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Various methods have been used to identify activated T cells such as binding of MHC tetramers and expression of cell surface markers in addition to cytokine-based assays. In contrast to these published methods, we here describe a strategy to identify T cells that respond to any antigen and track the fate of these activated T cells. We constructed a retroviral double-reporter construct with enhanced green fluorescence protein (EGFP) and a far-red fluorescent protein from Heteractis crispa (HcRed). LTR-driven EGFP expression was used to enrich and identify transduced cells, while HcRed expression is driven by the CD40Ligand (CD40L) promoter, which is inducible and enables the identification and cell fate tracing of T cells that have responded to infection/inflammation. Pax5 deficient pro-B cells that can give rise to different hematopoietic cells like T cells, were retrovirally transduced with this double-reporter cassette and were used to reconstitute the T cell pool in RAG1 deficient mice that lack T and B cells. By using flow cytometry and histology, we identified activated T cells that had developed from Pax5 deficient pro-B cells and responded to infection with the bacterial pathogen Listeria monocytogenes. Microscopic examination of organ sections allowed visual identification of HcRed-expressing cells. To further characterize the immune response to a given stimuli, this strategy can be easily adapted to identify other cells of the hematopoietic system that respond to infection/inflammation. This can be achieved by using an inducible reporter, choosing the appropriate promoter, and reconstituting mice lacking cells of interest by injecting gene-modified Pax5 deficient pro-B cells.

IFNγ is critical for host defence against various food-borne pathogens including Salmonella enterica and Listeria monocytogenes, the causative agents of salmonellosis and listeriosis, respectively. We investigated the impact of regional IFNγ expression at the intestinal epithelial barrier on host invasion by salmonellae and listeriae following oral challenge. Transgenic mice (IFNγ-gut), generated on an IFNγ knock-out (KO) background, selectively expressed IFNγ in the gut driven by the modified liver fatty acid-binding protein (Fabpl4× at −132) promoter. Infections with attenuated S. enterica Typhimurium or with L. monocytogenes did not differ significantly in IFNγ-KO, IFNγ-gut and wild-type mice. Further, Listeria-specific CD4+ and CD8+ T cells were not altered in IFNγ-gut mice. Thus, this model indicates that local IFNγ expression by non-immunological cells in the distal part of the small intestine, caecum and colon is insufficient for prevention of gut penetration by S. enterica Typhimurium and L. monocytogenes. [ABSTRACT FROM AUTHOR]