Abstract Book - Pathology and Laboratory Medicine - University of ...

POSTER presentationGRADUATE STUDENTBASIC SCIENCESABSTRACT #35The effect of PEGylation on intracellular signaling of lymphocytesDana Kyluik 1 , Dan Wang and Mark Scott1Department of Pathology and Laboratory Medicine, UBC; 2 Canadian Blood Services and UBCCentre for Blood Research, VancouverBackground /objectivesThe covalent attachment of non-immunogeneic polymers, such as methoxypoly(ethylene glycol) [mPEG] tothe surface of cells has been shown to dramatically reduce in vivo antigenic recognition and immunogenicity.Recently, our laboratory demonstrated that PEGylated allogeneic leukocytes induced an immunosuppressive state(increased Tregs and decreased Th17 cells) both in vitro (human and mouse) and in vivo (murine transfusionmodel). This finding was in stark contrast to the proinflammatory state induced by unmodified allogeneic cells.Thus, PEGylation may be a promising approach in the prevention of allorecognition and the induction ofimmunotolerance in transplantation. However, our studies to date have not defined the underlying molecularevents inducing the tolerogenic response in PEGylated cells. It is our hypothesis that the loss of allorecognition inPEGylated cells results in changes to intracellular signaling cascades giving rise to both altered cytokine secretionand changes in T-cell differentiation.MethodsJurkat cells were modified with or without activated 20 kDa mPEG at 0.5 or 2 mM for 60 minutes. Cells werestimulated for 12 minutes with mitogens to trigger proliferation (phytohaemagglutinin [PHA]). Cell lysates wereassessed for phosphorylation status using human phospho-kinase array kits. Membranes were analyzed for relativelevels of phosphorylated protein using Chemigenius software.ResultsPhosphorylated intermediates important in negative regulation of apoptosis such as Akt and Erk1/2 weresignificantly decreased in modified cells stimulated with PHA compared to PHA stimulated unmodified cells, aswell as unstimulated control cells. This may indicate a lack of inhibition of pro-apoptotic regulators such as Bimand Bad. In addition, the down-regulation of CREB, which is a mediator in the Ras/Raf pathway important fortranscription of anti-apoptotic proteins such as Bcl-2 and Bcl-xL, also occurred in modified Jurkat cells.ConclusionsThe initial phosphokinase screen indicates that various intermediates important in cell survival are decreased,suggesting a pro-apoptotic cell fate in modified cells stimulated with PHA. This initial data may indicateimportant signaling pathways and targets for further investigation.Oral/Poster Presentations * 2012 47