Inhibition of Antigen-Specific and Nonspecific Stimulation of Bovine T and B Cells by Lymphostatin from Attaching and Effacing Escherichia coli

Abstract

Enterohemorrhagic Escherichia coli (EHEC) and enteropathogenic E. coli (EPEC) are enteric bacterial pathogens of worldwide importance. Most EPEC and non-O157 EHEC strains express lymphostatin (also known as LifA), a chromosomally encoded 365-kDa protein. We previously demonstrated that lymphostatin is a putative glycosyltransferase that is important in intestinal colonization of cattle by EHEC serogroup O5, O111, and O26 strains. However, the nature and consequences of the interaction between lymphostatin and immune cells from the bovine host are ill defined. Using purified recombinant protein, we demonstrated that lymphostatin inhibits mitogen-activated proliferation of bovine T cells and, to a lesser extent, proliferation of cytokine-stimulated B cells, but not NK cells. It broadly affected the T cell compartment, inhibiting all cell subsets (CD4, CD8, WC-1, and γδ T cell receptor [γδ-TCR]) and cytokines examined (interleukin 2 [IL-2], IL-4, IL-10, IL-17A, and gamma interferon [IFN-γ]) and rendered T cells refractory to mitogen for a least 18 h after transient exposure. Lymphostatin was also able to inhibit proliferation of T cells stimulated by IL-2 and by antigen presentation using a Theileria-transformed cell line and autologous T cells from Theileria-infected cattle. We conclude that lymphostatin is likely to act early in T cell activation, as stimulation of T cells with concanavalin A, but not phorbol 12-myristate 13-acetate combined with ionomycin, was inhibited. Finally, a homologue of lymphostatin from E. coli O157:H7 (ToxB; L7095) was also found to possess comparable inhibitory activity against T cells, indicating a potentially conserved strategy for interference in adaptive responses by attaching and effacing E. coli.

Direct Manipulation of T Lymphocytes by Proteins of Gastrointestinal Bacterial Pathogens

“…Further, the effects of lymphostatin on T cells appear to be long-lived, even in the absence of continued incubation with the protein, preventing mitogenic activation for more than 18 h after transient exposure and withdrawal of the protein. Lymphostatin was also able to inhibit antigen-specific proliferation of bovine T cells using Theileria parva antigens presented on infected irradiated APCs to T. parva-specific T cells as a model antigen system (18). These findings suggest that lymphostatin might act to permanently desensitize T cells to a stimulus, possibly suppressing T cell responses, preventing or dampening a productive immune response, and delaying clearance of infection (18).…”

“…Lymphostatin was also able to inhibit antigen-specific proliferation of bovine T cells using Theileria parva antigens presented on infected irradiated APCs to T. parva-specific T cells as a model antigen system (18). These findings suggest that lymphostatin might act to permanently desensitize T cells to a stimulus, possibly suppressing T cell responses, preventing or dampening a productive immune response, and delaying clearance of infection (18). It would appear that the effects of lymphostatin interfere with signaling in a membrane-proximal way, as inhibition was not achieved in T cells stimulated with phorbol 12-myristate 13-acetate (PMA)/ionomycin, which bypass membrane signaling.…”

“…It has been demonstrated that lymphostatin is capable of inhibiting all major T cell subsets. In addition, lymphostatin has some activity against B cells but not natural killer cells (18). Further, the effects of lymphostatin on T cells appear to be long-lived, even in the absence of continued incubation with the protein, preventing mitogenic activation for more than 18 h after transient exposure and withdrawal of the protein.…”

“…There are a number of unresolved questions regarding the activity of lymphostatin, including its cellular target of glycosylation. Further, in O157:H7 strains of EHEC, where full-length lymphostatin is not expressed, there is a putative homologue, ToxB, that also shows T cellinhibitory activity, as well as homology at the N-terminal end of the molecule to TcdA/B (18). This suggests that lymphostatin and lymphostatin-like molecules may be a family of proteins expressed by E. coli to control T cell responses to infection.…”

The Role of Escherichia coli Shiga Toxins in STEC Colonization of Cattle

“…Lymphostatin is a putative glycosyltransferase implicated in intestinal colonization of cattle by STEC serogroup O5, O111 [120], and O26 strains [176] early after infection, before adaptive responses may be expected to have developed [177]. Lymphostatin inhibits mitogen-activated proliferation of bovine T cells and, to a lesser extent, proliferation of cytokine-stimulated B cells, but not NK cells [178]. It broadly affects the T cell compartment, inhibiting all cell subsets (CD4, CD8, WC-1, and γδT cells) and several cytokines (IL-2, IL-4, IL-10, IL-17A, and IFN-γ) and renders T cells refractory to mitogen.…”