A Role for Glycosylphosphatidylinositol Anchored Proteins in T Lymphocyte Homeostasis

The glycosylphosphatidylinositol (GPI) anchorage of proteins to the outer leaflet of the plasma membrane imparts unique properties and controversial functionality. While all GPI-anchored proteins (GPI-AP) contain a conserved GPI core structure, they represent a large class of functionally diverse proteins with known capacities for intracellular signalling, protein trafficking, and localization within membrane microdomains, termed lipid rafts (LR). Many studies have implicated GPI-AP as regulators of T cell receptor (TCR)-mediated cellular activation, however, as they are not transmembrane molecules, mechanisms underpinning their signalling capacity remain elusive. We have isolated a mutant clonal variant devoid of GPI-AP. This T cell clone produced 10-100-fold more TCR-induced interleukin (IL)-2 than the GPI-AP+ parent clone. As GPI-AP reside exclusively within LR, we hypothesized that within this signalling scaffold, they may function as essential regulators of TCR/CD3-mediated “on” and “off” signals. Model systems utilizing GPI-AP+ and GPI-AP- variants of the antigen specific, IL-2-dependent T cell clone, and primary GPI-AP- T cells were generated towards directly testing this hypothesis. In the clonal system, GPI-AP deficiency was shown to impart a prolonged TCR/CD3-induced clonal expansion, which correlated with both enhanced IL-2 messenger RNA (mRNA) and protein production. The re-expression of GPI-AP abrogated the prolonged growth/survival phenotype, consistent with a GPI-AP-dependent attenuation of TCR/CD3 signalling. The phenotype was recapitulated in primary GPI-AP- CD4+ T cells. Hypersensitive TCR/CD3 signalling in the primary model correlated with an enhanced basal kinase activity of the Src-family protein tyrosine kinase Fyn, essential for TCR/CD3 induced IL-2 production. The role of the GPI-anchor, as opposed to that of one or more GPI-anchored proteins in supporting this phenotype was assessed using GPI-anchor sufficient, GPI-AP deficient primary T cells. Preliminary results demonstrated that the TCR/CD3-induced IL-2 phenotype of GPI+/GPI-AP- closely resembles that of GPI-AP+, wild-type T cells. While a mixed genetic background in these GPI+/GPI-AP- T cells precludes formal proof of this conclusion, results thus far attribute a potentially novel role for GPI in regulating homeostatic T cell physiology. The biological significance of the results may provide insight into the mechanism underpinning the human disease paroxysmal nocturnal hemoglobinuria, the maintenance of cellular homeostasis and proliferative disease states writ large.Ph.D