Poster session 1Roles of calreticulin and its mutants associated with MPN disorders in immune responses

MHC class I-β2m heterodimers (MHC-I) in the ER are typically loaded with peptides derived from cytosolic proteolysis, facilitated by the peptide-loading complex (PLC). Calreticulin (CRT) is an ER-localized lectin chaperone that is also an established member of the PLC. CRT is comprised of an N-terminal lectin domain, a proline-rich central P domain, and a negatively charged C-terminal domain (CTD) that also contains an ER-retention motif. Somatic frameshift CRT mutations (CRT-FS) have been shown to be associated with myeloproliferative neoplasms, a type of chronic, incurable blood tumor that can transform to acute myeloid leukemia. All mutations generate a common novel C-terminal domain that lacks the ER-retention signal and renders it positively charged. To study the effect of this mutation on antigen presentation by MHC-I, we first generated CRT-null HEK293T and found reduced surface MHC-I levels and association of MHC-I with low-affinity pep- tides. This phenotype was rescued by expression of CRT but not CRT-FS. CRT-FS was found to be secreted and no interaction with the PLC was detected, along with reduced recruitment of MHC-I. On retaining CRT-FS in the ER, it was found to associate with the PLC but did not restore MHC-I interaction with the PLC or surface MHC-I levels. Based on biochemical analysis and analysis of the PLC structure, we hypothesize that the altered C-terminal domain of CRT affects protein-protein interactions in the PLC that that render the complex ineffectual. Thus, the downregulation of MHC-I in CRT-FS-expressing tumor cells allows them to evade immune surveillance. We have used a patient-derived MPN cell line to generate cell lines lacking CRT expression, expressing CRT or CRT-FS, and have recapitulated the phenotypes reported above. These cell lines will be used to identify tumor-associated antigens and to investigate how extracellular CRT and CRT-FS influence innate immune responses, thus identifying potential immunotherapy targets.