Original ArticlesEndogenous and exogenous factors contributing to the surface expression of HLA B27 on mutant APC
Introduction
The assembly of class I human leukocyte antigen (HLA) molecules involves a number of intracellular chaperones [1]. In normal antigen presenting cells (APC), nascent HLA class I heavy chain is targeted to the endoplasmic reticulum (ER) and is initially stabilized by interacting with the chaperone calnexin. Once β2-microglobulin (β2m) associates with the class I heavy chain, calnexin is exchanged for another ER resident molecule, calreticulin, leading to a further association of this complex with the recently identified protein ERp57 2, 3. The HLA class I heterodimer, in association with this complex of ER proteins, is then bound by a specialized chaperone called tapasin. Tapasin is a 48-kDa glycoprotein, believed to approximate peptide receptive class I heterodimers to the transporter associated with antigen processing (TAP) molecules [1]. The TAP molecules are responsible for translocating antigenic oligopeptides from the cytosol into the lumen of the ER. Thus, tapasin co-ordinates the delivery of properly conformed and peptide receptive HLA class I molecules to the site of peptide translocation, facilitating the loading of antigenic peptides into the binding cleft of class I HLA molecules. Once loaded with an appropriate antigenic peptide, the trimeric class I heavy chain/β2m/peptide complex traverses the ER-golgi apparatus, undergoes further glycosylation and is expressed on the surface of APC. These mature class I HLA complexes are then subjected to scrutiny by CD8+ T lymphocytes.
Studies utilizing mutant APC have revealed polymorphism in the surface expression of HLA A and HLA B gene products, reflecting their differential dependence on chaperone and TAP molecule function. One such cell line is T2, which was derived from a fusion of 721.174 B lymphoblastoid and CEM T cells [4]. T2 is hemizygous for the 721.174 haplotype containing HLA A∗0201, HLA B∗5101, and HLA Cw∗0102 and does not contain genes encoding for class II HLA, LMP and TAP proteins. The phenotype of T2 is similar to 721.174, with reduced surface levels of HLA A2 (approximately 30% of normal) and low levels of HLA B51 despite adequate intracellular synthesis of this molecule [5]. The lack of high levels of surface expression of the endogenous HLA B51 in T2 is due to a defect in antigen presentation, resulting from the deletion of the TAP molecule in this cell line. The expression of HLA A2 in T2 is predominantly a result of the presentation of signal sequence peptides 6, 7, derived from proteins destined for the ER and secretory pathways. These peptides are generated by signal sequence peptidase following translocation of the polypeptides through the signal recognition pore, a structure distinct from the TAP complex. HLA class I genes transfected into T2 display varied levels of surface expression 4, 8, 9, 10, 11, reflecting the stability of the class I alleles and their differential ability to bind to peptides delivered via alternative pathways to TAP.
Another example of altered class I expression is seen in the X-irradiation mutant 721.220, a cell line generated by multiple rounds of mutagenesis of the 721.45 B-lymphoblastoid cell line (LCL) [12]. Both HLA A and B are deleted in 721.220 and its sister cell 721.221 and a defect in tapasin has recently been elucidated in 721.220 [13]. The genetic lesion in the tapasin gene expressed in 721.220 is the result of a single nucleotide substitution in the 5′ splicing site for exon 2. Mature tapasin transcripts in 721.220, therefore, have exon 1 ligated directly to exon 3, resulting in translation of a polypeptide product which lacks 49 of the N-terminal amino acid residues and the last 8 residues of the signal peptide. This product is poorly expressed and inefficiently translocated into the ER yet still interacts with the TAP heterodimer [13]. The lack of the 49 N-terminal amino acids of the mature polypeptide, however, ablates interaction with class I heavy chain. Thus, the 721.220 cell line provides a means to delineate the role of the tapasin-class I interaction from other functions of tapasin, such as its interaction with the TAP heterodimer and peptide translocation. In contrast, antigen processing in 721.221 appears to be normal.
Expression of mature class I HLA molecules in the 721.220 cell line appears to be more complex than the case for T2. Studies that have examined introduced class I molecules have revealed significant polymorphism in the dependence of class I-tapasin interaction for the surface expression of given alleles. Grandea et al. 14, 15 have demonstrated that several class I alleles are expressed at significantly reduced levels in this mutant APC. The alleles tested included HLA A1, B8, B35, HLA C, and HLA G and all of were expressed, typically, at around 20–25% of the level observed when these same genes were transfected into non-defective APC such as C1R [15]. In contrast, HLA A2 was expressed at considerably higher levels when introduced into 721.220 (50–60% of levels observed in C1R.A2) suggesting this allele is relatively stable in the tapasin defective cell line [15].
Peh et al. [16] examined the expression of three HLA B alleles in 721.220 cells. In addition, the ability of these alleles to present viral epitopes delivered via a recombinant vaccinia virus (rVV) was compared to 721.220 transfected with HLA B8, B27 or B44 and co-transfected with human tapasin. HLA B27 was expressed independently of tapasin and CTL recognition of these cells was similar whether tapasin function was reconstituted or not (see below for further discussion). HLA B8 was expressed at levels around five-fold lower in the absence of tapasin compared to the same construct expressed in cells co-transfected with hTsn. The lysis of B8 transfected 721.220 cells by viral specific CTL was correspondingly lower (approximately 30%) compared to the same cell line super-transfected with the human tapasin gene. Much lower levels of surface expression were observed for HLA B44 transfected into 721.220 cells resulting in non-detectable B44-restricted killing by viral specific CTL. Super-transfection of hTsn into these cells restored normal surface expression and CTL recognition of HLA B44 [16].
