Stacy Reed

Advisor: Dr. N. Marano

SLU Festival of Science 2001 Poster Presentation

Functional effects of exogenous lipids in T cells

T cells are a type of lymphocyte that function as a part of the immune system.  On the surface of the cell are receptors, which are made up of proteins that bind to specific molecules.  T cells are capable of recognizing antigens (molecules identified as foreign by the immune system) by binding the receptors to extracellular ligands, which cause a series of changes inside the T cell, a process known as signal transduction.  Aggregations of the large T cell receptor (TCR) complexes are necessary for cell activation. T cells also have CD4, a coreceptor protein that increases binding interactions of the TCR and helps to activate T cells.

Glycolipids are also found on cell membranes.  They consist of polysaccharides bound to lipids and are important in membrane organization.  They are also associated with the aggregated TCR complexes.  One of the major glycolipids affiliated with T cells is GM3 (Neu5Aca3Galb4GlcCer), which causes the internalization of CD4 and affects T cell function.  Once a TCR has been activated, there is an increase in the calcium ion concentration in the cell.  This calcium response can be compared between treated and untreated T cells using spectrofluorimetry.

GM3 is an exogenous monosialoganglioside (a sialylated glycosphingolipid) that is associated with the membrane glycoprotein CD4.  Normally, GM3 is found bound to T cell membranes and serves to modulate activity of protein kinases, specifically tyrosine phosphorylation and the inhibition of tyrosine kinase activity.  The presence of GM3 causes the endocytosis of CD4.  It has also been shown to decrease levels of calcium flow into the activated T cell (Chen et al., 2000). Slide photographs were taken to compare the fluorescence of GM3 treated and untreated cells.

The leukemia T cells were maintained in RPMI medium with 10% fetal bovine serum.  The dye used for the CD4 experiments was fura-2 with DMSO.  The FL-WinLab program measures the data of the spectrofluorometer and calculates the ratio of the peak to the isospectic point, and displays these on a graph. Antibodies can help to crosslink glycolipids and help promote signal transduction.  The primary antibody used was aCD4, while the secondary was OKT3, ana-TCR antibody, which aggregates the TCR and causes an increase in the internal Ca²⁺.Triton detergent (TX-100) was used to lyse the cells, allowing the dye to escape and gave a reading for the maximum fluorescence.A run with Tris and EGTA would give a minimum value.  A second antibody was also tested, rabbit anti-mouse IgG, which showed no significant difference compared to the level of OKT3 alone.  This suggests that the value given is the actual maximum.