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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

Immunization with Ad-SCT-H74L induces strong antitumor protection in HHD mice. (a,b) In vivo cytotoxic T lymphocyte (CTL) assay with tumor cells. Mice (five to eight per group) were vaccinated with Ad-WT, Ad-FMP, Ad-SCT-HHD or Ad-SCT-H74L at various doses. One week later, RMA-HHD and RMA-HHD-FMP cells were labeled with 0.25 and 2.5 μmol/l of CFSE, respectively, mixed at 1:1, and injected i.v. into each mouse (1 × 107 cells per mouse). After 12–14 hours, spleen cells were prepared and stained with PE-conjugated anti-H-2Db mAb. After washing, H-2Db+ cells were analyzed for their expression of CFSE by flow cytometry. (a) Each symbol represents an individual mouse. Horizontal bars represent the mean. ***P < 0.001; NS, not significant, One-way analysis of variance. (b) Representative data of the in vivo CTL assay are shown. The numbers in the panels indicate the percentage of FMP-specific lysis. L: RMA-HHD labeled with 0.25 μmol/l CFSE; H: RMA-HHD-FMP labeled with 2.5 μmol/l CFSE. (c) Survival of mice. After 1 week following immunization with 1.6 × 106 TCID50 of either Ad-WT, Ad-FMP, Ad-SCT-HHD or Ad-SCT-H74L, mice (ten per group) were challenged i.v. with 5 × 106 RMA-HHD-FMP cells. The experiment was repeated twice with similar results. (d) Inhibition of tumor growth. Mice (five per group) were immunized with 1.6 × 106 TCID50 of Ad-WT, Ad-FMP, Ad-SCT-HHD or Ad-SCT-H74L. After 1 week, mice were challenged subcutaneously with 5 × 104 RMA-HHD-FMP cells/mouse and monitored for the tumor growth. Curves represent individual mice. The experiment was repeated twice with similar results.
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fig6: Immunization with Ad-SCT-H74L induces strong antitumor protection in HHD mice. (a,b) In vivo cytotoxic T lymphocyte (CTL) assay with tumor cells. Mice (five to eight per group) were vaccinated with Ad-WT, Ad-FMP, Ad-SCT-HHD or Ad-SCT-H74L at various doses. One week later, RMA-HHD and RMA-HHD-FMP cells were labeled with 0.25 and 2.5 μmol/l of CFSE, respectively, mixed at 1:1, and injected i.v. into each mouse (1 × 107 cells per mouse). After 12–14 hours, spleen cells were prepared and stained with PE-conjugated anti-H-2Db mAb. After washing, H-2Db+ cells were analyzed for their expression of CFSE by flow cytometry. (a) Each symbol represents an individual mouse. Horizontal bars represent the mean. ***P < 0.001; NS, not significant, One-way analysis of variance. (b) Representative data of the in vivo CTL assay are shown. The numbers in the panels indicate the percentage of FMP-specific lysis. L: RMA-HHD labeled with 0.25 μmol/l CFSE; H: RMA-HHD-FMP labeled with 2.5 μmol/l CFSE. (c) Survival of mice. After 1 week following immunization with 1.6 × 106 TCID50 of either Ad-WT, Ad-FMP, Ad-SCT-HHD or Ad-SCT-H74L, mice (ten per group) were challenged i.v. with 5 × 106 RMA-HHD-FMP cells. The experiment was repeated twice with similar results. (d) Inhibition of tumor growth. Mice (five per group) were immunized with 1.6 × 106 TCID50 of Ad-WT, Ad-FMP, Ad-SCT-HHD or Ad-SCT-H74L. After 1 week, mice were challenged subcutaneously with 5 × 104 RMA-HHD-FMP cells/mouse and monitored for the tumor growth. Curves represent individual mice. The experiment was repeated twice with similar results.

