Functional genome‐wide analysis of macrophage clearance of Francisella tularensis: novel roles for CD137 and CD137 ligand

We performed a genome‐wide shRNA screen to identify mediators of the enhanced killing of intracellular Francisella tularensis ( FT ) caused by interferon‐gamma. A top ‘hit’ was CD137, whose knockdown resulted in increased bacterial load in human macrophages 24 hours after infection with FT . CD137 and its ligand, CD137L, are transmembrane modulatory proteins expressed on many immune cell types, and comprise a bidirectional signaling system, wherein CD137L initiates signaling through CD137, and vice versa. , We used a panel of in vitro assays to further investigate the role of CD137 and CD137L in macrophage clearance of FT . Treatment with anti‐CD137 antibody resulted in markedly increased bacterial load at 24 hours post infection, as measured by flow cytometry (N=4, p<.01), scanning cytometry (N=7, p<.001) and cell lysate CFU (N=3,p<.05), indicating diminished bacterial killing (and reproducing results of the shRNA screen). Macrophages cultured in wells coated with CD137:Fc fusion protein to stimulate CD137L had sharply reduced uptake of GFP+ FT relative to controls (Fc‐coated plates), as determined by flow cytometry (N=6, p<.001). Conversely, treatment with blocking anti‐CD137L antibody resulted in increased phagocytosis (N=4, p<.01). These data demonstrate that both CD137 and CD137L modulate clearance of FT, with CD137 signaling enhancing bacterial killing, and CD137L signaling reducing phagocytosis. Supported by NIH ES00002 and HDTRA 1‐06‐C‐036