In vitro and in vivo evaluation of staphylococcal superantigen peptide antagonists

Abstract

Superantigen peptide antagonists failed to block T-cell activation and cytokine production as well as toxic shock induced by staphylococcal enterotoxin B (SEB) in HLA class II transgenic mice. They also failed to inhibit the binding of SEB to HLA class II molecules as well as activation of human T lymphocytes in vitro.

FIG. 1.

Single-cell suspensions of splenocytes from HLA-DQ8 (a) and HLA-DR3 (b and c) transgenic mice were cultured for 48 h in the presence of medium alone or various concentrations of SEB. SEB antagonistic peptides were also added at the indicated concentrations. (a and b) Splenocyte proliferation, as determined by measuring tritiated-thymidine uptake. (c) Interleukin-2 (IL-2) production in vitro by splenocytes from HLA-DR3 transgenic mice, as measured by sandwich enzyme-linked immunosorbent assay. Each bar represents the mean ± standard error from at least four independent experiments.

FIG. 2.

Single-cell suspensions of splenocytes from HLA-DQ8 transgenic mice were cultured for 48 h in the presence of medium alone or various concentrations of the indicated SAg. SEB P12A was added at the indicated concentrations. Cell proliferation was determined by measuring tritiated-thymidine uptake. Each bar represents the mean ± standard error from at least four independent experiments.

FIG. 3.

Single-cell suspensions of splenocytes from HLA-DR3 transgenic mice were cultured for 48 h in the presence of medium alone or various concentrations of the indicated SAg. SEB P12A was added at the indicated concentrations. Cell proliferation was determined by measuring tritiated-thymidine uptake. Each bar represents the mean ± standard error. Representative data from two of the four independent but similar experiments are given.

FIG. 4.

HLA-DR3 transgenic mice were challenged with a single (10-μg) dose of SEB. Simultaneously, they also received 100 μg of either SEB P1 or SEB P2 or PBS alone. Mice were sacrificed 3 days later, and the distributions of various T-cell subsets in spleen (a) and thymus (b) were determined by flow cytometry. Numbers indicate the absolute cell counts in thymus. Each bar represents the mean ± standard deviation from at least three mice.

FIG. 5.

HLA-DR3 transgenic mice were challenged with a single (10-μg) dose of SEB alone or SEB with 100 μg of SEB P12A. Mice were sacrificed 3 days later, and the distributions of various T-cell subsets in spleen (top) and thymus (bottom) were determined by flow cytometry. Each bar represents the mean ± standard deviation from at least four or five mice. SP, single positive.

FIG. 6.

PBMC from healthy individuals were cultured in vitro with the indicated concentrations of SEB along with different concentrations of SEB antagonistic peptides for 48 h. Cell proliferation was determined by measuring thymidine incorporation. Each bar represents the mean ± standard error from at least five different individuals.

FIG. 7.

PBMC from healthy individuals were incubated with 10 μg of nonbiotinylated SEB (A) or 10 μg of biotinylated SEB (B to F) along with the indicated concentrations of SEB P1 (C) or SEB P2 (D) or nonbiotinylated SEB (E and F). The extent of SEB binding to cells was determined by using avidin-labeled phycoerythrin for flow cytometry. Representative data from two similar experiments are given.