Investigation of CD8+ T Cell Cytotoxicity Within the Central Nervous System when Infected with Cryptococcus neoformans

Two slightly different methodologies were used in this study to determine whether CD8+ T cells exhibit a cytotoxic effect towards microglial cells when infected with Cryptococcus neoformans. The line of CD8+ T cells used was a TK1 cell line (ATCC Catalog No. CRL-2396), and the microglial cells used were EOC cells (ATCC Catalog No. CRL-2469). Both of these lines were from mice. One method used to discover the activity of cytotoxicity involved adding beads to activated EOC cells. The bead laden EOC cells were then combined with TK1 cells, and the control setups were not mixed with TK1 cells. If cytotoxicity occurred, the supernatant of the TK1 and EOC combination would contain more beads due to the lysing of the infected EOC cells by the TK1 cells. This was quantitatively determined by using an analytical scale to measure the differing masses between the setups. DVPP, a common pesticide, was also added to some setups since it is an inhibitor of cytotoxicity activity (Li et al. 2004 ). The other methodology performed involved CytoTox 96 Non-Radioactive Cytotoxicity Assay [Pro mega TB 163]. This assay determined whether cytotoxicity occurred by measuring the release of lactate dehydrogenase, LDH, upon cell lysis. The EOC cells and TK1 cells were combined along with the Cryptococcus antigen. When the LDH was released, a red product formed. The degree of redness between the different setups was quantitatively measured with a spectrometer. A significant red absorbance within the combined cell setup would indicate a cytotoxic effect. This experimental approach has never been utilized for central nervous system derived cells. This research will increase knowledge pertaining to CD8+ T cell cytotoxic response to microglial cells laden with medically important non-viral pathogens