The motivation behind the work was elegant in its simplicity. Somatic mutations in the CALR gene produce oncogenic calreticulin variants that drive certain myeloproliferative neoplasms by binding the thrombopoietin receptor MPL and activating it without ligand. These variants lose their KDEL endoplasmic reticulum retention signal, allowing them to be secreted or displayed on the cell surface. Because calreticulin was believed to be a lectin-like chaperone within the peptide loading complex of MHC class I, and because previous reports had documented decreased MHC class I surface expression, defective peptide loading, and an altered presented repertoire in cells expressing oncogenic calreticulin, the authors hypothesized that oncoCALR might be a uniquely sinister oncogene, one that simultaneously induces malignant transformation and immune evasion by corrupting the very machinery the immune system uses to detect it.

To test this hypothesis, the team engineered the murine pro-B cell line Ba/F3, derived from an H-2Kb-negative C3H mouse strain, to express murine MHC class I molecules fused to a mCherry reporter carrying ovalbumin-derived peptides of high, medium, and low binding affinity. These target cells, carrying either wild-type calreticulin, a calreticulin knockout, heterozygous or homozygous oncogenic mutations, or deletions of exon 9, were co-cultured with OT-I T cells that recognize the ovalbumin peptide in the context of H-2Kb. Flow cytometric assessment of the T cell activation marker CD69 after 6 and 24 hours revealed no defects at any time point in any mutant group. Even removal of the N-linked glycosylation sites N86 and N176 from H-2Kb, which should abolish the lectin-mediated interaction between calreticulin and the nascent MHC class I molecule, left antigen recognition by T cells entirely intact.

The researchers then extended their analysis to primary cells. Haematopoietic stem cells harvested from mice carrying oncogenic calreticulin or a calreticulin knockout in their haematopoietic system were lentivirally transduced to present the ovalbumin peptide by their endogenous H-2Kb complex and co-cultured with OT-I T cells for 72 hours. T cell activation, measured as the percentage of CD8-positive CD25-positive cells, and target cell killing were indistinguishable across all genotypes. The team did confirm previous reports of lower surface H-2Kb expression in homozygously oncoCALR-expressing and calreticulin-deficient cells, but this quantitative reduction did not translate into any functional impairment, consistent with the observation that even a single MHC class I molecule can trigger a T cell response through the MHC/TCR interaction.

The most ambitious test came in vivo. The researchers infected mice carrying heterozygous or homozygous oncogenic calreticulin, a calreticulin knockout, or wild-type calreticulin in their haematopoietic system, or in the liver under an AlbCre promoter, with 2 million focus-forming units of lymphocytic choriomeningitis virus strain Armstrong. Eight days post-infection, at the peak of the adaptive immune response, flow cytometric analysis of blood and spleen revealed no significant differences in the relative abundance of effector T cells or LCMV-specific effector T cells across genotypes. Cytokine production of splenic CD8-positive T cells restimulated with the LCMV gp33 peptide was unaltered in calreticulin knockout cells and, remarkably, significantly increased in heterozygous oncoCALR cells, while the cytotoxic protein granzyme B remained steady in all groups. The absence of calreticulin or the presence of its oncogenic variant did not compromise the antiviral T cell response in any organ system examined.

Finally, the team turned to human samples. CD34-positive haematopoietic stem cells were isolated from ten HLA-A02:01-positive and oncoCALR-positive myeloproliferative neoplasm patients and eight HLA-A02:01-positive healthy control donors. These target cells were co-cultured with a Jurkat T cell line engineered to express a chimeric antigen receptor recognizing the endogenous WT1-derived HLA-A*02:01-restricted peptide RMFPNAPYL. WT1 was selected as the antigen because it is equally expressed by healthy donor and patient-derived CD34-positive stem cells. CAR Jurkat activation, measured by CD69 expression after 48 hours, showed only a marginally lower response to patient-derived cells, and the researchers could find no differences in HLA surface expression, WT1 antigen levels, or expression of the T cell-suppressing ligand PD-L1 that would explain this slight decrease. MHC class I function in oncoCALR-expressing human cells was intact or, at most, minimally impaired.

