Listeria. monocytogenes (LM-OVA) clearance assay

Ova Expressing Listeria monocytogenes (LM-OVA) clearance was examined in two models: 1) direct injection of bacteria to 6PGD^−/− mice (and their corresponding 6PGD^fl/fl mice), and 2) immunization of WT mice (CD45.1⁺) one day after adoptive transfer of OTI⁺ CD8⁺ T cells (CD45.2⁺). In both models mice were immunized by intravenously (i.v.) transfer of recombinant attenuated Lm-OVA (2 × 10³ CFU [colony-forming units]). Adoptive transfer included i.v. transfer of 1 × 10⁵ OTI cells from either 6PGD^fl/fl CD4^cre mice or 6PGD^fl/fl mice. Three days post infection, splenocytes were isolated and analyzed for bacterial load (CFUs) or frequency of OVA-specific CD8⁺ T cells. To check the bacterial load (CFUs), harvested splenocytes were cultured at 37°C on Brain Heart Infusion (BHI) culture agars and colonies were counted 24 hr later. The frequency of transferred cells in spleen was determined flow cytometry staining of CD45.2⁺ cells. PE-labeled OVA-specific MHC class I (H2Kb/OVA257–264) tetramer was used to determine OVA-specific CD8⁺ T cells in LM-OVA infected 6PGD^−/− or 6PGD^fl/fl mice.