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

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.

Keywords: antigen presentation; antigen processing; calreticulin; major histocompatibility complex (MHC); myeloproliferative neoplasms; peptide-loading complex; protein export; protein secretion; tumor immunology/immunotherapy.

Figure 1.

Generation and characterization of CRT-null–, CRT-WT–, and CRT-FS–expressing cell lines. A, organization of the calreticulin gene and protein. Nine exons encode CRT, which is composed of an N-terminal lectin domain, a central, proline-rich P-domain, and an acidic CTD that contains an ER retention sequence, KDEL. Insertion and deletion mutants in exon 9 of the calreticulin gene lead to a +1-bp frameshift, resulting in a unique amino acid sequence of the C-terminal tail. B, CRT mRNA levels in CRT-null cells and CRT-null cells transduced with the indicated CRT constructs were assessed by qPCR via normalization to the housekeeping gene β-actin. CRT mRNA expression in HEK293T cells was used to calculate -fold change. C, Western blot analysis of expression of CRT-WT and CRT-FS in cell lysates using antibodies that recognize all forms of CRT (α-CRT) or only mutant CRT (α-CRT-FS). D, secretion of CRT was assessed by incubating cells expressing either CRT-WT or CRT-FS with 10 μg/ml BFA for 8 h, followed by Western blot analysis of CRT expression in cell lysates and culture supernatants. E, cells expressing CRT-FS^DEL or CRT-FS^INS were labeled with [³⁵S]Met for 15 min followed by immunoprecipitation of CRT from the lysates or culture supernatant at the indicated time points of chase, and samples were visualized after separation on nonreducing SDS-polyacrylamide gels by autoradiography. F, interaction of CRT and MHC-I with the PLC in cells expressing CRT-WT or CRT-FS was carried out by immunoprecipitation of tapasin from cell lysates and Western blot analysis of co-immunoprecipitated proteins. #, a nonspecific band in the right panel at 49 kDa. G, MHC-I expressed on the surface of cells expressing no CRT or CRT-WT or CRT-FS was estimated by staining intact cells with the W6/32 antibody followed by FACS. Histograms from G are summarized in bar format. Qualitative data shown are a representation of at least three independent experiments. Quantitative data shown are mean ± S.D. (error bars) of three experiments. Statistical significance was evaluated using the unpaired Student's t test; *, p < 0.05; **, p < 0.01; ***, p < 0.005; ns, not significant.

Figure 2.

The effect of retaining CRT-FS in the ER on peptide loading and surface MHC-I levels. A, Western blot analysis of the expression of CRT in cell lysates. B, Western blot analysis of the expression of CRT-WT and mutants in cell lysates and culture supernatant from control cells and cells treated with 10 μg/ml BFA for 8 h. C, MHC-I expressed on the surface of cells expressing no CRT or all CRT variants was estimated by staining intact cells with the W6/32 antibody followed by FACS. Histograms from C are summarized in bar format. D, the interaction of all CRT variants with the PLC was assessed by immunoprecipitation (IP) of tapasin, followed by Western blot analysis of associated proteins. E, purified GST and GST-fusion CRT proteins from E. coli used in the pulldown assay (left). Lysates from CRT-null cells were incubated with immobilized GST, GST-CRT-WT, GST-CRT^Y92A, and GST-CRT-FS. Associated proteins were analyzed by Western blotting. Qualitative data shown are a representation of at least three independent experiments. Quantitative data shown are mean ± S.D. (error bars) of at least three experiments. Statistical significance was evaluated using the unpaired Student's t test; *, p < 0.05; **, p < 0.01; ***, p < 0.005; ns, not significant.

Figure 3.

