ABCMdb

ABCC8 p.Pro815Ala

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Publications

PMID: 10975847 [PubMed] Haicheur N et al: "The B subunit of Shiga toxin fused to a tumor antigen elicits CTL and targets dendritic cells to allow MHC class I-restricted presentation of peptides derived from exogenous antigens."

No. Sentence Comment

37 Recombinant Shiga B fusion proteins and peptide The Shiga B-P815A fusion protein was constructed by inserting a PCR cassette containing the P815A sequence into the NotI site of pB-Glyc- KDEL (28). Login to comment
44 Synthetic peptides SIINFEKL (SL8) and H-2Ld -restricted peptide P815A, LPYLGWLVF, encompassing the 257-264 residues of OVA and the 35-43 residues of P1A, respectively, were obtained from Altergen (Schiltigheim, France) and stored in PBS. Login to comment
56 For the P815A-Ld -restricted presentation, the cells were plated in 24-well flat-bottom microplates at 5 ϫ 104 cells/well, pulsed at 37°C with Ag, and cocultured for 24 h with 3 ϫ 104 anti-P815 A CTL obtained from mice immunized with the highly immunogenic L1210-P1A-B7 leukemic cell line. Login to comment
61 Immunization protocols Female DBA/2 (H-2d ) mice (aged 6-8 wk) from Iffa Credo were immunized by i.p. injection on days 0, 8, and 15 with 40 ␮g of purified recombinant Shiga B protein fused with the P815A peptide and mixed, or not, with IFA. Login to comment
62 Eight days later spleen cells were stimulated in vitro with L1210-P1A-B7, and CTL activity was measured 5 days later on target P815 or P1-204 (a P815A-negative variant of the P815 mastocytoma). Login to comment
63 Results P815A-specific CTL activity in mice immunized with Shiga B-P815A We chose to fuse the immunodominant epitope of P1A protein, P815A, to the Shiga B subunit because this Ag represents a murine model for the human tumor Ags belonging to the Mage family. Login to comment
65 DBA2 mice were immunized three times by i.p. injection with Shiga B-P815A in the presence or the absence of IFA. Login to comment
68 To confirm the specificity of these results, we used a P815A-negative variant of P815, P1-204, as a control and subtracted the cytotoxicity measured against P815 and P1-204, as previously described (35). Login to comment
69 Marked induction of P815A-specific CTL (Ͼ20% cytotoxicity) was again demonstrated in mice immunized with the highly immunogenic L1210-P1A-B7 cells or with Shiga B-P815A in the presence or the absence of IFA (Fig. 1B). Login to comment
71 Indeed, when we mixed the P815A peptide with the Shiga B subunit alone, no CTL induction was elicited (Fig. 1C). Login to comment
72 Shiga B targets exogenous Ags into the MHC class I pathway BM-DC from DBA/2 mice (H-2d ) were incubated with the Shiga B-P815A fusion protein. Login to comment
73 As shown in Fig. 2A, the BM-DC then efficiently presented the P815A peptide in a dose-dependent manner to specific anti-P815A CTL obtained after immunization of mice with the L1210-P1A B7 leukemic cell line. Login to comment
75 The specificity of the anti-P815A CTL was controlled by their ability to 4 Abbreviations used in this paper: PPMP, 1-phenyl-2-hexa-decanoylamino-3-morpholino-1-propanol; BM-DC, bone marrow-derived dendritic cells; DTAF, 5-(4,6-dichlorotriazin-2-yl)amino)fluorescein. Login to comment
76 3302 lyse BM-DC only when the target cells were pulsed with the P815A peptide (Fig. 2A). Login to comment
82 To exclude extracellular processing of the protein, we showed that paraformaldehyde-fixed dendritic cells did not allow presentation of Shiga FIGURE 1. Induction of CTL response in mice after immunization with Shiga B subunit fused to the P815A peptide. Login to comment
83 A, DBA2 mice were immunized by i.p. injection on days 0, 8, and 15 with 40 ␮g (1 nmol) of purified recombinant Shiga B subunit fused with the P815A peptide either alone or mixed with IFA. Login to comment
85 CTL activity was measured 5 days later on the target mastocytoma P815 expressing the P815A peptide. Login to comment
86 As positive and negative controls, mice were immunized with the L1210-P1A-B7 leukemic cell line or the P815A peptide (1 nmol). Login to comment
87 B, Data are expressed as P815A-specific cytotoxicity obtained after subtraction of lysis measured against P815 and P1-204, a P815A-negative variant of the P815 mastocytoma. Login to comment
88 The cytotoxicity observed on the P815A-negative target was usually low and always Ͻ25%. Login to comment
90 C, DBA2 mice were immunized by i.p. injection on days 0, 8, and 15 with 40 ␮g (1 nmol) of purified recombinant Shiga B subunit fused with the P815A peptide or with the Shiga B subunit mixed with the P815A peptide (1 nmol). Login to comment
92 CTL activity was measured 5 days later on the target mastocytoma P815 expressing the P815A peptide. Login to comment
93 As positive and negative controls, mice were immunized with the L1210-P1A-B7 leukemic cell line or the P815A peptide. Login to comment
94 The cytotoxicity observed on the P815A-negative target was Ͻ5% in these experiments. Login to comment
97 A, BM-DC (5 ϫ 104 ) from DBA/2 mice were pulsed at 37°C with various concentrations of Shiga B-P815A protein or peptide P815A and cocultured for 24 h with 3 ϫ 104 anti-P815A CTL obtained from mice immunized with L1210-P1A-B7. Login to comment
105 Because the in vitro expansion of anti-SL8 T cell hybridoma is much easier than that of anti-P815A CTL, we selected the OVA model for the next experiments. Login to comment
138 Discussion We have shown that immunization of mice with recombinant nontoxic Shiga toxin B subunit fused to the tumor Ag P815A reproducibly elicits CTL against this Ag. Login to comment
141 As reported by other groups, the P815A peptide alone or mixed with IFA failed to induce any specific CTL activity (44, 45). Login to comment
142 The poor immunogenicity of the P815A peptide was reinforced by the inability of dendritic cells pulsed with this peptide to significantly elicit specific CTL. Login to comment