Anthrax toxin-mediated delivery in vivo and in vitro of a cytotoxic T-lymphocyte epitope from ovalbumin

Abstract

We reported earlier that a nontoxic form of anthrax toxin was capable of delivering a cytotoxic T-lymphocyte (CTL) epitope in vivo, such that a specific CTL response was primed against the epitope. The epitope, of bacterial origin, was fused to an N-terminal fragment (LFn) from the lethal-factor component of the toxin, and the fusion protein was injected, together with the protective antigen (PA) component, into BALB/c mice. Here we report that PA plus LFn is capable of delivering a different epitope--OVA(257-264) from ovalbumin. Delivery was accomplished in a different mouse haplotype, H-2Kb and occurred in vitro as well as in vivo. An OVA(257-264)-specific CTL clone, GA-4, recognized EL-4 cells treated in vitro with PA plus as little as 30 fmol of the LFn-OVA(257-264) fusion protein. PA mutants attenuated in toxin self-assembly or translocation were inactive, implying that the role of PA in epitope delivery is the same as that in toxin action. Also, we showed that OVA(257-264)-specific CTL could be induced to proliferate by incubation with splenocytes treated with PA plus LFn-OVA(257-264). These findings imply that PA-LFn may serve as a general delivery vehicle for CTL epitopes in vivo and as a safe, efficient tool for the ex vivo expansion of patient-derived CTL for use in adoptive immunotherapy.

FIG. 1

CTL-mediated lysis of OVA_257–264 peptide-coated EL-4 cells. Mice were injected i.p. with LFn-OVA_257–264 plus PA (A) or LFn-OVA_257–264 without PA (B). After in vitro stimulation, samples were assayed for their ability to lyse ⁵¹Cr-labeled EL-4 cells coated with OVA_257–264 peptide (solid circles) or not coated (open circles). Targeting was evaluated by measuring the amount of ⁵¹Cr release. The effector-to-target-cell ratios (E:T ratios) examined were 10:1, 3:1, and 1:1. Similar levels of lysis were observed in each of five replicates.

FIG. 2

GA-4-mediated lysis of EL-4 cells treated with LFn-OVA_257–264 plus PA. EL-4 cells were treated with 100 ng of LFn-OVA_257–264 (A), 10 ng of LFn-OVA_257–264 (B), or 1 ng of LFn-OVA_257–264 (C). Each sample was treated in the presence (solid circles) or absence (open circles) of PA. Following treatment with the fusion protein, the target cells were loaded with ⁵¹Cr. The target cells were incubated with OVA_257–264-specific CTL to give E:T ratios of 10:1, 3:1, and 1:1. Targeting was evaluated by measuring the amount of ⁵¹Cr release. Similar levels of lysis was observed in each of three repeat experiments.

FIG. 3

GA-4 targeting of EL-4 cells treated with LFn-OVA_257–264 and PA mutants. EL-4 cells were treated with LFn-OVA_257–264 plus wild-type PA (solid circles), --D mutant PA (solid squares), SSSR mutant PA (open squares), or no PA (open circles). Following treatment with the fusion protein and PA, the target cells were loaded with ⁵¹Cr. The target cells were added to OVA_257–264-specific CTL at E:T ratios of 10:1, 3:1, and 1:1. Targeting was evaluated by measuring the amount of ⁵¹Cr release. Similar levels of lysis was observed in each of three repeat experiments.

FIG. 4

CTL-mediated lysis of OVA_257–264 peptide-coated EL-4 cells. Mice were injected i.p. with LFn-OVA_257–264 plus PA (A), LFn-OVA_257–264 without PA (B), LFn-OVA_257–264 plus PA mutant SSSR (C), or LFn-OVA_257–264 plus PA mutant --D (D). After in vitro stimulation, the samples were assayed for their ability to lyse ⁵¹Cr-labeled EL-4 cells coated with OVA_257–264 peptide (solid circles) or not coated (open circles). Targeting was evaluated by measuring the amount of ⁵¹Cr release. The E:T ratios examined were 10:1, 3:1, and 1:1. Similar levels of lysis were observed in each of three replicates.

FIG. 5

In vitro expansion of OVA_257–264 specific CTL. Aliquoted mouse splenocytes were treated for 4 h with medium alone, 1 μM OVA_257–264 synthetic peptide, 100 ng of LFn-OVA_257–264 fusion plus PA, 100 ng of LFn-OVA_257–264 minus PA, 10 ng of LFn-OVA_257–264 fusion plus PA, or 10 ng of LFn-OVA_257–264 minus PA. OVA_257–264-specific CTL were added to each sample. Following 48 h of incubation, [³H]thymidine was added to the samples. At 14 h later, the samples were harvested and thymidine incorporation into the cells was assessed. Statistical analysis was performed by Student’s t test.