Having failed to demonstrate a pivotal role for calreticulin in MHC class I functionality, the researchers sought to identify what calreticulin actually does at the proteome level. They performed tandem mass tag mass spectrometry on primary murine neutrophil granulocytes and cytotoxic T cells from calreticulin knockout, homozygous oncoCALR, and wild-type mice, restricting their analysis to proteins of the secretory pathway that could potentially interact with the endoplasmic reticulum-based chaperone. Five proteins were absent from samples of both test genotypes and both cell types: acyloxyacyl hydrolase, eosinophil peroxidase, neutral alpha-glucosidase AB, myeloperoxidase, and phospholipidase B-like 1. The abundance of murine MHC class I, H-2Kb, remained steady in the proteome despite its decreased surface expression, likely because the molecule accumulates in the Golgi apparatus rather than being degraded. These findings establish calreticulin as an indispensable chaperone for a specific subset of secretory pathway proteins, but not for MHC class I.

How, then, did calreticulin’s role in MHC class I function become so massively overestimated? The authors point to the history of its discovery. Calreticulin was found associated with MHC class I through co-immunoprecipitation assays from human cell lines in 1996 and later identified as part of the peptide loading complex by cryo-electron microscopy of affinity-purified extracts. The researchers argue that calreticulin, as a lectin chaperone capable of interacting with any glycoprotein bearing a Glc1Man9GlcNAc2 oligosaccharide, likely contaminates affinity purification-based analyses nonspecifically. Indeed, calreticulin is a member of the so-called CRAPome, a repository listing binding partners frequently found in co-immunoprecipitation and mass spectrometry protein interaction studies. Calreticulin may maintain a physical association with MHC class I, but this interaction does not influence its function. Moreover, some HLA alleles have been reported to be completely independent of the entire peptide loading complex.

The implications of this work are considerable. For patients with myeloproliferative neoplasms, the finding that MHC class I functionality is intact in oncoCALR-expressing cells encourages the feasibility of cancer vaccination approaches that previous studies had abandoned, claiming defects in antigen presentation as a cause. The identification of myeloperoxidase and other antibacterial immune proteins as calreticulin-dependent chaperone clients suggests that patients carrying oncogenic calreticulin mutations may harbor a subtle immune defect in the antibacterial response, an area that warrants further investigation. Calreticulin itself remains biologically essential: pan-calreticulin-deficient mice are not viable due to a defect in cardiogenesis, and female mice expressing oncogenic calreticulin homozygously in their haematopoietic system do not breed. The functional role of calreticulin in the peptide loading complex, the authors conclude, now needs to be redefined and its significance reevaluated, a conclusion that will force immunologists to reconsider a molecular relationship they had taken for granted since the mid-1990s.

Subject of Research: The role of calreticulin and its oncogenic variants in MHC class I antigen presentation and T cell immune responses

Article Title: Haematopoietic Cells Deficient for Calreticulin or Expressing Its Oncogenic Variants Maintain Functional Antigen Presentation

Article References: Höhrhan, M., Antonio‐Herrera, L., Colaço, H. G., Wagner, A., Unterwurzacher, M., Varga, C., Kratochwill, K., Bergthaler, A., & Kralovics, R. (2026). Haematopoietic Cells Deficient for Calreticulin or Expressing Its Oncogenic Variants Maintain Functional Antigen Presentation. Journal of Cellular and Molecular Medicine, 30(19), Article e71399. https://doi.org/10.1111/jcmm.71399

Image Credits: AI Generated

DOI: 10.1111/jcmm.71399

Keywords: calreticulin, MHC class I, antigen presentation, myeloproliferative neoplasms, oncogenic CALR mutations, T cell response, peptide loading complex, immunopeptidomics, cancer vaccination, chaperone biology, Haematopoietic, Cells