Mentions: We next performed tumor challenge experiments. After immunization with either Ad-WT, Ad-FMP, Ad-SCT-HHD or Ad-SCT-H74L, HHD mice were challenged by receiving the adoptive cell transfer of carboxyfluorescein diacetate succinimidyl ester (CFSE)-labeled RMA-HHD tumor cells expressing the full-length FMP (RMA-HHD-FMP) along with RMA-HHD cells as an internal control. Twelve hours later, % lysis specific for RMA-HHD-FMP cells in each spleen was measured by flow cytometry. At a virus inoculation dose of 1.6 × 107 TCID50, there was no statistical difference in % specific lysis between Ad-SCT-H74L-infected mice and either Ad-SCT-HHD- or Ad-FMP-injected mice (Figure 6a). However, a significant difference between them was observed at an inoculation dose of 1.6 × 106 TCID50. That is, vaccination with Ad-SCT-H74L gave significantly higher clearance of RMA-HHD-FMP tumor cells expressing endogenous FMP58-66 in mice than that with Ad-SCT-HHD or Ad-FMP (Figure 6a,b). We next determined whether Ad-SCT-H74L vaccination could mediate prophylactic protection against a lethal challenge of RMA-HHD-FMP tumor cells. After immunization with 1.6 × 106 TCID50 of either Ad-WT, Ad-FMP, Ad-SCT-HHD or Ad-SCT-H74L, mice were challenged intravenously (i.v.) with 5 × 106 cells/mouse of RMA-HHD-FMP cells. As shown in Figure 6c, all Ad-WT-infected mice died by day 29, whereas many of Ad-SCT-H74L-immunized mice (7 out of 10 mice) survived for over 60 days after tumor inoculation. On the other hand, vaccination with 1.6 × 106 TCID50 of either Ad-FMP or Ad-SCT-HHD did not induce sufficient protection against i.v. challenge with a high dose of tumor cells (Figure 6c). We also tested whether vaccination with Ad-SCT-H74L could inhibit the growth of tumor at the skin. To this end, HHD mice were immunized with various recombinant adenoviruses at a dose of 1.6 × 106 TCID50, and then inoculated subcutaneously with a low number (5 × 104/mouse) of RMA-HHD-FMP tumor cells. Vaccination with Ad-SCT-H74L inhibited tumor growth more efficiently than that with Ad-FMP or Ad-SCT-HHD, while immunization with Ad-WT had no effect on the reduction of tumor growth (Figure 6d). Overall, these data demonstrate that vaccination with Ad-SCT-H74L provides better protection from tumor challenge than that with Ad-FMP or Ad-SCT-HHD.


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)

