ABCMdb

PMID: 10975847

Haicheur N, Bismuth E, Bosset S, Adotevi O, Warnier G, Lacabanne V, Regnault A, Desaymard C, Amigorena S, Ricciardi-Castagnoli P, Goud B, Fridman WH, Johannes L, Tartour E
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.
J Immunol. 2000 Sep 15;165(6):3301-8., [PubMed]

Sentences

No. Mutations Sentence Comment

37 ABCC8 p.Pro815Ala ABCC8 p.Pro815Ala 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 ABCC8 p.Pro815Ala 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 ABCC8 p.Pro815Ala 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 ABCC8 p.Pro815Ala 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 ABCC8 p.Pro815Ala 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 ABCC8 p.Pro815Ala ABCC8 p.Pro815Ala ABCC8 p.Pro815Ala 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 ABCC8 p.Pro815Ala 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 ABCC8 p.Pro815Ala 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 ABCC8 p.Pro815Ala ABCC8 p.Pro815Ala 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 ABCC8 p.Pro815Ala Indeed, when we mixed the P815A peptide with the Shiga B subunit alone, no CTL induction was elicited (Fig. 1C). Login to comment 72 ABCC8 p.Pro815Ala 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 ABCC8 p.Pro815Ala ABCC8 p.Pro815Ala 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 ABCC8 p.Pro815Ala 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 ABCC8 p.Pro815Ala 3302 lyse BM-DC only when the target cells were pulsed with the P815A peptide (Fig. 2A). Login to comment 82 ABCC8 p.Pro815Ala 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 ABCC8 p.Pro815Ala 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 ABCC8 p.Pro815Ala CTL activity was measured 5 days later on the target mastocytoma P815 expressing the P815A peptide. Login to comment 86 ABCC8 p.Pro815Ala 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 ABCC8 p.Pro815Ala ABCC8 p.Pro815Ala 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 ABCC8 p.Pro815Ala The cytotoxicity observed on the P815A-negative target was usually low and always Ͻ25%. Login to comment 90 ABCC8 p.Pro815Ala ABCC8 p.Pro815Ala 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 ABCC8 p.Pro815Ala CTL activity was measured 5 days later on the target mastocytoma P815 expressing the P815A peptide. Login to comment 93 ABCC8 p.Pro815Ala As positive and negative controls, mice were immunized with the L1210-P1A-B7 leukemic cell line or the P815A peptide. Login to comment 94 ABCC8 p.Pro815Ala The cytotoxicity observed on the P815A-negative target was Ͻ5% in these experiments. Login to comment 97 ABCC8 p.Pro815Ala ABCC8 p.Pro815Ala ABCC8 p.Pro815Ala 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 ABCC8 p.Pro815Ala 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 ABCC8 p.Pro815Ala 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 ABCC8 p.Pro815Ala 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 ABCC8 p.Pro815Ala 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