In analogous studies, HLA A∗0201 was shown to present TAP-dependent viral epitopes to CTL when transfected into 721.220, reflecting the ability of this molecule to load TAP-translocated peptides in the absence of tapasin-mediated co-localization [17]. Importantly, HLA A2 introduced into the 721.220 cell line was not as efficiently loaded as the same molecules expressed in the tapasin competent 721.221 cell line. When these molecules were examined biochemically, pulse-chase experiments revealed a significant pool of suboptimally loaded A2 molecules egressing to the surface of tapasin deficient 721.220 cells [17]. These observations imply a role for tapasin in securing optimally fitting peptides for loading into class I molecules. Interestingly, Lehner et al. [18] have shown that introduction of a soluble tapasin gene, which encoded for the lumenal N-terminal domain of the mature protein, fully restored HLA B8 expression in 721.220. HPLC analysis of radiolabeled peptides eluted from B8 purified from 721.220 transfectants revealed a relatively normal peptide profile suggesting that the soluble tapasin had restored the peptide repertoire of HLA B8. These observations indicate that stabilization of class I molecules by interaction with the N-terminal domain of tapasin was more critical than the co-localization of the class I molecules to TAP, a function normally mediated through the deleted C-terminal regions present in wild type tapasin. The authors suggest that tapasin may, therefore, be critically involved in the stabilization and loading of peptides into class I molecules rather than simply a means of co-localizing these molecules to the TAP heterodimer [18].
As described, HLA B27 is stably expressed on the surface of 721.220 cells [16] with comparable surface levels of HLA B27 observed on 721.220 cells transfected with B27 (B27.220) or co-transfected with B27 and human tapasin (B27.220.hTsn). Furthermore, HLA B27 restricted viral killing of B27.220 targets was comparable to that of B27.220.hTsn cells when a rVV containing the viral epitope was used to infect these cells. Some reduction in the relative killing of B27.220 relative to B27.220.hTsn cells was observed if the cells were infected for shorter periods with the rVV prior to the CTL assay. This observation suggested that the assembly of HLA B27 with viral peptides was not fully tapasin-independent and was less efficient in absence of functional tapasin. This study also examined the conformation of HLA B27 using flow cytometry and a restricted panel of B27-specific monoclonal antibodies (mAbs). Although staining with the mAbs ME1 and HLA-ABCm3 was very similar for B27.220 and B27.220.hTsn, the mAb B27M1 was able to differentiate between the B27 molecules expressed on the surface of these two transfectants [16]. Increased expression of “free” heavy chain (HC-10 staining) was also detected on B27.220 cells compared with B27.220.hTsn [16].
Here we examine the nature of surface HLA B27 expression more closely by extending the range of mAbs used to probe the conformation and peptide repertoire of HLA B27 expressed in the presence and absence of wild type tapasin. In addition, we examined the potential role of exogenous factors, present in the culture media, on the tapasin-independent expression of HLA B27. Because TAP molecules are functional in 721.220, we examined the contribution of TAP-independent determinants by examining the expression of HLA B27 in the T2 cell line. Finally, the stability of the B27 molecules expressed on 721.220 transfectants was studied by following the cell surface decay of the complexes over time.
Section snippets
Cell lines and culture
The 721.220 is a human LCL in which HLA A and B genes have been deleted and a non-functional tapasin protein is expressed [13]. The transfection of HLA B∗2705 and human tapasin into 721.220 has been described previously [16]. The T2 cell line is a TAP-negative cell line and transfection with B∗2705 is reported elsewhere [19]. The EBV transformed B-LCL Jesthom is a B cell line homozygous for HLA A∗0201, HLA B∗2705, and HLA Cw1 [20]. All cells were grown in RPMI-1640 (Life Technologies,
Results and discussion
We have reported previously that the HLA B27 specific mAb, B27M1, differentially stains HLA B27 molecules expressed on the surface of cells with and without functional tapasin expression [16]. Other B27-specific mAbs previously tested stained these cells with comparable fluorescence intensity, suggesting that similar steady state levels of HLA B27 were expressed in the presence and absence of tapasin. We have extended the range of B27-specific mAbs used to screen the 721.220 transfectants. The
Acknowledgements
The authors thank the following colleagues for gifts of mAbs; Dr. Jack Strominger for MARB4, Dr. Richard Raybourne for Ye-2 and B27M2, and Dr. Xiaoning Xu for HC-10. This work was supported by grants from the National Health & Medical Research Council of Australia, the Australian Kidney Foundation, the J.H. and J.D. Gunn Medical Research Foundation, Clive and Vera Ramaciotti Foundation, the Rebecca L. Cooper Medical Research Foundation, and the Arthritis Foundation of Australia. A.J.K. was the
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