Delivery of a MalE CD4(+)-T-cell epitope into the major histocompatibility complex class II antigen presentation pathway by Bordetella pertussis adenylate cyclase

Abstract

Recombinant adenylate cyclase toxoids are shown to deliver inserted foreign CD4(+)-T-cell epitopes into the major histocompatibility complex class II presentation pathway, inducing a specific CD4(+)-T-cell response in vivo and yielding in vitro stimulation of specific CD4(+) T cells at a 100-times-higher molar efficiency than the free peptide containing the epitope.

FIG. 1.

SDS-PAGE analysis of the purified ACT/MalE preparations. The proteins were purified from urea extracts by DEAE- and phenyl-Sepharose chromatography as previously described (8), and 1 to 3 μg of each was analyzed together with the wild-type ACT on a 7.5% acrylamide gel stained with Coomassie blue.

FIG. 2.

Most of the ACT/MalE proteins efficiently deliver the inserted CD4⁺-T-cell epitope for presentation to specific T cells. Splenocytes from C57BL/6 (A) or BALB/c (C) mice were used as APCs. Various concentrations of the AC toxoids, harboring the MalE CD4⁺-T-cell epitope at different sites, were added to APCs. The APCs were then cocultured with 10⁵ cells of CRMC3 (A) or FBCD1 (C) anti-MalE CD4⁺-T-cell hybridoma, which selectively recognize complexes of the H-2^b or H-2^d MHC class II molecules with bound MalE peptide (NGKLIAYPIAVEALS), respectively. As positive controls, C57BL/6 (B) or BALB/c (D) splenocytes were incubated with various concentrations of the MalE peptide and CRMC3 (B) or FBCD1 cells (D). After 18 h of culture, supernatants were frozen for at least 2 h at −80°C. The amount of IL-2 produced by the stimulated CRMC3 and FBCD1 cells was then determined by the CTLL proliferation method. Briefly, 10⁴ cells of the IL-2-dependent CTLL line per well were cultured with 100 μl of supernatant in 200 μl of final volume. Twenty-four hours later, [³H]thymidine (50 μCi/well) was added and cells were harvested 6 h later with an automated cell harvester. Incorporated thymidine was detected by scintillation counting. Each point was done in duplicate, and the experiment was repeated four times. Results are expressed in change in counts per minute of incorporated [³H]thymidine (counts per minute in the presence − counts per minute in the absence of ACT). WT, wild type.

FIG. 3.

In vivo induction of CD4⁺-T-cell responses by MalE/ACT proteins. C57BL/6 mice were intravenously injected with 50 μg of MalE/ACT proteins bearing the MalE epitope at site 108 (•) or 336 (▪). Mice were injected with mock AC toxoid (asterisks) as negative control. One week later, the mice were sacrificed and the splenocytes were in vitro stimulated with various concentrations of the MalE peptide. After 72 h of culture, [³H]thymidine (50 μCi/well) was added and cells were harvested 6 h later with an automated cell harvester. Incorporated thymidine was detected by scintillation counting. Results show the antigen-specific proliferation obtained for one representative mouse out of four tested in two independent experiments. Each point was done in triplicate and standard deviations are indicated by the bars (n = 3). Results are expressed in changes in counts per minute of incorporated [³H]thymidine (counts per minute in the presence of peptide − counts per minute in the absence of peptide).