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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

HHD-H74L improves the presentation of exogenous peptides. (a) Structure of HHD-H74L. LS: leader sequence; huβ2m: human β2m; α1, α2, α3, Tm, Cyt: α1, α2, α3, transmembrane, cytoplasmic domains. (b) Diagram of HHD-H74L. (c) Enhanced cytotoxic T lymphocyte (CTL) recognition of HHD-H74L-expressing targets. CTL lines were examined for killing activities against RMA, RMA-HHD and RMA-H74L in the presence of a peptide (FMP58–66, HPV-E629–38, HTLV-tax11–19 or HIV-pol476–484) by 51Cr-release assays. Data are shown as the mean ± SD of triplicate wells. The experiments were repeated three times. *P < 0.05; **P < 0.01 compared to RMA-HHD, Student’s t-test. (d) Enhanced peptide-binding by HHD-H74L. RMA-S-HHD or RMA-S-H74L cells were cultured with a peptide, and examined for their surface expression of HHD or HHD-H74L by flow cytometry. The experiment was repeated twice with similar results. Data are standardized as the % relative binding, and shown as the mean ± SD of triplicate wells. *P < 0.05; **P < 0.01 compared to RMA-S-HHD, Student’s t-test. (e) CTL recognition of various HHD mutants. FMP-specific CTL lines were examined for killing activities against RMA expressing HHD mutants with various mutations at position 74 in the presence of 100 nmol/l FMP58-66 by 51Cr-release assays. The data are representative of one of three independent experiments. Data are standardized as the % relative lysis and shown as the mean ± SD of triplicate wells. *P < 0.05; **P < 0.01; ***P < 0.001; NS, not significant, One-way analysis of variance.
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fig1: HHD-H74L improves the presentation of exogenous peptides. (a) Structure of HHD-H74L. LS: leader sequence; huβ2m: human β2m; α1, α2, α3, Tm, Cyt: α1, α2, α3, transmembrane, cytoplasmic domains. (b) Diagram of HHD-H74L. (c) Enhanced cytotoxic T lymphocyte (CTL) recognition of HHD-H74L-expressing targets. CTL lines were examined for killing activities against RMA, RMA-HHD and RMA-H74L in the presence of a peptide (FMP58–66, HPV-E629–38, HTLV-tax11–19 or HIV-pol476–484) by 51Cr-release assays. Data are shown as the mean ± SD of triplicate wells. The experiments were repeated three times. *P < 0.05; **P < 0.01 compared to RMA-HHD, Student’s t-test. (d) Enhanced peptide-binding by HHD-H74L. RMA-S-HHD or RMA-S-H74L cells were cultured with a peptide, and examined for their surface expression of HHD or HHD-H74L by flow cytometry. The experiment was repeated twice with similar results. Data are standardized as the % relative binding, and shown as the mean ± SD of triplicate wells. *P < 0.05; **P < 0.01 compared to RMA-S-HHD, Student’s t-test. (e) CTL recognition of various HHD mutants. FMP-specific CTL lines were examined for killing activities against RMA expressing HHD mutants with various mutations at position 74 in the presence of 100 nmol/l FMP58-66 by 51Cr-release assays. The data are representative of one of three independent experiments. Data are standardized as the % relative lysis and shown as the mean ± SD of triplicate wells. *P < 0.05; **P < 0.01; ***P < 0.001; NS, not significant, One-way analysis of variance.

Mentions: Previous studies demonstrated that the mutant HLA-A*02:01 molecule with the H74L mutation possessed the unique ability to enhance the presentation of exogenously loaded peptides to peptide-specific CTLs.5,18 To confirm these results in the HHD system, we firstly constructed a mutant HHD gene that could generate this mutation in the HHD molecule (HHD-H74L) (Figure 1a,b) by the inverse polymerase chain reaction (PCR)-based site-directed mutagenesis. The HHD-H74L gene was then transfected and expressed in mouse lymphoma, RMA cells (RMA-H74L). The expression level of the H74L mutant molecules on RMA-H74L cells was almost equivalent to that of the HHD molecules on RMA cells expressing the WT HHD (RMA-HHD). To test the ability of the H74L mutant, HLA-A*02:01-restricted, peptide-specific CTL lines were generated from HHD mice, and examined for their killing activities against RMA-HHD and RMA-H74L in the presence of synthetic peptides at various concentrations in standard 51Cr-release assays. Consistent with the previous data,5 RMA-H74L cells were lysed by influenza A matrix peptide (FMP58-66)-specific CTLs at much lower peptide concentrations than RMA-HHD cells (Figure 1c). Similar results were obtained with different HLA-A*02:01-restricted peptides derived from human papillomavirus (HPV)-E6 (HPV-E629-38), human T-lymphotropic virus (HTLV)-tax (HTLV-tax11-19) and human immunodeficiency virus (HIV)-pol (HIV-pol476-484) (Figure 1c), indicating that this phenomenon was not peptide-specific as described in the previous studies.18


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)

