MHC class I molecules present peptide to CD8+ cytolytic T lymphocytes with brilliant specificity, thus ensuring accurate identification of virus-infected and tumor cells. However, several of the functions of class I molecules are limited by peptide and/or beta2m dissociation from the heavy chain. Indeed blocking class I heavy chain assembly with peptide and/or beta2m are major pathways used by tumors and viruses to evade detection by CD8+ T cells. To circumvent limitation of class I as a consequence of their propensity to disassemble, we have engineered class I molecules as single chain trimers (SCTs) with the composition of peptide--spacer--beta2m--spacer--heavy chain. Our recently published findings have shown that SCT have remarkable properties including extraordinary stability at the cell surface and potent stimulation of class I/peptide-specific T cells and antibodies. In this grant we will define the mechanisms by which SCT are potent immune stimulators and test their unique advantages over native class I molecules in DC based immunotherapies and when used as DNA vaccines against tumors or viruses. Of particular significance, these findings will determine whether SCT present antigen following DNA vaccination by cross priming or direct presentation to CD8+ T cells. Furthermore we will test whether the keen ability of SCT to expand CD8+ T cells ex vivo results from impaired interactions with inhibitory receptors and/or less dependency on costimulation. In addition we will exploit the unique ability of SCT to elicit MHC-restricted antibodies to raise mAbs to disease-relevant class I/peptide complexes. Such mAb will be invaluable for quantifying expression of specific class I/peptide complexes during disease progression and determine how this correlates with the activation and effector function of CD8+ T cells against viruses and tumors.
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