Tumor-associated calreticulin variants functionally compromise the peptide loading complex and impair its recruitment of MHC-I

Abstract

Major histocompatibility complex-I-βm 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 eptideoading omplex (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.

Immune Checkpoint Blockade Enhances Shared Neoantigen-Induced T-cell Immunity Directed against Mutated Calreticulin in Myeloproliferative Neoplasms

“…Even a heterozygous expression of Research. the mut-CALR protein, as is the case with majority of patients with CALR + MPN, has been shown to lead to a significant, albeit small, reduction in surface expression of MHC class I molecules (44). However, we did not observe a reduction in the cell-surface expression of MHC class I molecules in either PBMCs or CD34 + cells from patients with CALR + MPN as compared with HDs.…”

The Expression of Myeloproliferative Neoplasm-Associated Calreticulin Variants Depends on the Functionality of ER-Associated Degradation

“…The low expression level of CALR mutant proteins compared to their WT counterpart was previously reported in numerous studies [18,19,21,22], but the mechanisms involved in this phenomenon have generally not been addressed. Other groups have reported a secretion of CALR variant proteins in the extracellular medium [20,22,34,35]. In these studies, as in ours, CALR variants were detectable in the extracellular medium while the WT form was absent, as seen in Figure 1A.…”

Calreticulin del52 and ins5 knock-in mice recapitulate different myeloproliferative phenotypes observed in patients with MPN

“…b To investigate the effect of CALR del52 and CALR ins5 on HSCs we engrafted different ratios of homozygous del52/del52 (yellow, pink, red, brown and black squares and solid lines) and ins5/ins5 (turquoise, blue, green, dark blue and black squares with solid lines) CD45. that CALR del52 has an immunosuppressive effect 23 and causes a down-regulation of MHC-I endogenous antigen loading and presentation 24 but CALR mutant epitopes were also shown to stimulate CD4 + and CD8 + T cells in patients 25,26 . Importantly, in contrast to JAK2 V617F , which is detected at a low level in lymphoid cells and rarely in T lymphocytes from MPN patients,…”