Non-canonical epitope presentation and antigen processing by MHC-E

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Project Summary In the course of developing cytomegalovirus (CMV) as a new vaccine platform for eliciting effector differentiated T cell immunity, we observed that rhesus CMV (RhCMV) expressing simian immunodeficiency (SIV) antigens elicit immune responses that control and ultimately clear highly pathogenic SIV. Surprisingly however, protection was only observed with genetically modified vectors eliciting CD8+ T cells restricted by non-polymorphic, highly conserved MHC-E instead of classical MHC-I. Targeting HIV peptides presented by HLA-E thus represents a novel, unexpected and unconventional approach to AIDS vaccine development that has recently entered the clinical phase. However, we have only a very limited understanding of the molecular mechanisms that render CMV the only vaccine vector capable of eliciting these unconventional responses to any antigen and that render lentivirus-infected cells vulnerable to T cell control. Our findings that MHC-E presents a wide variety of antigens is unexpected because MHC-E predominantly binds the nonameric VMAPRTL(L,I,V,F)L (VL9) self-peptide contained in the cleavable leader sequence of MHC-I. Here, our goal is to elucidate how MHC-E is loaded with diverse non-canonical peptides in uninfected cells or in cells infected with HIV or CMV in vitro and to identify the RhCMV-infected cells and molecular mechanisms required for the priming of MHC-E restricted CD8+ T cells in vivo. These objectives will be accomplished by an international team of investigators with relevant experience. Aim 1 is to use a unique set of MHC-E/peptide specific reagents to monitor MHC-E peptide loading and presentation by myeloid and HIV-infected cells upon inhibiting specific cellular proteins and pathways. Cellular targets will be selected from hits of preliminary CRISPR/cas9 screens or based on their known function in vesicular traffic or peptide loading of classical MHC molecules. In aim 2, we will investigate how the extensive reorganization of intracellular vesicular structures observed in CMV-infected cells contributes to the loading of MHC-E with non-canonical peptides. A particular emphasis will be on the role of viral microRNAs which redirect vesicular traffic by targeting vesicular sorting proteins and which seem to be required for MHC-E restricted T cell stimulation by CMV-infected cells. The role of viral microRNAs as well as selected host pathways for the induction of these T cells in vivo will be examined in aim 3. This will be accomplished by generating recombinant RhCMV lacking microRNAs or expressing host gene-targeting small hairpin RNAs. Since recent results suggest that priming of MHC-E restricted T cells requires infection of myeloid cells expressing micro-RNA142, we will identify the infected cell type and characterize neighboring T cells by combining sophisticated imaging and spatial transcriptomic techniques. We expect that the results of this research will impact basic and translational immunology including the design of novel vaccines and immunotherapeutics.