Development of a cell based assay to assess antigen specific tolerance

Project Summary Despite advances in the field of transplantation that have curbed acute rejection through immunosuppressive drugs and better control of infection and ischemia reperfusion injurychronic allograft rejection is still a major obstacle for successful organ transplantationSuccessful organ transplantation appears to require a balanced function of effector and regulatory T cellsTregto prevent the emergence of Thbased fibrosis and fibro obliterative processes in the allograftThcells have been strongly associated with autoimmune diseaseincluding lupusrheumatoid arthritispsoriasis and multiple sclerosisin additionto its key role in the chronic rejection of lung and heart transplantsAn assay that can quantitate potential Treg Thsuppression in an antigen specific manner would be invaluable in predicting monitoring the development and maintenance of tolerance that is perturbed in chronic inflammatory auto immune pathologies such as chronic rejection of allograftExisting animal transplantation models and the trans vivo delayed type hypersensitivitytvDTHassay have provided tremendous insight into tolerance mechanismsStilltwo big hurdles keep these experimental approaches from achieving commercial and clinical implementationThe use of an animal modeltvDTH and transplantationThe high number of cells required for the assaytvDTHThe first hurdle is addressed by the development of the T Cell Based Cytokine assayT CBCat the University of WisconsinThis assay detects tolerogenic factorsincluding ILCDand TGFin cultures of human PBMCsPreliminary data from the University of Wisconsin suggests that the level of tolerogene gEbiILCDand TGFsignaling between Tregs and other immune cells strongly correlates with tvDTH endpointsindicating that the presence of these signaling factors can be used as an in vitro biomarker of immune suppression activationwithout the need for a mouseAdditional preliminary data from Salus Discovery addresses the second hurdle by demonstrating that the T CBC assay can be miniaturized onto a microfluidic platformrequiring onlytopatient cellsBy combining microfluidic technology with the T CBC assay we can decrease the number of cells required per assay while maintainingand possibly improvingsee preliminary dataperformance moving the assay towards commercial use within the auto immunity and transplant fields PROJECT NARRATIVE The goal of this proposal is to further the development of a new assay system that can measure antigenspecific tolerance in individuals with end stage organ dysfunctionthose undergoing acute chronic allo graft rejection in addition to individuals with auto immune pathologies resulting in loss of self toleranceThe lab of DrBurlingham has more thanyears of studying cellular immune responses that lead to allo graft rejectionIn conjunction with the lab of DrDario Vignalithey have designed an ex vivo assayT CBC assayto directly measure antigen specific increases of tolerogenic factorsincluding ILas assessed by surface eBiexpressionand TGFfrom human bloodSalus Discovery has developed a novel microplate fluidic system that can reduce the scale of the T CBC assay by two orders of magnitudethereby making the assay more commercially relevant to clinical and research centers who need to assess individuals for potential antigenspecific loss of toleranceThe end result of this proposaland a subsequent phase II studyis to fully characterize and assess the feasibility and commercial viability of the T CBC microfluidic plate system technology and its clinical implementation in multiple areas of antigen specific tolerance investigation including but not limited torheumatoid arthritispsoriasis and organ transplantation