Studying the function of antigen experienced CD4+GITR+T cells
 Xiaosung Liu
The aim of the project is to investigate the suppressive function of antigen experienced CD4+GITR+ T cells and identify surface markers unique to antigen experienced CD4+GITR+ T cells.
Antigen experienced and naïve CD4+GITR+ T cells will be purified and incubated with dendritic cells pulsed with VLPs overnight; surface markers on dendritic cells will be checked by FACS analysis. We will check activation markers such as MHC II, CD80 and CD86; surface inhibitory markers such as PD-1, PD-L1 will also be checked.
In another experiment, CD8 T cells from transgenic mice specific with TCR for HPV 16 E7 specific CTL epitope will be added to DC presenting E7 and VLPs /CD4+GITR+ co-cultures. CD8 T cell function will be checked for proliferation, cytokine production and their in vitro killing abilities.
To identify the surface markers of antigen experienced CD4+GITR+ T cells, mice will be immunized with BPVL1 VLPs, on the right side of hind legs intramuscularly on day 0 and 14, draining lymph nodes and control non draining lymph nodes will be collected 7 days after final immunization and single cell suspension prepared. The isolated cells will be enriched for CD4 by negative selection using commercially available kit (what?), and then positively selected for GITR. mRNA from CD4+GITR+ T cells either from draining or non draining lymph nodes will be purified and microarray performed at University of Queensland SRC microarray facility. Microarray results will be analyzed by bio-informatics professionals within the institute and special attention will focus on the cytokine activation and regulation pathway and cell surface gene expression. Gene expression changes of interest will be verified by real time PCR and finally by western blot.
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