Fu, H.; (2007) Functional role of calreticulin in MHC class I antigen presentation. Doctoral thesis , University of London.

Preview

Text U592866.pdf Download (9MB) | Preview

Abstract

MHC class I antigen presentation to cytotoxic T cells (CTLs) is critically important to maintain immunity against pathogens and tumour-transformed cells. In this conventional class I pathway, peptides generated from endogenous proteins in the cytosol by proteasomes are transported into the ER lumen and loaded onto class I heavy chain- p2microglobulin heterodimers with the assistance of peptide loading complex (PLC) to form MHC class I complexes, which then traffic to the cell surface for the inspection by CTLs. The PLC contains transporter associated with antigen processing (TAP), tapasin, calreticulin (CRT), ERp57, and possibly calnexin. CRT is a glycoprotein chaperone and can be divided into three structural and functional domains: N-terminus domain, central P domain and C-terminus domain. CRT plays a critical role in MHC Class I antigen processing and presentation and can elicit peptide-specific CD8+ T cell responses against tumours when exogenously administered with peptides. However, how CRT contributes to class I antigen presentation and the mechanism of its adjuvant effect in anti-tumour responses remain to be elucidated. To study the conventional MHC class I pathway, the protein antigen of interest has to be expressed inside cells. First, a novel method was described to deliver protein antigen directly into the cell cytosol. Mouse fibroblasts, lymphoma cell line RMA and its mutant counterpart cell line RMA-S were permeabilized with streptolysin O (SLO) first and chicken ovalbumin (OVA) was successfully introduced into both adherent and suspension cells. The OVA was properly processed and OVA-derived peptide OVA258- 265 (or SIINFEKL) was successfully presented via MHC class I molecule (Kb) to SIINFEKL- Kb specific T cell hybridoma-B3Z, while in RMA-S cell with TAP-2 deficient, the defect for MHC class I antigen processing was demonstrated. Next, using the SLO antigen delivery system, OVA was introduced into the cytoplasm of both CRT deficient mouse fibroblasts (K42) and CRT competent cells (K41), and K42 cells transfected with full length human CRT to investigate how CRT contributes to class I antigen presentation and the mechanism of its adjuvant effect in anti-tumour responses. It was showed that reduced class I expression in CRT deficient cells can be restored by the direct delivery of peptides into the ER or by incubation at low temperature. CRT deficient cells exhibit a TAP deficient phenotype in terms of class I assembly, without loss of TAP expression or functionality. Furthermore, a higher concentration of antigen in the cytosol is required for specific T cell stimulation in CRT deficient cells than that in wild type cells. The data indicates that CRT has a functional role in the maintenance of the low peptide concentration threshold required in the ER for efficient antigen presentation to T cells. Finally, to investigate the functional role of CRT related to its structure and the possible mechanism of CRT lowering the peptide concentration threshold required in the ER for efficient class I antigen presentation, I have made a series of constructs containing P-, NP-, PC-domains and full length CRT and established their expression in both K42 and K41 cells. While full length CRT restores MHC class I surface expression on K42 cells to the levels seen in the wild-type K41 cells, the expression of P-, NP-, PC-domains of CRT down-regulates MHC class I expression in K42 cells. In contrast, K41 cells transfected with truncated CRT exhibits a normal class I surface expression, indicating that disruption to MHC class I assembly only happens in the absence of CRT. In further experiments, I have demonstrated that the defect is due to empty or peptide suboptimally loaded class I complex while direct delivery of peptides into the ER or incubated at 26 where class I stabilized the class I surface expression recover to nearly normal level. After excluding several possible defects during antigen processing and presentation, such as negative effects on expression of components of PLP, TAP function, class I assembly, I propose that calnexin may compensate for the loss of CRT function in cells without CRT. The expression of truncated forms of CRT in CRT deficient cells might block the redundant function of calnexin, leading to further reduced cell surface MHC class I expression.

Download activity - last month

Export as XMLCSVJSON

Download activity - last 12 months

Export as XMLCSVJSON

Downloads by country - last 12 months

Export as CSVXMLJSON

Archive Staff Only

View Item