Defective Human Leukocyte Antigen Class I-associated Antigen Presentation Caused by a Novel β2-Microglobulin Loss-of-function in Melanoma Cells

Chien-Chung Chang,Janine L. Oliver,Soldano Ferrone,Galina V. Yamshchikov,Takeshi Ogino,Craig L. Slingluff,Nobuyuki Bandoh,David W. Mullins

doi:10.1074/jbc.m511525200

Chien-Chung Chang, Janine L. Oliver + Show 6 more

Open Access

https://doi.org/10.1074/jbc.m511525200

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Abstract

The major histocompatibility complex class I molecules consist of three subunits, the 45-kDa heavy chain, the 12-kDa beta(2)-microglobulin (beta(2)m), and an approximately 8-9-residue antigenic peptide. Without beta(2)m, the major histocompatibility complex class I molecules cannot assemble, thereby abolishing their transport to the cell membrane and the subsequent recognition by antigen-specific T cells. Here we report a case of defective antigen presentation caused by the expression of a beta(2)m with a Cys-to-Trp substitution at position 25 (beta(2)m(C25W)). This substitution causes misfolding and degradation of beta(2)m(C25W) but does not result in complete lack of human leukocyte antigen (HLA) class I molecule expression on the surface of melanoma VMM5B cells. Despite HLA class I expression, VMM5B cells are not recognized by HLA class I-restricted, melanoma antigen-specific cytotoxic T lymphocytes even following loading with exogenous peptides or transduction with melanoma antigen-expressing viruses. Lysis of VMM5B cells is restored only following reconstitution with exogenous or endogenous wild-type beta(2)m protein. Together, our results indicate impairment of antigenic peptide presentation because of a dysfunctional beta(2)m and provide a mechanism for the lack of close association between HLA class I expression and susceptibility of tumor cells to cytotoxic T lymphocytes-mediated lysis in malignant diseases.

Highlights

These processes are dependent on a functional antigen processing machinery (APM), which includes the proteasome subunits, the peptide transporters TAP1 and TAP2, and a number of ER-resident chaperons such as calnexin, calreticulin, ERp57, and tapasin [2, 3]. ␤2m plays an integral part in the assembly and transport of human leukocyte antigen (HLA) class I molecules because it stabilizes the HC-␤2m heterodimer through noncovalent protein-protein interactions, thereby allowing binding of endogenous antigenic peptides with the help of TAP and tapasin [4]
Marked HLA Class I Down-regulation on VMM5B Melanoma Cells— Fluorescence-activated cell sorting analysis of cells stained with mAb W6/32 showed that HLA class I molecules were barely detectable on melanoma cells VMM5B as compared with autologous VMM5A melanoma cells (Fig. 1A)
The HC-␤2mC25W dimer does not appear to constitute a conventional peptide-receptive conformation because neither exogenous nor endogenous peptides can be presented on it, as indicated by the lack of induction of lysis by the cognate cytotoxic T lymphocytes (CTLs). These findings represent the first example of a ␤2m structural abnormality that does not cause total HLA class I loss but causes defects in antigen presentation associated with their down-regulation

Summary

Introduction

This possibility is supported by the restoration of the staining with mAb W6/32 following loading of low pH-treated VMM5B cells with wild-type ␤2m along with a HLA-A2-binding peptide (HER-2/neu369–397, KIFGSLAFL, t1⁄2 ϭ 481.2 min). To determine whether the low level of HLA class I molecules on the membrane of VMM5B cells is caused by a reduced level of all HCs and/or by a defect in ␤2m, HC and ␤2m expression in VMM5A and VMM5B cell lysates were analyzed by Western blotting.

Results

Conclusion