Endotoxin (lipopolysaccharide; LPS) and superantigens (exotoxins) have been identified as potent inducers of lethal shock. While endotoxin primarily interacts with CD14 receptors on macrophages, superantigens like the staphylococcal enterotoxin B (SEB) preferentially activate T cells. Both cell types are triggered to release pro-inflammatory cytokines that in turn induce lethal shock. We analyzed whether endotoxin and superantigen interact during the induction phase of lethal shock. We report that LPS and SEB operate synergistically. Lethal doses of both inducers were reduced 100-fold when given in combination. The induced serum levels of tumor necrosis factor, interleukin-6, and interferon-gamma (IFN-gamma) were elevated and remained high for a prolonged period. Moreover, synergistic action of LPS and SEB induced lethal toxic shock even without presensitization of mice with D-galactosamine (D-GalN). Opposed to D-GalN-pretreated mice, mice injected with LPS and SEB showed less liver damage, but rather apoptosis of epithelial cells in the bowel. Cyclosporin A and treatment with anti-IFN-gamma monoclonal antibody blocked the synergistic action of LPS and SEB, indicating that T cell-derived IFN-gamma is the mediator of the observed synergism. Concomitant injection of LPS and SEB had no influence on SEB-induced T cell deletion and anergy induction. Since Gram-positive and Gram-negative bacteria can be recovered from septic blood samples, the synergistic action of endotoxin and superantigens might be relevant during lethal septicemia.

CpG-containing oligodeoxynucleotides (CpG-ODN) act as powerful adjuvant during in vivo induction of T cell responses. While CpG-ODN directly activate antigen-presenting cells (APC) and thus exert an extrinsic activity on T cells, it is unclear whether they directly affect T cells (intrinsic activity). Here we analyze the effects of CpG-ODN on T cells in an APC-free cell culture. We report that CpG-ODN co-stimulate T cells provided they were triggered via their TCR. CpG-ODN induced IL-2 production, IL-2 receptor expression and thus proliferation. Proliferation was blocked by cyclosporin A or anti-IL-2 monoclonal antibodies (mAb) but not by anti-IL-4 mAb. Moreover, CpG-co-stimulated T cells differentiated into cytolytic T lymphocytes in vitro. Of note, IL-2-driven growth of primed T cells was not affected by CpG-ODN. Co-stimulation was also operative in T cells from CD28 −/− mice and in TCR-transgenic T cells stimulated with peptide. CpG-ODN-mediated co-stimulation of T cells in vitro may thus explain part of the potent adjuvant effects of CpG-ODN in vivo.

Summary Pathogen‐derived pattern recognition ligands like lipopolysaccharide (LPS) and bacterial cytidine–guanosine (CpG)‐DNA not only activate dendritic cells and macrophages but are also mitogenic for B cells. Less clear are the claimed effects of CpG‐DNA on T cells, which range from direct activation, costimulation, or indirect transient activation via antigen‐presenting cell (APC)‐derived interferon type I (IFN type I). Here we demonstrate that CpG‐DNA sequence specifically triggers macrophages to produce IFN type I, interleukin (IL)‐12, IL‐6 and tumour necrosis factor (TNF), but lacks the ability to directly costimulate T cells. Strikingly, poly‐guanosine (poly‐G) extensions to CpG‐containing oligonucleotides (ODN) abolished the macrophage stimulatory potential yet generated T‐cell costimulatory activities. In fact, independently of CpG‐motifs, poly‐G‐ODN displayed the ability to costimulate T cells. Costimulation was operative on CD8 T cells but not CD4 T cells. Poly‐G‐mediated costimulation resulted in IL‐2‐driven T‐cell proliferation and induced cytolytic T cells. Overall the data imply that poly‐G motifs costimulate antigen reactive CD8 T cells, while CpG‐DNA motifs fail to do so but may affect T‐cell activation via APC derived cytokines such as IFN type I.