HHD-H74L improves the presentation of exogenous peptides. (a) Structure of HHD-H74L. LS: leader sequence; huβ2m: human β2m; α1, α2, α3, Tm, Cyt: α1, α2, α3, transmembrane, cytoplasmic domains. (b) Diagram of HHD-H74L. (c) Enhanced cytotoxic T lymphocyte (CTL) recognition of HHD-H74L-expressing targets. CTL lines were examined for killing activities against RMA, RMA-HHD and RMA-H74L in the presence of a peptide (FMP58–66, HPV-E629–38, HTLV-tax11–19 or HIV-pol476–484) by 51Cr-release assays. Data are shown as the mean ± SD of triplicate wells. The experiments were repeated three times. *P < 0.05; **P < 0.01 compared to RMA-HHD, Student’s t-test. (d) Enhanced peptide-binding by HHD-H74L. RMA-S-HHD or RMA-S-H74L cells were cultured with a peptide, and examined for their surface expression of HHD or HHD-H74L by flow cytometry. The experiment was repeated twice with similar results. Data are standardized as the % relative binding, and shown as the mean ± SD of triplicate wells. *P < 0.05; **P < 0.01 compared to RMA-S-HHD, Student’s t-test. (e) CTL recognition of various HHD mutants. FMP-specific CTL lines were examined for killing activities against RMA expressing HHD mutants with various mutations at position 74 in the presence of 100 nmol/l FMP58-66 by 51Cr-release assays. The data are representative of one of three independent experiments. Data are standardized as the % relative lysis and shown as the mean ± SD of triplicate wells. *P < 0.05; **P < 0.01; ***P < 0.001; NS, not significant, One-way analysis of variance.
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Related In: Results  -  Collection

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fig1: HHD-H74L improves the presentation of exogenous peptides. (a) Structure of HHD-H74L. LS: leader sequence; huβ2m: human β2m; α1, α2, α3, Tm, Cyt: α1, α2, α3, transmembrane, cytoplasmic domains. (b) Diagram of HHD-H74L. (c) Enhanced cytotoxic T lymphocyte (CTL) recognition of HHD-H74L-expressing targets. CTL lines were examined for killing activities against RMA, RMA-HHD and RMA-H74L in the presence of a peptide (FMP58–66, HPV-E629–38, HTLV-tax11–19 or HIV-pol476–484) by 51Cr-release assays. Data are shown as the mean ± SD of triplicate wells. The experiments were repeated three times. *P < 0.05; **P < 0.01 compared to RMA-HHD, Student’s t-test. (d) Enhanced peptide-binding by HHD-H74L. RMA-S-HHD or RMA-S-H74L cells were cultured with a peptide, and examined for their surface expression of HHD or HHD-H74L by flow cytometry. The experiment was repeated twice with similar results. Data are standardized as the % relative binding, and shown as the mean ± SD of triplicate wells. *P < 0.05; **P < 0.01 compared to RMA-S-HHD, Student’s t-test. (e) CTL recognition of various HHD mutants. FMP-specific CTL lines were examined for killing activities against RMA expressing HHD mutants with various mutations at position 74 in the presence of 100 nmol/l FMP58-66 by 51Cr-release assays. The data are representative of one of three independent experiments. Data are standardized as the % relative lysis and shown as the mean ± SD of triplicate wells. *P < 0.05; **P < 0.01; ***P < 0.001; NS, not significant, One-way analysis of variance.
Mentions: Previous studies demonstrated that the mutant HLA-A*02:01 molecule with the H74L mutation possessed the unique ability to enhance the presentation of exogenously loaded peptides to peptide-specific CTLs.5,18 To confirm these results in the HHD system, we firstly constructed a mutant HHD gene that could generate this mutation in the HHD molecule (HHD-H74L) (Figure 1a,b) by the inverse polymerase chain reaction (PCR)-based site-directed mutagenesis. The HHD-H74L gene was then transfected and expressed in mouse lymphoma, RMA cells (RMA-H74L). The expression level of the H74L mutant molecules on RMA-H74L cells was almost equivalent to that of the HHD molecules on RMA cells expressing the WT HHD (RMA-HHD). To test the ability of the H74L mutant, HLA-A*02:01-restricted, peptide-specific CTL lines were generated from HHD mice, and examined for their killing activities against RMA-HHD and RMA-H74L in the presence of synthetic peptides at various concentrations in standard 51Cr-release assays. Consistent with the previous data,5 RMA-H74L cells were lysed by influenza A matrix peptide (FMP58-66)-specific CTLs at much lower peptide concentrations than RMA-HHD cells (Figure 1c). Similar results were obtained with different HLA-A*02:01-restricted peptides derived from human papillomavirus (HPV)-E6 (HPV-E629-38), human T-lymphotropic virus (HTLV)-tax (HTLV-tax11-19) and human immunodeficiency virus (HIV)-pol (HIV-pol476-484) (Figure 1c), indicating that this phenomenon was not peptide-specific as described in the previous studies.18

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