Malaria infects 200 million people and causes more than 500 000 deaths worldwide annually. Infection by Plasmodium falciparum can lead to development of cerebral malaria, a fulminant and often fatal complication. In the mouse model termed experimental cerebral malaria (ECM), C57BL/6J mice develop a severe neurological complication when infected with Plasmodium berghei strain ANKA (PbA) that shares many features with cerebral malaria in humans.
CD8+ T cells are essential for development of experimental cerebral malaria (ECM), but the epitopes recognized by these CD8+ T cells were unknown. To search for novel epitopes, we constructed a PbA cDNA library and expressed it in EL4 cells (C57BL/6 background) . A novel method involving phosphorothioate-linked random primers and translational coupling was developed to enrich the cDNA library for in-frame inserts. In the EL4 cells, potential PbA epitopes are processed and presented on the cell surface bound to MHC class I molecules. Concurrently, TCRs from CD8+ T cells sequestered in the brains of mice with ECM were cloned and expressed in separate T reporter cell lines harbouring a LacZ cassette controlled by an NFAT promoter. Each T reporter cell line was used to screen the PbA cDNA library, to identify and clone transduced EL4 cells able to trigger TCR ligation and hence lacZ expression. Sequencing the cDNA inserts led to the identification of GAP50 and bergheilysin as two antigens that are recognized by CD8 + T cells in mice during infection. The epitopes were found to be SQLLNAKYL (Pb1) and IITDFENL (Pb2) respectively, by generating and testing candidate peptide-MHC tetramers.
With the peptide-MHC tetramers for Pb1, Pb2 and F4 (an epitope reported in the literature midway through this project), we were able to characterize the antigen-specific CD8+ T cell response during ECM. CD8 + T cells specific for each of the three epitopes were induced in infected mice, expressing IFNγ, granzyme B, LFA–1high, and CXCR3 in the spleen, with downregulation of CXCR3 in cells in the brain. These markers are associated with pathogenicity, thus their expression by CD8+ T cells in mice not succumbing to ECM seems contradictory. The apparent contradiction was resolved when we discovered that only mice with ECM cross-present high levels of malaria epitopes in the brain microvasculature, showing that this is the essential feature that determines ECM susceptibility. Accordingly, CD8+ T cells recognizing Pb1, Pb2 and F4 have the capacity to damage these cells and disrupt the blood-brain barrier, presumably leading to onset of ECM development during infection. Tolerizing mice towards these three peptides could not protect infected mice against ECM, suggesting that ECM is caused by multiple malaria-specific CD8 + T cells.
|School:||National University of Singapore (Singapore)|
|School Location:||Republic of Singapore|
|Source:||DAI-B 77/06(E), Dissertation Abstracts International|
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