q600Y substitution in the context of an S510 CTL escape mutation (S510W513R+Q600Y). (B) Equivalent in vitro growth kinetics among rJ and rJ.SQ600Y viruses. 17Cl-1 cells were infected with rJ or rJ.SQ600Y at a multiplicity of infection (MOI) of 1.0. Cells and supernatants were harvested at the indicated times and titers were measured by plaque assay as described in Materials and Methods. Data are representative of two independent experiments. (C) Equivalent in vivo fitness among rJ and rJ.SQ600Y viruses. Five week old B6 and BALB/c mice were infected with virus mixtures consisting of equal PFU of rJ and rJ.SQ600Y variant virus. Seven days p.i., total RNA was harvested from the brains of mice and relative representation of virus template was determined via RT-PCR and direct sequencing of PCR products. The relative proportion of animals in which only rJ, only rJ.SQ600Y, or a mixture of the two viruses is shown. (D) High-magnitude, unidirectional cross-reactivity. Representative dot plots demonstrating the frequency of epitope-specific CD8 T cells in a mouse infected with rJ (top panels) or rJ.SQ600Y (bottom panels). Numbers represent the frequency of epitope-specific CD8 T cells among total CD8 T cells recovered from the brains of mice 7 days p.i. (E) Summaries of the frequency (left panel) and absolute number (right panel) of epitope-specific CD8 T cells recovered from the brains of rJ and rJ.SQ600Y-infected mice 7 days p.i. Data shown in D represent mean±SEM for 4 independent experiments.
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ppat-1000186-g002: Fitness of and CTL responses elicited by rJ and rJ.SQ600Y.(A) Schematic depictions of recombinant wild type JHMV (rJ), recombinant JHMV encoding the Q600Y substitution (rJ.SQ600Y) and recombinant JHMV encoding the S598Q600Y substitution in the context of an S510 CTL escape mutation (S510W513R+Q600Y). (B) Equivalent in vitro growth kinetics among rJ and rJ.SQ600Y viruses. 17Cl-1 cells were infected with rJ or rJ.SQ600Y at a multiplicity of infection (MOI) of 1.0. Cells and supernatants were harvested at the indicated times and titers were measured by plaque assay as described in Materials and Methods. Data are representative of two independent experiments. (C) Equivalent in vivo fitness among rJ and rJ.SQ600Y viruses. Five week old B6 and BALB/c mice were infected with virus mixtures consisting of equal PFU of rJ and rJ.SQ600Y variant virus. Seven days p.i., total RNA was harvested from the brains of mice and relative representation of virus template was determined via RT-PCR and direct sequencing of PCR products. The relative proportion of animals in which only rJ, only rJ.SQ600Y, or a mixture of the two viruses is shown. (D) High-magnitude, unidirectional cross-reactivity. Representative dot plots demonstrating the frequency of epitope-specific CD8 T cells in a mouse infected with rJ (top panels) or rJ.SQ600Y (bottom panels). Numbers represent the frequency of epitope-specific CD8 T cells among total CD8 T cells recovered from the brains of mice 7 days p.i. (E) Summaries of the frequency (left panel) and absolute number (right panel) of epitope-specific CD8 T cells recovered from the brains of rJ and rJ.SQ600Y-infected mice 7 days p.i. Data shown in D represent mean±SEM for 4 independent experiments.

Mentions: Next, we used the consensus H-2Kb binding motif [24] to engineer a novel, high-avidity S598 CTL epitope. Importantly, Gln-3 diverges from the consensus H-2Kb-restricted ligand binding motif, in which a tyrosine is often present at position 3 [24],[25]. Therefore, we substituted a glutamine residue for tyrosine (Q600Y, CAA to TAT, RCYIFANI) at position 3 of the determinant with the aim of creating a peptide that bound more tightly to H-2Kb. Stability of the H-2Kb/S598 and H-2Kb/S598Q600Y complexes was assessed by circular dichroism (CD). As shown in Figure 1, H-2Kb/S598Q600Y was considerably more thermostable than the native complex (Tm 54°C vs 64°C). To probe the biological properties of the Q600Y substitution, we used reverse genetics to engineer a recombinant version of JHMV expressing the S598Q600Y epitope (Figure 2A). Recombinant viruses encoding this substitution replicated as efficiently as wild type JHMV (rJ) in vitro during one-step growth kinetics analyses and in vivo virus competition assays (Figure 2B, C). The immunogenicity of S598Q600Y was assessed by intracellular expression of IFN-γ by central nervous system (CNS)-derived lymphocytes. Since Cys-containing peptides are often diminished in ability to elicit a CTL response, we included a reducing agent (TCEP, Tris[2-carboxyethyl] phosphine) in the cultures. TCEP enhanced the stimulatory capacity of the S598 peptides (Figure S1B), indicating that a proportion of the unmodified S598 peptide stock had undergone oxidation. Thus, we stimulated CNS- and spleen-derived CTL ex vivo in the presence of 500 µM TCEP, a concentration consistent with other work with Cys containing peptides [23],[26].


