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Introduction of a point mutation into an HLA class I single-chain trimer induces enhancement of CTL priming and antitumor immunity.

Matsui M, Kawano M, Matsushita S, Akatsuka T - Mol Ther Methods Clin Dev (2014)

Bottom Line: We previously discovered one particular HLA-A*02:01 mutant that enhanced peptide-specific cytotoxic T lymphocyte (CTL) recognition in vitro compared to wild-type HLA-A*02:01.To investigate the effect of the H74L mutation on the in vivo CTL priming, we took advantage of the technology of the HLA class I single-chain trimer (SCT) in which three components involving a peptide, β2 microglobulin and the HLA class I heavy chain are joined together via flexible linkers.These data present the first evidence that a simple point mutation in the HLA class I heavy chain of SCT is beneficial for improving CTL-based immunotherapy and prophylaxis to control tumors.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology, Saitama Medical University , Iruma-gun, Saitama, Japan.

ABSTRACT
We previously discovered one particular HLA-A*02:01 mutant that enhanced peptide-specific cytotoxic T lymphocyte (CTL) recognition in vitro compared to wild-type HLA-A*02:01. This mutant contains a single amino acid substitution from histidine to leucine at position 74 (H74L) that is located in the peptide-binding groove. To investigate the effect of the H74L mutation on the in vivo CTL priming, we took advantage of the technology of the HLA class I single-chain trimer (SCT) in which three components involving a peptide, β2 microglobulin and the HLA class I heavy chain are joined together via flexible linkers. We generated recombinant adenovirus expressing SCT comprised influenza A matrix protein (FMP)-derived peptide, β2 microglobulin and the H74L heavy chain. HLA-A*02:01 transgenic mice were immunized with the adenovirus, and the induction of peptide-specific CTLs and antitumor immunity was investigated. It was clearly shown that the H74L mutation enabled the HLA-A*02:01 SCT molecule to dramatically enhance both in vivo priming of FMP-specific CTLs and protection against a lethal challenge of tumor cells expressing FMP. These data present the first evidence that a simple point mutation in the HLA class I heavy chain of SCT is beneficial for improving CTL-based immunotherapy and prophylaxis to control tumors.

No MeSH data available.


Related in: MedlinePlus

The SCT molecule comprises an epitope, human β2m and HHD with the H74L mutation. (a) Structure of the SCT molecule with the H74L mutation (SCT-H74L). LS: leader sequence; huβ2m: human β2m; α1, α2, α3: α1, α2, α3 domains; Tm: transmembrane domain; Cyt: cytoplasmic domain. (b) Diagram of SCT-H74L on the cell surface. (c) Expression of SCT-HHD or SCT-H74L on the surface of RMA-S cells. RMA-S and RMA-S transfectants were stained with the anti-HLA-A*02:01 mAb, followed by FITC-labeled goat antimouse IgG antibody. Thick lines: RMA-S-HHD, peptide-pulsed RMA-S-HHD (pulsed with 1 μmol/l FMP58–66), RMA-S-expressing SCT-HHD (RMA-S-SCT-HHD), and RMA-S-expressing SCT-H74L (RMA-S-SCT-H74L); Thin lines in all panels: RMA-S.
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fig2: The SCT molecule comprises an epitope, human β2m and HHD with the H74L mutation. (a) Structure of the SCT molecule with the H74L mutation (SCT-H74L). LS: leader sequence; huβ2m: human β2m; α1, α2, α3: α1, α2, α3 domains; Tm: transmembrane domain; Cyt: cytoplasmic domain. (b) Diagram of SCT-H74L on the cell surface. (c) Expression of SCT-HHD or SCT-H74L on the surface of RMA-S cells. RMA-S and RMA-S transfectants were stained with the anti-HLA-A*02:01 mAb, followed by FITC-labeled goat antimouse IgG antibody. Thick lines: RMA-S-HHD, peptide-pulsed RMA-S-HHD (pulsed with 1 μmol/l FMP58–66), RMA-S-expressing SCT-HHD (RMA-S-SCT-HHD), and RMA-S-expressing SCT-H74L (RMA-S-SCT-H74L); Thin lines in all panels: RMA-S.

Mentions: It is well documented that the SCTs are potent stimulators of peptide-specific CTLs in vivo.6,9,20 The SCT polypeptide comprises three MHC class I components including an antigenic peptide, β2m and the MHC class I heavy chain attached sequentially with flexible linkers.6 Given the unique property of the H74L mutant, we hypothesized that the SCTs of HHD-H74L could prime peptide-specific CTLs in vivo more efficiently than the SCTs composed of WT HHD. To prove this hypothesis, we produced SCT constructs consisting of the following elements beginning with the amino terminus: the leader sequence of β2m, the FMP58–66 peptide, the first flexible linker of 15 residues, the mature portion of human β2m, the second flexible linker of 15 residues, and, finally, the heavy chain of the HHD (SCT-HHD) or HHD-H74L (SCT-H74L) molecule (Figure 2a,b). When the SCT-HHD or SCT-H74L construct was stably expressed in the TAP-2-deficient cell line, RMA-S, there was an apparent increase of the expression level of MHC class I molecules on the cell surface at 37°C in comparison with RMA-S-HHD in the absence of an exogenous peptide (Figure 1c). These results implied that SCT-HHD or SCT-H74L was expressed on the cell surface even in the absence of endogenous peptides, suggesting that the SCT molecules are likely to bypass MHC class I antigen processing due to their preassembled forms.


