Accumulation of a potent γδ T‐cell stimulator after deletion of the lytB gene in Escherichia coli

Abstract

Activation of human Vγ9/Vδ2 T cells by many pathogens depends on the presence of small phosphorylated non-peptide compounds derived from the 2-C-methyl-d-erythritol 4-phosphate (MEP) pathway of isoprenoid biosynthesis. We here demonstrate that in Escherichia coli mutants deficient in lytB, an essential gene of the MEP pathway, a potent Vγ9/Vδ2 T-cell activator accumulates by a factor of approximately 150 compared to wild-type E. coli. The compound responsible for the strong immunogenicity of this E. coli mutant was subsequently characterized and identified as a small pyrophosphorylated metabolite, with a molecular mass of 262 Da, derived from the MEP pathway. Stimulation of human peripheral blood mononuclear cells (PBMC) with extracts prepared from the lytB-deficient E. coli mutant led to upregulation of T-cell activation markers on the surface of Vγ9/Vδ2 T cells as well as proliferation and expansion of Vγ9/Vδ2 T cells. This response was dependent on costimulatory growth factors, such as interleukin (IL)-2, IL-15 and IL-21. Significant levels of interferon-γ (IFN-γ) and tumour necrosis factor-α (TNF-α) were secreted in the presence of IL-2 and IL-15, but not in the presence of IL-21, demonstrating that proliferating phosphoantigen-reactive Vγ9/Vδ2 T cells do not necessarily produce proinflammatory cytokines.