Effect of CRT-FS on antigen presentation. A, the delivery of MHC-I to the surface of cells expressing various constructs of CRT was measured by acid-denaturing surface MHC-I at pH 3. 5, followed by neutralization and incubation at 37 °C in the absence (left) or presence (middle) of dynasore. The appearance of assembled, surface MHC-I over time was monitored by FACS using the W6/32 antibody. The activity of dynasore was verified by estimating surface EGFR on cells treated with EGF in the presence or absence of dynasore (right) by FACS. B, the thermostability of surface MHC-I in the various cell lines was assessed by incubating intact cells for 10 min at room temperature in PBS + 0.02% sodium azide, followed by incubation at the indicated temperatures for 10 min. The heat-resistant MHC-I remaining on the surface of cells was estimated by FACS using the W6/32 antibody. C, free heavy chain on the surface of cells at steady state was estimated by staining intact cells with the HC10 antibody followed by FACS analysis. D, free heavy chain on the surface of cells was generated by acid-denaturing surface MHC-I at pH 3.5 followed by neutralization. Levels of free heavy chain were estimated by FACS using the HC10 antibody. E, the stability of MHC-I complexes was also evaluated by a BFA-decay assay. The various cell lines were cultured overnight at 27 °C, treated with 10 μg/ml BFA, and incubated at 37 °C for the indicated periods of time. The decay of surface MHC-I over time was monitored by FACS using the W6/32 antibody. F, LMP1 mRNA levels in the seven cell lines were assessed by qPCR via normalization to the housekeeping gene β-actin. LMP1 mRNA expression in LMP1-expressing WT HEK293T cells was used to calculate -fold change. G, presentation of an Epstein–Barr virus–derived, high-affinity peptide (YLLEMLWRL) was estimated in cells expressing various CRT constructs and the LMP1 protein by staining intact cells with the MHC-I–YLLEMLWRL–specific antibody, followed by FACS. H, surface expression of the MHC-I allele that presents the peptide YLLEMLWRL, HLA-A2, was estimated by staining intact cells with the HLA-A2–specific antibody BB7.2 followed by FACS. Total surface MHC-I was determined by staining intact cells with the W6/32 antibody followed by FACS. Data shown are mean ± S.D. (error bars) of at least three experiments. Statistical significance was evaluated using the unpaired Student's t test: *, p < 0.05; **, p < 0.01; ***, p < 0.005; ns, not significant. Statistical significance in A, B, and E is from comparing data from cells expressing CRT-WT and the other six cell lines.

Figure 4.

Effect of co-expression of CRT-FS and CRT-WT on MHC-I function. A, Western blot analysis of the expression of CRT-WT and mutants in lysates and culture supernatant from HEK293T cells treated or not with 10 μg/ml BFA for 8 h. B, the effect of co-expressing CRT-FS on surface MHC-I levels was measured by staining intact cells with W6/32 antibody followed by FACS. Histograms from B are summarized in bar format. C, the stability of MHC-I complexes was also evaluated by a BFA-decay assay. The various cell lines were cultured overnight at 27 °C, treated with 10 μg/ml BFA, and incubated at 37 °C for the indicated periods of time. The reduction in surface MHC-I over time was monitored by staining intact cells with the W6/32 antibody followed by FACS. Qualitative data shown are a representation of three independent experiments. Quantitative data shown are mean ± S.D. (error bars) of three experiments. Statistical significance was evaluated using the unpaired Student's t test: *, <p 0.05; **, p < 0.01; ***, p < 0.005; ns, not significant. Statistical significance in C is from comparing data from cells expressing CRT-FS^DEL and cells expressing VC.

Figure 5.

Structural analysis of the interaction between CRT and tapasin. A, the structure of the PLC was obtained from the Protein Data Bank (entry 6ENY), and a cartoon representation of the structure of CRT in pink and tapasin in cyan was rendered in UCSF Chimera. The region of interaction between CRT and tapasin has been enlarged on the right. B, surface charge of the rodlike α-helix of CRT and the cradle-like structure of tapasin was calculated using coulombic surface coloring. Red, negative charge; blue, positive charge.

Figure 6.

Effect of tumor-associated CRT mutations on antigen presentation by MHC-I. Peptides derived from the cytosol are transported into the ER lumen by the peptide TAP. Left, the PLC and TAP facilitate peptide loading onto MHC-I. In cells expressing nonmutant CRT, CRT associates with MHC-I via the glucose present on its glycan and also interacts with the core PLC. UGT1 can act on the MHC-I glycan to preserve the glucose and facilitate reassociation with the PLC (a). MHC-I loaded with an appropriate peptide is trafficked via the Golgi (b) to the surface of cells (c). Right, in cells expressing CRT-FS, interaction with the PLC is reduced (A) as CRT-FS is secreted (B), but the resulting PLC is nonfunctional. Interaction of MHC-I with other proteins, such as UGT1, may be disrupted, causing inefficient loading and/or increased degradation of empty MHC-I complexes, resulting in lower surface MHC-I levels and an altered repertoire of peptides presented on the surface of cells expressing CRT-FS (C).