Abstract
Major histocompatibility complex-I-β2m dimers (MHC-I) bind peptides derived from intracellular proteins, enabling the immune system to distinguish between normal cells and those expressing pathogen-derived or mutant proteins. The peptides bind to MHC-I in the endoplasmic reticulum (ER), and this binding is facilitated by the peptide loading complex (PLC), which contains calreticulin (CRT). CRT associates with MHC-I via a conserved glycan present on MHC-I and recruits it to the PLC for peptide binding. Somatic frameshift mutations in CRT (CRT-FS) drive the proliferation of a subset of myeloproliferative neoplasms, which are chronic blood tumors. All CRT-FS proteins have a C-terminal sequence lacking the normal ER-retention signal and possessing a net negative charge rather than the normal positive charge. We characterized the effect of CRT-FS on antigen presentation by MHC-I in human cells. Our results indicate that CRT-FS cannot mediate CRT's peptide loading function in the PLC. Cells lacking CRT exhibited reduced surface MHC-I levels, consistent with reduced binding of high-affinity peptides, and this was not reversed by CRT-FS expression. CRT-FS was secreted and not detectably associated with the PLC, leading to poor MHC-I recruitment, although CRT-FS could still associate with MHC-I in a glycan-dependent manner. The addition of an ER-retention sequence to CRT-FS restored its association with the PLC but did not rescue MHC-I recruitment or its surface expression, indicating that the CRT-FS mutants functionally compromise the PLC. MHC-I down-regulation permits tumor cells to evade immune surveillance, and these findings may therefore be relevant for designing effective immunotherapies for managing myeloproliferative neoplasms.
Highlights
Major histocompatibility complex-I–2m dimers (MHC-I) bind peptides derived from intracellular proteins, enabling the immune system to distinguish between normal cells and those expressing pathogen-derived or mutant proteins
ERp57 is a thiol-reductase that functions together with calretitransporter associated with antigen processing; CRT, calreticulin; CTD, C-terminal domain; UGT1, UDP-glucosyltransferase 1; CRT-FS, CRT with frameshift mutations; myeloproliferative neoplasms (MPNs), myeloproliferative neoplasm; PV, polycythemia vera; ET, essential thrombocythemia; PMF, primary myelofibrosis; JAK, Janus kinase; STAT, signal transducers and activators of transcription; MPO, myeloperoxidase; BFA, brefeldin A; ER, endoplasmic reticulum; EGFR, epidermal growth factor receptor; guide RNAs (gRNAs), guide RNA; VC, vector control; qPCR, quantitative PCR; GST, glutathione S-transferase; TCR, T-cell receptor
Western blot analysis indicated that whereas CRT-WT was present at levels similar to that of normal HEK293T cells, no CRT-FSDEL and CRT-FSINS was detected by ␣-CRT (Fig. 1C, top)
Summary
Each MHC-I molecule is a heterodimer composed of a transmembrane heavy chain and 2-microglobulin (2m) that is associated with a short 8 –11-amino acid peptide These peptides are derived from cellular proteins and are presented on the surface of cells in a process called antigen presentation [1], which is integral to immune surveillance by cytotoxic CD8ϩ T lymphocytes. ERp57 is a thiol-reductase that functions together with calretitransporter associated with antigen processing; CRT, calreticulin; CTD, C-terminal domain; UGT1, UDP-glucosyltransferase 1; CRT-FS, CRT with frameshift mutations; MPN, myeloproliferative neoplasm; PV, polycythemia vera; ET, essential thrombocythemia; PMF, primary myelofibrosis; JAK, Janus kinase; STAT, signal transducers and activators of transcription; MPO, myeloperoxidase; BFA, brefeldin A; ER, endoplasmic reticulum; EGFR, epidermal growth factor receptor; gRNA, guide RNA; VC, vector control; qPCR, quantitative PCR; GST, glutathione S-transferase; TCR, T-cell receptor. We demonstrate that ablation of CRT expression in human cells causes inefficient peptide loading, leading to reduced levels of surface MHC-I and defective antigen presentation. In cells that co-express both CRT-WT and CRT-FSDEL, a small but significant reduction in surface MHC-I levels was observed
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