Immunization with Ad-SCT-H74L induces strong antitumor protection in HHD mice. (a,b) In vivo cytotoxic T lymphocyte (CTL) assay with tumor cells. Mice (five to eight per group) were vaccinated with Ad-WT, Ad-FMP, Ad-SCT-HHD or Ad-SCT-H74L at various doses. One week later, RMA-HHD and RMA-HHD-FMP cells were labeled with 0.25 and 2.5 μmol/l of CFSE, respectively, mixed at 1:1, and injected i.v. into each mouse (1 × 107 cells per mouse). After 12–14 hours, spleen cells were prepared and stained with PE-conjugated anti-H-2Db mAb. After washing, H-2Db+ cells were analyzed for their expression of CFSE by flow cytometry. (a) Each symbol represents an individual mouse. Horizontal bars represent the mean. ***P < 0.001; NS, not significant, One-way analysis of variance. (b) Representative data of the in vivo CTL assay are shown. The numbers in the panels indicate the percentage of FMP-specific lysis. L: RMA-HHD labeled with 0.25 μmol/l CFSE; H: RMA-HHD-FMP labeled with 2.5 μmol/l CFSE. (c) Survival of mice. After 1 week following immunization with 1.6 × 106 TCID50 of either Ad-WT, Ad-FMP, Ad-SCT-HHD or Ad-SCT-H74L, mice (ten per group) were challenged i.v. with 5 × 106 RMA-HHD-FMP cells. The experiment was repeated twice with similar results. (d) Inhibition of tumor growth. Mice (five per group) were immunized with 1.6 × 106 TCID50 of Ad-WT, Ad-FMP, Ad-SCT-HHD or Ad-SCT-H74L. After 1 week, mice were challenged subcutaneously with 5 × 104 RMA-HHD-FMP cells/mouse and monitored for the tumor growth. Curves represent individual mice. The experiment was repeated twice with similar results.
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fig6: Immunization with Ad-SCT-H74L induces strong antitumor protection in HHD mice. (a,b) In vivo cytotoxic T lymphocyte (CTL) assay with tumor cells. Mice (five to eight per group) were vaccinated with Ad-WT, Ad-FMP, Ad-SCT-HHD or Ad-SCT-H74L at various doses. One week later, RMA-HHD and RMA-HHD-FMP cells were labeled with 0.25 and 2.5 μmol/l of CFSE, respectively, mixed at 1:1, and injected i.v. into each mouse (1 × 107 cells per mouse). After 12–14 hours, spleen cells were prepared and stained with PE-conjugated anti-H-2Db mAb. After washing, H-2Db+ cells were analyzed for their expression of CFSE by flow cytometry. (a) Each symbol represents an individual mouse. Horizontal bars represent the mean. ***P < 0.001; NS, not significant, One-way analysis of variance. (b) Representative data of the in vivo CTL assay are shown. The numbers in the panels indicate the percentage of FMP-specific lysis. L: RMA-HHD labeled with 0.25 μmol/l CFSE; H: RMA-HHD-FMP labeled with 2.5 μmol/l CFSE. (c) Survival of mice. After 1 week following immunization with 1.6 × 106 TCID50 of either Ad-WT, Ad-FMP, Ad-SCT-HHD or Ad-SCT-H74L, mice (ten per group) were challenged i.v. with 5 × 106 RMA-HHD-FMP cells. The experiment was repeated twice with similar results. (d) Inhibition of tumor growth. Mice (five per group) were immunized with 1.6 × 106 TCID50 of Ad-WT, Ad-FMP, Ad-SCT-HHD or Ad-SCT-H74L. After 1 week, mice were challenged subcutaneously with 5 × 104 RMA-HHD-FMP cells/mouse and monitored for the tumor growth. Curves represent individual mice. The experiment was repeated twice with similar results.
Mentions: We next performed tumor challenge experiments. After immunization with either Ad-WT, Ad-FMP, Ad-SCT-HHD or Ad-SCT-H74L, HHD mice were challenged by receiving the adoptive cell transfer of carboxyfluorescein diacetate succinimidyl ester (CFSE)-labeled RMA-HHD tumor cells expressing the full-length FMP (RMA-HHD-FMP) along with RMA-HHD cells as an internal control. Twelve hours later, % lysis specific for RMA-HHD-FMP cells in each spleen was measured by flow cytometry. At a virus inoculation dose of 1.6 × 107 TCID50, there was no statistical difference in % specific lysis between Ad-SCT-H74L-infected mice and either Ad-SCT-HHD- or Ad-FMP-injected mice (Figure 6a). However, a significant difference between them was observed at an inoculation dose of 1.6 × 106 TCID50. That is, vaccination with Ad-SCT-H74L gave significantly higher clearance of RMA-HHD-FMP tumor cells expressing endogenous FMP58-66 in mice than that with Ad-SCT-HHD or Ad-FMP (Figure 6a,b). We next determined whether Ad-SCT-H74L vaccination could mediate prophylactic protection against a lethal challenge of RMA-HHD-FMP tumor cells. After immunization with 1.6 × 106 TCID50 of either Ad-WT, Ad-FMP, Ad-SCT-HHD or Ad-SCT-H74L, mice were challenged intravenously (i.v.) with 5 × 106 cells/mouse of RMA-HHD-FMP cells. As shown in Figure 6c, all Ad-WT-infected mice died by day 29, whereas many of Ad-SCT-H74L-immunized mice (7 out of 10 mice) survived for over 60 days after tumor inoculation. On the other hand, vaccination with 1.6 × 106 TCID50 of either Ad-FMP or Ad-SCT-HHD did not induce sufficient protection against i.v. challenge with a high dose of tumor cells (Figure 6c). We also tested whether vaccination with Ad-SCT-H74L could inhibit the growth of tumor at the skin. To this end, HHD mice were immunized with various recombinant adenoviruses at a dose of 1.6 × 106 TCID50, and then inoculated subcutaneously with a low number (5 × 104/mouse) of RMA-HHD-FMP tumor cells. Vaccination with Ad-SCT-H74L inhibited tumor growth more efficiently than that with Ad-FMP or Ad-SCT-HHD, while immunization with Ad-WT had no effect on the reduction of tumor growth (Figure 6d). Overall, these data demonstrate that vaccination with Ad-SCT-H74L provides better protection from tumor challenge than that with Ad-FMP or Ad-SCT-HHD.

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