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 deri ved from Escherichia coli that binds to ganglioside GM(1), a glycosphi ngolipid on the surface of mammalian cells. In this study, the effects of recombinant LTB (rLTB) on murine lymphocytes were examined in vitr o. T and B cells readily bound fluorescein isothiocyanate-labeled rLTB . CD8(+) T cells bound twice as much as CD4(+) T cells and B cells. Ex posure of T-cell subsets and B cells to rLTB abrogated mitogen-driven proliferation. CD8(+) T cells were more susceptible to rLTB than eithe r CD4(+) T cells or B cells. There were differences in the sensitivity of lymphocytes from various strains of mice to rLTB. This,vas attribu ted to qualitative and quantitative differences in the CD4(+) T cells. rLTB induced apoptosis in both T-cell subsets, but the level was sign ificantly higher in CD8(+) T cells. Apoptosis peaked at around 8 h aft er exposure to rLTB and incubation at 37 degrees C. Binding to ganglio side GM(1) was essential for suppression, since rLTB/G33D, a mutant wh ich does not bind GM(1), failed to inhibit proliferation or induce apo ptosis. Naive T cells, which were acutely sensitive to rLTB, became mo re resistant after activation. Conversely, activated T cells regained their sensitivity to rLTB when they reverted back to a resting state. A I-h pulse with rLTB was sufficient to inhibit T-cell proliferation a nd cytotoxic-T-lymphocyte generation in primary mixed lymphocyte react ion cultures. CD8(+) T cells were preferentially depleted in these cul tures. rLTB also induced functional modifications in T cells as indica ted by inhibition of gamma interferon secretion after polyclonal activ ation. Thus, rLTB may have immunomodulatory properties independent of its ability to induce apoptosis.