Simultaneous Quantification of Anti-vector and Anti-transgene-Specific CD8+ T Cells Via MHC I Tetramer Staining After Vaccination with a Viral Vector.

Upon viral infection, antigen-specific CD8+ cytotoxic T cells (CTLs) arise and contribute to the elimination of infected cells to prevent the spread of pathogens. Therefore, the frequency of antigen-specific CTLs is indicative of the strength of the T cell response against a specific antigen. Such analysis is important in basic immunology, vaccine development, cancer immunobiology and the adaptive immunology. In the vaccine field, the CTL response directed against components of a viral vector co-determines how effective the generation of antigen-specific cells against the antigen of interest (i.e., transgene) is. Antigen-specific CTLs can either be detected by stimulation with specific peptides followed by intracellular cytokine staining or by the direct staining of antigen-specific T cell receptors (TCRs) and analysis by flow cytometry. The first method is rather time-consuming since it requires sacrificing of animals to isolate cells from organs. Also, it requires isolation of blood from small animals which is difficult to perform. The latter method is rather fast, can be easily done with small amounts of blood and is not dependent on specific effector functions, such as cytolytic activity. MHC tetramers are an ideal tool to detect antigen-specific TCRs. Here, we describe a protocol to simultaneously detect antigen-specific CTLs for the immunodominant peptides of the viral vector VSV-GP (LCMV-GP, VSV-NP) and transgenes (OVA, HPV 16 E7, eGFP) by MHC I tetramer staining and flow cytometry. Staining is possible either directly from blood or from single cell suspensions of organs, such as spleen. Blood or single cell suspensions of organs are incubated with tetramers. After staining with antibodies against CD3 and CD8, antigen-specific CTLs are quantified by flow cytometry. Optionally, antibodies against CD43, CD44, CD62L or others can be included to determine the activation status of antigen-specific CD8+T cells and to discriminate between naïve and effector cells.

Hepatotoxicity of carbon tetrachloride: protective effect of Gongronema latifolium.

The protective effect of the ethanolic extract of Gongronema latifolium (GLE) on carbon tetrachloride (CCI4) induced hepatic toxicity was studied. Liver enzymes studied included alanine aminotransferase (ALT), aspartate aminotraferase (AST), and alkaline phosphates (ALP). Hepatic injuries involved with possible necrosis which may have contributed to its possible pathogenesis was explored. Administration of toxicant only showed that the ALT level was significantly (P<0.05) increased to 345.83% when compared to control. Pretreatment with Gongronema latifolium extract (GLE) non-significantly (P<0.05) decreased to 13.08% when compared to those treated with toxicant only. Also under experimental conditions, increasing the concentration of Gongronema latifoluim extract (GLE) non-significantly (P<0.05) decreased dose-dependently the level of ALT to 18.20%. The AST level was non-significantly (P<0.05) increased to 41.55% on treatment with toxicant only. Pretreatment with GLE decreased the AST level non-significantly (P<0.05) to 25.76%. No evident increase or decrease in the level of ALP was observed. Treatments with toxicant showed liver cells filled with uniformly distributed dense small fat droplets, large nuclei, inflamed cells and evidence of necrosis and fibrosis. Pretreatment with 100mg/kg of the extract showed microvesicular fatty change with no evidence of inflammation, necrosis or fibrosis. The protective effect of the GLE was more pronounced in ALT and AST. However, the GLE has a strong modulatory effect against the hepatocellular damage induced by carbon tetrachloride.