Introduction of a point mutation into an HLA class I single-chain trimer induces enhancement of CTL priming and antitumor immunity.

Matsui M, Kawano M, Matsushita S, Akatsuka T - Mol Ther Methods Clin Dev (2014)

The SCT molecule comprises an epitope, human β2m and HHD with the H74L mutation. (a) Structure of the SCT molecule with the H74L mutation (SCT-H74L). LS: leader sequence; huβ2m: human β2m; α1, α2, α3: α1, α2, α3 domains; Tm: transmembrane domain; Cyt: cytoplasmic domain. (b) Diagram of SCT-H74L on the cell surface. (c) Expression of SCT-HHD or SCT-H74L on the surface of RMA-S cells. RMA-S and RMA-S transfectants were stained with the anti-HLA-A*02:01 mAb, followed by FITC-labeled goat antimouse IgG antibody. Thick lines: RMA-S-HHD, peptide-pulsed RMA-S-HHD (pulsed with 1 μmol/l FMP58–66), RMA-S-expressing SCT-HHD (RMA-S-SCT-HHD), and RMA-S-expressing SCT-H74L (RMA-S-SCT-H74L); Thin lines in all panels: RMA-S.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4362367&req=5

fig2: The SCT molecule comprises an epitope, human β2m and HHD with the H74L mutation. (a) Structure of the SCT molecule with the H74L mutation (SCT-H74L). LS: leader sequence; huβ2m: human β2m; α1, α2, α3: α1, α2, α3 domains; Tm: transmembrane domain; Cyt: cytoplasmic domain. (b) Diagram of SCT-H74L on the cell surface. (c) Expression of SCT-HHD or SCT-H74L on the surface of RMA-S cells. RMA-S and RMA-S transfectants were stained with the anti-HLA-A*02:01 mAb, followed by FITC-labeled goat antimouse IgG antibody. Thick lines: RMA-S-HHD, peptide-pulsed RMA-S-HHD (pulsed with 1 μmol/l FMP58–66), RMA-S-expressing SCT-HHD (RMA-S-SCT-HHD), and RMA-S-expressing SCT-H74L (RMA-S-SCT-H74L); Thin lines in all panels: RMA-S.
Mentions: It is well documented that the SCTs are potent stimulators of peptide-specific CTLs in vivo.6,9,20 The SCT polypeptide comprises three MHC class I components including an antigenic peptide, β2m and the MHC class I heavy chain attached sequentially with flexible linkers.6 Given the unique property of the H74L mutant, we hypothesized that the SCTs of HHD-H74L could prime peptide-specific CTLs in vivo more efficiently than the SCTs composed of WT HHD. To prove this hypothesis, we produced SCT constructs consisting of the following elements beginning with the amino terminus: the leader sequence of β2m, the FMP58–66 peptide, the first flexible linker of 15 residues, the mature portion of human β2m, the second flexible linker of 15 residues, and, finally, the heavy chain of the HHD (SCT-HHD) or HHD-H74L (SCT-H74L) molecule (Figure 2a,b). When the SCT-HHD or SCT-H74L construct was stably expressed in the TAP-2-deficient cell line, RMA-S, there was an apparent increase of the expression level of MHC class I molecules on the cell surface at 37°C in comparison with RMA-S-HHD in the absence of an exogenous peptide (Figure 1c). These results implied that SCT-HHD or SCT-H74L was expressed on the cell surface even in the absence of endogenous peptides, suggesting that the SCT molecules are likely to bypass MHC class I antigen processing due to their preassembled forms.

Bottom Line: We previously discovered one particular HLA-A*02:01 mutant that enhanced peptide-specific cytotoxic T lymphocyte (CTL) recognition in vitro compared to wild-type HLA-A*02:01.To investigate the effect of the H74L mutation on the in vivo CTL priming, we took advantage of the technology of the HLA class I single-chain trimer (SCT) in which three components involving a peptide, β2 microglobulin and the HLA class I heavy chain are joined together via flexible linkers.These data present the first evidence that a simple point mutation in the HLA class I heavy chain of SCT is beneficial for improving CTL-based immunotherapy and prophylaxis to control tumors.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology, Saitama Medical University , Iruma-gun, Saitama, Japan.

ABSTRACT
We previously discovered one particular HLA-A*02:01 mutant that enhanced peptide-specific cytotoxic T lymphocyte (CTL) recognition in vitro compared to wild-type HLA-A*02:01. This mutant contains a single amino acid substitution from histidine to leucine at position 74 (H74L) that is located in the peptide-binding groove. To investigate the effect of the H74L mutation on the in vivo CTL priming, we took advantage of the technology of the HLA class I single-chain trimer (SCT) in which three components involving a peptide, β2 microglobulin and the HLA class I heavy chain are joined together via flexible linkers. We generated recombinant adenovirus expressing SCT comprised influenza A matrix protein (FMP)-derived peptide, β2 microglobulin and the H74L heavy chain. HLA-A*02:01 transgenic mice were immunized with the adenovirus, and the induction of peptide-specific CTLs and antitumor immunity was investigated. It was clearly shown that the H74L mutation enabled the HLA-A*02:01 SCT molecule to dramatically enhance both in vivo priming of FMP-specific CTLs and protection against a lethal challenge of tumor cells expressing FMP. These data present the first evidence that a simple point mutation in the HLA class I heavy chain of SCT is beneficial for improving CTL-based immunotherapy and prophylaxis to control tumors.

No MeSH data available.


Related in: MedlinePlus