Glycosphingolipids as Novel Targets for T-Cell Suppression by the B Subunit of Recombinant Heat-Labile Enterotoxin

Heat-labile enterotoxin subunit B (LTB) is a noncatalytic protein derived from Escherichia coli that binds to ganglioside GM(1), a glycosphingolipid on the surface of mammalian cells. In this study, the effects of recombinant LTB (rLTB) on murine lymphocytes were examined in vitro. T and B cells readily bound fluorescein isothiocyanate-labeled rLTB. CD8(+) T cells bound twice as much as CD4(+) T cells and B cells. Exposure of T-cell subsets and B cells to rLTB abrogated mitogen-driven proliferation. CD8(+) T cells were more susceptible to rLTB than either CD4(+) T cells or B cells. There were differences in the sensitivity of lymphocytes from various strains of mice to rLTB. This was attributed to qualitative and quantitative differences in the CD4(+) T cells. rLTB induced apoptosis in both T-cell subsets, but the level was significantly higher in CD8(+) T cells. Apoptosis peaked at around 8 h after exposure to rLTB and incubation at 37°C. Binding to ganglioside GM(1) was essential for suppression, since rLTB/G33D, a mutant which does not bind GM(1), failed to inhibit proliferation or induce apoptosis. Naive T cells, which were acutely sensitive to rLTB, became more resistant after activation. Conversely, activated T cells regained their sensitivity to rLTB when they reverted back to a resting state. A 1-h pulse with rLTB was sufficient to inhibit T-cell proliferation and cytotoxic-T-lymphocyte generation in primary mixed lymphocyte reaction cultures. CD8(+) T cells were preferentially depleted in these cultures. rLTB also induced functional modifications in T cells as indicated by inhibition of gamma interferon secretion after polyclonal activation. Thus, rLTB may have immunomodulatory properties independent of its ability to induce apoptosis.