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)

Fitness of and CTL responses elicited by rJ and rJ.SQ600Y.(A) Schematic depictions of recombinant wild type JHMV (rJ), recombinant JHMV encoding the Q600Y substitution (rJ.SQ600Y) and recombinant JHMV encoding the S598Q600Y substitution in the context of an S510 CTL escape mutation (S510W513R+Q600Y). (B) Equivalent in vitro growth kinetics among rJ and rJ.SQ600Y viruses. 17Cl-1 cells were infected with rJ or rJ.SQ600Y at a multiplicity of infection (MOI) of 1.0. Cells and supernatants were harvested at the indicated times and titers were measured by plaque assay as described in Materials and Methods. Data are representative of two independent experiments. (C) Equivalent in vivo fitness among rJ and rJ.SQ600Y viruses. Five week old B6 and BALB/c mice were infected with virus mixtures consisting of equal PFU of rJ and rJ.SQ600Y variant virus. Seven days p.i., total RNA was harvested from the brains of mice and relative representation of virus template was determined via RT-PCR and direct sequencing of PCR products. The relative proportion of animals in which only rJ, only rJ.SQ600Y, or a mixture of the two viruses is shown. (D) High-magnitude, unidirectional cross-reactivity. Representative dot plots demonstrating the frequency of epitope-specific CD8 T cells in a mouse infected with rJ (top panels) or rJ.SQ600Y (bottom panels). Numbers represent the frequency of epitope-specific CD8 T cells among total CD8 T cells recovered from the brains of mice 7 days p.i. (E) Summaries of the frequency (left panel) and absolute number (right panel) of epitope-specific CD8 T cells recovered from the brains of rJ and rJ.SQ600Y-infected mice 7 days p.i. Data shown in D represent mean±SEM for 4 independent experiments.
© Copyright Policy
Related In: Results  -  Collection

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

ppat-1000186-g002: Fitness of and CTL responses elicited by rJ and rJ.SQ600Y.(A) Schematic depictions of recombinant wild type JHMV (rJ), recombinant JHMV encoding the Q600Y substitution (rJ.SQ600Y) and recombinant JHMV encoding the S598Q600Y substitution in the context of an S510 CTL escape mutation (S510W513R+Q600Y). (B) Equivalent in vitro growth kinetics among rJ and rJ.SQ600Y viruses. 17Cl-1 cells were infected with rJ or rJ.SQ600Y at a multiplicity of infection (MOI) of 1.0. Cells and supernatants were harvested at the indicated times and titers were measured by plaque assay as described in Materials and Methods. Data are representative of two independent experiments. (C) Equivalent in vivo fitness among rJ and rJ.SQ600Y viruses. Five week old B6 and BALB/c mice were infected with virus mixtures consisting of equal PFU of rJ and rJ.SQ600Y variant virus. Seven days p.i., total RNA was harvested from the brains of mice and relative representation of virus template was determined via RT-PCR and direct sequencing of PCR products. The relative proportion of animals in which only rJ, only rJ.SQ600Y, or a mixture of the two viruses is shown. (D) High-magnitude, unidirectional cross-reactivity. Representative dot plots demonstrating the frequency of epitope-specific CD8 T cells in a mouse infected with rJ (top panels) or rJ.SQ600Y (bottom panels). Numbers represent the frequency of epitope-specific CD8 T cells among total CD8 T cells recovered from the brains of mice 7 days p.i. (E) Summaries of the frequency (left panel) and absolute number (right panel) of epitope-specific CD8 T cells recovered from the brains of rJ and rJ.SQ600Y-infected mice 7 days p.i. Data shown in D represent mean±SEM for 4 independent experiments.
Mentions: Next, we used the consensus H-2Kb binding motif [24] to engineer a novel, high-avidity S598 CTL epitope. Importantly, Gln-3 diverges from the consensus H-2Kb-restricted ligand binding motif, in which a tyrosine is often present at position 3 [24],[25]. Therefore, we substituted a glutamine residue for tyrosine (Q600Y, CAA to TAT, RCYIFANI) at position 3 of the determinant with the aim of creating a peptide that bound more tightly to H-2Kb. Stability of the H-2Kb/S598 and H-2Kb/S598Q600Y complexes was assessed by circular dichroism (CD). As shown in Figure 1, H-2Kb/S598Q600Y was considerably more thermostable than the native complex (Tm 54°C vs 64°C). To probe the biological properties of the Q600Y substitution, we used reverse genetics to engineer a recombinant version of JHMV expressing the S598Q600Y epitope (Figure 2A). Recombinant viruses encoding this substitution replicated as efficiently as wild type JHMV (rJ) in vitro during one-step growth kinetics analyses and in vivo virus competition assays (Figure 2B, C). The immunogenicity of S598Q600Y was assessed by intracellular expression of IFN-γ by central nervous system (CNS)-derived lymphocytes. Since Cys-containing peptides are often diminished in ability to elicit a CTL response, we included a reducing agent (TCEP, Tris[2-carboxyethyl] phosphine) in the cultures. TCEP enhanced the stimulatory capacity of the S598 peptides (Figure S1B), indicating that a proportion of the unmodified S598 peptide stock had undergone oxidation. Thus, we stimulated CNS- and spleen-derived CTL ex vivo in the presence of 500 µM TCEP, a concentration consistent with other work with Cys containing peptides [23],[26].

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