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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

Induction of long-lasting memory CTLs. HHD mice (six per group) were immunized once with 1.6 × 105 TCID50 of Ad-WT or Ad-SCT-H74L. At day 60 after the immunization, spleen cells were prepared, and stimulated in vitro twice with peptide-pulsed syngeneic spleen cells. 51Cr-release assays were then carried out at various E:T ratios, using RMA-HHD cells pulsed with (+) or without (−) FMP58-66 as targets. The labels 1, 2, 3 indicate data from three different representative mice. Representative data are shown as the mean ± SD of triplicate wells. *P < 0.05; **P < 0.01; ***P < 0.001 compared to RMA-HHD pulsed without a peptide, Student’s t-test.
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fig7: Induction of long-lasting memory CTLs. HHD mice (six per group) were immunized once with 1.6 × 105 TCID50 of Ad-WT or Ad-SCT-H74L. At day 60 after the immunization, spleen cells were prepared, and stimulated in vitro twice with peptide-pulsed syngeneic spleen cells. 51Cr-release assays were then carried out at various E:T ratios, using RMA-HHD cells pulsed with (+) or without (−) FMP58-66 as targets. The labels 1, 2, 3 indicate data from three different representative mice. Representative data are shown as the mean ± SD of triplicate wells. *P < 0.05; **P < 0.01; ***P < 0.001 compared to RMA-HHD pulsed without a peptide, Student’s t-test.

Mentions: It was also examined whether long-lasting FMP-specific CTLs could be elicited in HHD mice immunized with Ad-SCT-H74L at a low dose of 1.6 × 105 TCID50. At this inoculation dose, neither Ad-SCT-HHD nor Ad-FMP induced detectable FMP-specific CTL responses in mice (Figures 3 and 4). After 60 days following immunization, 51Cr-release assays were then performed at various effector to target cell (E:T) ratios. As shown in Figure 7, FMP58–66-specific CTLs were detected in mice even on day 60 after immunization with Ad-SCT-H74L, but not in mice injected with Ad-WT. These data indicate that immunization with Ad-SCT-H74L at a low dose can effectively generate long-lasting memory CTLs in mice.


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)

Induction of long-lasting memory CTLs. HHD mice (six per group) were immunized once with 1.6 × 105 TCID50 of Ad-WT or Ad-SCT-H74L. At day 60 after the immunization, spleen cells were prepared, and stimulated in vitro twice with peptide-pulsed syngeneic spleen cells. 51Cr-release assays were then carried out at various E:T ratios, using RMA-HHD cells pulsed with (+) or without (−) FMP58-66 as targets. The labels 1, 2, 3 indicate data from three different representative mice. Representative data are shown as the mean ± SD of triplicate wells. *P < 0.05; **P < 0.01; ***P < 0.001 compared to RMA-HHD pulsed without a peptide, Student’s t-test.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig7: Induction of long-lasting memory CTLs. HHD mice (six per group) were immunized once with 1.6 × 105 TCID50 of Ad-WT or Ad-SCT-H74L. At day 60 after the immunization, spleen cells were prepared, and stimulated in vitro twice with peptide-pulsed syngeneic spleen cells. 51Cr-release assays were then carried out at various E:T ratios, using RMA-HHD cells pulsed with (+) or without (−) FMP58-66 as targets. The labels 1, 2, 3 indicate data from three different representative mice. Representative data are shown as the mean ± SD of triplicate wells. *P < 0.05; **P < 0.01; ***P < 0.001 compared to RMA-HHD pulsed without a peptide, Student’s t-test.
Mentions: It was also examined whether long-lasting FMP-specific CTLs could be elicited in HHD mice immunized with Ad-SCT-H74L at a low dose of 1.6 × 105 TCID50. At this inoculation dose, neither Ad-SCT-HHD nor Ad-FMP induced detectable FMP-specific CTL responses in mice (Figures 3 and 4). After 60 days following immunization, 51Cr-release assays were then performed at various effector to target cell (E:T) ratios. As shown in Figure 7, FMP58–66-specific CTLs were detected in mice even on day 60 after immunization with Ad-SCT-H74L, but not in mice injected with Ad-WT. These data indicate that immunization with Ad-SCT-H74L at a low dose can effectively generate long-lasting memory CTLs in mice.

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