Mechanisms of immune tolerance following genetic manipulation and transplantation of bone marrow-haematopoietic stem cells
Creators: name: Joglekar, Amit
Publisher: name: Monash University
Year: 2017
Subjects: Uncategorized

description: Autoimmune disorders affect approximately 5-6% of the population and result from aberrant responses from the immune system that targets self-tissues. Multiple sclerosis (MS) is an autoimmune disease where the myelin sheath surrounding axons of the central nervous system (CNS) is targeted by a destructive adaptive immune response, causing loss of motor and/or cognitive function. Experimental autoimmune encephalomyelitis (EAE) is a mouse model of MS and can be induced by immunisation with myelin antigen such as myelin oligodendrocyte glycoprotein (MOG) and other identified peptides. Our laboratory has been interested in promoting antigen specific tolerance by transplanting genetically manipulated bone marrow (BM) haematopoietic stem cells (HSCs) that are retrovirally transduced to express an autoantigen. Previous studies from our lab have shown that transplantation of BM-HSCs retrovirally transduced to express MOG, renders mice resistant to MOG35-55 peptide induced EAE. In this model it has been shown that thymic development of MOG35-55 specific CD4 T cells from 2D2 TCR transgenic mice was impaired and indicative of clonal deletion as a mechanism of immune tolerance. Given the immune repertoire to any defined antigen may consist of a polyclonal population of T cells that express a range of TCRs with differing affinities, the fate of autoantigen specific T cells in an environment of ectopic antigen expression is not clear and whether all will be deleted or some driven to become Tregs. Hence, a major hypothesis in my studies is that T cells with the highest affinity TCR will be deleted while those with lower affinity TCR will become Tregs. Using retrogenic technology and the availability of number of MOG specific TCR clones with varying affinities, mixed BM chimeras were generated by mixing different MOG35-55 specific TCRs with BM expressing MOG. We observed that the majority of MOG35-55 specific T cells were deleted regardless of their TCR affinities, but also some development into FoxP3 expressing Tregs in mice expressing MOG. The peripheral population of MOG35-55 specific T cells from all TCR clones were significantly reduced in mice expressing MOG compared to other lymphoid cell populations. A closer examination of these T cells revealed an increase in proportion of FoxP3 expression in mice expressing MOG. The second results chapter was aimed at testing whether peripheral tolerance mechanisms can exist independent of central tolerance mechanisms. This involved adoptive transfer of MOG35-55 specific CD4+ T cells labelled with cell tracking dye 5(6) - Carboxyfluorescein succinimidyl ester (CFSE), into mice expressing MOG, thus bypassing the thymus and then analysed to determine their fate in the periphery. We found that adoptively transferred MOG35-55 specific CD4+ T cells proliferated and expressed activation markers. Further analysis of these cells indicated that the adoptively transferred CD4+ T cells expressed FoxP3 in mice that expressed MOG. Overall the study suggested that peripheral expression of MOG resulted in the generation of MOG35-55 specific Tregs. The third results chapter was aimed at observing if tolerance induced following the transfer of bone marrow which has been genetically manipulated to encode a single antigen will be non-specific and able to suppress immune response to another associated but unrelated autoantigens. We used C57Bl/6 X SJL F1 mice that are known to be susceptible to EAE following immunisation with both MOG35-55 and PLP139-151 peptide and develop remitting-relapsing EAE (RR-EAE). Eight weeks following transplantation of BM encoding MOG, mice were immunised with MOG35-55 and PLP139-151 peptides followed by monitoring of disease status. It was observed that MOG mice immunised with PLP139-151 peptide were not protected and displayed remitting-relapsing-EAE (RR-EAE) with 100% incidence of EAE as opposed to MOG35-55 peptide immunised mice which remained disease free. There was no difference in the proportion of polyclonal Tregs in the MOG mice and the control groups (data not shown). The serum antibody levels suggested epitope spreading might have occurred in MOG mice when immunised with PLP139-151 peptide, which is similar to the literature. Overall our system of combining gene therapy with bone marrow transplantation caused thymic deletion of the TCR clones regardless of their affinity and also generated antigen-specific Tregs in the periphery.