Abstract A060: The effects of tumor-associated calreticulin mutants on antigen presentation

Abstract

The phenomenon of antigen processing and presentation aids the immune system in distinguishing between normal and malignant cells. In cancer cells, Major Histocompatibility Complex (MHC) proteins present tumor- associated antigens (TAAs) that may serve as targets of immunotherapy. Antigen presentation by MHC class I-β2m heterodimers (MHC-I) requires peptide association to occur in in the ER. Binding is facilitated by the Peptide Loading Complex (PLC), which consists of the TAP transporter, tapasin, the thiol-oxidoreductase ERp57, and a lectin chaperone, calreticulin (CRT). CRT associates with MHC-I via the single, conserved, glycan present on MHC-I, drawing it into the PLC to facilitate peptide loading.

CRT is comprised of an N-terminal lectin domain, a proline-rich central P domain, and a negatively charged C-terminal domain (CTD) that is also contains an ER retention motif. Two recent studies report a mutant CRT associated with myeloproliferative neoplasms (MPNs). These MPNs carry a somatic frame-shift CRT mutation (CRT_FS) that generates a novel CTD that lacks the ER-retention signal and renders it positively charged. Previous studies have shown that ablation of CRT expression in mouse embryonic fibroblast cells result in decreased surface expression of MHC-I, which is reversed on reconstitution of full-length CRT. Interestingly, reconstitution of CRT lacking the CTD did not restore MHC-I expression, indicating a role for the CTD in MHC-I expression. We therefore suspect that antigen presentation in MPNs carrying the CRT_FS is altered.

To address the role of CRT_FS in antigen presentation in a human cell line, we have generated a CRT knockout of HEK293T cells using CRISPR/Cas9 technology. The CRT^−/− cells exhibit reduced surface levels of MHC-I. We used these calreticulin-deficient cells to generate stable cell lines expressing CRT or CRT_FS. We find that expression of CRT restores surface MHC-I, but expression of CRT_FS does not. We are currently assessing the reason behind the alterations in MHC-I expression in these cell lines using various biochemical techniques. Future experiments include analysis of the peptide repertoire displayed on the surface of these cells as well as changes in the molecular interactions of the mutant CRT with other members of the PLC. After a preliminary study in HEK293T cells, we will study the effect of CRT_FS in antigen presentation in MPN cell lines to identify unique TAAs to facilitate the specific targeting of these cells by immunotherapy.

Citation Format: Najla Arshad, Peter Cresswell. The effects of tumor-associated calreticulin mutants on antigen presentation [abstract]. In: Proceedings of the Second CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; 2016 Sept 25-28; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(11 Suppl):Abstract nr A060.